(XCoH) in an analogous manner to the definition of

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(XCoH) in an analogous manner to the definition of Powered By Docstoc
					                          PROCEEDINGS OF THE BIOCHEMICAL SOCIETY                                                51P
This was shown by chromatography of the tri-                   Gas-Liquid Chromatography               of    Mono-
ethylamine salts of phosphatidic acid on silicic               glycerides and Diglycerides
acid-impregnated glass-fibre paper, with the solvent
system chloroform-methanol-0-73 M-ammonia soln.                By R. WATTS and R. DILS. (Department of Bio-
 (200:45:6, by vol.). When the phosphate group was             chemi8try, Univer8ity of Birmingham)
removed with L-a-phosphatidate phosphohydrolase
 (EC (Weiss, Kennedy & Kiyasu, 1960),                     Qualitative parameters defining the gas-liquid-
diglyceride accounted for 85% of the phosphatidate              chromatographic behaviour of trimethylsilyl (TMS)
hydrolysed. The remainder was monoglyceride                     ethers of mono- and di-glycerides are largely lacking
 (Hill, Husbands & Lands, 1968). A twofold                      in the literature (see Kuksis, 1967). We therefore
purification of the enzyme was achieved by ultra-               extended our studies of factors affecting the
sonic treatment in 0.01% deoxycholate. Further                 separation of triglycerides on SE-30 and QF-1
purification was not successful. The apparent Km                (Watts & Dils, 1968) to the TMS ethers of a wide
for L-glycerol 3-phosphate was 50,4M. This is                  range of mono- (C2-C18) and di-glycerides (C4-C36).
similar to the value calculated from the results of               Both gas-liquid and thin-layer chromatography
Tzur, Tal & Shapiro (1964) for rat liver, but is lower         were used to determine completeness of silylation,
than the value for either the yeast enzyme (Kuhn &             and conditions that have been used (Tallent &
Lynen, 1965) or the E. coli enzyme (Ailhaud &                  Kleiman, 1968) were shown to yield partially
Vagelos, 1966).                                                silylated products.
   The enzyme can be protected from inhibition by                 Isothermal analysis showed that on SE-30 or
the thiol-binding agent N-ethylmaleimide (NEM)                 QF-1, log(retention volume) was directly propor-
by prior incubation with acyl-CoA. The amount of               tional to carbon-atom number and inversely
thiol group so protected was determined by using               proportional to absolute temperature. However,
[14C]NEM. When 42% protection of the enzyme                    the TMS ethers of lower monoglycerides (C2-C6) and
activity was achieved by incubation of the enzyme              diglycerides (C4-C12) deviated from these relation-
with palmitoyl-CoA before the addition ofNEM, the              ships. Since no single temperature could be em-
amount of thiol group so protected was determined              ployed to analyse the full range ofeither mono- or di-
to be 0-1 m,umole/mg. of enzyme protein. This was              glyceride TMS ethers, temperature-programmed
calculated from the difference in binding of [14C]-            analysis was used. The temperature-programmed
NEM to the protected enzyme in the presence and                conditions that enabled triglycerides to be separated
absence of L-glycerol 3-phosphate. These thiol                 (Watts & Dils, 1968) gave good separation of both
groups are essential for the activity of the enzyme.           mono- and di-glyceride TMS ethers. Relative
There relationship to the active site of the enzyme is         elution temperatures (TRE) were therefore used to
very close; we were unable to separate the thiol               compare the chromatographic properties of different
groups from the enzyme activity.                               glyceride classes. We defined the carbon equivalent
                                                               of a TMS group (ACTMs) as the difference in carbon-
  This work was supported in part by grant AM-05310 from       atom number (per TMS group) between a mono- or
the United States Public Health Service.
                                                               di-glyceride TMS ether and the triglyceride with
Ailhaud, G. P. & Vagelos, P. R. (1966). J. biol. Chem.         the same TRE under the same conditions of tempera-
   241, 3866.                                                  ture programming. Though the TMS group is
Cheniae, G. M. (1965). Plant Physiol. 40, 235.                 equivalent to about 5*3 carbon atoms, the apparent
Hill, E. E., Husbands, D. R. & Lands, W. E. M. (1968).         increase in molecular weight, as indicated by
  J. biol. Chem. 243, 4440.                                    ACTms, was found to be only 1-7-2-5 for mono-
Kornberg, A. & Pricer, W. E., jun. (1953). J. biol. Chem.      glycerides, and varied from - 20 to 4-8 for di-
   204, 345.
Kuhn, N. J. & Lynen, F. (1965). Biochem. J. 94,240.            glycerides. This indicates specific interactions
Lands, W. E. M. & Hart, P. (1965). J. biol. Chem. 240, 1905.   between these solute molecules and column
Tzur, R., Tal, E. & Shapiro, B. (1964). Biochim. biophys.      components.
  Acta, 84, 18.                                                  To compare the behaviour of free diglycerides, we
Weiss, S. B., Kennedy, E. P. & Kiyasu, J. Y. (1960).           defined the carbon equivalent of hydroxyl group
  J. biol. Chem. 235, 40.                                      (/XCoH) in an analogous manner to the definition of
                                                               ACTMS. Values of ACOH (3.1-4.4) showed that free
                                                               and silylated diglycerides are eluted at the same
                                                               TRE. Thus the usefulness of silylation for di-
                                                               glycerides appears to be solely in minimizing
                                                               adsorption on to column walls or supports (or
                                                                 By using weight and molar correction factors, the
                                                               composition of the TMS ethers of diglycerides
derived from egg and ox brain lecithins was                  tion explains the higher amounts of palmitate
determined.                                                  incorporated into glycerides in spite of a decreased
                                                             phosphatidate formation.
Kuksis, A. (1967). In Lipid Chromatographic Arnalysi8,          The increase of phosphatidate phosphohydrolase
  p. 332. Ed. by Marinetti, G. V. New York: Marcel
  Dekker, Inc.                                               activity in starvation is probably related to the in-
Tallent, W. H. & Kleiman, R. (1968). J. Lipid Res. 9, 146.   creased flux offree fatty acids into liver (Olivecrona,
Watts, R. & Dils, R. (1968). J. Lipid Re8. 9,40.             1962) and increased glyceride content observed in
                                                             livers of starved animals (Mayes, 1962). Our results
                                                             do not explain the changes in fatty acid esterification
                                                             in starvation found in liver slices (Vavrecka,
The Incorporation of [14C]Palmitate into                     Poledne & Petrasek, 1967) and perfused liver
Glycerides and Phospholipids of Liver Homo-                  (Mayes & Felts, 1967).
genates from Fed and Starved Rats
                                                               This work was supported by grants from the Wellcome
By M. VAVpE&A, M. P. MITcirEL and G.                         Trust (M.V.) and the M.R.C. (M.P.M.).
HitBSCHER. (Department of Biochemi8try, Univer8ity
of Birmingham)                                               Mayes, P. A. (1962). Metabolism, 11, 781.
                                                             Mayes, P. A. & Felts, J. M. (1967). Nature, Lond., 215, 716.
   The incorporation of labelled palmitate into              Olivecrona, Th. (1962). Acta physiol. scand. 54, 295.
glycerides and total phospholipids of rat liver homo-        Smith, M. E. & Hfibscher, G. (1966). Biochem. J. 101, 308.
genates was measured in the presence of optimum              Smith, M. E., Sedgwick, B., Brindley, D. N. & Hiubscher, G.
amounts of cofactors and albumin (Smith &                      (1967). Europ. J. Biochem. 3, 70.
                                                             Vavrecka, M., Poledne, R., & PetrAsek, R. (1967). Ab8tr.
Hiubscher, 1966; Smith, Sedgwick, Brindley &                   4th Meet. Fed. Europ. Biochem. Soc., 08o, p. 50.
Hubscher, 1967). Adult female rats, fed ad lib. or
starved for 36-40hr., were used in these experiments.
   The ratio of labelled glycerides to labelled phos-        Extraction and Assay of the Fat-Mobilizing
pholipids changed significantly in starvation (0*82          Substance from Urine of Normal Adult
in fed and 3-0 in starved animals). Over 80% of the          Human Subjects in Various Physiological
label incorporated into total phospholipids was              States
found in phosphatidate. When calculated per total            By G. L. S. PAWAN. (Department of Medicine and
liver, the incorporation of palmitate into glycerides        Institute of Clinical Research, Middlesex Hospital
was slightly increased in starvation, but the labelling      Medical School, London, W. 1)
of phosphatidate was decreased to 34 % of the
control value. The sum of the label in glycerides               A fat-mobilizing substance (FMS) has been
and phosphatidate decreased to 75% of the fed                detected in urine of man and some other mammals
control.                                                     during conditions of active fat catabolism (Chalmers,
   The latter decrease might be explained by a               Kekwick, Pawan & Smith, 1958; Beaton, Szlavko &
decreased phosphatidate formation on subcellular             Stevenson, 1966). This material produces marked
particles. When a total particulate fraction                 effects on fat metabolism, body weight and energy
prepared from liver homogenates was assayed as               loss in certain animal species (Kekwick & Pawan,
above, the rate of incorporation of palmitate into           1963, 1965) and in human subjects (Kekwick &
phosphatidate, which was the major lipid fraction            Pawan, 1968). It has been shown to increase
labelled, was decreased in starved animals to 68%            lipolysis in adipose tissue both in vivo and in vitro
of the control value. This decrease was of the same          (Chalmers, Kekwick & Pawan, 1960; Cahill,
order as that of total proteins (64% of fed control).        Pawan & Chalmers, 1961). This communication
   The increased glyceride/phospholipid ratio                reports results of a study of the relative activity of
observed with homogenates from starved animals               FMS extracted from urine of normal subjects.
might be due to an increased phosphatidate                      Urine collections were obtained from the following
phosphohydrolase activity. The phosphohydrolase              groups: (1) 72 normal adults (48 men, 24 women;
participating in glyceride biosynthesis is known to          aged 18-72, mean age 38 years; all within 85-115%
be present in the particle-free supernatant (Smith           of ideal body weight) before, during, and after a
et al. 1967). This enzyme was assayed by using               36hr. fast; (2) 20 normal pregnant women (mean
membrane-bound biosynthetically produced phos-               age 29 years) eating ad lib., at different stages of
phatidate as substrate. In starvation, the activity          pregnancy; (3) 18 athletic men (mean age 27 years)
per total liver was increased to 190% and the specific       before and after periods of exercise (1000 yards swim
 activity to 320% of the corresponding values of             or 3 mile run, in 25min.). FMS was extracted from
fed control animals. As phosphatidate phospho-               each urine specimen as outlined by Kekwick &
hydrolase activity was found to be rate-limiting in          Pawan (1967), special precautions being taken, to
homogenates, the increase of its activity in starva-         avoid loss from inactivation, and because of the

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Description: (XCoH) in an analogous manner to the definition of