Acetyltransferase activity in human platelet microsomes and washed platelets

It has been demonstrated that human platelets form platelet-activating factor (PAF) when stimulated by thrombin, collagen and ionophore A-23187, but the mechanism of its formation has not been elucidated. In this study we demonstrated increased acetyltransferase activity (i.e., transfer of the acetyl moiety of [3H]acetyl-CoA to lyso-PAF (1-alkyl-sn-glycero-3-phosphocholine) to form PAF) occurring in human platelet microsomes made from platelets stimulated by thrombin or ionophore A-23187. This stimulation resulted in a 2-4-fold increase in acetyltransferase activity over unstimulated platelets. Acetyltransferase activity was also demonstrated by incubating [3H]acetate with whole platelets and stimulating with thrombin or ionophore A-23187. Radioactive PAF was detected when the platelets were stimulated. None was formed without stimulation. These findings indicate that acetyltransferase may play a role in the biosynthesis of PAF by human platelets.     

Metabolism of gammalinolenic acid by human blood platelet microsomes

The microsomal fraction was used to test the ability of human platelets to metabolize gammalinolenic acid. The microsomal delta 6 and delta 5 fatty acid desaturase activities were measured and the incorporation of [14C]malonyl CoA into prostaglandins was also determined. The results indicate that human platelets have the capacity to elongate gammalinolenic acid (18:3 n-6) to dihomogammalinolenic acid (20:3 n-6) precursor of PGE1. Labeled PGE1 could be detected when human platelets microsomes were incubated with [14C]malonyl CoA in the presence of gammalinolenic acid. The results also show that human platelet microsomes have little delta 6 or delta 5 desaturase enzyme activity.     

Release of arachidonate from diglyceride in human platelets requires the sequential action of a diglyceride lipase and a monoglyceride lipase

Release of arachidonate from 2-arachidonyl diglyceride by human platelet microsomes was investigated. Diglycerides labeled with ¹⁴C-stearate at sn-1 and with ³H-arachidonate at sn-2 were used as a substrate for microsomal diglyceride lipase. Diglyceride was deacylated first at sn-1 as evidenced by the accumulation of 2-arachidonyl monoglyceride but not of 1-stearoyl monoglyceride. Subsequent release of arachidonate from monoglyceride required the action of a monoglyceride lipase. Studies on substrate specificity indicated that diglyceride lipase utilized 2-arachidonyl diglyceride as the best substrate.     

Hydrolysis of neutral glycerides by lipases of rat brain microsomes.

The hydrolysis of monoacylglycerol and diacylglycerol by rat brain microsomes was followed by measuring the release of glycerol and monooleylglycerol from dispersions of water insoluble glyceryl esters of oleic acid. The microsomes showed three lipolytic activities. One activity, optimal at pH 4.8, catalyzed the hydrolysis of diacylglycerol but not monoacylglycerol. Two other lipolytic activities, optimal at pH 8.0-8.6, catalyzed the hydrolysis of both diacylglycerol and monoacylglycerol. The pH 8.0-8.6 activities were sensitive to heat and SH-reagents. Detergents were inhibitory in all cases. Extraction of the microsomes with KCl, KSCN, urea or Triton X-100 did not change the ratio of diacylglycerol hydrolysis at pH 4.8 and 8.0. The results of subcellular fractionation studies showed that there was no significant enrichment of the acid lipase in any fraction.     

Reversal of platelet aggregation by aortic microsomes

The microsomal fraction of dog aortas inhibited human platelet aggregation induced by arachidonic acid, ADP, or thrombin. When aortic microsomes were added to a preparation of irreversibly aggregated platelets, the aggregates dispersed after 4–6 minutes. The fact that aortic microsomes inhibit platelet aggregation induced by ADP suggests that its effect is probably on the cellular function of platelets and not in direct competition against thromboxane A₂.     

Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice

Fluorescent triacylglycerols containing pyrenedecanoic (P10) and pyrenebutanoic (P4) acids were synthesized and their hydrolysis by lipases from human gastric juice and stomach homogenate was investigated. The existence in stomach homogenate of four different lipolytic enzymes hydrolyzing fluorescent triacylglycerols is suggested by the comparison of various enzymatic properties: acyl chain length specificity, heat inactivation and effect of detergents (Triton X-100 and taurocholate), serum albumin, diethyl-para-nitrophenyl phosphate (E600) and other inhibitors. (1) The acid pH4-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol and exhibited the characteristic properties of the lysosomal lipase: the maximal activating effect of detergents occurs at relatively high concentrations (the substrate/detergent optimal molar ratios were 1:5 and 1:25 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively); its activity was strongly inhibited by para-chloromercuribenzoate (2.5 mmol/l), but was not significantly affected by serum albumin and E600 (10(-2) mmol/l). (2) The neutral pH7-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol. It is resistant to E600 and heat-stable, similarly to the acid pH4-lipase, but it is well discriminated from the acid enzyme by its substrate/detergent optimal molar ratios (1:2 and 1:3 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively), whereas higher detergent concentrations, optimal for the acid lipase, are strongly inhibitory for the neutral enzyme. (3) The pH5-lipase present in gastric juice as well as in stomach homogenate exhibited properties obviously discriminating it from the other lipolytic enzymes from stomach homogenate: broad substrate specificity for P10- as well as P4-triacylglycerols, activation by low concentrations of amphiphiles (with optimal ratios triacylglycerols/taurocholate, triacylglycerols/taurocholate and triacylglycerols/phosphatidylcholine around 1:1, 1:3 and 1:0.1, respectively), heat-lability, strong activation by serum albumin and inhibition by E600 (10(-2) mmol/l). This pH5-lipase is the sole lipolytic enzyme present in gastric juice and is probably identical with the well-known 'gastric' lipase. (4) A pH7.5-enzyme is characterized by its specificity for P4-triacylglycerols, its heat-lability at 50 degrees C and its strong inhibition by E600 (10(-2) mmol/l).     

Enzyme activities and isozyme composition of triglyceride,diglyceride and monoglyceride lipases in Periplaneta americana,Locusta migratoria and Polia adjuncta

• 1.1. Measurements of maximal enzyme activities were combined with an electrophoretic study of isozyme make-up in an examination of triglyceride, diglyceride and monoglyceride lipases from the flight muscle, fat body and gut of the cockroach, Periplaneta americana and the locust, Locusta migratoria and from the flight muscle and fat body of the moth, Polia adjuncta.
• 2.2. Tri-, di- and mono-glyceride lipases were present in all tissues of the insects with diglyceride lipase ≥ triglyceride lipase activity in all cases and monoglyceride lipase ≥ diglyceride lipase activity in locust and moth.
• 3.3. In the flight muscle, a strong correlation was found between the activities of lipases and the known use of lipid as a fuel for flight in these insects. Lipase activities were lowest in the cockroach (a carbohydrate-based flight metabolism), intermediate in the locust (both carbohydrate and lipid-fueled flight), and highest in the moth (a non-feeding, lipid-catabolizing adult) flight muscle.
• 4.4. Polyacrylamide gel electrophoresis, using substrate-impregnated gels and stained for fatty acids released by lipase action, demonstrated the presence of tissue specific isozymes of tri-, di- and mono-glyceride lipases in the three insects. In addition, some, but not all, tissues showed multiple molecular forms of one or more of the lipases.
• 5.5. Diglyceride and monoglyceride lipase activities in both flight muscle and fat body of the insects coelectrophoresed suggesting the possibility that these two lipase activities might be catalyzed by a single enzyme protein.

     

Purification of steroid sulphohydrolase from human placenta microsomes

A procedure for purification of oestrone sulphate sulphohydrolase from human placenta microsomes was elaborated. The use of Concanavalin-A-Sepharose chromatography made it possible to separate, for the first time, oestrone sulphate sulphohydrolase (Mr 36,000, optimum pH 7.0, Km 5.5 X 10(-5) M, specific activity 1563 nmol X min-1 X mg protein-1) from arylsulphatase C (Mr 45,000, optimum pH 7.6, Km 0.96 X 10(-3) M). The observed third subfraction showed both arylsulphate C and oestrone sulphate sulphohydrolase activity. Sigmoidal kinetics of oestrone sulphate sulphohydrolase after DEAE-cellulose chromatography (Mr 130,000) points to the allosteric character of the enzyme.     

Degradation of monogalactosyl diglyceride and diagalactosyl diglyceride by sheep pancreatic enzymes

1. Saline extract of sheep pancreas acetone-dried powder was shown to catalyse acyl ester hydrolysis of spinach leaf galactosyl diglycerides and also galactosylglucosyl diglyceride of Lactobacillus casei. 2. Sodium deoxycholate stimulated the enzyme activity. Ca(2+) had no effect on the hydrolysis of monogalactosyl diglyceride, but it enhanced that of digalactosyl diglyceride. When added together, there was considerably less activity with both the substrates. 3. Optimal hydrolysis was observed at pH7.2. 4. The initial point of hydrolysis was at position-1, leading to the formation of monogalactosyl monoglyceride and digalactosyl monoglyceride. Further hydrolysis to the corresponding galactosylglycerols and later to galactose and glycerol was also observed, indicating the presence of alpha- and beta-galactosidases in the enzyme preparation. 5. Formation of monogalactosyl diglyceride from digalactosyl diglyceride by the action of alpha-galactosidase was noted. 6. Monogalactosyl diglyceride was also hydrolysed by beta-galactosidase to a limited extent, giving rise to diacylglycerol and galactose. 7. Attempts at purification of monogalactosyl diglyceride acyl hydrolase by using protamine sulphate treatment, Sephadex G-100 filtration and DEAE-cellulose chromatography gave a partially purified enzyme which showed 9- and 81-fold higher specific activity towards monogalactosyl diglyceride and digalactosyl diglyceride respectively. This still showed acyl ester hydrolysis activity towards methyl oleate, phosphatidylcholine and triacylglycerol. 8. When sheep, rat and guinea-pig tissues were compared, guinea-pig tissues showed the highest activity towards both monogalactosyl diglyceride and digalactosyl diglyceride. In all the species pancreas showed higher activity than intestine.     

Effect of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine on calcium fluxes by human platelet microsomes

Under conditions where optimal concentrations of arachidonic acid, phosphatidic acid, or the calcium ionophore A23187 caused release of 50-95% of calcium from preloaded platelet microsomes, basophil platelet activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, AGEPC) did not cause the release of calcium at concentrations as high as 2 X 10(-5) M. The failure to stimulate calcium release was not due to metabolism or inactivation of AGEPC. These results show that AGEPC is not a calcium ionophore and is unable to directly effect the release of calcium from microsomes by mechanisms other than ionophoric action. The increase in intracellular levels that occurs during AGEPC-induced platelet aggregation must be an indirect effect of the AGEPC.     

Characterization of lipases from Staphylococcus aureus and Staphylococcus epidermidis isolated from human facial sebaceous skin

Two staphylococcal lipases were obtained from Staphylococcus epidermidis S2 and Staphylococcus aureus S11 isolated from sebaceous areas on the skin of the human face. The molecular mass of both enzymes was estimated to be 45 kDa by SDS-PAGE. S2 lipase displayed its highest activity in the hydrolysis of olive oil at 32 degrees C and pH 8, whereas S11 lipase showed optimal activity at 31 degrees C and pH 8.5. The S2 lipase showed the property of cold-adaptation, with activation energy of 6.52 kcal/mol. In contrast, S11 lipase's activation energy, at 21 kcal/mol, was more characteristic of mesophilic lipases. S2 lipase was stable up to 45° C and within the pH range from 5 to 9, whereas S11 lipase was stable up to 50 degrees C and from pH 6 to 10. Both enzymes had high activity against tributyrin, waste soybean oil, and fish oil. Sequence analysis of the S2 lipase gene showed an open reading frame of 2,067 bp encoding a signal peptide (35 aa), a pro-peptide (267 aa), and a mature enzyme (386 aa); the S11 lipase gene, at 2,076 bp, also encoded a signal peptide (37 aa), pro-peptide (255 aa), and mature enzyme (399 aa). The two enzymes maintained amino acid sequence identity of 98-99% with other similar staphylococcal lipases. Their microbial origins and biochemical properties may make these staphylococcal lipases isolated from facial sebaceous skin suitable for use as catalysts in the cosmetic, medicinal, food, or detergent industries.     

Endothelial and lipoprotein lipases in human and mouse placenta

Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin-Sepharose affinity chromatography, the EL protein eluted as a single peak without detectable phospholipid or triglyceride (TG) lipase activity. The major portion of LPL protein eluted slightly after EL. This peak also had no lipase activity and most likely contained monomeric LPL. Fractions eluting at a higher NaCl concentration contained small amounts of LPL protein (most likely dimeric LPL) and had substantial TG lipase activity. In situ hybridization studies showed EL mRNA expression in syncytiotrophoblasts and endothelial cells and LPL mRNA in syncytiotrophoblasts. In contrast, immunohistochemistry showed EL and LPL protein associated with both cell types. In mouse placentas, lack of LPL expression resulted in increased EL mRNA expression. These results suggest that the cellular expression of EL and LPL in human placenta is different. Nevertheless, the two lipases might have overlapping functions in the mouse placenta. Our data also suggest that the major portions of both proteins are stored in an inactive form in human term placenta.