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The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidycholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A2 of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphinogomyelin which is not hydrolysed by the former enzyme.

1. The secretion from sheep pancreas and a supernatant fraction prepared from the gland contained an EDTA-insensitive acid phospholipase A1 which readily deacylated phosphatidylinositol (pH optimum, 5.3), 1-acylglycerophosphoinositol and phosphatidic acid, but had limited action of phosphatidylcholine and phosphatidylethanolamine even with deoxycholate present. The enzyme was not a triacylglycerol lipase. 2. The action of the phospholipase A1 on phosphatidylinositol was inhibited effectively by Ca2+ and Mg2+, probably by interaction of these ions with the substrate. 3. In the presence of calcium the decomposition of phosphatidylinositol and lysophosphatidylinositol by the supernatant fraction was overwhelmingly by phosphodiesterase action (EC 3.1.4.10), producing inositol monophosphate and its cyclic derivative. Its pH optimum was about 6.0 but with considerable activity extending to pH 8.5. 4. The phosphodiesterase was not secreted in the pancreatic juice.

1. Rabbit intestinal brush border membrane vesicles were prepared either from frozen or fresh tissue and the lipid composition was analysed. 2. Lipids extracted from membranes prepared by the Ca2+-precipitation method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98--112; Kessler, M., Acuto, O., Storelli, C., Murer, H., Müller, M. and Semenza, G. (1978) Biochim. Biophys. Acta 506, 136--154) had exceptionally high levels of lysophospholipids and free fatty acids. An intrinsic, Ca2+-activated phospholipase A is responsible for the lipid decomposition. 3. It was necessary to modify the preparation of brush border membranes. Essentially, EGTA is used to keep the free Ca2+ concentration low so that phospholipases are inactivated, and Mg2+ instead of Ca2+ is employed to aggregate selectively contaminating membranes. The modified procedure gives membranes suitable for lipid analysis. 4. The molar ratio of neutral, phospholipid and glycolipid is about 1 : 1 : 1. The major neutral lipids are free cholesterol and fatty acids. The major phospholipids are phosphocholine-containing (approx. 45%) and phosphatidyl-ethanolamine (approx. 40%). The remainder (15--20%) is made up ppf acidic phospholipids, mainly phosphatidylserine and phosphatidylinositol. The major glycolipid is ceramide monohexodise. 5. The major fatty acids of total lipids and phospholipids are palmitic, stearic, oleic and linoleic acids. Individual phospholipids are characterised by distinct fatty acid compositions and differ markedly in the ratio of unsaturated-to-saturated fatty acid. X

Departure from first-order kinetics was observed for the inactivation of bovine erythrocyte and electric eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) by n-propanol. This was attributed to the presence of isoenzymes in the commercial preparations, although inactivation via a two-step process cannot be eliminated. The rate of inactivation of bovine erythrocyte enzyme by propanol or sodium dodecyl sulphate was decreased by the presence of added protein and increased by Triton X-100. The presence of a substrate (acetylthiocholine) decreased the rate of inactivation of bovine erythrocyte acetylcholinesterase by sodium dodecyl sulphate, but increased the rate of inactivation by propanol. Re-interpretation of earlier data on the inactivation and reversible inhibition of acetylcholinesterase by simple aliphatic alcohols indicated that they do not bind to hydrophobic regions of the active site.