Soma Kumar

BIOSYNTHESIS OF SHORT-CHAIN FATTY ACIDS IN LACTATING MAMMARY SUPERNATANT.

Abstract 1. 1. Acetate, acetoacetate, β-hydroxybutyrate and butyrate are effective precursors for the synthesis of butyric, hexanoic, octanoic, decanoic and longer-chain acids by the mammary supernatant obtained from lactating goats and rabbits. 2. 2. The distribution of 14 C in the acids synthesized from specifically labeled substrates indicates that prior cleavage of acetoacetate and β-hydroxybutyrate is not necessary for the synthesis of butyric acid. 3. 3. Avidin had little effect on the synthesis of butyrate. The synthesis of hexanoic and longer-chain acids, however, was inhibited indicating the involvement of malonyl-CoA only in the stepwise elongation of the butyryl chain. 4. 4. Citrate was seen to enhance the synthesis of butyrate as well as the longerchain acids. It was also found to be helpful in preserving the enzyme system during storage.

Incorporation of short- and long-chain fatty acids into glycerides by lactating-goat mammary tissue

Abstract 1. 1. A particulate system obtained from lactating-goat mammary gland catalyzed the incorporation fo [I- 14 C]acyl-CoA into glycerides with dl -α-glyceroophosphate or α,β-diglyceride as acyl acceptor. 2. 2. The pathway for the triglyceride synthesis in this system appears to involve the formation of diglyceride form α-glycerophosphate and its subsequent conversion to triglyceride. 3. 3. Oleic, hexanoic, octanoic, and palmitic acids were incorporated into glycerides to different but considerable extents while the incorporation of butyrate was relatively low. 4. 4. The incorporation of palmitate into α-glycerophosphate resulted in the formation of considerable proportion of triglycerides. On the other hand, the incorporation of hexanoate or octanoate into α-glycerophosphate did not proceed beyond the formation of diglycerides to any appreciable extent. 5. 5. Utilization of glycerol for glyceride synthesis was not appreciable in this system.

Biosynthesis of fatty acids in mammary tissue: I. Purification and properties of fatty acid synthetase from lactating-goat mammary tissue☆

Abstract Fatty acid synthetase of a high degree of purity has been prepared from the particle-free supernatant fraction of lactating-goat mammary tissue. Ultracentrifugation and polyacrylamide gel electrophoresis revealed heterogeneity of the preparation. However, the enzyme complex was sufficiently free of malonyl-CoA decarboxylase activity to enable the demonstration of the requirement for the “primer” acyl-CoA in addition to malonyl-CoA and NADPH. The enzyme appeared to utilize butyryl-CoA more efficiently than acetyl-CoA as “primer.” The nature and extent of the fatty acids synthesized using these two “primers” were characteristically different and were pronouncedly influenced by the concentration of malonyl-CoA.

Biosynthesis of fatty acids in mammary tissue: II. Synthesis of butyrate in lactating rabbit mammary supernatant fraction by the reversal of β-oxidation☆

Abstract An enzyme fraction has been obtained from lactating rabbit mammary particlefree supernatant fraction which synthesizes butyrate from acetyl-CoA by the reversal of β-oxidation. This fraction has been shown to contain acetoacetyl-CoA thiolase, NADH-dependent acetoacetyl-CoA reductase, and crotonyl-CoA hydratase. The reduction of crotonyl-CoA appears to be brought about by the fatty acid synthetase using NADPH or NADH. The two reducing reactions are believed to overcome the thermodynamically unfavorable reaction involved in the condensation of two molecules of acetyl-CoA.

Production and utilization of butyryl-CoA by fatty acid synthetase from mammalian tissues

Abstract Butyrate constitutes 25 to 40% of the fatty acids formed by fatty acid synthetase purified from cow mammary gland, rat mammary gland, rat liver, and rat adipose tissue. In contrast, the extent of formation of this acid by the enzyme complex from chicken liver, pigeon liver, goose uropygial gland, and yeast is only 2 to 5%. Most of the butyrate produced is esterified to CoA, while most of the longer-chain acids formed are as free acids. The formation of butyrate appears to be a physiological process as judged from its formation in high amounts in rat liver slices. Butyrate formation is correlated with the ability of the enzyme from the different sources to utilize butyryl-CoA as primer. It is argued that butyryl- S -Enzyme formed as an intermediate during the course of fatty acid synthesis either undergoes chain elongation by condensation with malonyl-CoA or has the butyryl group transferred to CoA by a transacylation reaction. The concentration of malonyl-CoA or free CoA then determines which of the two competing processes is favored.

Regulation of the expression of α-amylase gene by sodium butyrate.

SummarySodium butyrate exerts a pronounced inhibition on the gibberellic acid-induced synthesis and secretion of α-amylase by aleurone cells of barley seeds. This inhibition, which is reversible and non-competitive with cespect to gibberellic acid, is concentration dependent, with virtually total inhibition being accomplished between 4 and 5 mM sodium butyrate. The pattern of inhibition of α-amylase formation correlates well with a decrease in the accumulation of its messenger RNA. The addition of butyrate 12 h after the addition of gibberellic acid to half-seeds, has no effect on the formation and secretion of α-amylase. It has been shown in earlier studies that the synthesis of α-amylase mRNAs takes about 12 h for completion. The conclusion that butyrate interferes with some step in the transcriptional process is supported by a decrease observed in the RNAs that hybridize with a cloned α-amylase cDNA. The results of in vitro translation confirm the inhibition of the formation of several translatable mRNAs. Further, immunological probing detected only trace amounts of α-amylase proteins in the secretion of butyrate-treated seeds. Translation of functional mRNAs, post-translational modifications and the secretion α-amylase are not affected by sodium butyrate. It is concluded that butyrate selectively inhibits the transcription of several genes that are under the influence of gibberellic acid. This report is the first one documenting the inhibitory effect of sodium butyrate on a hormone-induced synthesis and accumulation of mRNAs in a plant system.

Formation of acids shorter than palmitic by rat liver cytosol

Abstract Particle-free liver supernatant of rats maintained on stock diet synthesizes fatty acids of average chain lengths 9–11 carbon atoms when the protein concentration is high while palmitic acid is the main product when protein concentration is low. This ability to synthesize acids shorter than palmitic is lost on purification of fatty acid synthetase or by starvation of the rats followed by the ingestion of a high sucrose diet. The results are consistent with the presence of a factor in the cytosol, similar to that in lactating mammary glands, which shortens the chain length of the products of fatty acid synthetase.

Incorporation of fatty acids into milk glycerides

Abstract Palmitic, oleic, octanoic and hexanoic acids were incorporated by lactating goat mammary particulate fraction into glycerides. The distribution of radio-activity in the different glyceride fractions shows that the synthesis follows the scheme suggested by Weiss, Kennedy and Kiyasu (12). The data further indicate that the incorporation of the third molecule of fatty acid is influenced by the nature of the diglyceride acceptor.

Transacylase activity of lactating bovine mammary fatty acid synthase.

An assay for the transacylation reaction catalyzed by fatty acid synthase was developed which does not require model substrates or labelled acyl‐derivatives of CoA. It involves the transfer of the acyl group from unlabelled CoA to [3H]CoA. This assay shows the occurrence of transacylation at a relatively high rate with a variety of substrates that the enzyme is able to utilize. The activity is unaffected by dissociation of the enzyme or modification by iodoacetamide or 2‐chloroacetyl‐CoA.

Incorporation of palmitate into glycerides by mammary mitochondrial and microsomal fractions.

Abstract Investigation of the extent and nature of the glycerides formed from DL-α-glycerophosphate and 1,2-dipalmitin by the mammary mitochondrial and microsomal fractions failed to reveal any fundamental difference in the synthesis of glycerides.