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Vitamin I. and Biosynthesis of Protein and Prothrombin

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Vitamin I. and Biosynthesis of Protein and Prothrombin Powered By Docstoc
					                    OF
      THE JOURNAL UIOLOGIC~L       CHEMKWRY
                                              19F8
 Vol. 243,No. 14,Issueof July 25,pp. 3939-3939,
                 P&&d in U.S.A.



Vitamin               I. and              Biosynthesis                      of Protein                 and        Prothrombin”
                                                                                                                (Received for publication,    August l&1967)

               ROBERTA B. HILL,~              SANCIA GAETANI,~ ANNA                   MARIA    PAOLUCCI,$       P. B. RAMARAO,~
               ROSEMARIE ALDEN,              AND G. S. RANHOTRA
               From the Division of Nutritional Biocherristry, University of Illinois, Urbana, Illinois 61803
               D. V. SHAH,~( V. Ii.            SHAH,~[ AND B. CONNOR JOHNSON
               From the Department of Biochemistry, University of Oklahoma School of Medicine and Oklahoma Medical
               ResearchFoundation, Oklahoma City, Oklahoma 73104




                                 SUMMARY                                               gave an essentially normal prothrombin        response.     At higher
    The role of vitamin K in the synthesis of prothrombin                      has     cycloheximide     levels the response to vitamin KI, while much
been examined with the following results.                                              less complete, was clear and definite.
    1. Vitamin K deficiency has no effect on general protein                              6. The response of vitamin K-deficient        rats to vitamin K1,




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 synthesis as studied by amino acid incorporation                  into protein        administered     following   treatment   with blocking       doses of
in vivo and in vitro and by tryptophan                pyrrolase production             puromycin,    appears significant.
following tryptophan feeding.                                                             7. These data appear to indicate that the site of function
    2. Prothrombin        activity was found in normal rat liver                       of vitamin K is not at the genetic level, as has been reported,
microsomes         with increased         release following           ultrasonic       but at a late stage in translation     of prothrombin     messenger
treatment,     but could not be found in microsomes                     from the       RNA to form a functional prothrombin         molecule.
livers of vitamin K-deficient           rats or dicumarol-treated             rats.
Administration        of suboptimal levels of vitamin K to vitamin
K-deficient     rats resulted in detectable microsomal                prothrom-
bin in 2 hours and essentially normal values within 3 hours.
    3. Adequate       vitamin KI, given by injection,                completely            Despite the numerous possible functions which have been de-
restored the blood prothrombin              levels of vitamin K-deficient              scribed for vitamin K in the living cell, e.g., in oxidative phos-
rats in 1 hour, and of dicumarol-              or warfarin-treated         rats in     phorylation     (l-4), in electron transport (5-8), and steroid de-
5 to 7 hours depending               on the amounts of warfarin                and     hydrogenation        (9), the only recognized symptom of vitamin
vitamin K1 given.                                                                      K deficiency in man and experimental                animals is lengthened
    4. Following      administration      of actinomycin       D or ethionine          clotting time of the blood, with subsequent internal hemorrhag-
to vitamin       K-deficient       rats, treatment        with vitamin           K1    ing. The increased clotting time in blood or plasma is due to a
markedly       stimulated       prothrombin        production,        indicating       decreased concentration          of prothrombin      and of certain other
that the site of action of vitamin K is beyond the level of                            clotting factors, e.g. VII (extrinsic), IX, and X. There is no
transcription     of DNA to prothrombin           messenger RNA.                       evidence that vitamin K1 occurs in the prothrombin                molecule
    5. Treatment       with vitamin K1, following              administration
                                                                                        (10) ’
to vitamin K-deficient          rats of cycloheximide          at a level just             Prothrombin       is synthesized in liver parenchymal cells (ll),
sufficient to block synthesis of prothrombin                for 6 to 8 hours,          and upon incubation of a cell-free system from rat liver, its con-
    * These investigations were supported in part by Grants AM                         tent in microsomes has been shown to increase (12, 13). Our
06005 and AM 10282 from the National Institutes of Health and by                       laboratory has been particularly           interested in the problem of
Contract DA-49-193 MD-2830 between the Office of the Surgeon                           vitamin K function, and these studies are a part of a continuing
General, Department of the Army, and the University of Illinois.
The opinions expressed in this publication are those of the authors                    program on the role of vitamin K in prothrombin                 formation.
and not necessarily those of the Army.                                                 Since these clotting factors are proteins, the possibility was first
    $ Present address, Department of Nutrition and Food Science,                       considered that vitamin K was involved generaliy in all protein
University of Kentucky, Lexingt,on, Kentucky 4050G.
    0 Present address, Department     of Nutrition,  University     of                 synthesis, even though the postulation              of Martius and Nitz-
Rome, Rome, Italy.                                                                     Litzow (l), that this was at the level of ATP synthesis, seemed to
    1 Present address, Central Food Technological     Research In-                     be untenable (4, 5). We have investigated the possible involve-
stitute, Mysore-2, India.
    )I Present address, Faculty of Science, Maharaja      Sayajirao,                   ment of vitamin K in general protein synthesis by studying the
University of Baroda, Baroda, India.                                                   incorporation      in &JO and in vitro of radioactive L-(1J4C)-leucine

                                                                                  3930
Issue of July 25, 1968                                            Hill    et al.                                                                    3931

into protein and the induction in vitro of tryptophan pyrrolase             were washed twice with 10% trichloracetic      acid, twice with               hot
(14).                                                                       95% ethanol, once with ethanol-ether       (3 : I), and twice                with
   In view of the specificity of response of vitamin K of only cer-         ether. Weighed amounts (2 to 3 mg) of dried sample                           were
tain blood-clotting     proteins, it was logical to consider the possi-     dissolved in 1 ml of 1 M hyamine hydroxide in methanol at                     50”,
bility that the vitamin might function at the genetic level in the          and 14 ml of scintillation   grade toluene containing 6 g of                 2,5-
specific biosynthesis of the clotting factors (15-19).       Studies on     diphenyloxazole  per liter were added.
prothrombin      activity in normal and vitamin K-deficient rats
after treatment with agents which block protein synthesis at                       Incorporation    in     Vitro of L-(1 J4C)-Leucine     into   Liver
                                                                                                           Microsomal Protein
different levels indicate, however, that vitamin K functions at a
site later than transcription from DNA (20, 21).                               Microsomes and pH 5 enzymes, prepared from the livers of
                                                                            control and vitamin K-deficient     rats, were incubated with L-
                      EXPERIMENTAL     PROCEDURE                            (I-14C)-leucine, following the procedure of Keller and Zamecnik
   Male Sprague-Dawley          rats were fed a vitamin K-deficient         (31). The proteins precipitated      wit,h 10% trichloracetic  acid
diet (22). Controls were given, in addition, 40 pg of the diphos-           were washed twice with hot 10% trichloracetic        acid, hot 95%
phosodium ester of menadione orally, weekly. The deficient                  ethanol, and acetone-ether (1:l) and then dried. The samples
animals were housed in tubular coprophagy-preventing              cages     were subsequently handled like those from the studies of in-
 (23). Plasma was obtained from oxalated blood samples for the              corporation in viva.
determination      of prothrombin    time (24-27) and for the studies
of the incorporation        in vivo of radioactive leucine into blood                 Experiments         Involving     Treatment  with   Protein
proteins.    Animals were killed by decapitation        and all organs                                   Synthesis-blocking Agents
were rinsed twice in ice-cold isotonic solutions of sodium chloride             Vitamin K Treatment Experiments-nn-Ethionine                  and puro-
or sucrose, followed by immediate homogenization          with approxi-     mycin dihydrochloride       were Nutritional      Biochemicals products.
mately 3 volumes of cold 0.25 M sucrose in a Potter-Elvehjem




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                                                                            Warfarin was obtained from K and K Laboratories,                   Inc., New
type homogenizer.         Protein concentrations   were measured by         York, and vitamin K1 from General Biochemicals.                     Actidione
the method of Lowry et’al. (a), except in the incorporation                  (cycloheximide) was supplied by Upjohn Company, Kalamazoo,
studies in viuo, in which total nitrogen was determined by the              Michigan.      Simplastin    was obt’ained from Warner-Chilcott
boric acid modification of the micro-Kjeldahl          procedure (29).      Institute Division, Morris Plains, New Jersey.
Radioactivity      measurements were made in a Packard Tri-Carb                 Control, vitamin K-deficient, and warfarin-treated              rats were
liquid scintillation spectrometer.                                          used to investigate prothrombin      time after treatment with actino-
                                                                            mycin D, ethionine, cycloheximide            (Actidione),     or puromycin.
           Prothrombin     Activity in Rat Liver Microsomes
                                                                            Warfarin-treated     animals were controls receiving intraperitoneal
   Microsomal    fractions were obtained     by centrifugation      of      injections of sodium warfarin (0.1 mg or 1 mg/lOO g of body
homogenates prepared in 0.25 M sucrose from livers of control,              weight) 20 to 24 hours before injection of the protein synthesis-
vitamin K-deficient, and dicumarol-treated     rats, after removal          blocking agent. The doses of actinomycin                D used were 500,
of cell debris, nuclei, and mitochondrial        fractions.     Heavy        1000, and 2000 pg/lOO g of body weight, and those of cyclo-
microsomes were obtained by centrifugation      at 17,500 x g for           heximide were 50, 250, 750, and 1500 pg/lOO g of body weight.
60 min; light microsomes were obtained by centrifugation            at      They were suspended in wat.er, such that the intraperitoneal
105,000 x g for 60 min; “total microsomes” indicates heavy plus             injection never exceeded 0.5 ml.
light. Heavy microsomes were resuspended and recentrifuged                      Normal and vitamin K-deficient rats received injections of
twice in 0.25 M sucrose, while the surfaces of the light and total          ethionine intraperitoneally     in two doses (100 mg/lOO g of body
microsomal pellets were rinsed twice with a sucrose solution.               weight each), one at zero time, and the other 3 hours later. The
Finally, the fractions were suspended in the citrate buffer of              amount administered at each injection was dissolved in warm
Helgeland and Laland (30). After ultrasonic treatment in a                  0.9% NaCl immediately before injection and was injected at
lo-kc ultrasonic oscillator, the suspensions were recentrifuged at           body temperature.        For comparative purposes, in each group
105,000 x g for 60 min before the prothrombin         activities were       two or three rat.s received inject,ions of the same volume of 0.9%
determined in the supernatant.      Results are reported in units           NaCl.
per g of protein in the microsomal fraction used, on the basis that             Puromycin was given to normal and vitamin K-deficient rats
1 ml of standard human plasma contains 100 units, the minimum               at the rate of 20 mg, 30 mg, and 40 mg/lOO g of body weight.
for our procedure being 2.5 units per g of protein.                         Of a freshly prepared solution containing 20 mg of puromycin
                                                                            dihydrochloride     in 0.9% NaCl and brought to pH 5.5 with
        Incorporation     in    Vivo of L-(i-%J-Leucine    into             NaOH, 1 ml was injected intraperitoneally.
                   Proteins    of Organs and Plasma                             Vitamin K1 solutions were prepared by mixing well 50 mg of
    Male rats, 12 vitamin K-deficient and 12 control, were paired           vitamin K1 into 0.6 ml of Tween 80 and bringing the solution to
according to time of birth and weight.     After the intraperitoneal         10 ml wit.h 0.90/, NaCl.        This solution could then be diluted
injection of L-(1-I%)-leucine   (5 C/l00 g of body weight), three           with 0.9% NaCl for lesser concentrations             of vitamin K1. The
animals from the control group and three from the vitamin K-                usual curative dosages used were 40 pg for vitamin K-deficient
deficient group were killed 1, 2, 4, and 6 hours following injection        rats and 100 pg and 1 mg for warfarin-treated            rats, given sodium
of the labeled amino acid. The livers, spleens, kidneys, and                warfarin in doses of 0.1 and 1 mg/lOO g of body weight, respec-
hearts were homogenized in demineralized water.         The proteins        tively.     The vitamin was given at various times (0 to 24 hours)
of the organs and the plasma were precipitated overnight at 2”              after injection of the protein synthesis-blocking              agent, as in-
with equal volumes of 20% trichloracetic        acid. The proteins           dicated in the figures. In some experiments the Quick (24, 27)
3932                                                    Vitamin      K and Biosynthesis             of Protein             and Prothrombin                                        Vol. 243, No.                  14

prothrombin     time assay was used to follow prothrombin          activity                                                                     TABLE         II
over a period of time in the same animal.         The tips of the tails                                         Plasma        and liver      microsomal prothrombin                  activity        in
of the rats were cut off, blood was withdrawn        in a capillary tube,                                                                 dicumarol-treated  rats
the volume was marked and silicone-treated for microprocedures,
                                                                                                                                                                      Plasma pro-                 Tot~aJmm$ro-
and the tails were cauterized.      One volume of blood was trans-                                  No.    of rats    Dicumarol    treatment”    TiTFvEp
                                                                                                                                                                     thrombin        time         prothrombin
ferred quickly into 2 volumes of a thromboplastin-calcium-
sodium chloride mixture (Simplastin)         in a partially hollowed                                -I                                                  hr                  set                   mits/gfiroteinb
glass plate, heated continuously to 37.5”. Time required for the                                           10          None                                                15                         16
clotting in these experiments is expressed as percentage of normal                                          6          Fed for 3 days                                  80->500                         0
 (Figs. 1, 3a, 3b, and 5) based on the Quick prothrombin               time                                 2          Injected                          2             14-14                          16.9”
from control animals (15 f 1 set).                                                                          1          Injected                          6                 14                          9.8
     In those experiments in which the Allington (25) method for                                            3          Injected                         12             25-25-75                        0
prothrombin      time was used, the data are expressed as deter-                                            1          Injected                         24                 61                          0
 mined in set (Figs. 2, 4, 6, and 7). Both methods of expressing                                       a The rats were fed 0.25yo dicumarol in vitamin K-free ration
prothrombin       time have their merits. The partial cures ob-                                     or they received intraperitoneal  injections of a Tween 80 suspen-
 tainable following high levels of blocking agents cannot be clearly                                sion containing 25 mg/lOO g of body weight.
 seen in the “percentage of normal” plots, while they can be seen                                      b After sonic treatment.
 on a direct plot. On the other hand, the complete response of                                         c Pooled livers.
 the vitamin K-deficient animal to vitamin K is most dramatically
 shown on the percentage of normal plot.                                                                                                        TABLE        III
     Radioactive iZmino Acid Incorporation      Experiments-Two           or                        Prothrombin activity in plasma and in total microsomes from livers
 four hours after treatment with actinomycin D or cycloheximide,                                           of vitamin K-dejicient    rats (two rats in each group)  at
normal rats received intraperitoneal         injections of uniformly                                            various times after intraperitoneal    injection of




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 labeled mixed W-amino acids (5 C/100 g of body weight) and                                                                        vitamin K,
were killed 1 hour later. Control animals received the labeled
                                                                                                                                                                                Microsomal       prothrombin
amino acids but no antibiotic.       The proteins of plasma and of                                        Vitamin     KI                                  Plasma                      after ultrasonic
                                                                                                             injected                              prothrombin      time                   treatment
heavy microsomes, prepared in 0.25 M sucrose as previously
 described, were precipitated    with equal volumes of cold 20%                                                 Pg                                            set                      units/g     protein
 trichloracetic acid, containing 25 mg/lOO ml of cold mixed amino                                               10                                            92                            0
acids (Beckman standard calibration         mixture, type 1). After                                             20                                            52                            0
 15 min the samples were centrifuged.        The protein precipitates                                           10                                            20                             2.0,3.0
were then each washed once with 10% trichloracetic               acid con-                                      10                                            16                             4.6, 3.4
taining 25 mg/lOO ml of cold mixed amino acids, 10% trichlor-                                                   20                                            14                            15.7,9.3
acetic acid, water, and absolute ethanol; then twice with ethanol-
ether (3:1), and twice with anhydrous ether. Of the dried                                           phenyloxazolyl)]benzene                  (dimethylPOPOP))                      were          added,         and
samples, 5 to 10 mg, dissolved in 0.2 ml of 2.5 N NaOH, were                                        the vials were quickly                shaken.
pipetted into a scintillation    vial filled with a thixotropic          gel
                                                                                                                                                 RESULTS
powder (Cab-0-Sil, Packard).       Following this, 15 ml of scintillator
 (800 ml of scintillation grade toluene, 200 ml of ethanol, 5 g of                                                                   Microsomal         Prothromlrin
2,5-diphenyloxazole      (PPO), and 0.3 g of 1,4-bis-[2-(4-methyl-5-
                                                                                                        It has been shown that liver microsomal prothrombin              can be
                                                                                                     “completed”     or activated or released by incubation (12) or ultra-
                                       TABLE   I
                                                                                                    sonic treatment (30), and that ultrasonic treatment is the more
Prothrombin           activity in liver microsomes and cell sap before                        and   efficient (13). This apparent “release” of prothrombin                 from
                 after sonic treatment from control and vitamin
                                                                                                    microsomes led us to study the effect of vitamin K deficiency on
                                      K-dejicient       rats
                                                                                                    prothrombin      activity at the microsomal level. These data are
  Average values are given, and ranges are shown in parentheses.                                    given in Tables I through III, in which the designation “0”
                      <a of animalr                       Control               K-deficient         signifies that the prothrombin         activity was unmeasurable in our
                                                                                                    system.
   Cell   fraction                                                           Before         After
                                           Before sonic        After sonic    sonic         sonic
                                                                                                        Heavy microsomes contained more prothrombin             activity than
                        +K      -K          treatment           treatment    treat-        treat-   light microsomes (Table I) and no activity was found in mito-
                                                                              ment          merit
                                                                                                    chondria, in agreement with the results of Goswami and Munro
                                                                                                     (12). No prothrombin         activity could be detected in liver micro-
Total micro-            15        5         (4.7813.4)         (11.:!23.9)      0                   somes from vitamin K-deficient rats.
     somes                                                                                              Prothrombin     activity was not found in microsomes from rats
  Heavy                  6        5         (7.2!%2)           (17.z7.3)        0                   fed a dicumarol-containing         diet for 3 days (Table II).      Twelve
                                                                                                    hours after an injection of dicumarol            (25 mg/lOO g of body
  Light                  2        3                 6          (l&2.2)          0                   weight) to control rats, microsomal prothrombin              activity was
                                                                                                    unmeasurable,      while that of plasma had decreased to 30% of
Cell sap                                            5                           0
                                                                                                    normal.      Plasma prothrombin         levels thus decreased as micro
Mitochondria                                                         0
                                                                                      -I            somal prothrombin        decreased.
Issue of July      25, 1968                                           Hill et al.                                                                                           3933

    The data in Table III show the opposite process. Vitamin                                                       TABLE    IV
 K-deficient rats with plasma prothrombin               times greater than   E$ect of actinemycin D and cycloheximide      on incorporation in vivo
 100 set were given suboptimal quantities of vitamin K1. A dose                      of mixed 14C-amino acids (5 C/l00 g of body weight,
of 40 wg will restore plasma prothrombin            activity of vitamin K-            injected 1 hour prior to sacrijice) into microsomal
deficient rats to normal in 1 hour; doses in 10 and 20 pg require a                     and plasma proteins of vitamin    K normal rats
longer time interval.           Simultaneously     with the decreases in       Figures in parentheses indicate the number of rats.
 plasma prothrombin         times, increases in microsomal prothrombin                                         Incorporation     into              Incorporation           into
 were observed (Table III).                                                                      Dose of            microsomes                              pl~SIllil
                                                                                    Treatment   blocking
     The fact that vitamin K has not been found in the prothrombin                               agentQ
                                                                                                               3 hrs             5 hrs             3 hrs                  5 hrs
 molecule (10) does not eliminate the possiblity that it might be
 specifically responsible for “activating”         or for joining together
 subunits of the protein, or that it might be needed for the syn-
 thesis of the peptide chains or for assembling the molecule in its          Actinomycin          500      114.2      (2)   80.7         (3)   116.8       (2)          80.9      (3)
 entirety.      It is also possible that the vitamin may be required            D                1000       55.4      (1)   66.2         (3)   103.8       (2)          70.1      (3)
                                                                                                 2000       44.1      (1)   61.8         (3)    85.3       (2)          64.2      (3)
 in all protein syntheses (e.g. for ATP synthesis, as suggested by
 Martius and Nitz-Litzow           (1)) but that this effect is observed     Cyclohexi-           750       36 (1)          12.5         (1)     5 (1)                   6 (1)
 in the synthesis of the clotting proteins because of their very               mide              1500       20 (1)          11 (1)               4 (1)                   9 (1)
 rapid rate of turnover.         To study the effect of vitamin K defi-
  ciency on general protein synthesis, the induction of tryptophan              5 Protein synthesis-blocking     agents given 3 and 5 hours prior
 pyrrolase activity by tryptophan             (14) and the incorporation     to sacrifice.
  of L-(l-i4C)-leucine    into proteins, both in tivo and in z&o, were          b Controls (3 rats) did not receive any antibiotic.   Mean numer-
 examined.                                                                   ical values for l-hour incorporations     were 377 cpm per mg of pro-
                                                                             tein for microsomes and 300 cpm per mg of protein for plasma.




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                       Amino Acid Incorporation
    When rats were injected with L-(1-14C)-leucine, the radioac-             normal rats at zero time and 5 PC of mixed 14C-amino acids were
tivity found per mg of protein in heart, spleen, kidney, liver, and          injected intraperitoneally   1 hour prior to decapitation at 3 or 5
plasma did not differ between vitamin K-deficient animals and                hours, the incorporation   of radioactivity into microsomal protein
controls at any time from 1 to 6 hours after injection of the                was essentially unaltered compared to that in controls not in-
labeled amino acid. Liver microsomes plus pH 5 enzymes (31)                  jected with actinomycin D. At a level of 2000 pg of actinomycin
from vitamin K-deficient rats incorporated         L-(l-14C)-leucine as      D per 100 g of body weight, incorporation       of radioactivity    into
well as did those from control animals, even though the deficiency           both microsomal and plasma proteins was somewhat decreased.
was severe, as shown by plasma prothrombin           times (70 to over       In contrast, injections of cycloheximide at levels of 750 pg and
 120 set).                                                                    1500 pg/lOO g of body weight decreased incorporation          of radio-
    The induction of tryptophan pyrrolase by tryptophan feeding              activity from the mixed 14C-amino acid into plasma proteins to
was also found to be unaffected by vitamin K deficiency, both                less than 10% of the controls and into microsomal proteins to
in the case of intact and adrenalectomized       rats (14).                  low values. These differences are consistent with the site of
    Thus the effects of vitamin K deficiency on protein synthesis            action of actinomycin D in blocking synthesis of RNA (32) and
appear to be confined to prothrombin      and related clotting pro-          of cycloheximide in preventing the growth of nascent polypeptide
teins. Vitamin K-deficient rats respond completely in 1 hour,                chains (33) and peptide bond formation (34).
and warfarin- or dicumarol-treated    rats in 5 to 7 hours, to ade-
 quate injections of vitamin K1 (Fig. lc). Only when there has               Response of Hypoprothrombinemic    Animals     to Vitamin                                  K1 Given
been extensive internal hemorrhaging,     resulting in a marked de-            after Administration   of Protein Synthesis-blocking                                     Agents
 crease in blood cell volume, will the vitamin K-deficient animal                Control Experiments with Normal Animals to Establish Levels
 sometimes fail to respond immediately t.o treatment with vitamin            of Protein Synthesis-blocking Agents Required to Block Prothrombin
 6.                                                                          Synthesis-Fig.     1 gives the data on the effects of actinomycin D
                                                                             and cycloheximide on blood prothrombin        levels. After injection
     Experiments     with Agents Which Block Protein      Synthesis          of actinomycin D into control rats, decreased prothrombin            ac-
   Possible hereditary relationships     between the vitamin K-de-           tivity in blood can be observed. This is shown in Fig. la for
pendent blood-clotting  factors led Olson (15, 19) to postulate that         doses of 500 and 2000 pg/lOO g of body weight of actinomycin D
vitamin K acted as an effector molecule at the genetic level in the           (the curve for doses of 1000 fig lies between the two given). At
synthesis of these blood-clotting    factors. It was reported that           all these levels of actinomycin D, all animals died within 8 to 24
menadione given to vitamin K-deficient           chicks (15, 17) and         hours.
vitamin K1 given to dicumarol-treated       rats (16, 17) were unable            Cycloheximide, since it blocks after mRNA, caused an immedi-
to restore plasma prothrombin       activity after protein synthesis         ate decrease in prothrombin        activity, which reflects the rapid
had been blocked by actinomycin D. The results of our experi-                turnover rate of prothrombin      (21). Blood prothrombin      activity
ments with control, vitamin K-deficient, and warfarin-treated                decreased, essentially linearly, to low levels in about 6 hours
rats, in which protein synthesis has been blocked by actinomycin             following all dose levels of cycloheximide (Fig. lb).      The rate of
D or by cycloheximide (Actidione), are shown in Table IV and                 decrease was only slightly slower following 50 pg of cycloheximide
Fig. 1 through 3.                                                            per 100 g of body weight than that following 750 or 1500 g. All
   From the data given in Table IV, it can be seen that when                 animals that received 50 pg of cycloheximide per 100 g of body
500 pg of actinomycin D were injected intraperitoneally           into       weight recovered and had normal prothrombin           levels 24 hours
3934                                                 Vitamin       K and Biosynthesis              of Protein     and Prothrombin                                 Vol. 243, No. 14

after treatment; at 250 pg all animals lived and prothrombin              re-
coveries, ranging from 30 to 100% of normal, were found 30
hours after treatment, although prothrombin           levels had remained
low for all these rats from the 6th to the 15th hour after treat-
ment. Following        the administration     of 750 and 1500 fig of
 cycloheximide per 100 g of body weight, all animals were dead
 within 6 to 16 hours after treatment.        The toxicity of these pro-
 tein synthesis-blocking     agents is apparently not related to their
 effect on prothrombin     activity, but rather to their general block-
 ing of protein synthesis, since the dead animals showed no
 hemorrhagic symptoms.
     Similar control data on the blocking of prothrombin           synthesis
 in normal rats by ethionine and puromycin are given in Fig. 4,
 Curve A and Fig. 7A, respectively.
     Control Experiments to Establish Recovery Times of Dejkient
 and Warfarin-treated Animals       following  Vitamin      K1 Administra-
 tion-It     was found that 50 mg of sodium warfarin injected per
 100 g of body weight would kill a rat in 20 min without decreasing
 prothrombin activity, yet a rat given 20 mg/lOO g of body weight
 may live 30 hours or longer, while prothrombin                activity de-
creases to less than 1% of normal (prothrombin                  time being
 greater than 180 set). In Fig. lc, prothrombin          curves are shown
 for 13 rats given injections of 1 mg of sodium warfarin per 100 g of




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 body weight, 24 hours before zero time. At zero time, five of the
rats received injections of 1 mg of vitamin K, and the pro-                                                                 I              I              1               I


 thrombin times returned to 100% of normal within 8 hours                                                                  2             4               6               8              I
  (1~5) ; eight rats remained untreated       (1~8). When 0.1 mg of                                                                            HOURS
 sodium warfarin per 100 g of body weight was given to four rats                                       FIG. 2. Effect of vitamin       K1 treatment       on vitamin      K-deficient
 24 hours before zero time, prothrombin        activity did not decrease                           and warfarin-treated         rats.    Curve   A, four vitamin          K-deficient
 below 20% of normal, and 0.1 mg of vitamin K1 restored pro-                                       rats given 40 rg of vitamin         Kt per rat at zero time; Curve B, five
                                                                                                   normal   rats given sodium warfarin           (0.1 mg/lOO     g of body weight)
 thrombin activity to normal levels within 6 hours (1~4). In                                       24 hours before zero time and given vitamin               K, (0.1 mg/lOO           g of
                                                                                                   body weight)      at zero time; Curve C, seven normal          rats given sodium
                                                                                                   warfarin    (1 mg/lOO      g of body weight)       24 hours before        zero time
                   a                             b                           C                     and given vitamin        KI (1 mg/lOO     g of body weight)        at zero time.

                                                                                                   addition, 40 pg of vitamin K1 restored prothrombin           activity
                                                                                                   of vitamin K-deficient rats in 1 to 1s hours (leg), and 500 pg
                                                                                                   of vitamin Ki will act even more rapidly.
                                                                                                      Similar data obtained by the Allington        (25) method of pro-
                                                                                                   thrombin determination      are plotted in Fig. 2 and show again an
                                                                                                   almost complete response to vitamin K1 injection in 1 hour in the
                                                                                                   case of the vitamin K-deficient animals (Curve A), and a much
                                                                                                   more delayed response in the case of animals treated with
                                                                                                   warfarin (Curves B and C).
                                                                                                      Experiments with Hypoprothrombinemic         Animals-With    these
                             IO0                                                                   controls available, it was then possible to study the response of
       0          5                          5           IO 0            5           lo
                                                                                                   vitamin K-deficient or warfarin-treated     rats to vitamin Ki given
                                                                                                   simultaneously or at various time intervals following administra-
    FIG.    1. Prothrombin           activity,      expressed as percentage of
 normal     and also expressed           in set, plotted      against    treatment        time     tion of the different protein synthesis-blocking     agents.
in hours.        a, actinomycin          D was given intraperitoneally                 to con-
trol rats, of which five were given 500 pg/lOO g of body weight                             and          Response to Vitamin          K1 in Presence of Actinomycin               D
five were given 2000 pg/lOO g of body weight.                          b, cycloheximide
was given intraperitoneally                 to control     rats, of which          five were           In Fig. 3a (Curves 1 and 9) it is shown that vitamin K-deficient
given 50 pg/lOO g of body weight,                   10 were given 250 pg/lOO               g of    rats given vitamin Ki, simultaneously with or 3 hours after an
body weight          and 11 were given 750 fig/100 g of body weight.                      c, 10,   injection of 500 pg of actinomycin D per 100 g of body weight,
vitamin      K-deficient      rats, not treated        with vitamin       K.    3, vitamin
K-deficient        rats treated      at zero time with          40 rg of vitamin            K1;    recover their prothrombin          activity.    Warfarin-treated      rats
8, rats given 1 mg of warfarin                per 100 g of body weight               24 hours       given actinomycin D also responded to vitamin Ki (Curves 4-W
before zero time, but not treated                with vitamin         K; 6, rats given 1           and I-W), but less rapidly, corresponding to the time curve for
mg of warfarin           per 100 g of body weight          24 hours before zero time               recovery after warfarin injections shown in Fig. 1~5. Obviously,
and given 1 mg of vitamin               K, at zero time; 4, rats given 0.1 mg of
                                                                                                    100% of normal cannot be reattained since prothrombin           activity
warfarin      per 100 g of body weight              24 hours before         zero time and
given 0.1 mg of vitamin             KI at zero time.           Numbers       on the graph          is slowly lost in control rats given the same quantity of actino-
indicate     the number         of rats.                                                            mycin D. Recovery of prothrombin            activity after injection of
Issue of July 25, 1968                                              Hill   et al.

2000 pg of actinomycin D was also studied because of the results
of incorporation     of mixed 14C-amino acids into microsomal and
plasma protein (Table IV).        From the data on control rats
given 2000 pg/lOO g of body weight of actinomycin D (Fig. 3b,
Curve C-5), it appeared that prothrombin     synthesis was continu-
ing at a rate sufficient to keep blood prothrombin    above 50% of
normal for 6 hours, but that after 6 hours, a sharp break in the
curve occurs presumably due to exhaustion of mRNA for pro-
thrombin synthesis; and thereafter a rapid fall in blood pro-
thrombin,     similar to that seen after cycloheximide,      occurs.




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                                                                                FIG. 4. Effect of vitamin K1 treatment on rats treated with two
                                                                             equal doses of 100 mg of ethionine per 100 g of body weight each.
                                                                             Curve A, seven normal rats given ethionine at 0 and 3 hours;
                                                                             Curve B, five vitamin K-deficient rats given ethionine at 0 and 3
                             HOURS                                           hours and vitamin K1 (40 pg per rat) at 5 hours; Curve C, three
                                                                             vitamin K-deficient rats given ethionine at 0 and 3 hours and
                                                                             vitamin K1 (40 pg per rat) at 24 hours.

                                                                             Nonetheless, as can be seen from Fig. 3b (Curves 5, 2 and S)
                                                             b               vitamin K-deficient rats (5% or less of normal prothrombin     levels,
                                                                             prothrombin    time being greater than 80 set) responded to vitamin
                                                                             Kr by increased blood prothrombin levels up to the control values,
                                                                             even when (Curve S) the vitamin was given 5 hours after the high
                                                                             actinomycin D dose and no further mRNA for prothrombin            ap-
                                                                             peared available.     These data indicate that vitamin K functions
                                                                             after prothrombin    mRNA formation in the regulation of the syn-
                                                                             thesis of active prothrombin.
                                                                                 It has been stated that ethionine blocks protein synthesis by
                                                                             blocking conversion of nucleolar RNA to cytoplasmic ribosomal
                                                                             RNA rather than by blocking mRNA synthesis (35, 36). While
                                                                             ethionine thus blocks at a step later than does actinomycin D, it
                                                                             can be seen from Fig. 4 that vitamin Kr treatment at 5 hours and
                                                                             even at 24 hours after starting ethionine injection in vitamin K-
                                                                             deficient rats still gave a definite prothrombin    response.
     Ot2t4t6                  HOURS
                                                 8         ‘O                       Response to Vitamin   K1 in Presence of Cycloheximide
   FIG. 3. Prothrombin       activity of control, vitamin K-deficient,          Since the above data indicated that the site of vitamin K action
and warfarin-treated      rats given 500 (Fig. 3~) and 2000 (Fig. 36)        was definitely beyond the transcription level, further experiments
fig of actinomycin D per 160 g of body weight at zero time, fol-
lowed by vitamin K1 at times as indicated.           C, control rats; W,     were carried out with cycloheximide, which blocks protein syn-
warfarin-treated    rats (1 mg/lOO g of body weight, 24 hours before         thesis at the translation    level (33, 34). Restoration      of pro-
zero time). Numbers on graphs represent the number of rats.                  thrombin levels upon vitamin Kr administration        to vitamin K-
Vitamin K1 (40 pg) was given to vitamin K-deficient rats, except             deficient animals treated with cycloheximide would indicate that
for the group in Fig. 3b, Curve 3, which was given 500 pg (5 hours
after actinomycin D). Vitamin Kl (1 mg) was given to the                     the vitamin acts at a site later than the site of action of cyclo-
warfarin-treated    rats.                                                    heximide.
3936                                      Vitamin K and Biosynthesis             of Protein and Prothrombin                                   Vol. 243, No.     14

   From Fig. 5, it is clearly seen that vitamin K1 (100 pg), given
1 hour after cycloheximide (50 fig/100 g of body weight) ad-
ministration,    resulted in an essentially complete restoration of
prothrombin       levels in the case of vitamin             K-deficient   rats
(Curve 4) and warfarin-treated         rats (Curve 9). At the same time,
control rats, given the same level of cycloheximide, showed an
almost 80% reduction in prothrombin               level in about 7 hours
(Curve 5), while warfarin-treated          and vitamin K-deficient rats,
given cycloheximide and no vitamin K, showed no increase in
prothrombin     (curves not shown).
    Data on warfarin-treated        animals given higher levels of cyclo-
heximide are presented in Fig. 6. These animals received in-
jections of 0.1 mg/lOO g of body weight (Fig. 6A) and 1 mg/lOO
g of body weight (Fig. 6@ of sodium warfarin 24 hours before the
cycloheximide        injections.   Groups received 250 pg (Fig. 6A)
or 750 pg (Fig. 6B) of cycloheximide and 0.1 mg (Fig. 6A, Curve
A) or 1 mg (Fig. 6B, Curve A) of vitamin K1 per 100 g of body
weight.     Control groups were given 250 pg (Fig. 6A, Curve B)
or 750 pg (Fig. 6B, Curve B) of cycloheximide per 100 g of body
weight, but no vitamin K1. It can be seen from Fig. 6, A and B,
that the prothrombin          time of the warfarin-treated     animals given
                                                                                           201             I                 I            1      I
only cycloheximide increased steadily, while, in contrast, the                                   0        2           4                  6      8




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prothrombin        time of warfarin-treated          animals given cyclo-                            t                           HOURS
heximide and vitamin Ii1 showed marked decreases over several
hours.
    Because of the rapid fall in prothrombin           after the administra-
 tion of cycloheximide to normal rats and because of the possible




                                                                                            01             I             I                I     I
                                                                                                          2          4                   6     8            1
                                                                                              t                        HOURS
                                                                                      FIG. 6. Effect of vitamin     K1 administration       on warfarin-treated
                                                                                 rats given cycloheximide.          A, Curve A, five      rats given 0.1 mg of
                                                                                 sodium warfarin       per 100 g of body weight 24 hours before zero time,
                                                                                 250 fig of cycloheximide        per 100 g of body weight      at zero time, and
                                                                                 0.1 mg of vitamin        K, per 100 g of body weight        at 1 hour; Curve B,
                                                                                 three rats given the same warfarin           and cycloheximide          treatments
                                                                                 but no vitamin       K1.     B, Curve A, five rats given        1 mg of sodium
                                                                                 warfarin    per 100 g of body weight         24 hours before      zero time, 750
                                                                                 pg of cycloheximide         per 100 g of body weight       at zero time, a.nd 1
                                                                                 mg of vitamin      K1 per 100 g of body weight         at zero time; Curve B,
                                         HOURS                                    three rats given the same warfarin           and cycloheximide           treatment
   FIG.    Effect of cycloheximide
          5.                           treatment  on prothrombin                  but no vitamin      KI.
response to vitamin K,. Curve 5, five normal rats given 50 rg of
cycloheximide at zero time; Curve 4, four vitamin K-deficient rats               structural antagonism between cycloheximide and vitamin K,
given 50 pg of cycloheximide at zero time and 100 Mg of vitamin  K,              large doses of vitamin K1 were given to cycloheximide-treated
at 1 hour; Curve 9, nine rats given 0.1 mg of sodium  warfarin            per
100 g of body weight 24 hours before zero time, 50 pg of               cyclo-    hypoprothrombinemic,    but not vitamin K-deficient, rats; how-
heximide   at zero time, and 100 pg of vitamin KI at 1 hour.                     ever, no response was obtained.
Issue of July 25, 1968                                                                Hill     et al.                                                                   3937

                                                                                                tion by the two-stage procedure of Ware and Seegers (26) was
                                                                                                attempted in recovery experiments following administration                   of
                                                                                                 cycloheximide and ethionine to vitamin K-deficient rats. In
                                                                                                these cases, blood for plasma preparation was obtained by heart
                                                                                                 puncture from control and experimental animals at different time
                                                                                                intervals.       The recovery patterns for the animals studied con-
                                                                                                firm entirely the foregoing data; however, since blood could not
                                                                                                be repeatedly drawn by heart puncture, the two-stage prothrom-
                                                                                                bin assay procedure was used only for confirmation of isolated
                                                                                                 points.
                                                                                                     The results of amino acid incorporation         and tryptophan pyrro-
                                                                                                lase induction experiments (14) show that general protein syn-
                                                                 HOUR
                                                                                                 thesis is not interfered with in vitamin K deficiency, even though
                                                                                                 the protein prothrombin          (and other vitamin K-dependent           pro-
                                                                                                teins) almost disappear from the blood and can no longer be
                                                                                                 found in the microsomes.
                                                                                                     The data show that vitamin K1 treatment of the deficient ani-
                                                                                                 mal shortly after administration         of high doses of actinomycin D
                                                                                                 will stimulate prothrombin         production.      Reversal of vitamin K
                                                                                                 deficiency was also obtained within 1 hour, when vitamin K1 was
                                                                                                 given 1 hour after an injection of cycloheximide at a level which
                                                                                                 had been shown to block prothrombin              synthesis in control ani-
                                                                                                 mals for more than 6 hours. Following much higher levels of




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                I       I      I      I

                2    4    6          8                                                           cycloheximide, a less complete but still clear response to vitamin
                    HOURS                                                                        K was obtained in vitamin K-deficient rats. Vitamin K-de-
    FIG.  7. Effect of puromycin          treatment       on prothrombin          response       ficient animals also responded to vitamin K1 administered 5 or
 to vitamin        K1. A, Curve a, three            normal     rats given 20 mg of               24 hours after ethionine injection.          Vitamin Ki given to normal
puromycin        per 100 g of body          weight.      Curve b, two normal            rats     rats 24 hours after ethionine injection has no effect on prothrom-
given 30 mg of puromycin              per 100 g of body weight.             Curve c, two
normal     rats given 40 mg of puromycin               per 100 g of body weight;                 bin level.
Curve d. five vitamin         K-deficient       rats given 20 mg of puromycin                        In the case of puromycin, vitamin K1 given simultaneously
per 1OO’g of body weight             at zero time and vitamin             KI-(40     pg per      with the antibiotic provoked a slight response in vitamin K-
rat) at zero time.         B, Curve a, one vitamin            K-deficient        rat given       deficient rats in the presence of 40 mg of puromycin per 100 g of
40 mg of puromycin           per 100 g of body weight               at zero time and
                                                                                                 body weight and more response in the presence of 20 mg. There
vitamin     K1 (40 pg per rat) at zero time; Curve b, two vitamin                         K-
deficient    rats given 40 mg of puromycin               per 100 g of body weight                was little response to vitamin Ki administered 1 or 3 hours after a
at zero time and vitamin            Kr (4Opg per rat) at 1 hour; Curve c, two                    proven prothrombin          synthesis-blocking     level (40 mg/lOO g of
vitamin     K-deficient     rats given 40 mg of puromycin                   per 100 g of         body weight) of puromycin had been given to vitamin K-de-
body weight         at zero time and vitamin              Kr (40 .ng per rat) at 3               ficient rats. These latter data are in agreement with the results
hours.
                                                                                                  obtained by Suttie (37) in liver perfusion experiments.                Prydz
                                                                                                   (38) has found that puromycin and warfarin both inhibit Factor
        Response to Vitamin               K1 in Presence of Puromycin                             VII biosynthesis in suspensions of rat liver cells; however, he has
   The determination     of the amount of puromycin required to                                   not examined the effect of puromycin on vitamin K-initiated
block prothrombin     synthesis in normal rats is presented in Fig.                               formation of Factor VII.
7A which shows that with a puromycin dose of 40 mg/lOO g of                                           In interpreting  these results, several factors need to be taken
body weight the prothrombin         time increases rapidly and the                                into consideration.     We determined the amount of protein syn-
blood prothrombin     level drops to about 15% of normal (Fig. 7A,                                thesis-blocking agent to be used in recovery experiments on the
Curve c). A puromycin dose of 50 mg/lOO g of body weight to                                       basis of the amount required in the vitamin K-adequate animal
normal rats was found to be lethal, the rats dying in 5 to 5+                                     to reduce active circulating blood prothrombin             to a low level.
hours. The data on puromycin treatment of vitamin K-deficient                                    The use of such control animals appears essential, since tech-
rats are presented in Fig. 7A, Curve d and Fig. 7B, Curves a, b,                                  niques are not available to measure the incorporation             of labeled
and c. While it is evident from Fig. 7A, Curve d that animals                                     nucleic acid into specific prothrombin         messenger RNA and since
do show some response to a dose of 40 pg of vitamin K1 given                                      the rat has insufficient isolatable blood prothrombin            to readily
simultaneously    with a suboptimal level of puromycin (20 mg/                                    permit the measurement of the incorporation              of labeled amino
100 g of body weight), when the puromycin dose is increased to                                    acids into prothrombin.         In any case, since it is the formation of
the complete prothrombin        synthesis-blocking     level of 40 mg/                            active prothrombin     following vitamin K administration          that was
100 g of body weight (Fig. 7B, Curves a, b, and c), the response                                  being studied, a level of protein synthesis-blocking          agent proven
to subsequent vitamin Kr administration         is very small (Fig. 7B,                           to block synthesis of this particular protein (or group of proteins)
Curves b and c) (negligible if plotted as percentage of normal).                                  was established and was used. Such controls have been lacking
                                                                                                  in other studies (15, 37). If low doses of blocking agent are
                                      DISCUSSION
                                                                                                  given to vitamin K-deficient rats over a 24-hour period, the
  Since the prothrombin data reported here were obtained by the                                   animals frequently die of hemorrhage within that period due to
Quick (24, 27) or Allington (25) single-stage methods, confuma-                                   the vitamin K deficiency even though the antibiotic dose given
3938                                      Vitamin K and Biosynthesis            of Protein and Prothrombin                              Vol. 243, No.      14

 may be too low to completely block prothrombin             synthesis. On       or puromycin should not block their conversion to prothrombin
 the other hand, the fact that a vitamin K-deficient rat responds               by vitamin K, given either simultaneously                   or 1 hour later.
 within 1 or 2 hours to vitamin Ki treatment makes possible the                 Losito (48) was unable to confirm the presence of this prothrom-
 use of essentially totally blocking doses of these agents, instead             bin inhibitor      using vitamin K-deficient and dicumarol-treated
 of the low levels used in many longer term animal experiments.                 chicks.
     The blocking of prothrombin      synthesis, primarily at one site in          Our data are in agreement with the report of Anderson and
 protein synthesis, by a protein synthesis-blocking               agent still   Barnhart (ll), who found that no fluorescence due to prothrom-
 leaves all sites between the block and the final “activation”           open   bin is seen in the liver of the dicumarol-treated                animal upon
 for vitamin K action, if it functions beyond the blocking site.                examination with specific prothrombin           fluorescent antibody, but
 However, while the site of action of vitamin K and all subsequent              that fluorescence appears within 2 hours after treatment of such
 sites are still open, lack of prothrombin      regeneration upon treat-        animals with vitamin K, indicating a rapid “turnon”                     of pro-
 ment with vitamin K could also mean that the blocking agent                    thrombin formation in the liver by vitamin K.
 acting at an earlier site had blocked the availability          of required        It is assumed that vitamin K deficiency blocks only one site of
 precursors or enzymes. On the other hand, good responses ob-                   prothrombin      synthesis and that prothrombin           synthesis proceeds
 tained with vitamin K do indicate that the level of action of the              normally up to that step in the vitamin K-deficient animal.
 vitamin is beyond the site blocked.                                                Puromycin has been reported at various times to completely
     Warfarin- or dicumarol-treated      rats are less likely to die from       block vitamin K-induced Factor VII formation (Suttie (37).\, not
 hemorrhage during antibiotic treatment, but are considered less                to block vitamin K-induced prothrombin                  formation (Olson et
 useful experimental animals because (a) these vitamin K antago-                al. (43)) in liver perfusions, and not to block vitamin K-induced
 nists have let’hal effects unrelated to blood clotting, as demon-              Factor VII formation in liver slices (Babior (49)). Our data
 strated by 2- to 4-hour nonhemorrhagic              death following high       indicate an effect similar to that of high levels of cycloheximide,
  doses (20 mg or more) of warfarin, and (b) because the presense               i.e. inhibition but not a complete block. Since puromycin blocks
  of the antagonist makes the cure with vitamin K more difficult,               protein synthesis by terminating            peptide synthesis, becoming




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 requiring higher vitamin K levels and longer times, due pre-                   itself incorporated     into the growing peptide chain (50-52), it is
 sumably to competition for binding sites occupied by the drug.                 thus inactivated and large amounts must be used. This action
     The data from the actinomycin D and ethionine experiments                  of puromycin is accompanied by a rapid breakdown of rat liver
 indicate clearly that the site of vitamin K function is beyond the             polysomes to a new steady state level, characterized by a shift to
 the transcription level, and the data from the cycloheximide ex-               smaller aggregates (53). These data and those obtained at high
  periments indicate that the vitamin functions beyond the site at              levels of cycloheximide         indicate that polysomal breakdown
 which cycloheximide blocks protein synthesis.                                  interferes with the prothrombin         response to vitamin K adminis-
     Cycloheximide acts at a site beyond the attachment of the                  tration.
 amino acid-charged tRNA to the ribosome and appears to block                      On the basis of the data presented in this paper, it appears that
 transfer of amino acids from aminoacyl-tRNA                to form poly-       a possible model for the role of vitamin K in the synthesis of
 peptide (34). Its activity appears to depend on the character of               prothrombin      (and by inference, of the other vitamin K-dependent
 the microsomes (39-41).        Cycloheximide has been found to not             clotting proteins, Factors VII, IX, and X) is as a precursor of a
 inhibit polyribosome formation,         but to prevent conversion of           cofactor of a protein (the binding site of which is competitive
 polyribosomes to single ribosomes under the usual stimuli (33,                 with warfarin) which is involved in the removal of the precursor
 42). This may be further evidence that it blocks at the level of               peptide from the polysome into its proper folded tertiary struc-
 peptide synthesis (33). Godchaux, Adamson, and Herbert (42)                    ture. It may be that formation of this folded structure (8 S-S
 have shown the ribosomal changes to be very much greater at                    bonds have been found in canine prothrombin                (54) and in bovine
  higher cycloheximide concentrations, and this may well explain                prothrombin       (55)) is an integral part of the complete synthesis
  the relatively poor cures obtained at the higher dose levels of               of the molecule at the level of peptide bond formation and that
 cycloheximide.                                                                 this is the site of translation regulation of the synthesis of pro-
     Subsequent to the first repor& (20,21) on the ability of vitamin           thrombin.      This model bears some resemblance to that proposed
 K to bring about restoration of blood prothrombin             levels in the    by Cline and Bock (56) for translation regulation of specific pro-
 vitamin K-deficient or warfarin-treated          rat in the presence of        tein synthesis.
 actinomycin D, confirmation         with the use of intact animals,               Fieser (57) showed that the vitamin K quinones are active
 liver perfusion, and liver slice techniques has been reported by               -SH reagents and vitamin K-dependent                  clotting proteins are
 J. P. Olson, Miller, and Troup (43), Suttie (37), and Lowenthal                inactivated     by sulhydryl compounds; thus it is possible that
 and Simmons (44). Recently R. E. Olson et al. (45, 46) have                    vitamin K functions by virtue of its ability to bind -SH com-
 withdrawn      their earlier conclusion of transcription        control by     pounds.
 vitamin K (15-19) and are now in agreement with the findings                      From the duration of the plateau level for blood prothrombin
 that vitamin K exerts its control at a later level.                            at the highest level of actinomycin D (Fig. la), the turnover time
    The proposal of Hemker et al. (47) that a preprothrombin             syn-   of prothrombin        messenger RNA can be est.imated as approxi-
 thesized in the liver is converted to prothrombin            in a vitamin      mately 6 hours. From the cycloheximide data (Fig. lb), one can
 K-dependent step appears unlikely in view of the cycloheximide                 estimate the turnover time of final active prothrombin                  as also
data. If, as Hemker has proposed, this preprothrombin               piles up    about 6 hours. These data, of course, give no evidence as to
to the extent that it spills over into the blood, where it acts as an           whether the decay of blood prothrombin             activity is due to degra-
inhibitor of prothrombin      conversion, these high levels of prepro-          dation or deactivation of the protein.
thrombin would have been present in our vitamin K-deficient                        The data on the blocking of estrogen-induced                  reactions by
rats and the administration      of even high levels of cycloheximide           cycloheximide (58) is similar to the data on vitamin K-induced
Issue of July 25, 1968                                                                Hill et al.                                                                                          3939

prothrombin       synthesis and may indicate               a similarity      in sites of       28. LOWRY, 0. H., ROSEBROUGH,     N. J., FARR, A. L., AND RANDALL,
                                                                                                     R. J., J. Biol. Chem., 193, 265 (1951).
action (59).                                                                                   29. SCALES, F. M., AND HARRISON,                           A. P., Ind. Eng. Chem.,               12,
                                                                                                       350 (i920).
   ilcknowZedgments-Actinomycin         D and all vitamins were gen-                           30. HELGELAND,            L., AND LALAND,             S., Biochim.        Biophys.      Acta, 62,
erously supplied by Merck Sharpe and Dohme Laboratories,                                               200 (1962).
Rahway, New Jersey, through the courtesy of Dr. Laurent                                        31. KELLER, E: B., AND ZAMECNIK,                          P. C., J. Biol. Chem., 221, 45
Michaud.     Actidione    (cycloheximide)    was generously supplied                                   (1956).
                                                                                               32. TRAKATELLIS.            A. C., AXELROD.           A. E.. AND MONTJAR,M.,               Nature,
by Upjohn & Company, Kalamazoo,               Michigan,   through the
                                                                                                       203, 1134 (1964).
courtesy of Dr. George Savage.                                                                 33. WETTSTEIN.            F. 0.. NOLL.             H.. AND PENMAN,                S.. , Biochim.
                                                                                                       Biophys.      ‘Acta, Si, 525 (1964j.
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       phys. Acta, 63, 550 (1962).                                                             41. VAZQUEZ,         D., AND MONRO,                 R. E., Biochim.            Biophys.       Acta,
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       Biol. Med., 121, 1287 (1966).                                                           42. GODCHAUX,          W., ADAMSON,             S., AND HERBERT,            E., J. Mol. Biol.,
 8. MARTIUS,         C., Vitamins      Hormones,       24, 341 (1966).                                 27, 57 (1967).
 9. GALE, P. H., PAGE, A. C., STOUDT, T. H., AND FOLICERS, K.,                                 43. OLSON, J. P., MILLER,                L. L. AND TROUP, S. B., J. Clin. Invest.,
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