Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase.

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.

A role for phosphoinositide 3-kinase in bacterial invasion.

Listeria monocytogenes is a bacterial pathogen that invades cultured nonphagocytic cells. Inhibitors and a dominant negative mutation were used to demonstrate that efficient entry requires the phosphoinositide (PI) 3-kinase p85alpha-p110. Infection with L. monocytogenes caused rapid increases in cellular amounts of PI(3, 4)P2 and PI(3,4,5)P3, indicating that invading bacteria stimulated PI 3-kinase activity. This stimulation required the bacterial protein InlB, host cell tyrosine phosphorylation, and association of p85alpha with one or more tyrosine-phosphorylated proteins. This role for PI 3-kinase in bacterial entry may have parallels in some endocytic events.

Room-temperature single-photon emitters in titanium dioxide optical defects.

Fluorescence properties of crystallographic point defects within different morphologies of titanium dioxide were investigated. For the first time, room-temperature single-photon emission in titanium dioxide optical defects was discovered in thin films and commercial nanoparticles. Three-level defects were identified because the g(2) correlation data featured prominent shoulders around the antibunching dip. Stable and blinking photodynamics were observed for the single-photon emitters. These results reveal a new room-temperature single-photon source within a wide bandgap semiconductor.

Overexpression of the myristoylated alanine-rich C kinase substrate in human choroidal melanoma cells affects cell proliferation.

Reduced expression of the myristoylated alanine-rich C kinase substrate (MARCKS) has been described in various cell lines after oncogenic or chemical transformation, leading to the question of whether this protein may be involved in cell proliferation. Here we compare the expression of MARCKS in human tumor-derived choroidal melanoma cells (OCM-1) and in primary cultures of normal choroidal melanocytes. We found an important down-regulation of the protein in the melanoma cell line. Stable transfection of these cells with the cDNA coding for MARCKS led to the selection of several clones expressing variable levels of the protein. Proliferation experiments performed with four of these clones revealed that cell growth was reduced by 35-40% when compared with control cells. Upon serum starvation, cell proliferation was almost abolished when the expression level of MARCKS was high, whereas it was only partially reduced in the controls. MARCKS overexpression induced a higher percentage of cells in the G0-G1 phase of the cell cycle upon serum starvation, as well as the inhibition of colony formation in soft agar. Finally, the expression of the CDK inhibitor p27 was increased in the cells presenting a high level of MARCKS protein. Altogether, these data suggest that the expression of this protein kinase C substrate affects the proliferation and partially reverts the transformed phenotype of the OCM-1 cells.

Stimulation by epidermal growth factor of inositol phosphate production in plasma membranes from A431 cells.

Plasma membranes were isolated from A431 cells previously labelled with myo-[3H]inositol during exponential growth, using a rapid procedure on Percoll gradients. They displayed a significant phospholipase (PLC) activity against phosphoinositides, which was stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), epidermal growth factor (EGF) and fetal calf serum (FCS) (24%, 11% and 97% over controls, respectively). The effect of EGF was not significantly increased by GTP gamma S. Upon addition of cytosol, EGF promoted an almost 100% stimulation of inositol 1,4,5-trisphosphate and inositol bisphosphate generation, which displayed an absolute requirement for GTP gamma S. This dose-dependent effect of cytosol was linear until 60 micrograms/ml of cytosolic protein and decreased afterwards; it was abolished by heat treatment and trypsin hydrolysis, and it was not reproduced by an identical amount of bovine serum albumin. The same biphasic stimulation was observed with phosphotyrosyl proteins immunopurified from cytosol of A431 cells previously stimulated by EGF. Since phosphotyrosyl proteins displayed PLC activity, our data suggest that soluble protein substrates of EGF receptor tyrosine kinase, including PLC, could be involved in the regulation of phosphoinositide hydrolysis in response to EGF. Using phosphatidyl[3H]inositol 4,5-bisphosphate (PIP2) dispersed with unlabelled phosphatidylethanolamine and phosphatidylserine as an exogenous substrate, no stimulation of PLC activity by EGF could be detected, either with membranes or with membranes plus cytosol. It is concluded that EGF might stimulate hydrolysis of phosphoinositides by PLC through complex interactions between plasma membrane and cytosolic factors which still remain to be identified.

Impaired secretion of heart lipoprotein lipase in cyclophosphamide-treated rabbit.

Cyclophosphamide administration into fasted rabbits induces a hypertriglyceridaemia and a defect in vascular lipoprotein lipase. Heart LPL activity was more than 50% decreased after antimitotic treatment in fasted animals. The tissue distribution of lipoprotein lipase activity was followed in heart using recycling perfusion. Cyclophosphamide administration resulted in a profound decline in the heparin-releasable lipoprotein lipase activity, concordant with a higher recovery in the residual heart tissue. The effects were more pronounced in fasted than in fed animals. In agreement, the proportion of neosynthesized [35S]methionine-labelled lipoprotein lipase released by heparin was decreased by 50% following antimitotic treatment. The lipolysis of very low density lipoprotein-labelled triacylglycerols was found 2.5-fold reduced in hearts from cyclophosphamide-treated rabbits as compared to controls. These results suggest that a defective secretion of lipoprotein lipase may contribute to the poor expression of lipolytic activity in the vascular bed and to the occurrence of hypertriglyceridaemia during cyclophosphamide treatment.

Possible origins of PAF-acether and lyso-PAF-acether in rat lung alveoli secondary to hypoxia.

After 4 h hypoxia, platelet activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and its deacetylated derivative, lyso-PAF-acether, accumulate in rat lung surfactant, the latter in a 1000-fold excess (Prévost, M.C., Cariven, C., Simon, M.F., Chap, H. and Douste-Blazy, L. (1984) Biochem. Biophys. Res. Commun. 119, 58-63). In order to determine the origin of these two phospholipids, rat lung alveolar lavages and rat lung macrophages were examined for phospholipid composition before and after 4 h of hypoxic treatment. Our data indicate an activation of phospholipase A2 in both compartments, as detected by the accumulation of lysophosphatidylcholine. The main effect was observed in lung surfactant, where phosphatidylcholine hydrolysis attained 13%. This change was concomitant with the activation of a calcium-independent phospholipase A2 present in lung alveolar lavages, which might be responsible for the accumulation of some lyso-PAF-acether, alkylacylcholine glycerophospholipids being present in low but significant amounts in lung surfactant. However, the main source of PAF and lyso-PAF-acether appears to be alveolar macrophages, which secreted significant amounts of the two phospholipids upon in vitro hypoxic treatment, although the participation of other cells, such as type II pneumocytes, cannot be excluded. The relative amounts of the two compounds might be regulated by both an intracellular and an extracellular acetylhydrolase, the two enzymes being distinct proteins on the basis of their different isoelectric points.

Asymmetric distribution of arachidonic acid in the plasma membrane of human platelets. A determination using purified phospholipases and a rapid method for membrane isolation.

1. Non-lytic degradation of human platelet phospholipids have been performed using a combination of bee venom phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) and Staphylococcus aureus sphingomyelinase C (sphingomyelin choline phosphohydrolase). Under these conditions, 25.4% of total phospholipds are degraded and 6.4% of total platelet arachidonic acid is released. 2. A new method for rapid isolation of platelet plasma membrane is described, based on the use of [3H]concanavalin A as a membrane marker and of self-generating gradients of Percoll. Plasma membranes are enriched 5.2 fold in lectin marker and 0.43 in N-acetyl-beta-D-glucosaminidase, the main contaminant. This method allows to estimate that 57% of the total cell phospholipids and 61% of the total arachidonic acid content are located in the plasma membrane. 3. The distribution of phospholipids and arachidonic acid between the two leaflets of the plasma membrane has been deduced by using these values and those obtained from non-lytic treatment of intact platelets by phospholipases. It is concluded that 45% of plasma membrane phospholipids, comprising 93% of sphingomyelin, 45% of phosphatidylcholine, 9% of phosphatidylserine, 16% of phosphatidylinositol and 20% of phosphatidylethanolamine form the outer half of the human platelet plasma membrane. The phospholipids appear to bear only 10% of the total membrane arachidonic acid.

Selective inhibition of human platelet phospholipase A2 by buffering cytoplasmic calcium with the fluorescent indicator quin 2. Evidence for different calcium sensitivities of phospholipases A2 and C.

Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076).

Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver.

Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.

Behaviour of phospholipase modified-HDL towards cultured hepatocytes. II. Increased cell cholesterol storage and bile acid synthesis.

Human total HDL (hydrated density 1.070-1.210), HDL2 (1.070-1.125), HDL3 (1.125-1.210) or HDL separated by heparin affinity chromatography were treated with or without purified phospholipase A2 from Crotalus adamanteus. Control and treated HDL were reisolated and were then incubated with cultured hepatocytes. 1. Mass measurements evidenced a time-dependent cholesterol enrichment in hepatocytes cultured in the absence of lipoproteins. Addition of HDL2 still enhanced by 25% the cell cholesterol content and down-regulated endogenous sterol synthesis in similar proportions. Conversely, HDL3 slightly decreased the amount of free cholesterol in hepatocytes (-12%). 2. Incubations with phospholipase A2-treated HDL resulted in a 35%-50% increase of both the cellular cholesterol esterification and the cholesterylester accumulation, when compared to cells cultured in the presence of control-HDL. This effect was observed with HDL2, HDL3 and combining the data with all subfractions. 3. Cultured hepatocytes secreted cholic and beta-muricholic acids as major bile acids and HDL2 showed a tendency to stimulate their secretion. Phospholipase treatment of HDL again induced an increased production by hepatocytes of those two bile acids. Thus, whereas HDL2 and HDL3 display different behaviours with respect to cell cholesterol content, neosynthesis and bile acid secretion, their modifications by phospholipases always orientate the cell sterol metabolism in the same direction: increased cholesterylester accumulation and bile acid production.

Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients.

Two kinds of membranes (plasma membranes and intracellular membranes) have been separated from human platelets by fractionation on Percoll gradients (successively at pH 7.4 and pH 9.6). On alkaline Percoll gradient, plasma membranes floated at low density, as shown with specific markers such as [3H]concanavalin A and monoacylglycerol lipase, whereas intracellular membranes sedimented in the higher densities and displayed a 5.6-12.4-fold enrichment in NADH diaphorase, antimycin insensitive NADH-cytochrome-c oxidoreductase and Ca2+-ATPase. Another criterion allowing differentiation of two membrane populations of human platelets was their lipid composition, which showed a cholesterol/phospholipid molar ratio of 0.5 in plasma membranes against 0.2 in intracellular membranes. Phospholipid analysis of the two kinds of membranes displayed also quite different profiles, since phosphatidylcholine increased from 30-32% in the plasma membrane to 52-66% in the intracellular membranes. This was at the expense of sphingomyelin (20-23% in plasma membrane, against 6.8-7.7% in intracellular membranes) and of phosphatidylserine (12-13% in plasma membrane, against 2-6% in intracellular membranes). Other striking differences between plasma membranes and intracellular membranes were obtained by SDS-polyacrylamide gel electrophoresis, which revealed the absence of actin and myosin in the intracellular membrane, whereas both proteins were present in significant amounts in plasma membranes. Finally, intracellular membranes but not plasma membranes were able to incorporate calcium. These results suggest that intracellular membrane fractions are derived from the dense tubular system and plasma membranes should correspond to the whole surface membrane of human platelets.

Phospholipase A2-treated human high-density lipoprotein and cholesterol movements: exchange processes and lecithin: cholesterol acyltransferase reactivity.

Human HDL3 (d 1.125-1.21 g/ml) were treated by an exogenous phospholipase A2 from Crotalus adamenteus in the presence of albumin. Phosphatidylcholine hydrolysis ranged between 30 and 90% and the reisolated particle was essentially devoid of lipolysis products. (1) An exchange of free cholesterol was recorded between radiolabelled erythrocytes at 5-10% haematocrit and HDL3 (0.6 mM total cholesterol) from 0 to 12-15 h. Isotopic equilibration was reached. Kinetic analysis of the data indicated a constant rate of free cholesterol exchange of 13.0 microM/h with a half-time of equilibration around 3 h. Very similar values of cholesterol exchange, specific radioactivities and kinetic parameters were measured when phospholipase-treated HDL replaced control HDL. (2) The lecithin: cholesterol acyltransferase reactivity of HDL3, containing different amounts of phosphatidylcholine, as achieved by various degrees of phospholipase A2 treatment, was measured using a crude preparation of lecithin: cholesterol acyltransferase (the d 1.21-1.25 g/ml plasma fraction). The rate of esterification was determined between 0 and 12 h. Following a 15-30% lipolysis, the lecithin: cholesterol acyltransferase reactivity of HDL3 was reduced about 30-40%, and then continued to decrease, though more slowly, as the phospholipid content was further lowered in the particle. (3) The addition of the lecithin: cholesterol acyltransferase preparation into an incubation medium made of labelled erythrocytes and HDL3 promoted a movement of radioactive cholesterol out of cells, above the values of exchange, and an accumulation of cholesteryl esters in HDL. This reflected a mass consumption of free cholesterol, from both the cellular and the lipoprotein compartments upon the lecithin: cholesterol acyltransferase action. As a consequence of a decreased reactivity, phospholipase-treated HDL (with 2/3 of phosphatidylcholine hydrolyzed) proved much less effective in the lecithin: cholesterol acyltransferase-induced removal of cellular cholesterol.

Phospholipase C from human sperm specific for phosphoinositides.

Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization.

Pathways of arachidonic acid liberation in thrombin and calcium ionophore A23187-stimulated human endothelial cells: respective roles of phospholipids and triacylglycerol and evidence for diacylglycerol generation from phosphatidylcholine.

Cultured endothelial cells from human umbilical vein were incubated for 20 h at 37 degrees C in the presence of [U-14C]arachidonic acid. Around 60-70% of the radioactive fatty acid was incorporated into cell lipids and was predominantly found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerol (39%, 33%, 13% and 6.5% of total incorporated radioactivity, respectively). Stimulation of the cells with human thrombin (2 U/ml) or calcium ionophore A23187 (5 microM) promoted the release into supernatants of arachidonic acid, 6-ketoprostaglandin F1 alpha, prostaglandins E2 and F2 alpha, in decreasing order of importance. The amount of secreted material was 4-fold higher with A23187, compared to thrombin. Parallel to the liberation process, phosphatidylcholine underwent a rapid decrease of radioactivity with both agonists, suggesting the involvement of a Ca2+-dependent phospholipase A2. Phosphatidylethanolamine displayed a minor decrease with A23187, whereas some reacylation was observed at 10 min with thrombin. Phosphatidylinositol was non-significantly affected in thrombin-stimulated cells, whereas A23187 promoted an early but minor decrease, followed by resynthesis. In contrast to A23187, thrombin was also able to promote a significant hydrolysis of triacylglycerol, which might thus be implicated in the process of arachidonate liberation. Finally, radioactive phosphatidic acid and diacylglycerol appeared in endothelial cells, in response to the two agonists. However, diacylglycerol formation did not parallel that of phosphatidic acid, especially with A23187. Determination of the 14C/3H ratio of the different lipids upon cell labelling with both [14C]arachidonic acid and [3H]palmitic acid revealed that diacylglycerol and phosphatidic acid are hardly derived from inositol-phospholipid breakdown by phospholipase C. Other possible pathways involving for instance phospholipase C splitting of phosphatidylcholine are discussed.

Guinea pig intestinal phospholipase B: protein expression during enterocyte maturation and effects of N-oligosaccharide removal on enzymatic activities and protein stability.

Guinea pig phospholipase B (PLB) is an intestinal brush-border hydrolase displaying a broad substrate specificity towards various dietary lipids. PLB was detected by immunoblotting as a single 140-kDa polypeptide in all cell populations isolated from guinea pig intestinal mucosa, but increased in parallel to its activity from undifferentiated to mature cells, the specific activity of the enzyme remaining constant. Moreover, N-glycosylation, which contributed to 23% of the apparent molecular mass, was identical along the cell differentiation axis. In all cell fractions, N-linked sugar chains were of the complex type, since they were removed by N-glycosidase F, whereas PLB remained insensitive to endoglycosidase H. Moreover, lack of O-glycosylation was demonstrated by the insensitivity of PLB to O-glycosidase and by its failure to interact with Helix pomatia lectin after prior treatment with neuraminidase or alpha-fucosidase. Enzymatic removal of sugar chains reduced phospholipase A2, lysophospholipase and diacylglycerol lipase activities by 27-35%, kinetic analysis indicating a decrease in apparent Vmax values for the three enzymatic activities, whereas the Km remained unchanged. Finally, the carbohydrate-depleted form of PLB did not display gross changes in thermal stability, in contrast to PLB from microorganisms previously investigated. Our data indicate that the high level of PLB N-glycosylation is poorly related to its biological function. Whether carbohydrate chains are involved in proper targeting of the enzyme to the brush-border membrane remains to be established.

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells.

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.

Studies on ether phospholipids. II. Comparative composition of various tissues from human, rat and guinea pig.

The ether phospholipid composition of various tissues (brain, heart, lung, liver, kidney, testis, erythrocytes and plasma) has been investigated in human, rat and guinea pig, using a new method of determination (El Tamer, A., Record, M., Fauvel, J., Chap, H. and Douste-Blazy, L. (1984) Biochim. Biophys. Acta 793, 213-220). This is based on the selective removal of diacyl phospholipid species by phospholipase A1 degradation followed by acidolysis of the plasmalogens. Our results fit rather well with other literature data available for human and rat tissues, illustrating the good reliability of the method. Among various differences noted between the three mammalian species, guinea pig is characterized by a relatively higher content of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) and of ethanolamine plasmalogens in blood plasma. Alkylacyl-GPC, a putative precursor of platelet activating factor (PAF-acether or 1-alkyl-2-acetyl-GPC), is also more abundant in guinea pig lung and in human kidney. This study also revealed a striking parallelism between the tissue content of alkylacyl-GPC and alkylacyl-GPE (1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine). This new observation is discussed in relation to a possible metabolic link between these two phospholipids.

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. I. Studies on neutral glycerides.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various neutral glycerides. Triolein hydrolysis proceeded in the absence of di- and monoolein accumulation. Optimal conditions for di- and monoolein hydrolysis included an alkaline pH (9-10), a substrate concentration of 10 mM, and the presence of sodium deoxycholate (12 and 24 mM, respectively). Pancreatic colipase (bovine) had no effect on the activity of the two lipases. The comparison between the rates of hydrolysis of various substrates revealed the following order of decreasing enzyme activity: diolein greater than 1(3)-monoolein greater than tributyrin = triacetin greater than or equal to triolein = 2-monoolein. No hydrolysis of p-nitrophenylacetate and cholesteryloleate could be detected. Using 1-[3H]palmitoyl-2-[14C]linoleoyl-sn-glycerol, both enzymes displayed a strong preference for the 1-position, leading to the accumulation of 2-[14C]linoleoyl-sn-glycerol. Identical activities were found for the two lipases. It is concluded that the two cationic lipases from guinea pig pancreas represent a unique group of lipolytic enzymes different from other previously described enzymes, including classical pancreatic lipase, gastric and lingual enzymes, mold lipases and carboxylesterhydrolase.

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. II. Studies on glycerophospholipids.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various natural and synthetic phospholipids. Natural glycerophospholipids carrying a 1-acyl-bond were degraded in the following order of decreasing activity: phosphatidylcholine = phosphatidylinositol greater than 1-acyl-sn-glycero-3-phosphocholine greater than phosphatidylethanolamine greater than phosphatidylglycerol. Sodium deoxycholate was an activator with all the phospholipids tested, each one requiring its own optimal concentration of detergent. Whereas 1-alkyl-2-acyl-sn-glycero-3-phosphocholine remained fully insensitive to enzyme degradation, 2-acyl-sn-glycero-3-phosphocholine was hydrolysed to some extent. However, additional experiments involving time-course hydrolysis revealed that this was entirely due to the migration of the 2-acyl-chain to the sn-1 position. From studies using racemic or enantiomeric phosphatidylcholines, it was concluded that the enzymes are not stereospecific. Activity against 1-acylpropanediolphosphocholine was much lower than with 1-acyl-sn-glycero-3-phosphocholine, indicating that the 2-hydroxyl group (or the 2-acyl-ester group) participates in the substrate reactivity through a strong inductive effect. Some activity could be detected against 1,3-diacylglycero-2-phosphocholine (beta-phosphatidylcholine) and 1-acylglycol-2-phosphocholine. It is thus concluded that the failure of the lipases to hydrolyse the 2-acyl-bond in a natural phospholipid is due to the steric hindrance brought about by the acyl, alkyl or hydroxyl group present in the sn-1 position. The lipases might also be unable to hydrolyse acyl-ester bonds involving a secondary alcohol.