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					THE   .JOURNAL O F BIOLOGICAI. CHEMISTRY
Vd    255,N U 5 . lwue of March IO. pp. 1950-1955. 19XU
Prrnted tn   [   SA


D-Mannitol Oxidationin theLand Snail, Helix aspersa *
                                                               (Received for publication, June 18, 1979, and in revised form, September 25, 1979)


                      Jean E. Vorhaben, Jerry F. Scott, and James W. Campbell
                      From the Department of Biology, William MarshRice University, Houston, Texas 77001



   Respiration by mitochondrialpreparations from he-                                           MATERIALS A N D METHODS
patopancreas tissue of the land snail Helix aspersa is                        D-[U-“C]Mannose, D-[ l-,’H]mannose, and ~-[5-:’H]fructose were
stimulated by D-mannitol. Therate of mannitol-stimu-                       purchased from New England Nuclear. ~-[U-’~C]Mannitol syn-       was
lated respiration is approximately one-half that given                     thesized from [‘4C]mannose by reduction with sodium borohydride
by succinate, the most effective substrate thus far                        essentially as described by Isbell et al. (11). The specific radioactivity
tested with these preparations. Mannitol-stimulated                        was 280 pCi/pmol. Chromatographic and electrophoretic analyses of
respiration is cyanide-insensitive but is not inhibited                    the productindicated a greater than99.5%radiochemical purity. With
                                                                           the exception of salicylhydroxamate, which was a gift from Dr. W. D.
                                        the
by salicylhydroxamate.The product of membrane-                             Bonner, Jr.,University of Pennsylvania, andthenoyltrifluoroacetone,
bound mannitol-oxidizing activity was shown to be D-                       which was purchased from Aldrich Chemical Co., other reagents and
mannose by thin layer chromatography, high voltage                         enzymes were obtained from the Sigma Chemical Co. This included
electrophoresis of the germanate and          com-
                                         borate                            the sugar alcohols, sugars, oligomycin, rotenone, antimycin A, yeast
plexes, gas chromatography of the trimethylsilyl deriv-                    phosphoglucoseisomerase, phosphomannose isomerase, glucose-6-




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ative, low resolution mass spectrometry of the trimeth-                    phosphate dehydrogenase,hexokinase, and bovine serum albumin
ylsilyl derivative, and by an enzymatic method depen-                      (essentially fatty acid-free).
dent upon phosphomannose isomerase. The reaction                             H . aspersa were collected locally or in San Diego, Calif. through
mannitol + O2 + mannose is stoichiometric; however,                        the courtesy of Dr. J. M. Cregg, University of California, San Diego.
                                                                           They were fed lettuce ad libitum in the laboratory.
it is not known whether O2 is the immediate electron
acceptor. The activity in Helix mitochondria is thus                                             Isolation of Mitochondria
uniqueamong most alditol-oxidizing enzymes in not
                                                                              Previous attempts toisolate coupled mitochondria from gastropod
being pyridine nucleotide linked and in acting on car-                     hepatopancreas tissue have given negative results (12-14). Because
bon 1 rather than carbon2.                                                 of this, several procedures were tried including those used to obtain
                                                                           coupled mitochondria from other invertebrate tissues such as crus-
                                                                           tacean hepatopancreas (15).   None were successful.During thesetrials,
                                                                           the acid phosphatase content (indicative of lysosomal contamination)
  D-Mannitol is the most    common alditol in fungi, algae, and            and specific activity of cytochrome oxidase were determined for the
higher plants (1, 2 ) . In several higher plant species, it is a           different fractions and the final preparations were checked for their
major product of photosynthesis and, like sucrose and other                morphological integrity and purity by electron microscopy and for
nonreducing oligosaccharides, is translocated in the phloem.               their ability to couple electron transport from succinate to ADP
Despite the fact that mannitol may represent        a significant          phosphorylation as indicated by acceptor control ratios. The proce-
                                                                           dure used yielded a preparation containing a t least 60% intact mito-
storage carbohydrate in plants, studies on its utilization by
                                                                           chondria, less than 0.4% of the homogenateacid phosphatase activity,
animals, especially herbivors, have been limited. However, in              and a t least a &fold increase in the specific activity of cytochrome
at least one instance, it has shown to support the
                              been                        growth           oxidase. The details are as follows. Hepatopancreas tissue was ho-
and development of two phytophagous insects when present                   mogenized by hand in a TenBroeck homogenizer in 9 volumes of 0.5
as the sole dietary carbohydrate (3).Any utilization of dietary            M sucrose containing 5 mM Hepes,’ pH 7.4, 2.5 mM EDTA, and 0.5%
mannitol by animals has heretofore    been assumed toproceed               bovine serum albumin. The homogenate was centrifuged a t 1000 X g
via its initial conversion to fructoseby the nonspecific action            for 15 min. The supernatant fluid was collected and centrifuged a t
                                                                           6000 X g for 15 min. The resulting pellet was collected and resus-
of “sorbitol dehydrogenase” (EC 1.1.1.14, L-iditol NAD+ oxi-               pended in an original volume of homogenizing medium and recentri-
doreductase) (4, 5). In bacteria, yeast, and fungi, conversion is          fuged a t 6000 X g for 15 min. This latter washing procedure was
generally by more specific pyridine nucleotide-linkedmannitol              repeated twice using 0.5 andthen 0.25 volume of homogenizing
ormannitol-1-phosphate dehydrogenases, theproducts of                      medium. The final pellet was resuspended in the homogenizing me-
which are fructose or fructose 6-phosphate(e.g.Refs. 6 to 10).             dium to give 5 to 10mg of protein/&.Nosucraseactivity             was
                                                                           detectable in this preparation. All of the above manipulations were
                 we
In this report, show that mitochondrial preparations         from
                                                                           carried out at 0 to 3°C.
the garden snail Helix aspersa contain an oxidase that con-
verts D-mannitol to D-mannose in the absence of pyridine                                               Enzyme Assays
nucleotides. The rateof mannitol oxidation, relative to that    of            The oxidation of mannitol was determined by two procedures. For
other substrates, suggests that mannitol and possibly other                the polarographic assay, oxygen uptake was measured a t 25°C using
                                                     for
alditols may be important dietary carbohydrates herbivo-                   a Clark electrode with a Gilson model K-IC oxygraph. The respiration
rous gastropod molluscs.                                                   medium contained 0.5 M sucrose, 5 mM Hepes, pH 7.4,2.5 m~ EDTA
                                                                           or EGTA, 5 mg/ml of bovine serumalbumin, 2.5 m~ potassium
                                                                           phosphate, pH 7.4, and 2.5 m~ magnesium chloride in a final volume
                                                                           of 2 ml. Substrateandproteinconcentrationsare         given in table
   * This researchwas supported by Grant AI 05006 from the United          legends.
States Public HealthService National Institute of Allergy and Infec-          For the radiometric assay, mitochondria were incubated with 10
tious Diseases. The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore            The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-pipera-
be herebymarked “aduertisement” in accordance with 18 U.S.C.               zineethanesulfonic acid; EGTA,ethylene glycol bis(P-aminoethyl
Section 1734 solely to indicate this fact.                                 ether) (N,N’)-tetraacetic acid; MenSi, trimethylsilyl.

                                                                       1950
                       Oxidation                          D-Mannitol                                                                          1951
mM n-[U-"C]mannitol(0.4 pCi/pmol) in the medium described above material was removed by centrifugation at 100,000 x g for 1 h; the
except for the deletion of potassium phosphate and    magnesium chlo- supernatant was taken to dryness andredissolved in 70% ethanol for
ride. The total volume was 0.2 ml. A control reaction contained heat- chromatography as describedabove. Separation of mannose from
treated enzyme. The reaction was terminated by the addition of 0.1 unreacted mannitol was sufficiently clean so that additional separa-
ml of 0.5 M perchloric acid and precipitated protein was removed by tion by electrophoresis was unnecessary. This was determined by
centrifugation. The supernatant fluid was neutralized with 0.1 ml of           cutting anarrow strip from the center      of the chromatogram and
1 N potassium hydroxide and centrifuged again to remove the per-               staining for reducing sugar. Acontrol was prepared with heat-treated
chlorate salt. Isolation of [I4C]mannose from the supernatant fluid            enzyme. The product was eluted with waterfromtheunstained
was accomplished by descending chromatography and electrophore-                sections of the chromatogram, taken to dryness, and   allowed to stand
sis. Details of these procedures are described below. The total super- overnight at room temperature in 1 ml of 0.1 N hydrochloric acid to
natant fraction was used in the chromatography step, and following equilibrate anomeric forms. The hydrochloric acid was removed by
development, ["Clmannose was eluted with water and brought to a vacuum distillation at 40 to 45OC. To prepare the Me:Si derivatives,
volume of 1 ml. Aliquots of this solution were then subjected to               0.2 ml of a mixture of pyridine/hexamethyl disilazone/trimethyl-
electrophoresis to ensure complete separation of mannose and man- chlorosilane (17:2:1) was added to thedried samples which were then
nitol. T h e corresponding area from the control strip was also eluted left desiccated overnight a t room temperature.
and electrophoresed. Since mannitolmoved slightly ahead of fructose               Gas chromatography of the MeaSi derivatives was with a Hewlett-
in the electrophoretic procedure,   ['H]fructose was applied with each         Packard model 402 gas chromatograph equipped with a flame ioni-
sample as a marker. Following electrophoresis in sodium germanate, zation detector. Other details are given in Fig. 2.
the strips were b e d , cut into I-cm pieces, and counted in a Perma-             Mass spectra of the Me:,Si derivatives were obtained with an LKB
blend I/toluenemixture(PackardInstruments)           usinga Packard 9000 gas chromatograph-mass spectrometer equipped with a 6-foot
model PLD PRIAS counter. Efficiency of counting was determined                 1% OV17 column. The temperature programmerwas run from 120 to
by the external standard channels ratio. Quenched (nitromethane)               250°C a t 8 to lO"C/min. The ionizing voltage was 70 eV, the accel-
and unquenched standards     were prepared from["CI- a n d ~ H l t o l u e n e eration voltage 3.5kV, and the anode current        60 FA. Ion source
 (Packard Instruments).                                                        temperature was 270'C.

                     Additional Enzyme Assays                                                              RESULTS




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   Cytochrome oxidase was determined asdescribed by Wharton and
Tzagoloff (16) using a AE,,,  of
                               19,600       cm-l. Acid phosphatase                Succinate Oxidation by Mitochondrial Preparations
activity was followed by measuring hydrolysis of p-nitrophenyl phos-
                                                                               We, like other investigators (12, 13), found the utilization of
phate. The assay mixture contained 10 mM p-nitrophenyl phosphate
and 0.2 M sodium acetate, pH 5, in a volume of 1.4 ml. The reaction         pyruvate and   tricarboxylic acidcycle intermediates by isolated
was stopped by adding 0.1 ml of M sodium hydroxide and the absorb-          hepatopancreasmitochondriato         be limited. For example,
ance was measured at 410 nm. Aspartate transaminase was deter-                                                   of
                                                                            there was no detectable stimulation respiration by pyruvate,
mined as described by Karmen (17). Phosphomannose isomerase was                                             by
                                                                            isocitrate, a-ketoglutarate (or glutamate or glutamine), or
measured by the method of Gracy and Noltmann (18). This same                malate. Nor was P-hydroxybutyrate or proline oxidized. a-
general procedure was used for phosphoglucose isomerase by substi-
                                                                            Glycerophosphate gave some stimulation (-6 nmol of Os/mg
tuting fructose 6-phosphatefor mannose 6-phosphate. Theseenzyme
activities were assayed with the Helix cytosolic fraction ( 0 , O X g
                                                          1 0O O            of mitochondrial proteinemin-' at 10 mM concentration) but,
supernatant fluid) that hadbeen passed through a Bio-Gel P-2 column         of the substrates tested, succinate  was by far the most effec-
to remove low molecular weight compounds. Hydrogen peroxide was             tive. In oneseries of experiments, the ratewith succinate was
determined as described by Rancour et al. (19). Sucrase activity was        20.4 k 5.6 (S.D., w = 8) nmol of 02/mg of protein-min" for
determined according to Dahlqvist (20) using Sigma Co. Glucostat            active snails (1:00 a.m.) and 14.2 k 1.3 for snails in experimen-
reagents. Low molecularweightendogenous         compounds were re-
moved prior to assay by passage through Bio-Gel P-2 columns. All
                                                                            tally induced estivation (average individual O2 consumption
enzyme assays were carried out a t 25'C.                                    less than 20% that of active individuals). As expected, succi-
   Protein was estimated by the Lowry method (21) using crystallized        nate-stimulatedrespiration wasunaffectedby           rotenone (1
bovine serum albumin as the standard. Corrections were made for             ,UM) but was inhibited by azide and cyanide (2 m~ each). The
any albumin addedto the preparation.                                        latter is consistent with the presence of a relatively active
                                                                            cytochrome oxidase activity in the preparations (830 & 217
Isolation a n d Identification of the Product of Mannitol Oxidation
                                                                            (S.D., n = 8) nmol/mg of protein-min" for active snails). At
   Paper Chromatography-Although         no sucrase activity was de-        0.25 and 2.5 pg/mg of mitochondrial protein, antimycin gave
tected, removalof the sucrose present in the  reaction mixture became
a critical step for product identification since its presence in high
                                                                            87.5 and 91.8%inhibition, respectively,of succinate-stimulated
concentration interferedwith subsequent analytical procedures. This         respiration. Thenoyltrifluoroacetone, another site11-directed
was achieved by descending chromatography using strips of Whatman           inhibitor, also inhibited (95.4 and 100% at 0.2 and 2 mM,
NO.3MM paper (5 X 56 cm). Development was for 30 to 36 h with n-            respectively). Both of theseinhibitors were addedasthe
butyl alcohol/pyridine/water (60a.30, by volume). Compounds      were       methanolic solutions; the amount of methanol added (5 p1/2
detected by their reaction with alkaline silver nitrate (22) or perio-      ml total volume) was without effect. These results indicate
date/permanganate (23). Radioactive compounds wereinitially lo-
cated on chromatograms witha Packard Instruments model 7201
                                                                            that electron transport from succinate proceeds via sites I1
chromatogram scanner. The migration of sucrose and mannitol rela-                  1
                                                                            and 1 1 in the classical manner. However, there was no indi-
tive to that of mannose was 0.47 and 0.85, respectively. Compounds          cation of coupling in the Helix preparations   with succinate as
were readily eluted from the strips with wgter for additional separa-       substrate. Acceptor-control ratios were always near 1.0 and
tion by electrophoresis.                                                    succinate-stimulated respiration   was insensitiveto oligomycin
   Electrophoresis-High voltage    electrophoresis was carried out as       (2.5 ,ug/mgof mitochondrial protein). In addition to ADP,
described by Lindberg and Swan (24)using 0.05 M sodium germanate            several other compounds    were tested as  phosphoryl acceptors,
(pH 10.7) or 0.1 M sodium borate (pH 10) buffers a t 40°C. A Savant
model HV3000 power supply and model FP-22A tank were used for                L
                                                                            a l with negative results. These included GDP, IDP, CDP,
the electrophoresis.                                                        and arginine.
   Mass Spectrometry-Trimethylsilyl derivatives of the product and
standards were prepared for mass spectrometric analysis. T o circum-                                 Alditol Oxidation
vent the problem of high sucrose levels in the reaction mixture, the
                                                                              During initial studies on hepatopancreasmitochondria iso-
mitochondrial fraction (prepared in sucrose/Hepes medium without
bovine serum albumin) was extensively dialyzed against 10 mM po-            lated and made to respireGreenawalt's medium(25),a high
                                                                                                      in
tassium phosphate buffer, pH 7.4, so that the sucrose concentration         rate of nondepletable "endogenous" respiration was observed
was reduced to 2 X lo-' M. In addition, sucrose was deleted from the                                                     was
                                                                            which, unlikesuccinate-stimulated respiration, insensitive
reaction mixture. Following incubation, mitochondrial membranous            to cyanide. A study of the components of this isolation me-
1952                                                     D-Mannitol Oxidation
dium revealed that this "endogenous" respiration was due to                                               TABLE I1
mannitol. Compared with succinate,therate                 of mannitol         Submitochondrial localization of mannitol oxidase activity
oxidation was significant. In the same series of experiments                A mitochondrial preparation wassubjected to sonic disruption for
referred to above, the ratefor active snails was 14.9 +- 4 (S.D., 3 min (15 s on; 15 s off) at 0°C. The membrane and matrix fractions
n = 8 ) nmol of Odmg of proteinamin" or 73% the rate with were separated by centrifugation at 1 0 O x g for 60 min. NADH,
                                                                                                                   O
                                                                                                                0, O
                                                                        succinate, and mannitol oxidase activities were measured by Oa up-
succinate. These measurements          were made in the presence of take in the presence of each substrate (5,5, and 10 mM, respectively).
cyanide which, as shown in Table I, causes a stimulation of                                              Aspar-  Cyto-             Succi- Man-
mannitol oxidation. In the absence of cyanide, the rate with                                              tate  chrome NADH nate           nitol
mannitol was generally around 50% that with succinate.Ara-                                               tens-
                                                                                                         e0x1-           oxidase 0x1-      oxi-
                                                                                                        amlnase   dase              dase   dase
binitol was also oxidized by these preparations, usually at 25
                                                                                                                      ~moI.min"
to 30% the succinate rate. As with mannitol, this was also
                                                                             mitochondria
                                                                        "Intact"                         41.70   48.41     0.13     1.12 1.09
stimulated by cyanide. Ribitol, xylitol, and threitol also stim- (51.5)"
ulated respiration but to a much less extent than mannitol              Membrane   fraction (34.2) 3.10          55.00 0.910.32
                                                                                                                          0.88
and arabinitol. The rateof mannitol oxidation in thepresence Matrix         fraction         38.70
                                                                                           (17.1)                  0.30    0.01     0.01 0.02
of the other alditols was not additive and,in fact, was signifi-              Total milligrams of protein/fraction.
cantly less than with mannitol alone. This suggests that the
same activity in the preparations is responsible for the ob-
served oxidations of all the alditols although this has not been        oxidase activity of the preparations. NADH-stimulated O2
pursued in detail. Other related compounds not           oxidized were uptake was only 30% that of mannitol-stimulateduptake.
glycerol, meso-erythritol, D-glucitol (sorbitol), meso-dulcitol Assuming NADH is accessible to the site of transfer, this
 (galactitol), L-ascorbate, D-glycero-D-gdactoheptitol (persei- suggests that NAD' is not an immediate electron acceptor                       in
tol), and the  monosaccharides D-mannose, D-fructose, D-galac- the mannitol oxidase reaction. Neither NAD' nor NADP'
tose, and D-glucose.                                                    affected mannitol-stimulated O2 uptake when added to mem-




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   In addition to  being cyanide-insensitive, mannitol oxidation brane                        that been
                                                                               preparations had                        exhaustively dialyzed
was also insensitive to rotenone (1 p ~ ) antimycin (0.2 p ~ ) , against 0.1 M sucrose containing 5 mM Hepes, pH 7.4, and 2.5
                                                     ,
thenoyltrifluoroacetone ( 2 m ~ )oligomycin (5pg/ml), sodium mM EDTA. Nor wasa mannitol-dependent reduction of pyr-
                                        ,
benzoate (1 mM), azide ( 2 mM), arsenate (1 m),               and sali- idine nucleotides observed spectrophotometrically. Theseob-
cylhydroxamate (1 m), inhibitor of cyanide-insensitive servations,along withthe observedinsensitivity of intact
                              an
respiration in plants (26). Antimycin, like cyanide, also stim- mitochondrial preparations to rotenone, thus                  indicate that the
ulated mannitol oxidation. This suggests that there may be mannitol oxidase activity i s not pyridine nucleotide-linked.
an interaction between the pathway           of electron transport from
                                                                                      Product of Mannitol Oxidase Reaction
mannitol and the normal pathway, especially that from suc-
cinate.                                                                     Electrophoretic a n d ChromatographicSeparation-Ex-
                                                                        periments using several thin layer systems indicated that,
                                                                                                                           (27)
      Subcellular a n d Submitochondrial Localization of                unlike most othermannitol-oxidizing enzymes, the productof
                        Mannitol Oxidase                                Helix mannitol oxidase was not fructose. The product co-
   Preliminary tissue fractionation studies indicate that man- chromatographed with D-mannOSe in these systems and was
nitol oxidase activity is mainly mitochondrial. It has not been easily distinguished from other sugars such as galactose and
possible to establish whether or not is exclusively localized glucose. However, the separationof mannose from fructose in
                                              it
in this compartment because of mitochondrial fragility. In most of the systems tested was incomplete. The identity of
some studies, the microsomal fraction (100,000 x g,60 min) product with mannose was further confirmed by high voltage
contained a high percentage of the activity. However, this electrophoresis of the borate and germanate                         complexes. This
fraction also contained a high percentage of the cytochrome method also served to distinguish the product from other
c oxidase activity, indicating contamination with mitochon- common sugars. The mobilities of the borate complexes of
drial membranes which, as discussed below, also contain the galactose, mannitol, fructose, and glucose relative to that of
mannitol oxidase activity.                                              mannose were 1.3, 1.4, 1.4, and 1.6, respectively. Electropho-
   As shown inTable 11,the mannitoloxidase activity of Helix resisin germanate bufferreadily separated mannose from
mitochondrial preparationsis exclusively localized in the fructose and mannosefrom mannitol and was superior to the
membrane fraction. This fraction also contained the NADH borate system with regard to these separations. Fig. 1 shows
                                                                        the migration of the germanatecomplexes of the product and
                              TABLE      I                              of [3H]mannose and ["H]fructose. Not shown in the figure is
                                                                        the migration of mannitol whichmovesslightly                   ahead of
       Alditol oxidation by Helix mitochondrial preparations
                                                                        fructose.
   Details of mitochondrial isolation procedure and 0 uptake mea-
                                                         2                  Separation of the trimethylsilyl derivatives of mannose,
surements are given under "Materials and Methods." Succinate was
               M
present at 5 m concentration and the alditols at 10 m~ concentra-       fructose, and the productby gas chromatography is shown                 in
tion. Potassium cvanide was added to 2 m final concentration as Fig. 2. In addition to again showing that the trimethylsilyl
                                                 M
indicated and mannitol (third addition) to 10 m final concentration. derivative of the product is chromatographically identical
                                                   M
         Substrate
                            1st addition: 2nd addition: 3rd addition:    with that of mannose, it was also possible to show that the
                              substrate           KCN        mannitol
                                                                         ratio of its anomeric forms after equilibration more similar
                                                                                                                                is
                                     n n o l Odmgprotein.min"n           to thatof mannose thanof fructose. From the peak              areas, the
       Succinate                 22.6                0         14.5      CY form of mannose and the product        comprised 62.8 and 63.4%,
       D-Mannitol                10.4
                                14.1               14.1
                                  6.6               9.5        10.5
                                                                        respectively, of the mixture whereas         with fructose, 77.7%was
       D-Arabinitol
       meso-Ribitol               10
                                   .                            8.9      present as the CY form. In none of the chromatographic pro-
       meso-Xylitol               1.o                           8.3     cedures was there evidence for more than a single product.
       D-Threitol               -0.5                            7.3      This observation together with results of the time course of
    The endogenous rate was 1.5 nmol of OJmg of protein.min-I.           mannose formation discussed below indicate mannose is the
Values reported have been corrected for the endogenous rate.             primary product.
                         Oxidation                               D-Mannitol                                                                    1953
                                                                                                               I              I
          i                                           ' H -Fructose




     16
     I4 l
     I2
0
 I
     IO
 X

 3
 $
 0

     6

     4

                                                                                al
                                                                                in
     2                                                                          E
                                                                                0
                                                                                a
                                                                                in
                                                                                0)
              I   2 3 4 5 6   7 8             2 3 4 5 6 7 8 9
                                     9 IO I I 1 1 1 1 1 1 1 1 20                L


                                    Mlgration (crn)                             L
                                                                                0
                                                                                c
   FIG. 1. Electrophoresis of reaction product in germanate                     0
                                                                                al
buffer. Helix mitochondria (0.92 mg of protein) were incubated for              c
                                                                                                              \ I 3.5p/




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                                                                                0)
60 min at 25°C in a total volume of 0.2 ml of 0.5 M sucrose containing          n                                         frucjose
5 m Hepes, pH 7.4,2.5 m~ EDTA, 0.5% bovine serum albumin, and
   M
     M                     (0.4
10 m D-[U-14C]~annitOl pCi/pmol). The rate of mannitol-stim-
ulated 0 uptake by the preparation was 7.1 nmol/mg of protein.
        2
min". Data for the amount of mannitol oxidized during incubation
are given in Table IV. The reaction was stopped with 0.1 ml of 0.5 M
perchloric acid and, following removal of the latter as potassium
perchlorate, the product was isolated from the total reaction mixture
by descending paper chromatography as described in the text. The
product was eluted with water, brought to a volume of 1 ml, and an                      -
aliquot of 0.25 mlelectrophoresed on Whatman No. 1 paper (2.5 X 40                      2
cm) in 0.5 M sodium germanate, pH 10.7, for 90 min a t 1500 volts. D-
                                      were
[5-3H]fructoseand ~-[l-~H]mannose added as standards. After
drying, the paper was cut into I-cm sections which were counted                                                    I           I
for I4C and 'H. Migration is toward the anode. Thesubstrate                                          Retention t i m e
([14C]mannitol),which is initially separated from the product by                FIG. 2. Gas chromatography of the trimethylsilyl derivative
chromatography, migrates slightly ahead of fructose in this system.
                                                                              of the product. The product, formed essentially as described in Fig.
                                                                              1 except that nonlabeled mannitol was used as the substrate, was
                                                                              isolated and derivatized as described in the text. Analyses were carried
   Mass Spectrometry-A comparison of the mass spectra of                      out isothermally on a 6-foot 3% OV 17 column. Temperatures of the
the trimethylsilyl derivatives of the a form of the product and               column, flame detector, and injection port were 140°C, 215"C, and
of a-D-mannose and a-D-fruCtOSeis shown in Fig. 3. Typical of                 185"C, respectively.
the spectra of MeaSi derivatives of sugars, a peak  correspond-
ing to themolecular ion was absent from the mass spectra of                   and p anomers of both the product and mannose, a fragment
these compounds and the highest m/e value observed was at                     of low intensity at m/e 435 was observed which results from
525 (28). Since this fragment, characteristic of many hexoses,                the stepwise elimination of fist a methyl group from a MeaSi
appeared in the spectra of both mannose and fructose and was                  residue and then a trimethylsilanol molecule (28). With the
of such low intensity, it was omitted from Fig. 3. A major                    anomeric forms of fructose, a fragmentof low abundance was
difference between the spectra of the product (mannose) and                   seen at m/e 437 which arises from the loss of a
fructose is the absence of a major fragment at m/e 191 in the                 .CH20Si(CH3)a     group from the molecular ion (30). The spec-
spectrum of the latter.During fragmentationof the pyranose                    trum of the trimethylsilyl derivative of the substrate,D-man-
ring of mannose, this fragment arises through a rearrangement                 nitol, was essentially as previously described (31), giving major
involving a transfer of a trimethylsiloxyl group from C Sto CI                peaks at m/e 319, 204, 147, 217, 103, and 307 in order of
and subsequent formation of the fragment (Me3Si0)&H+                          decreasing relative intensity.
 (29). This rearrangement is prevented in the furanose ring                      Enzymatic Analysis-A final approach to the identity of
form of fructose. Other major differences include a higher                    the product as mannose was an enzymatic one. In this      coupled
relative intensity at m/e 217 with frucrose which was more                    system, NADP+ reduction is dependent upon the sequence:
 pronounced in a comparison of the spectraof the p forms of                   phosphorylation of mannose to form mannose &phosphate,
 the compounds (not shown). The relative intensityat m/e 217                  conversion of the latter tofructose 6-phosphate by phospho-
with P-D-mannose and thej3 form of the product was around                     mannose isomerase, isomerization of this to glucose 6-phos-
 20%;for p-fructose, it was 71%.There was also a lower relative               phate by phosphoglucose isomerase, and the conversion of
 intensity at m/e 204 in the spectrum of p-fructose compared                  glucose 6-phosphate to glucono-&lactone 6-phosphate by glu-
 with the other compounds (5%versus 98%).These relative
                 two                                                          cose-6-phosphate dehydrogenase concomitant with the reduc-
 differences a t m/e 217 and 204 reflect basic differences be-                tion of NADP+. As shown in Table 111, NADP+ reduction was
 tween the pyranose and furanose ring structures (29). An                      obtained with either added mannose or product. Reduction
 additional difference between the product and fructose can be                                                                  did
                                                                              was also obtained with added fructose, but this not require
 noted at m/e values above 400. In the spectra of both the a                  the presence of phosphomannose isomerase.
1954                                                         D-Mannitol Oxidation


            1 00   1                                    204
                                                         I
                                                                        OLPHA-D-RONNOSE




                                                                REOCTION PRODUCT - PEAK           1


            100    4                                    2414




                                                                                                                                                            Downloaded from www.jbc.org by guest, on October 12, 2011
                                                                        ALPHA-0-FRUCTOSE




                   50       1 00          150          200               250          300             350          400    450          500
                                                                               rn/e
         FIG. 3. Mass spectra of the trimethylsilyl derivatives of (I-D-manIIOSe,(I form of the product, and (I-D-fiuctoSe.

                              TABLE   I11                                                                        TABLE   IV
                   Enzymatic analysis ofproduct                                               Stoichiometry of the mannitol oxidase reaction
   The membrane fraction of Helix mitochondria was prepared as                       Components of the reaction mixture are given in Fig. 1, including
described in Table 11. This fraction was then dialyzed a t 0°C for 24 h           mitochondrial protein concentration. Incubation was for the times
against several changes of 0.1 M sucrose containing 5 m Hepes, pH
                                                          M                       indicated at 25°C. The reaction was stopped and ['4C]mannose was
              M
7.4, and 2.5 m EDTA. Following resedimentation, the membrane                      isolated and counted as described under "Materials and Methods"
fraction was washed with 50 m~ Tris-chloride, pH 7.4, by resuspen-                and in Fig. 1. For the electrophoresis step, aliquots of 0.25 ml were
sion are resedimentation. The membrane fraction (approximately 10                 spotted for the 40- and 60-min incubation periods, 0.5 ml for the 5-,
mg of protein) was finally suspended in 0.4 ml of Tris buffer containing          lo-, and 20-min periods, and the total 1 ml for the 1- and 2-min
0.4 M D-mannitol and incubated for 30 min at 25°C. The membranes                  periods and the control. Values are reported as nanomoles formed or
were removed by rapid centrifugation at room temperature (l0,OOO                  utilized in the total reaction mixture.
X g for 5 min) and 50 pl of the supernatant fluid were analyzed in the                                                                       Expected
coupled assay system. The basic reaction mixture contained in 1 ml                        Time         Mannitol
                                                                                                          utilized        Mannose formed
                                                                                                                                     based
                                                                                                                                      on               O2
volume, 1m NADP+, 2 m ATP, 6 m magnesium chloride, 50 m
            M               M           M                             M                                                                      uptake
Tris-chloride, pH 7.4, and either 50 pl of the supernatant fluid from                      rnin             nmol                nmol          nmol
above, 2 m D-mannose, 2 m D-fructose, or 50 m~ D-mannitol. The
           M                  M                                                              1            -6.4   (6.4)"      +6.5 (6.5)         6.5
coupling enzymes were added as indicated. NADPH formation was                               2            -12.9   (6.5)      +11.2 (5.6)        13.0
monitored at 340 nm.                                                                        5            -32.4   (6.5)      +29.7 (5.9)        32.6
                                                                                           10            -44.8   (4.5)      +40.7 (4.1)        65.2
            Additions                                                                      20           -129.8   (6.5)     +119.3 (6.0)       130.4
                                                                                           40           -259.8   (6.5)     +245.7 (6.1)       260.8
                                            nmol NADPH.min"                                60           -389.7   (6.5)     +377.2 (6.3)       391 .O
1st Gluocse-&phosphate de-            0          0          0             0           a   Rate as nanomoles.min".
      hydrogenase (1.4 units)
2nd Hexokinase (2.1 units)            0         0           0            0
3rd Phosphoglucoseisomerase           0         29.4        0            0                              Stoichiometry of the Reaction
      (3.4 units)
4th Phosphomannose isomer-           15.8                   11.3          0         The aboveobservationsconfirm        that D-mannose is the
      ase (2.2 units)                                   ~          ~~
                                                                                  product of the mannitol oxidase reaction in Helix mitochon-
   " Fifty microliters of supernatant fluid from the membrane fraction            drial preparations.The time course for mannose formation by
incubated in the absence of D-mannitol gave no reduction of NADP+.                these preparations is linear (Table IV), suggesting that man-
                      Oxidation                       D-Mannitol                                                                      1955
nose is theprimaryandnot         a secondaryproduct of the             lization would require phosphorylation followed by isomeri-
reaction. As shown in Table IV, the conversion of 1 mol of             zation of the resulting mannose 6-phosphate to fructose 6-
D-mannitOl to 1 mol of D-mannose occurs with the uptake of             phosphate for utilization via the glycolytic sequence. Consist-
1 mol of 02.  The 1:1 stoichiometry between O2 consumption             ent with this                          in
                                                                                    possibility is the presence the cytosolic fraction
and mannose formation was confirmed in a similar study in              of Helix hepatopancreas of a relatively active phosphoman-
which mannose was quantitated by the enzymatic procedure               nose isomerase. The level of this enzyme averages about 330
                                    A K,,,
described above (data not shown). of 10 mM for mannitol                pmol/g of tissue. h" (120 nmol/mg of protein -min") com-
was determined from polarographic assays using a mitochon-             pared with the level of phosphoglucose isomerase of around
drial preparation. The , for O2 has not yet
                        K                   been determined            1040 pmol/g of tissue.h" (370 nmol/mg of proteinamin").
nor has the immediate electron acceptor, if other than 0 2 ,
been identified. However, preliminary studies have shown a                                       REFERENCES
mannitol-dependent formation of hydrogen peroxide with a                1. Bourne, E. J. (1958) Handb. Pfzphysiol. 6,345-362
stoichiometry approaching 1:l; for every mole of hydrogen               2. Lewis, D. H., and Smith, D. C. (1967) New Phytologist 66, 143-
peroxide formed, 0.8 mol of 0 2 was consumed (observed values                 184
representing the average four determinations were 6.7 nmol
                          of                                            3. Dadd, R. H. (1960)J. Insect Physiol. 5, 201-316
of O2 consumed and 8.8 nmol of Hz02 produced/mg of protein).            4. Touster, O., and Shaw, D . R. D. (1962) Physiol. Rev. 42, 181-225
                                                                        5. Walsall, E. P., Lyons, S. A., and Metzger, R. P. (1978) Comp.
                                                                             Biochem. 59B, 213-218
                                                                        6. Kersters, K., Wood, W. A,, and De Ley, J. (1965) J . Biol. Chem.
                           DISCUSSION
                                                                              240,965-974
   The mannitol oxidase demonstrated here in Helix mito-                7. Ueng, S. T.-H., Hartanowicz, P., Lewandoski, C., Keller, J., Hol-
chondrial preparationsis unique among most        alditol-oxidizing          ick, M., and McGuinness, E. T. (1976) Biochemistry 15, 1743-
enzymes in not being pyridine nucleotide-linked and in form-                 1749
                                                                        8. Blumenthal, H. J. (1976) in The Fitamentous Fungi (Smith, J.
ing mannose and not fructose as the product. Mannitol-oxi-




                                                                                                                                                Downloaded from www.jbc.org by guest, on October 12, 2011
                                                                             E., and Berry, D. R., eds)Vol. 2, pp. 292-307, Wiley, New York.
dizing enzymes not requiring pyridine nucleotides have been             9. Barnett, J. A. (1976) Adu. Carbohydr. Chem. Biochem. 32, 125-
reported, but differ fundamentally from the Helix enzyme.                    234
Arcus and Edson (32), for example, described a cytochrome-             10. Ruffner, H. P., Rast, D., Tobler, H., and Karesch, H.(1978)
linked mannitol oxidase in Acetobacter. In addition to being                 Phytochemistry 17,865-868
inhibited by cyanide, this bacterial enzyme forms fructose as          11. Isbell, H. S., Frush, H. L., Moyer, J. D. (1960) J. Res. Nut.
                                                                                                        and
                                                                             Bur. Standards 64A, 359-362
the product of mannitol oxidation and shows a high level of            12. Rees, K. R. (1953) Biochern. J. 55,478-484
activity with D-glucitol as substrate.Inyeast,          specifically   13. Weinbach, E. C. (1956) Arch. Biochem. Biophys. 64, 129-143
 Torulopsis candida,mannose has been reported a product
                                                      as               14. Pietnykowski, A,, and Michejda, J. W. (1969) Bull. SOC. Amis
of mannitol oxidation along with fructose. However,       the pres-          Sci. Lettr. Posnon ( ) 193-201
                                                                                                    09,
ence of mannose in the mixture was attributed to the action            15. Chen, C.-H.,and Lehninger, A. L. (1973) Arch. Biochem. Bzophys.
of phosphomannose isomerase (33) and not to its direct for-                   154,449-459
                                                                       16. Wharton, D. C., and Tzagoloff, A. (1967) Methods Enzymol. 10,
mation by the NADP+-linked mannitol          dehydrogenase.                  245-250
   The metabolic significance of mannitol oxidation in Helix           17. Karrnen, A. (1955)J. Clin. Invest. 34, 131-133
and related snails is, at present, not known. Herbivorous land         18. Gracy, R. W., and Noltmann, E. A. (1968) J. Biot. Chem. 243,
snails are highIy adapted to utilizing complex plant polysac-                3161-3168
charides in that hydrolytic enzymes present in their digestive         19. Rancour, N. J., Hawkins, E. D., and Wells, W. W. (1979) Arch.
juice are capable splitting practically all naturally occurring
                  of                                                         Biochem. Biophys. 193, 232-241
                                                                       20. Dahlqvist, A. (1964) Anal. Biochem. 7, 18-25
glycosidic linkages (34). It is, therefore, perhaps not unex-
                                                                       21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J .
pected that they might    utilize alditols andespecially mannitol             (1951) J. Biol. Chem. 193,265-275
which occurs universally in higher        plants, in some species      22. Partridge, S. M. (1948) Biochem. J. 42,238-248
accounting for 8 to 10% of the dry weight (2). In one of the           23. Lernieux, R. U., and Bauer, H. F. (1954)Anal. Chem. 26,920-921
few nutritional studies on the    utilization of mannitol by phy-      24. Lindberg, B., and Swan, B. (1960) Acta Chem. Scand. 14, 1043-
tophagous species, Dadd (3) found it to support the growth                    1050
and development of two locusts, Schistocerca gregaria and              25. Greenawalt, J. W. (1974) Methods Enzymol. 31, 310-323
                                                                       26. Schonbaurn, G. R., Bonner, W. D., Jr., Storey, B. T., Bahr, J.
                                                                                                                                  and
Locusta rnigratoria. Based upon body weightattained within                   T. (1971) Plant Physwl. 47, 124-128
a fixed time interval, mannitol was 60 to 75% as effective as          27. Ghebregzabher, M., Rufini, S., Monaldi, B., and Lato, M. (1976)
melezitose, the most effective carbohydrate studied. Utiliza-                J. Chromatogr. 127,133-162
tion in these insectsis presumably via the nonspecific action          28. DeJongh, D. C., Radford, T., Hribar, J. D.,Hanessian, S., Bieber,
of sorbitol dehydrogenase (35) as in other animals (4). How-                 M., Dawson, G., and Sweeley, C. C. (1969) J. Am. Chem. SOC.
ever, in the studies of Dadd (3), mannitol was more effective                91, 1728-1740
                                                                       29. Radford, T., and DeJongh, D. C. (1972) in Biochemical Applz-
as a dietary carbohydrate in Schistocerca than sorbitol and                  cations of Muss Spectrometry (Waller, G. R., pp. 313-350,
                                                                                                                              ed)
the two were about equally effective in Locusta. If indeed                   Wiley-Interscience, New York
mannitol is utilized directly as a dietarycarbohydrate by              30. Curtius, H.-Ch., Muller, M., and Vollrnin, J. A. (1968) J. Chro-
Helix, its pathway of utilization differs from the main path-                matogr. 37,216-224
ways utilized by other species. In animals, fructose formed            31. Petersson, G. (1969) Tetrahedron 26,4437-4443
from mannitolby the actionof sorbitol dehydrogenase can          be    32. Arcus, A. C., and Edson, N. L. (1956) Biochem. J. 64,385-394
                                                                       33. Barnett, J. P. (1969) J . Gen. Microbiol. 52, 131-159
phosphorylated to enter theglycolytic sequence. In yeast (9)           34. Thirlwell, M. P., Stasdine, G. A,, and Whitaker, D. R. (1963) Can.
and fungi (8), mannitol may fwst be phosphorylated followed                  J. Biochem. Physiol. 41, 1603-1610
by oxidation to fructose 6-phosphate. In      Helix, mannose uti-      35. Chino, H. (1961) J . Insect Physiol. 6, 231-240

				
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