Triacylglycerol synthesis in goat mammary gland. The effect of ATP, Mg2+ and glycerol 3-phosphate on the esterification of fatty acids synthesized de novo.

Goat mammary-gland microsomal fraction by itself induces synthesis of medium-chain-length fatty acids by goat mammary fatty acid synthetase and incorporates short- and medium-chain fatty acids into triacylglycerol. Addition of ATP in the absence or presence of Mg2+ totally inhibits triacylglycerol synthesis from short- and medium-chain fatty acids, and severely inhibits synthesis de novo of medium-chain fatty acids. The inhibition by ATP of fatty acid synthesis and triacylglycerol synthesis de novo can be relieved by glycerol 3-phosphate. The effect of ATP could not be mimicked by the non-hydrolysable ATP analogue, adenosine 5'-[beta,gamma-methylene]triphosphate and could not be shown to be caused by inhibition of the diacylglycerol acyltransferase by a phosphorylation reaction. Possible explanations for the mechanism of the inhibition by ATP are discussed, and a hypothetical model for its action is outlined.

Triacylglycerol synthesis in goat mammary gland. Factors influencing the esterification of fatty acids synthesized de novo.

ATP alone had no effect on incorporation of fatty acids synthesized de novo and membrane-bound diacylglycerol into triacylglycerol. Combined addition of ATP and Mg2+ totally inhibits incorporation of fatty acids synthesized de novo and stimulated incorporation of membrane-bound diacylglycerol. ATP, Mg2+ and glycerol 3-phosphate stimulate incorporation of fatty acids synthesized de novo into triacylglycerol, but inhibited the incorporation of membrane-bound diacylglycerol. Diacylglycerol generated in situ was shown to be superior to diacylglycerols preloaded on the membrane as substrate for the diacylglycerol acyltransferase. A model is proposed to explain the effect of absorbed exogenous fatty acid on fatty acid synthesis de novo in goat mammary gland.

Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis.

Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.

Transacylation as a chain-termination mechanism in fatty acid synthesis by mammalian fatty acid synthetase. Synthesis of medium-chain-length (C8-C12) acyl-CoA esters by goat mammary-gland fatty acid synthetase.

1. Ruminant mammary-gland fatty acid synthetases can, in contrast with non-ruminant mammary enzymes, synthesize medium-chain fatty acids. 2. Medium-chain fatty acids are only synthesized in the presence of a fatty acid-removing system such as albumin, beta-lactoglobulin or methylated cyclodextrin. 3. The short- and medium-chain fatty acids synthesized were released as acyl-CoA esters from the fatty acid synthetase.

Fatty-acid synthesis in lactating-goat mammary gland. 1. Medium-chain fatty-acid synthesis.

Tissue slices from lactating goat-mammary gland synthesized short (C4:0 and C6:0), medium (C8:0 and C10:0) and long-chain (C12:0 to C16:0) fatty acids in proportions similar to that found in goat milk fat. In contrast, the particle-free supernatant fraction and the purified fatty acid synthetase from this tissue synthesized predominantly short-chain and long-chain fatty acids. Terminating acyl-thioesterases of low molecular weight could not be detected in the particle-free supernatant. Addition of the microsomal fraction to the particle-free supernatant induced the synthesis of medium-chain fatty acids in proportions which were similar to those found in goat milk fat.

Fatty-acid synthesis in lactating-goat mammary gland. 2. Medium-chain fatty acid synthesis.

Addition of goat, rat, rabbit and cow mammary microsomal fraction to fatty acid synthetase of goat mammary gland induced C10 fatty acid synthesis. When the microsomal fractions from rat, rabbit and cow mammary gland were incubated with their respective fatty acid synthetases only the cow enzyme synthesized significant amounts of C10 compared to the purified fatty acid synthetases alone. The goat mammary microsomal fraction was not able to induce C10 synthesis by rat and rabbit mammary-gland fatty acid synthetase, but only by the goat and cow enzyme. Both goat and cow mammary-gland fatty acid synthetase have in contrast to the rabbit and rat enzymes the ability to hydrolyse medium-chain acyl-thioesters. We therefore suggest a novel mechanism for medium-chain fatty acid synthesis in lactating-goat mammary gland. This mechanism involves an activation of the medium-chain acyl-thioester hydrolase within the fatty acid synthetase of goat mammary gland by an unknown microsomal factor.

Molecular weight and subunit size of rabbit mammary-gland fatty acid synthetase.

1. The molecular weights of fatty acid synthetases isolated from lactating rabbit, rat, cow and goat mammary glands were estimated by sucrose gradient centrifugation and compared by chromatography on Sepharose 6B. 2. The values obtained for all four enzymes were in the same range (0.40 X 10(6)-0.55 X 10(6)) as that found for other mammalian and avian fatty acid synthetases. The molecular weight found for the rabbit mammary enzyme therefore differs from published values of approx. 0.9 X 10(6). 3. The molecular weights of the subunits of these four synthetases were 225000-242000. Again, the value for the rabbit mammary enzyme differs from published values.

Prostaglandin E1 high affinity binding sites of rat thymocytes. Specificity and blockade by non-steroidal antiinflammatory drugs and localization in a plasma membrane-enriched fraction.

Great specificity is demonstrated for the prostaglandin E1 high affinity binding sites of rat thymocytes. Whereas prostaglandin E2 has the same affinity as prostaglandin E1, 13 other prostaglandin derivatives and antagonists are bound with 2-1000 times smaller affinities. 50% inhibition of the high affinity binding of prostaglandin E1 to rat thymocytes is demonstrated for three non-steroidal antiinflammatory drugs, indomethacin (3.6. 10(-5) M), salicylic acid (2.9. 10(-3) M) and acetylsalicylic acid (2.10(-2) M). The low affinity binding of prostaglandin E1 is enhanced by the same concentration of indomethacin, however, to a lesser degree and more variable than the inhibition of the high affinity binding of prostaglandin E1. Like intact cells a 50-fold purified plasma membrane fraction, isolated from a homogenate of rat thymocytes, shows reversible high affinity binding of prostaglandin E1 as well as irreversible binding of unidentified tritiated compounds. The binding data are compatible with a localization in the plasma membrane of high affinity sites for reversible binding with a considerably higher dissociation constant than that found for whole cells. Their identity remains to be demonstrated.