Regulation of rat hepatic lipase by the composition of monomolecular films of lipid.

The regulation of hepatic lipase (HL) by the lipid composition of monomolecular substrate films was examined using a monolayer technique at constant surface pressure. HL-catalyzed hydrolysis of triacylglycerol, a poor substrate for HL in pure monomolecular films, was activated by diradylglycerol and its phosphorylated derivatives in mixed films containing 10 mol % triacylglycerol. When triacylglycerol was progressively diluted with dialkylglycerol, triacylglycerol hydrolysis by HL was maximal between 90 and 98 mol % dialkylglycerol. The best activators, dialkylphosphatidic acid and dialkylphosphatidylethanolamine, increased triacylglycerol hydrolysis 13-14-fold, and the enhancement of HL-catalyzed triacylglycerol hydrolysis by the activator lipids was inversely related to the average mean molecular area of the mixed films. The hydrolysis of 5 mol % triacylglycerol in mixed films that also contained phosphatidylcholine and 0-20 mol % cholesterol was inhibited approximately 80% when the concentration of cholesterol was 10-13 mol %. Interestingly, between 15 and 17 mol % cholesterol the hydrolysis rate was restored to about 50% of the uninhibited rate, but at 20 mol % cholesterol this value decreased back to 80% inhibition of hydrolysis. The hydrolysis of phosphatidylethanolamine in mixed films with 0-20 mol % cholesterol decreased approximately 30% in films containing 10-12 mol % cholesterol. However, at 15 mol % cholesterol the hydrolysis rate was restored to the same level observed for a pure phosphatidylethanolamine film. This enhancement of HL activity occurred at about the same cholesterol concentration as the restoration of triacylglycerol hydrolysis observed for the triacylglycerol/phosphatidylcholine/cholesterol films.(ABSTRACT TRUNCATED AT 250 WORDS)

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 mumol/min/mg for 1-oleoyl-sn-glycerol, 163 mumol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 mumol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 mumol/min/mg for PA, 50 mumol/min/mg for PE and 4 mumol/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 the sn-1 and sn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereoselective with respect to 1,3-diacyl-sn-glycerol substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic lipase hydrolysis of lipid monolayers. Regulation by apolipoproteins.

A monolayer technique was used to study the substrate specificity of hepatic lipase (HL) and the effect of surface pressure and apolipoproteins on hydrolysis of lipid monolayers by this enzyme. HL hydrolyzed readily phosphatidylethanolamine monolayers. Pure trioctanoylglycerol was found to be a poor substrate but when progressively diluted with nonhydrolyzable 1,2-didodecanoylphosphatidylcholine hydrolysis of triacylglycerol by HL reached maximum at a molar ratio of 1:1 triacylglycerol to phosphatidylcholine. The activation of triacylglycerol hydrolysis was not due to altered penetration of HL. The surface pressure optimum of HL for the hydrolysis of phosphatidylethanolamine monolayers was broad between 12.5 and 25 mN/m. When apolipoprotein E was injected beneath the monolayer of phosphatidylethanolamine prior to enzyme addition, a 3-fold activation of HL was observed at surface pressures equal to or below 15 mN/m. Below surface pressures of 20 mN/m apolipoprotein E did not affect the penetration of HL into the lipid-water interface. Apolipoprotein E slightly activated the hydrolysis of triacylglycerol by HL at 10 mN/m. At a high surface pressure of 25 mN/m all apolipoproteins tested (apolipoproteins A-I, A-II, C-I, C-II, C-III, and E) inhibited the penetration into and HL activity on phosphatidylethanolamine At 18.5 mN/m all apolipoproteins except apolipoprotein E inhibited the hydrolysis of triacylglycerol in the triacylglycerol:phosphatidylcholine mixed film. Based on these results we present a hypothesis that phospholipid present in apolipoprotein E-rich high density lipoprotein-1 and triacylglycerol in intermediate density lipoprotein would be preferred substrates for HL.

Hydrolysis of thioester analogs by rat liver phospholipase A1.

The hydrolysis of thioester containing phospholipids by rat liver plasmalemma phospholipase A1 was measured in a continuous spectrophotometric assay. In this assay thioester substrates were employed which, upon hydrolysis, liberated a free thiol which was reacted with 4,4'-dithiopyridine to yield the product 4-thiopyridone that absorbs at 324 nm. Thioester substrates, prepared by chemical synthesis, were used in phospholipid and Triton X-100 micelles for kinetic analysis carried out according to the method of Hendrickson and Dennis (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739). Vmax, Ks, and Km values obtained for various isomers and racemic mixtures of the synthetic thioester analogs are compared with corresponding oxyester substrates. Unnatural sn-1 isomers competitively inhibited the hydrolysis of natural sn-3 isomers of phosphatidylethanolamine and phosphatidic acid. Furthermore, the sn-1 isomer of phosphatidic acid was hydrolyzed by phospholipase A1, but with lower catalytic efficiency than the sn-3 isomer. The presence of a thioester at the sn-1 position did not change the Vmax significantly, as compared to the oxyester phospholipids. When two thioesters were present on the phospholipid molecule, the Vmax was decreased significantly. A convenient synthesis of 1-monothioester analogs of phospholipids is reported. The results presented show the usefulness of the spectrophotometric assay for measuring phospholipase A1 activity as well as the influence of racemic mixtures and thioesters on the hydrolytic rate.

The degradation of platelet-activating factor and related lipids: susceptibility to phospholipases C and D.

1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is an ether-linked lipid that exhibits selective cytotoxicity toward several types of tumor cells and is relatively inactive toward normal cells under the same conditions of treatment. The mechanis of this selective cytotoxicity is unknown. We conducted studies to determine whether this compound is metabolized by phospholipases C and D and, if so, whether sensitive and resistant cells differ in their ability to degrade ET-18-OCH3 by these enzymes. We have examined the metabolism of the L-isomer of ET-18-OCH3, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (L-ET-18-OCH3), by lysophospholipase D of rat liver microsomes and by a phospholipase D from the marine bacterium Vibrio damsela. The metabolism of L-ET-18-OCH3 was also examined in cell culture using Madin-Darby canine kidney cells, human promyelocytic leukemia cells and human myelocytic leukemia cells. In these studies, L-ET-18-OCH3 and related 1-O-alkyl-linked phosphocholine analogs radiolabeled with 3H in the 1-O-alkyl chain were used. L-ET-18-OCH3 was not hydrolyzed by lysophospholipase D from rat liver microsomes under conditions where cleavage of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine was observed. However, phospholipase D from the marine bacterium V. damsela readily hydrolyzed L-ET-18-OCH3 to 1-O-[3H]octadecyl-2-O-methyl-sn-glycero-3-phosphate, demonstrating that L-ET-18-OCH3 can be degraded by a phospholipase D. Platelet-activating factor (PAF) and lyso-PAF were also substrates for the bacterial phospholipase D.(ABSTRACT TRUNCATED AT 250 WORDS)