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Treatment of the anaerobic, Gram negative general fatty acid auxotroph Butyrivibrio S2 with lysozyme in low molarity buffers resulted in the formation of protoplasts, some of which retained the original rod-shaped morphology of the organism. The protoplasts were stabilised by the presence of Mg2+ ions. Most of the phospholipase A and C and galactolipase activity of the cells was retained by the protoplasts. Electron microscopy and chemical markers were used to monitor the separation of plasma membrane and cell wall fragments by density gradient centrifugation after osmotic lysis of protoplasts. Phospholipase and galactolipase activities were demonstrated in a subcellular fraction which contained only fragments of plasma membrane.

1. 1. A method has been devised which enables the specific radioactivities of phosphatidyl ethanolamine, phosphatidyl serine and phosphatidyl choline, and also diphosphoinositide, to be determined in a single small sample of tissue (150–400 mg) which has been labelled with 32 P. 2. 2. A lipid extract of the tissue is hydrolysed under mild alkaline conditions, and identifiable phosphorus-containing hydrolysis products of the phospholipids are completely resolved by two-dimensional filter paper chromatrography. 3. 3. The method has been used to examine a lipid extract prepared from a guinea-pig brain dispersion after incubation with labelled phosphate. 4. 4. In such dispersions there is little incorporation of 32 P into phosphatidyl ethanolamine, -serine or, -choline, while there is an appreciable synthesis of diphosphoinositide and a lipid-solvent soluble substance which is probably phosphatidic acid.

Measurements have been made of the binding of radioactive Ca2+ to unimolecular films of purified phosphatidylserine and phosphatidylinositol at various film pressures. Three theoretical treatments based on the mass equation have been applied to the experimental data to evaluate the binding of Ca2+ in terms of K A , the apparent association constant, and n , the fraction of phospholipids molecules acting as binding sites for a Ca2+. At low film pressures (or low charge densities) the binding of Ca2+ follows the mass equation. Deviations from the mass equation are observed with increasing film pressure (charge density) and are attributed to electrostatic interaction between adjacent binding sites. The ratio of Ca2+ bound/lipid reaches a maximum at about 30 dynes/cm when the spacing between phospholipid molecules is most favourable for two-point electrostatic attachment of Ca2+. Ca2+ bound to phosphatidylserine and phosphatidylinositol form a Stern layer and do not penetrate into the plane of the Gouy potential.

Changes which occur in the concentrations of certain amino acids in rat brain during fluoroacetate poisoning, insulin hypoglycaemia and thiopentone anaesthesia have been measured. Fluoroacetate poisoning causes a reduction in the cerebral glutamic acid and aspartic acid levels, and an increase in the alanine concentration. Both insulin hypoglycaemia and thiopentone anaesthesia produce a reduced glutamic acid content and a rise in the aspartic acid level.

(1) An analysis has been conducted of the lipids present in three obligately anaerobic bacteria isolated from the ovine rumen belonging to the genus Butyrivibrio. Two of these organisms are rich in phospholipase (A1 + A2) activity, and appear to be different strains of the species fibrisolvens. (2) The only N-containing lipids comprise N-acyl-phosphatidylethanolamine occurring as a minor component in all organisms and a new lipid, diglyceride galactosylphosphorylethanolamine in one of these. (3) All three organisms contained the n-butyryl ester of phosphatidyl-glycerol and in one this represented the major phospholipid present. Valeryl, iso-valeryl, propionyl and myristoyl esters of phosphatidylglycerol were also detected. (4) Two organisms contained glycerylphosphorylgalactosyldiglyceride and one of these also contained a large proportion of a less polar galactophospholipid which is probably a diacyl derivative of the former lipid. (5) All three organisms contained monogalactofuranosyl diglyceride and from one a n-butyryl ester of this galactolipid was isolated. (6) In all of the lipids examined the "diglyceride' moiety consisted almost entirely of plasmalogenic diglyceride (alk-1-enyl, acyl, glycerol).

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. 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

Intercellular coupling between cumulus cells and oocytes has been measured in vitro using intracellular markers derived from 3H-labelled choline, uridine and inositol. These compounds can only enter oocytes through junctions with cumulus cells. Choline, selected for most detailed study, is metabolized to phosphocholine and lecithin by cumulus cells and is probably transported into oocytes after phosphorylation. The effect of gonadotropins on follicles, either in vivo or in culture is to reduce, but not totally eliminate, passage of labelled markers from cumulus cells into oocytes. The hormone primarily responsible for this change in coupling, which takes 12–15 h to become evident, is FSH. Inhibition of follicular steroid synthesis using aminoglutethimide has a small but significant effect on this hormone-dependent system of intercellular communication. The advantages are emphasized of using cumulus-oocyte complexes, together with intracellular markers, to study basic properties of intercellular coupling. Present results on cumulus cell-oocyte coupling are also discussed in relation to the regulation of meiosis.

The hydrolysis by trypsin of the bacterial phospholipid, lysylphosphatidyl-glycerol has been studied at the air-water interface. High specific activity [14C]-lysylphosphatidylglycerol was prepared biosynthetically and the trypsin action followed by measuring the loss of surface radioactivity from a monolayer of the phospholipid spread on a water surface. After a lag phase, hydrolysis proceeded at a constant rate proportional to the enzyme concentration until 50–85% of the substrate had been hydrolysed. Maximum hydrolysis occurred between 10–35 dynes/cm surface pressure and at pH 7.5–8.5. A slight fall in the surface pressure occurred but this was less than predicted from the force-area curves of lysylphosphatidylglycerol and phosphatidylglycerol. Ca2+ (0.5 mM) and to a lesser extent Mg2+ and Sr2+ stimulated the reaction. During the reaction 45Ca2+ became bound on the monolayer in proportion to the extent of the enzymic hydrolysis. The stimulation by calcium required the synergic presence of low concentrations of Tris buffer. High concentrations of CaCl2 (2 mM), NaCl (25 mM), Tris buffer (47 mM) almost completely inhibited the reaction. Chlorpromazine, which removes Ca2+ from phospholipid membranes, markedly stimulated hydrolysis at 2.5–6.0 μM, but only in the presence of added calcium: higher concentrations of chlorpromazine (0.125 mM) completely inhibited the reaction.

1. 1. Preparations of the phospholipase in P. notatum mycelium and ox pancreas were found to slowly hydrolyse monophosphoinositide liberating acid-soluble inositol. 2. 2. The P. notatum enzymic digest of monophosphoinositide contained glycerylphosphorylinositol, glycerophosphoric acid, inositol, inorganic phosphorus and free fatty acids. 3. 3. The pancreas enzymic digest of monophosphoinositide contained inositol monophosphate and very small amounts of glycerylphosphorylinositol; no glycerophosphoric acid, inorganic phosphorus or free inositol could be detected. 4. 4. The pH optimum of monophosphoinositide attack by the pancreas phospholipase preparation was 6.0 and by the P. notatum phospholipase preparation was 3.3. These are the optimal pH's at which the enzymes attack lysolecithin. 5. 5. Monophosphoinositide attack by the pancreas phospholipase preparation was markedly stimulated by 0.01 M Ca ++ , whereas lysolecithin breakdown was not affected nor the hydrolysis of monophosphoinositide or lysolecithin by the P. notatum enzyme. 6. 6. Lysolecithin breakdown by pancreas phospholipase was strongly inhibited by fluoride. 7. 7. Both lysolecithin and lecithin inhibited the breakdown of monophosphoinositide by the pancreas and P. notatum phospholipase preparations. 8. 8. The probable pathway of breakdown of monophosphoinositide by the P. notatum enzyme preparation is discussed.

The adsorption of 45Ca to monolayers of phosphatidylinositol and dicetylphosphoric acid has been measured as a function of subphase pH with simultaneous recordings of surface pressure and interfacial potential. Below pH 3 little calcium was adsorbed and the films are assumed to be unionized. With acid subphases between pH 3 and 6.5 adsorption of calcium occurred initially, but it was then gradually lost due to an ageing process in the films. This time dependent change in the properties of the film was independent of the presence of Ca2+, but was dependent on the H+ concentration in the subphase; it was however not due to an acid hydrolysis of the monolayer. Ca2+ was permanently adsorbed at pH values above 6.5 with an increasing affinity up to pH 11.

1. 1. α-Toxin from Cl. perfringens (phospholipase C) has been purified until, as judged by electrophoresis and agar diffusion, a single protein component was obtained. 2. 2. The α-toxin hydrolyses lecithin in the absence of Ca 2+ , provided that small amounts of a long-chain cation ( e.g. stearylamine) are present in the micelle. The rate of hydrolysis in the system is much slower than that which occurs in the presence of Ca 2+ . 3. 3. The addition of the long-chain cation produced a positive electrophoretic mobility of the substrate. Complete neutralization of this net positive charge, by addition of a long-chain anion, resulted in total inhibition of enzyme activity. 4. 4. With a substrate activated with a long-chain cation, hydrolysis ceased when 60% of the lecithin molecules had been hydrolysed. Evidence is presented which indicates that this reaction is associated with reversal of the charge of the micelle from the positive to the negative state, this reversal being due to diglyceride, which eventually accumulates on the surface. 5. 5. Calcium, magnesium and uranyl salts activated the hydrolysis of lecithin emulsions and at the same time caused the micelles to become positively charged. 6. 6. Conversely, ferricyanide ions were efficient inhibitors of the enzyme and at the same time caused a reversal of the charge of the lipid micelles from positive to negative. 7. 7. A small hydrolysis of phosphatidylethanolamine by α-toxin has been demonstrated. The rate of this hydrolysis is accelerated if lecithin is added, or of it is combined as a lipoprotein. 8. 8. High-pressure ( i.e. more than 40 dynes/cm) monomolecular films of [ 32 P]-lecithin are hydrolysed when Ca 2+ or UO 2 2+ are added to the bulk phase. However, the addition of long-chain cations to lecithin did not result in measurable hydrolysis of such films. Hydrolysis of low-pressure lecithin films (less than 30 dynes/cm) was observed in the complete absence of divalent cations. 9. 9. It is suggested that α-toxin can attack a phospholipid at a micelle-water interface only when the molecule is in close association with a free cationic group. When this cationic group is provided by a water-insoluble amphipathic molecule, the activation is directly related to the z-potential, i.e. net charge. On the other hand, limited hydrolysis can occur in the presence of divalent ions, e.g. Ca 2+ , even when the z-potential is net negative, possibly because of localized regions of positive charges at a molecular level.