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The formation of hydroxystearic acids during the conversion of stearic to oleic acid by liver and yeast preparations
SHORT COMMUNICATIONS
170
The formation of hydroxystearic acids during the conversion of stearic to oleic acid by liver and yeast preparations In the course of experiments designed to investigate the mechanism of oleic acid biosynthesis, Ez-14C]stearic acid was incubated with rat-liver homogenates, yeast cells and cell-free extracts of yeast. In every experiment, from I-2 °/e of the labelled stearate was converted to an acid having the chromatographic properties of hydroxystearic acid (Table I). After isolation of this acid by silicic acid chromatography 1 of the mixture of fatty acid methyl esters derived from a liver homogenate experiment, degradation of the labelled acid was carried out by the KMnO 4 procedure of JA,~ms AND WEBB~. The results of this degradation indicated that the labelled acid was a m~cture of isomeric hydroxystearic acids but it has not yet been possible to identify the individual components. Work is now in progress to clarify this points. TABLE I CHROMATOGRAPHICBEHAVIOUROF IO°HYDROXYSTEARICACIDAND UNKNOWN HYDROXYSTEARICACID Chroma~ographlc system
Gas-liquid chromatography on Apiezon L at x97° Gas-liquid chromatography on polyethylene glycol adipate at I73° Paper chromatography of free acid with 8o ~o ethanol Column chromatography of methyl ester on silicic acid
Behaviour of zo-hydroxystearate and unknown acid
Retention volume of 1.96 relative to methyl stearate Retention volume of 9.o relative, to methyl stearate Rs of o.94 Not eluted with xo ~o ether in petroleum ether; eluted with Ioo % ether
The hydroxystearic acids were formed by liver homogenates only under aerobic conditions. They were present when liver microsomes (which were shown to be the site of oleic acid biosynthesis) were incubated in the presence of supernatant solution but were not made b y the supernataut solution (cell sap) alone. The hydroxy acids were formed in the unesterified fatty acid fractton of the liver lipids. It is not likely that they arose by hydration of the labelled oleic acid which is formed from stearate in this system 3 since this would not explain the presence of the hydroxyl groups attached to carbon atoms other than C-9 or C-Io. Furthermore, when labelled oleate was incubated with the liver homogenates, only x.6 ~ of the label appeared in the region of hydroxystearate on the gas chromatogram. Since our liver homogenate converted an average of I 3 % of stearate to oleate, the subsequent conversion of this oleate to hydroxystearate would account for less than 2o°~ of the observed conversion of stearate to hydroxystearate. BLOOMFIELDAND BLOCH4 have shown that oxygen and reduced pyridine nucleotide are required for the formation of oleic acid from stearic acid. The observation that stearic acid is converted to hydroxystearate by liver and yeast preparations is compatible with the suggestion4,5 that a hydroxystearic acid may be an intermediate in oleic acid biosynthesis. LENNARZAND BLOCHe reported in a preliminary communication that labelled 9-hydroxystearate was converted to ~leic acid in good yield by cell-free yeast extracts. In experiments to be reported in detail elsewhere, we have B i o c h i m . B i o p h y s . AcJa,
57 (I062) x7o-x7x
SHORT COMMUNICATIONS
T7I
been able to demonstrate only very slight (1-4%) conversion of labelled 9- or Io-hydroxystearate to oleate by liver and yeast cell-free preparations. The precise role of hydroxystearic acid in the biosynthesis of oleic acid remains to be determined. 25% liver homogenates were prepared in 0.25 M sucrose and incubated for I h at 37 ° in air with a Tween-2o emulsion of [2-14C~stearic acid under the conditions described by BERNHARD et al. 3. Yeast cells (a strain of Saccharomyces cerevisiae) were grown and cell-free extracts prepared according to BLOOMFIELD AND BLOCH't. Silicic acid chromatography was carried out by the technique of HIRSCH ANn AHRENS1. Isolation and. simultaneous measurement of the a m o u n t s a n d radioactivity of the fatty acid methyl esters was carried out on the automatic gas-liquid radio-chromatogram described by JAMES AND PIPER7. The National Institute for Medical Research, M i l l Hill, London (Great Britain)
A . T . JAMES J . B . MARSH
i j. HIRSCHAND E. H. AHRENS,J. Biol. Chem., 233 (I958) 3zz. a A. T, JAMESAND J. P. W. WESB, Biocl~em. J., 66 (I957) 515 • a K. BERNHARD, J. VON BULOW-KOSTERAND H. ~,%'AGNER,Heir. Chim. :tcta, 42 (1959) 152. 4 D. K. BLOOMFIELDAND K. BLOCt~,J. Biql. Chem., 235 (z96o) 337• A. T. JAMES,J. P. W. WEEB ANDT. O. KELLOCR, Biochem. J., 78 (x96I) 333. 8 w. J. LENNARZAND K. BLOCH,J. Biol. Chem., 235 (I90O} PC 26. 7 A. T. JAMES AND E. A. PIPER, J. Chromatog., 5 (*-96I) 265. Received September 9th, 1961 BiochDn. Biophys. /Icta, 57 (I962) 17o-i71
Nature of the inhibition of yeast carboxylase by acetaldehyde In a previous communication from this laboratory, GRUBERAND WASSENAAR1 showed that acetaldehyde, either produced by the reaction or added, inhibits carbox3"lase in an apparent non-competitive or uncompetitive manner, one molecule of acetaldehyde combining with one enzymic site. They arrived at t,his conclusion by analysing the inhibition obtained at saturating levels of pyruvate and calculated a Ka (dissociation constant of the acetaidehyde-carboxwlase complex, which is not identical with the bound acetaldehyde intermediate:) of about 4 mM for acetaldehyde produced during the carboxylase re~tction. Added acetaldehyde was about 9o% as effective. From their data no decision could be taken between the two types of inhibition, and possible mixed types. Therefore experiments were carried out in which the concentration of added acetaldehyde was varied at four different pyruvate concentrations. The initial velocities were measured by the conventional "~'arburg technique taking intervals in which gas evolution was apparently linear or, in some cases, by drawing slopes. Air-dried brewer's yeast was used as enzyme source and the reaction was measured at pH 5.1 in o.1 M acetate buffer at a temperature of 27 °. The results of several typical e.cperiments are combined in Fig. I. In this figure 1/7; has been plotted against I/S for three acetaldehyde concentrations. A set of nearly parallel lines with a positive slope is obtained, which is in agreement with uncompetitive inhibition whereas non-competitive inhibition would have yielded a set of lines intersecting at a single point. Bwchim. Biophys. Acta, 57 (I962) I7I-z73
170
The formation of hydroxystearic acids during the conversion of stearic to oleic acid by liver and yeast preparations In the course of experiments designed to investigate the mechanism of oleic acid biosynthesis, Ez-14C]stearic acid was incubated with rat-liver homogenates, yeast cells and cell-free extracts of yeast. In every experiment, from I-2 °/e of the labelled stearate was converted to an acid having the chromatographic properties of hydroxystearic acid (Table I). After isolation of this acid by silicic acid chromatography 1 of the mixture of fatty acid methyl esters derived from a liver homogenate experiment, degradation of the labelled acid was carried out by the KMnO 4 procedure of JA,~ms AND WEBB~. The results of this degradation indicated that the labelled acid was a m~cture of isomeric hydroxystearic acids but it has not yet been possible to identify the individual components. Work is now in progress to clarify this points. TABLE I CHROMATOGRAPHICBEHAVIOUROF IO°HYDROXYSTEARICACIDAND UNKNOWN HYDROXYSTEARICACID Chroma~ographlc system
Gas-liquid chromatography on Apiezon L at x97° Gas-liquid chromatography on polyethylene glycol adipate at I73° Paper chromatography of free acid with 8o ~o ethanol Column chromatography of methyl ester on silicic acid
Behaviour of zo-hydroxystearate and unknown acid
Retention volume of 1.96 relative to methyl stearate Retention volume of 9.o relative, to methyl stearate Rs of o.94 Not eluted with xo ~o ether in petroleum ether; eluted with Ioo % ether
The hydroxystearic acids were formed by liver homogenates only under aerobic conditions. They were present when liver microsomes (which were shown to be the site of oleic acid biosynthesis) were incubated in the presence of supernatant solution but were not made b y the supernataut solution (cell sap) alone. The hydroxy acids were formed in the unesterified fatty acid fractton of the liver lipids. It is not likely that they arose by hydration of the labelled oleic acid which is formed from stearate in this system 3 since this would not explain the presence of the hydroxyl groups attached to carbon atoms other than C-9 or C-Io. Furthermore, when labelled oleate was incubated with the liver homogenates, only x.6 ~ of the label appeared in the region of hydroxystearate on the gas chromatogram. Since our liver homogenate converted an average of I 3 % of stearate to oleate, the subsequent conversion of this oleate to hydroxystearate would account for less than 2o°~ of the observed conversion of stearate to hydroxystearate. BLOOMFIELDAND BLOCH4 have shown that oxygen and reduced pyridine nucleotide are required for the formation of oleic acid from stearic acid. The observation that stearic acid is converted to hydroxystearate by liver and yeast preparations is compatible with the suggestion4,5 that a hydroxystearic acid may be an intermediate in oleic acid biosynthesis. LENNARZAND BLOCHe reported in a preliminary communication that labelled 9-hydroxystearate was converted to ~leic acid in good yield by cell-free yeast extracts. In experiments to be reported in detail elsewhere, we have B i o c h i m . B i o p h y s . AcJa,
57 (I062) x7o-x7x
SHORT COMMUNICATIONS
T7I
been able to demonstrate only very slight (1-4%) conversion of labelled 9- or Io-hydroxystearate to oleate by liver and yeast cell-free preparations. The precise role of hydroxystearic acid in the biosynthesis of oleic acid remains to be determined. 25% liver homogenates were prepared in 0.25 M sucrose and incubated for I h at 37 ° in air with a Tween-2o emulsion of [2-14C~stearic acid under the conditions described by BERNHARD et al. 3. Yeast cells (a strain of Saccharomyces cerevisiae) were grown and cell-free extracts prepared according to BLOOMFIELD AND BLOCH't. Silicic acid chromatography was carried out by the technique of HIRSCH ANn AHRENS1. Isolation and. simultaneous measurement of the a m o u n t s a n d radioactivity of the fatty acid methyl esters was carried out on the automatic gas-liquid radio-chromatogram described by JAMES AND PIPER7. The National Institute for Medical Research, M i l l Hill, London (Great Britain)
A . T . JAMES J . B . MARSH
i j. HIRSCHAND E. H. AHRENS,J. Biol. Chem., 233 (I958) 3zz. a A. T, JAMESAND J. P. W. WESB, Biocl~em. J., 66 (I957) 515 • a K. BERNHARD, J. VON BULOW-KOSTERAND H. ~,%'AGNER,Heir. Chim. :tcta, 42 (1959) 152. 4 D. K. BLOOMFIELDAND K. BLOCt~,J. Biql. Chem., 235 (z96o) 337• A. T. JAMES,J. P. W. WEEB ANDT. O. KELLOCR, Biochem. J., 78 (x96I) 333. 8 w. J. LENNARZAND K. BLOCH,J. Biol. Chem., 235 (I90O} PC 26. 7 A. T. JAMES AND E. A. PIPER, J. Chromatog., 5 (*-96I) 265. Received September 9th, 1961 BiochDn. Biophys. /Icta, 57 (I962) 17o-i71
Nature of the inhibition of yeast carboxylase by acetaldehyde In a previous communication from this laboratory, GRUBERAND WASSENAAR1 showed that acetaldehyde, either produced by the reaction or added, inhibits carbox3"lase in an apparent non-competitive or uncompetitive manner, one molecule of acetaldehyde combining with one enzymic site. They arrived at t,his conclusion by analysing the inhibition obtained at saturating levels of pyruvate and calculated a Ka (dissociation constant of the acetaidehyde-carboxwlase complex, which is not identical with the bound acetaldehyde intermediate:) of about 4 mM for acetaldehyde produced during the carboxylase re~tction. Added acetaldehyde was about 9o% as effective. From their data no decision could be taken between the two types of inhibition, and possible mixed types. Therefore experiments were carried out in which the concentration of added acetaldehyde was varied at four different pyruvate concentrations. The initial velocities were measured by the conventional "~'arburg technique taking intervals in which gas evolution was apparently linear or, in some cases, by drawing slopes. Air-dried brewer's yeast was used as enzyme source and the reaction was measured at pH 5.1 in o.1 M acetate buffer at a temperature of 27 °. The results of several typical e.cperiments are combined in Fig. I. In this figure 1/7; has been plotted against I/S for three acetaldehyde concentrations. A set of nearly parallel lines with a positive slope is obtained, which is in agreement with uncompetitive inhibition whereas non-competitive inhibition would have yielded a set of lines intersecting at a single point. Bwchim. Biophys. Acta, 57 (I962) I7I-z73