Raymond Dils

Fatty acid biosynthesis V. Purification and characterisation of fatty acid synthetase from lactating-rabbit mammary gland

1. 1. Fatty acid synthetase has been isolated from the particle-free supernatant fraction of lactating-rabbit mammary gland homogenates. 2. 2. Fatty acid synthetase was homogeneous based on the criteria of sedimentation as a single symmetrical peak on analytical ultracentrifugation and elution as a single peak with constant specific activity from Sephadex G-200 and DEAE-52 cellulose. However, traces of acetyl-CoA: COa ligase (ADP) and ATP: citrate lyase were associated with the synthetase. Malonyl-CoA carboxy-lyase activity, for which a modified assay is described, co-purified with fatty acid synthetase. 3. 3. The molecular weight of the fatty acid synthetase was 9.1·105 and its hydrated radius 120 A. It contained 58–59 sulphydryl groups per mole and the amino acid composition is given. 4. 4. The stability of fatty acid synthetase is described. The schlieren patterns of aged preparations showed that dissociation had occurred and this was not reversed on partial reactivation of the enzyme. 5. 5. In the absence of NADPH, fatty acid synthetase formed triacetic lactone from both acetyl-CoA and from acetyl-CoA plus malonyl-CoA. 6. 6. Using optimum substrate concentrations, fatty acid synthetase synthesised a wide range (C4:0−C18:0) of fatty acids. The major product was C16:0 and a significant amount of C4:0 was also synthesised.

Fatty acid biosynthesis VII. Substrate control of chain-length of products synthesised by rat liver fatty acid synthetase

1. 1. Gas-liquid and paper chromatography have been used to determine the chain-lengths of fatty acids synthesised by purified rat liver fatty acid synthetase from [1-14C]acetyl-CoA, [1,3-14C2]malonyl-CoA and from [1-14C]acetyl-CoA plus partially purified rat liver acetyl-CoA carboxylase. 2. 2. A wide range (C4:0–C18:0) of fatty acids was synthesised and the proportions were modified by substrate concentrations in the same manner as for purified rabbit mammary gland fatty acid synthetase. 3. 3. The relative amount of radioactivity incorporated from added acetyl-CoA and malonyl-CoA depended on the substrate concentrations used. At excess acetyl-CoA to malonyl-CoA, greater amounts of acetyl-CoA were incorporated than theoretically expected from the malonyl-CoA pathway. At excess malonyl-CoA, less acetyl-CoA was incorporated than theoretically expected. 4. 4. An increase in the chain-length of fatty acids synthesised from malonyl-CoA was observed with increasing malonyl-CoA concentrations and malonyl-CoA to acetyl-CoA ratios. A similar increase was observed when increasing amounts of a malonyl-CoA generating system were added. However, at the same overall rate of synthesis, a lower proportion of long-chain fatty acids was synthesised from carboxylated acetyl-CoA than from added malonyl-CoA. 5. 5. It is suggested that acetyl-CoA carboxylase may carboxylate acetate bound to fatty acid synthetase.

Fatty acid biosynthesis. VI. Specificity for termination of fatty acid biosynthesis by fatty acid synthetase from lactating-rabbit mammary gland.

1. 1. The extent to which the pattern of fatty acids synthesised by fatty acid synthetase from rabbit mammary gland could be modified by acetyl-CoA and malonyl-CoA concentrations was examined and the results compared with the composition of rabbit milk triglycerides, which contain a high proportion of medium-chain fatty acids. 2. 2. Evidence for two pathways of butyrate synthesis by fatty acid synthetase has been obtained, since butyrate was synthesised both from acetyl-CoA alone and acetyl-CoA plus malonyl-CoA. 3. 3. Increasing the malonyl-CoA concentration and the ratio of concentrations of malonyl-CoA to acetyl-CoA increased the proportion of long-chain (C14:0–C18:0) and decreased the proportion of short-chain (C4:0 and C6:0) fatty acids formed. Long-chain fatty acids were synthesised mainly as free acids and the short-chain fatty acids were predominantly in an esterified form. Medium-chain fatty acids (C8:0–C12:0) were only formed in these experiments in amounts approximately equimolar to the fatty acid synthetase protein. The modification in the pattern of fatty acids synthesised with increasing acetyl-CoA carboxylase activity was analogous to that with increasing concentrations of added malonyl-CoA. 4. 4. A specificity for the in vivo synthesis of medium-chain fatty acids by rabbit mammary gland must be superimposed on the specificity of isolated fatty acid synthetase for chain termination.

Acyl-CoA hydrolase(s) in rabbit mammary gland which control the chain length of fatty acids synthesised

Summary A factor (Fraction IV) is present in the cytosol of lactating rabbit mammary gland which interacts directly with fatty acid synthetase to terminate chain elongation. This causes the release from the synthetase of unesterified medium-chain fatty acids (C 8:0 –C 12:0 ) which are characteristic of rabbit milk. Fraction IV contains acyl-thioester hydrolase(s) which are active towards medium- as well as long-chain acyl-CoA esters. It is proposed that this activity could control chain termination by cleaving medium-chain acyl groups from the acyl carrier protein of fatty acid synthetase.

Immunological properties of medium-chain acyl-thioester hydrolase and fatty acid synthetase from lactating-rabbit mammary gland

The cytosol from lactating-rabbit mammary gland contains a medium-chain acyl-thioester hydrolase. This hydrolase terminates chain lengthening of the fatty acids synthesised by fatty acid synthetase so as to release C8:0 and C10:0 fatty acids which are characteristic of rabbit milk. The medium-chain hydrolase and the fatty acid synthetase present in this cytosol have been shown to be immunologically distinct. When fatty acid synthetase was purified from this cytosol it showed unexpected immunological reactivity towards antiserum raised to the medium-chain hydrolase. The precipitate formed was not due to fatty acid synthetase, but to medium-chain hydrolase contaminating the synthetase. However, the proportion of this medium-chain hydrolase which was recovered with the purified synthetase was too small to be detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was too small to elicit an antibody response in sheep. Immunological techniques have shown that the medium-chain hydrolase appears in rabbit mammary gland between days 17 and 22 of pregnancy. This coincides with the onset of milk-fat synthesis. The medium-chain hydrolase could not be detected in the cytosol from lactating-rabbit liver.

Fatty acid biosynthesis. VIII. The fate of malonyl-CoA in fatty acid biosynthesis by purified enzymes from lactating-rabbit mammary gland

Abstract 1. 1. We have investigated the formation and utilization of malonyl-CoA in fatty acid synthesis catalysed by preparations of partially purified acetyl-CoA carboxylase and purified fatty acid synthetase from lactating-rabbit mammary gland. 2. 2. Carboxylation of [1- 14 C]acetyl-CoA was linked to fatty acid synthesis by the presence of fatty acid synthetase and NADPH. The rate of fatty acid formation was equal to that of acetyl-CoA carboxylation, without the accumulation of free malonyl-CoA to a concentration required to obtain the same rate of fatty acid synthesis from added [1,3- 14 C 2 ]malonyl-CoA. 3. 3. The preparations of acetyl-CoA carboxylase and fatty acid synthetase were each able to decarboxylate [1,3- 14 C 2 ]malonyl-CoA. 4. 4. Both enzyme preparations acted as competitive inhibitors of 14 CO 2 fixation into acetyl-CoA catalysed by acetyl-CoA carboxylase in the absence of NADPH. This is attributed to a mechanism of product inhibition. The effect on the apparent activity of acetyl-CoA carboxylase assayed by malonyl-CoA formation is discussed. 5. 5. Our results suggest a metabolic compartmentation of the carboxylation step which facilitates the incorporation of carboxylated acetyl-CoA into fatty acids in the presence of NADPH and prevents its catabolism by side reactions.

Fatty acid synthase from rabbit mammary gland.

Publisher Summary This chapter discusses the fatty acid synthase from rabbit mammary gland. The fatty acid synthase complex isolated from lactating rabbit mammary gland catalyzes the synthesis of saturated even-numbered fatty acids from acetyl-CoA, malonyl-CoA, and nicotinamide adenine dinucleotide phosphate (NADPH). The activity of purified fatty acid synthase and of the enzyme during purification can be measured via the oxidation of NADPH. Optimum assay conditions at 37° are 200 m M potassium phosphate buffer, pH 6.6, 1 m M dithiolthreitol, 1 m M EDTA, 0.24 m M NADPH, 30 μ M acetyl-CoA, 40-50 μ M malonyl-CoA, and enzyme protein to produce an absorbance change of 0.05–0.15 unit/minute in a final volume of 1.0 ml. After measuring NADPH oxidation without added acetyl-CoA and malonyl-CoA, the reaction is started by adding these substrates. The reaction rate should be linear with time for at least 3 minutes. One unit of enzymatic activity oxidizes 1 μmole of NADPH per minute. Mammary tissue (about 100 g wet weight) is obtained from a lactating rabbit 4–20 days postpartum. Fatty acid synthase with maximum specific activity is obtained from animals at 4 days postpartum. From 4 to 20 days postpartum there is a gradual decline in the specific activity of the enzyme isolated. The purified enzyme has a specific activity at 37° of 880 ± 30 mμmoles of NADPH oxidized per minute per milligram of protein (mean of 15 preparations ± S.E.M.).

Partial purification from rabbit mammary gland of a factor whice controls the chain length of fatty acids synthesised

Summary A factor which controls the chain length of synthesised fatty acids has been partially purified from the cytosol of lactating rabbit mammary gland. The factor contains protein and has a molecular weight of 40,000–50,000. It alters the fatty acids synthesised by purified fatty acid synthetase from mainly C 14:0 and C 16:0 acids to C 8:0 , C 10:0 and C 12:0 acids. These mediumchain fatty acids are characteristic of rabbit milk.

Fatty acids synthesized by mammary gland slices from lactating guinea pig and rabbit

Abstract 1. 1. Mammary slices from lactating guinea pigs synthesize long- chain (C 16 -C 18 saturated and unsaturated fatty acids from acetate in the presence or absence of glucose. These are the predominant acids of guinea pig milk. The proportions of fatty acids synthesized varied with substrate concentration. 2. 2. In contrast, mammary slices from lactating rabbits synthesize octanoic (C 8:0 ) and decanoic (C 10:0 ) acids from acetate plus glucose and from glucose alone. These constitute up to 70 per cent of the fatty acids of rabbit milk. The proportions of fatty acids synthesized were constant even when the overall rate of synthesis varied 500-fold.

Fatty acid biosynthesis. X. Specificity for chain-termination of fatty acid biosynthesis in cell-free extracts of lactating rabbit mammary gland.

Abstract 1. 1. The pattern of fatty acids synthesised from [1−14C]acetate by subcellular fractions of lactating rabbit mammary gland fortified by appropriate cofactors was found to be dependent on the concentration of the fractions as measured by protein. 2. 2. At high concentrations (14–22 mg protein/ml), the cell-free homogenate, the microsomal plus particle-free supernatant, and the particle-free supernatant fractions all synthesised a high proportion of the medium-chain fatty acids (C8:0) and C10:0) which are characteristic of rabbit milk. 3. 3. Dilution of these fractions to less than 1 mg protein/ml caused a progressive increase in the proportion of long-chain acids (C14:0 and C16:0) synthesised. 4. 4. With concentrated subcellular fractions, synthesis of medium-chain fatty acids was not dependent on the rate of fatty acid synthesis, the concentration of malonyl-CoA, or the amount of fatty acid synthetase present. 5. 5. Though there was a correlation between the synthesis of medium-chain fatty acids and of triglyceride by particulate fractions, esterification of synthesised fatty acids did not influence the proportion of medium-chain acids formed. 6. 6. Evidence is presented that chain termination at C8:0 and C10:0 acids is controlled by a factor (or factors) present in the particle-free supernatant fraction. This factor is not lost on dialysis, and can be precipitated between 40 and 100% saturation with (NH4)2SO4 or by heating.