Patricia Sisson

Myocardial phospholipase a of microsomal and mitochondrial fractions

Abstract 1. 1. Phospholipase A1 and Ag2 activities of mitochondria and microsomes of myocardial tissue have been determined using exogenous [14C]phosphatidyl ethanolamine and [14C]phosphatidyl choline. Mitochondrial phospholipase A2 had a pH optimum of 9.5, required 2.0 mM Ca2+ for optimal activity and was markedly inhibited by 1.0 mM EDTA. Microsomal phospholipase A1 and A2 activities were optimal at pH 7.5 in the presence of 2.0 mM Ca2+ and were minimally inhibited by 1.0 mM EDTA. 2. 2. Incubations with 3H-14C mixed labeled phosphatidyl ethanolamine further confirmed the above observations and implicated lysophospholipase activity in the microsomal fractions. 3. 3. Phosphatidyl ethanolamine was preferred to phosphatidyl choline as substrate. 4. 4. This endogenous phospholipase A activity in the microsomal membranes of the myocardium may have relevance to in vitro studies of Ca2+ permeability and Ca2+-dependent ATPase changes previously noted during snake venom phospholipase A treatment of muscle membranes.

Activation of hepatic lipase catalyzed phosphatidylcholine hydrolysis by apolipoprotein E

The effect of apolipoproteins A-I, A-II, C-II, C-III and E on the hydrolysis of phosphatidylcholine and triacylglycerol by hepatic lipase was studied. Hepatic lipase catalyzed phospholipid hydrolysis was 1.8-fold activated by apolipoprotein E while the other apolipoproteins did not affect the hydrolysis by this enzyme. Triacylglycerol hydrolysis by hepatic lipase was 1.5-fold activated by apolipoprotein E while the other apolipoproteins inhibited hepatic lipase. These results suggest that lipoproteins containing apolipoprotein E may be preferred substrates for hepatic lipase.

Specificity of lipoprotein lipase and hepatic lipase toward monoacylglycerols varying in the acyl composition.

We report here that both the hepatic lipase and lipoprotein lipase demonstrate specificity towards the acyl group present on monoacylglycerols. We found that unsaturated glycerides are more readily degraded than saturated glycerides. However, the basis for this specificity appears to be different for each enzyme. The activity of the hepatic lipase, but not the lipoprotein lipase, could be stimulated by Triton X-100 and phosphoglycerides. We interpret these results to show that while both the lipoprotein lipase and hepatic lipase are sensitive to the physical state of the substrate (as shown by fluorescence depolarization), the lipoprotein lipase also has a low affinity for monoacylglycerols that contain a saturated acyl group. In the course of this study we also obtained evidence that some type of phase separation occurs when mixtures of saturated and unsaturated monoacylglycerols are prepared.

Hydrolysis of lipid mixtures by rat hepatic lipase

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and substrate specificity of rat hepatic lipase

Publisher Summary Hepatic lipase from rat liver is an acylhydrolase that cleaves the fatty acid from the 1(3)-position of a broad spectrum of glycerides. In addition to the hydrolytic reaction, the enzyme can catalyze transacylation reactions in which the free hydroxyl of an acceptor lipid molecule becomes acylated rather than water, which is the acyl acceptor in hydrolysis. Routine assay of hepatic lipase is carried out with 1-oleoyl-[2- 3 H]glycerol ([ 3 H]MO) for the steps in purification and to check enzyme stability. This has the advantage of eliminating the chromatographic separation of products since both hydrolysis and transacylation reactions release [ 3 H]glycerol that partitions into the methanol-water phase during extraction. As the remaining substrate partitions into the chloroform layer, enzyme activity is measured by scintillation counting of an aliquot of the upper phase. The method of enzyme purification involves perfusion of the liver followed by two chromatographic steps. Two important points should be considered, first, the liver should be thoroughly washed by perfusion prior to the addition of heparin to remove as much contaminating protein as possible and second, a minimum of time should be used in the preparation. The adoption of this procedure saves nearly two days in enzyme isolation and increases the specific activity of the final preparation by two- to three-fold. Proteolysis may also occur during preparation that may alter substrate specificity.

Utilization of serum lipoprotein lipids by the monoacylglycerol acyltransferase

The plasma membrane of rat liver contains an enzyme which is stimulated by heparin and hydrolyzes liposomes composed of phosphoglycerides as well as mono-and diacylglycerol (Waite, M. and Sisson, P (1973) J.Biol, Chem. 248, 7985-7902). Further, in liposomes this enzyme catalyzes a transacylation in which the acyl group is removed from position 1 of an acyl glyceride donor, and combined with the hydroxy group of a variety of acyl acceptors. To investigate the possible role of this enzyme in lipoprotein metabolism, we have incorporated specific labeled glycerides into lipoproteins. The high density, low and very low lipoproteins were then separated by molecular sieving and characterized by their physical and chemical properties. Using the labeled substrates as model lipoproteins, we found the following: 1)The enzyme is capable of hydrolyzing monoacylglycerol, diacylglycerophosphoethanolamine and diacylglycerophosphocholine; monoacylglycerol however is the preferred substrate in all three lipoprotein fractions. 2)Relative to the activity found on liposomes, the transacylation activity is low. 3)The specific radioactivity of the substrates in fraction B (low density lipoprotein) did not change during the reaction, which indicates that the labeled lipid is not a pool separate from the endogenous lipid of the lipoprotein.

The hydrolytic and transacylation activity of the phospholipase A1 purified from human post-heparin plasma.

This study demonstrated that the phospholipase A1 purified from human post-heparin plasma catalyzes the same reactions (hydrolysis and transacylation) and utilizes the same substrates as the phospholipase A1 obtained by heparin treatment of the plasmalemma of rat liver (Waite, M. and Sisson, P. (1973) J. Biol. Chem. 248, 7985). 1-acylglycerol was the preferred acyl donor and transacylation was the predominate reaction. The results strongly support our earlier conclusions that the phospholipase in plasma originates from the liver and that this enzyme is capable of using a variety of acyl acceptors, including water.

Hydrolysis of neutral lipid substrates by rat hepatic lipase

Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Vmax values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The Vmax values obtained were 144 μmol/min/mg for 1-oleoyl-sn-glycerol, 163 μmol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 μmol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 μmol/min/mg for PA, 50 μmol/min/mg for PE and 4 μmol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at thesn-1 andsn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereo-selective with respect to 1,3-diacyl-sn-glycerol substrates. To determine if the presence of one lipid could affect the hydrolysis of the other, all possible dual combinations of 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol, in the same micelle were made and the hydrolysis rate of each substrate was determined. Interaction occurred only for the 1,2-dioleoyl-sn-glycerol/1,3-dioleoyl-sn-glycerol mixture where the hydrolysis of 1,2-dioleoyl-sn-glycerol was slightly inhibited and that of 1,3-dioleoyl-sn-glycerol slightly activated compared to the predicted theoretical rate. These findings demonstrate that when presented in similar physical states, the neutral lipid substrates tested were hydrolyzed at a higher rate by hepatic lipase than the phospholipid substrates.

Monoacylglycerol acyltransferase activity in the rat liver plasmalemma fractions

Abstract Bile canalicular membranes and plasma membranes free of bile canalicular membranes were prepared from rat livers and their lipolytic activities were measured. Both preparations catalyzed hydrolysis and transacylation when monoacylglycerol and phosphatidylethanolamine were used as substrates. The specific enzymatic activity in the plasmalemma free of bile canalicular membranes was slightly higher than that in bile canalicular membranes. Neither preparation attacked the triacylglycerol of chylomicra, which indicates the lack of a lipoprotein lipase. Heparin and CaCl 2 stimulated the activities in both preparations. On the basis of these data, we suggest that monoacylglycerol acyltransferase can serve two distinct roles in the liver cell, depending upon the mumbrane fraction of association.