Membrane bound phosphatidate

The synthesis of triacylglycerol takes place in the endoplasmic reticulum (ER). In liver and adipose tissue, fatty adds in the cytosol obtained from the diet or from de novo synthesis of palmitic add become inserted into the ER membrane. The reactions are shown in Fig. 13-10. Membrane-bound acyl-CoA synthetase activates two fatty acids, and membrane-bound acyl-CoA transferase esterifies them with glycerol 3-phosphate, to form phosphatidic acid. Phosphatidic acid phosphatase releases phosphate, and in the membrane, 1,2-diacylglycerol is esterified with a third molecule of fatty acid. 

In addition to membrane-bound PLAiS, mammals have cytosolic PLAjS. Cytosolic PLAi activities have been studied in various tissues including heart, brain and testis. PA-PLAi has been purified from brain [45] and testes [39, 46]. Like other lipolytic enzymes, PLAi is affected by the assay conditions. Using a mixed micelle system Glomset and his colleagues [46] found that a 110 kDa enzyme from testes preferentially hydrolyzed phosphatidic acid. They cloned the PA-PLAi from bovine [16]. This PLAi lacks sequence similarity to type I PLAi, lysophospholipase, LCAT, and triacylglycerol lipases. 

A membrane-derived a-amylase was solubilized from membrane vesicles by treatment with Triton X-100 and was highly purified by chromatography on an anti-a-amylase-protein A-Sepharose column. Membrane-derived a-amylase was indistinguishable from the soluble extracellular enzyme by sodium dodecyl sulphate-gel electrophoresis and radioimmunoassay. The membrane-derived enzyme contains phospholipid. Approximately 30 to 80% of the phospholipid was extracted from the purified enzyme by chloroform-methanol. The extracted phospholipid was predominantly phosphatidylethanolamine. Treatment with phospholipase D released phosphatidic acid. Membrane-bound a-amylase was latent in membrane vesicles. Release of membrane-bound a-amylase from vesicles by an endogenous enzyme was maximal at pH 8.5, was inhibited by metal chelators and di-isopropyl fluorophosphate and was stimulated by Ca and Mg. The amount of membrane-bound a-amylase was related to the level of secretion. 

In higher plants, the lipolytic enzymes and their physiological functions are not well characterized [1]. iMost reports demonstrated that phospholipid catabolism in plants is achieved by the concerted actions of membrane-bound enzymes including phospholipase D, phosphatidate phosphatase, lipolytic acyl hydrolases and lipoxygenases [1,2]. With the exception of the phospholipase D, the literature on plant phospholipases is still very limited. We previously reported that tonoplast from Acer pseudoplatanus cells contains small amounts of phosphatidic acid and lysophospholipids, which were produced together with free fatty acids, particularly after addition of Ca " [31. These data suggested the possible involvement of phospholipase D and phospholipase A in the metabolism of vacuolar membrane lipids. The phospholipase activities were studied by following the hydrolysis of added sn-2-[14c]linoleyl-PC to tonoplast vesicles. Tonoplast was obtained by osmotic lysis of pure preparations of vacuoles isolated from protoplasts derived from Acer pseudoplatanus cells [4]. This present work demonstrated clearly the presence of phospholipase D and phospholipase Ai activities associated with the tonoplast of Acer, The phospholipase Ai showed an optimal activity at pH about 6-6.5, did not necessarily require divalent cations, but was stimulated by Mg- and particularly by Ca. This work presents the first evidence for the presence of phospholipases A in plant cells. 

The pathways diverge at phosphatidate. In the synthesis of triacylglycerols, phosphatidate is hydrolyzed by a specific phosphatase to give a diacylglycerol (DAG). This intermediate is acylated to a triacylglycerol in a reaction that is catalyzed by diglyceride acyltransferase. Both enzymes are associated in a triacylglycerol synthetase complex that is bound to the endoplasmic reticulum membrane. 

Kai M, Wada I, Imai S, Sakane F, Kanoh H (1996) Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase (type 2) bound to plasma membranes. Polymerase chain reaction amplification of mouse H2O2-inducible hic53 clone yielded the cDNA encoding phosphatidic acid phosphatase. J Biol Chem 271 18931-18938... 

Fig. 10. Mechanism explaining the acylation of -glycerol 3-phosphate in the envelope membranes. El, Acyl-CoA synthetase E2, acyl-CoA in-glycerol 3-phosphate acyltransferase E3, acyl-CoA acyl-sn-glycerol 3-phosphate acyltransferase E4, phosphatidic acid phosphatase. El, E3, and E4 are firmly bound to the chloroplast envelope. E2 is probably loosely bound to the envelope and is released into the medium during the isolation (see Fig. 12) (reproduced from Douce and Joyard, 1979a, by permission).

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