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Biomedical subjects

K Shirai

Publications and source records attributed to K Shirai.

At least 217 records · Page 12Linked to original sources
PubMed1982

Lipoprotein lipase-catalyzed hydrolysis of tri[14C]oleoylglycerol in a phospholipid interface. A monolayer study.

The lipoprotein lipase-catalyzed hydrolysis of triacylglycerol was determined in a lipid monolayer containing egg phosphatidylcholine and tri[14C]oleoylglycerol. In the presence of purified bovine milk lipoprotein lipase and fatty acid-free albumin, the rate of hydrolysis of tri[14C]oleoylglycerol, as determined by the decrease in surface activity, was dependent upon enzyme concentration and was enhanced by the addition of apolipoprotein C-II, the activator protein for the enzyme. Increasing the triacylglycerol content of the phospholipid monolayer from 1 to 6 mol% (relative to phospholipid) enhanced the rate of catalysis in the presence and absence of apolipoprotein C-II. However, at low substrate concentrations (less than 4 mol% tri[14C]oleoylglycerol), the activation factor for apolipoprotein C-II was greater than at high (4-6 mol%) triacylglycerol concentrations. The addition of sphingomyelin to the phosphatidylcholine monolayer decreased lipoprotein lipase activity. Based on these monolayer studies, we conclude that lipoprotein lipase catalyzes the hydrolysis of triacylglycerol at a phospholipid interface and that the rate of catalysis is dependent on the lipid composition of the monolayer.

Animals↗
PubMed1982

Effect of apolipoproteins on the hepatic lipase-catalyzed hydrolysis of human plasma high density lipoprotein2-triacylglycerols.

The effect of apolipoproteins on the hepatic lipase-catalyzed hydrolysis of high density lipoprotein (HDL) triacylglycerols was studied in an in vitro system consisting of purified human post-heparin hepatic lipase, HDL2 and albumin. The apparent values of the Michaelis constant (Km) and maximal velocity (Vmax) for the hepatic lipase-catalyzed hydrolysis of HDL2-triacylglycerols were 0.18 mM and 86 nmol free fatty acids released/mg hepatic lipase per min, respectively. The addition of purified human plasma apolipoprotein A-I, A-II, E, C-I or C-III2 (containing 2 mol of sialic acid) to HDL2 caused inhibition of hepatic lipase activity. At a 1:1 weight ratio of added apolipoprotein to HDL2-protein, inhibition was 50% for apolipoprotein E and over 75% for the other apolipoproteins tested. Inhibition of enzyme activity occurred with both the unfractionated HDL2 and the HDL which were reisolated by ultracentrifugation. The major alteration in the composition of the reisolated HDL was an increase in the protein to phospholipid ratio. Based on these results, we speculate on the possible role of the apolipoproteins in the metabolism of HDL2 by hepatic lipase.

Apolipoproteins↗
PubMed1982

Reciprocal effect of apolipoprotein C-II on the lipoprotein lipase-catalyzed hydrolysis of p-nitrophenyl butyrate and trioleoylglycerol.

Interaction of purified bovine milk lipoprotein lipase (LpL) with sonicated vesicles of dipalmitoyl phosphatidylcholine in the gel phase is associated with an increase in the rate of the LpL-catalyzed hydrolysis of p-nitrophenyl butyrate. There is a 6-fold increase in Vmax. Apolipoprotein C-II, the activator protein for LpL, inhibits the LpL-catalyzed hydrolysis of p-nitrophenyl butyrate. With 0.5 mol % tri[14C]oleoylglycerol present in the dipalmitoyl phosphatidylcholine vesicles and in the presence of 20 mM Ca2+, the rate of p-nitrophenyl butyrate hydrolysis is decreased reciprocally compared to trioleoylglycerol hydrolysis and is dependent on apolipoprotein C-II. These results suggest that apolipoprotein C-II enhances the activity of LpL by increasing the affinity of the active site of LpL for triacylglycerol.

Animals↗
PubMed1982

Immunological studies on bovine milk lipoprotein lipase. Effects of Fab fragments on enzyme activity.

Rabbit antiserum was prepared against purified bovine mild lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The gamma-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activity from adipose tissue of human and pig, but not that of rat and dog. Fab fragments were prepared by papain digestion of the gamma-globulin fraction. Fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The Fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol by the Fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the Fab fragments. However, preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the Fab fragments was associated with an increased protective effect against inhibition of enzyme activity; remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the Fab fragments. These immunological studies suggest that the interaction of lipoprotein lipase with apolipoprotein C-II in the presence of lipids is associated with a conformational change in the structure of the enzyme such that the Fab fragments are less inhibitory. The consequence of a conformational change in lipoprotein lipase may be to facilitate the formation of an enzyme-triacylglycerol complex so as to enhance the rate of the lipoprotein lipase-catalyzed turnover of substrate to products.

Animals↗
PubMed1982

Effect of dextran sulfate on the interaction between very low density lipoprotein and lipoprotein lipase.

The effect of dextran sulfate on the interaction between very low density lipoprotein (VLDL) and purified bovine milk lipoprotein was studied. Dextran sulfate increased VLDL-triacylglycerol hydrolysis by lipoprotein lipase about 2-fold, but did not alter the Km value for triacylglycerol in VLDL. Strong association of dextran sulfate with the VLDL-lipoprotein lipase complex was demonstrated by gel filtration on BioGel A-5m, although dextran sulfate did not bind to VLDL and only very slightly to lipoprotein lipase. These findings suggest that dextran sulfate increases triacylglycerol hydrolysis in VLDL by binding to the VLDL-lipoprotein lipase complex.

Animals↗
PubMed1982

Studies on CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity in rat arterial wall.

The properties of CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT) (EC 2.7.8.2.), which catalyzes de novo synthesis of phosphatidylcholine, were studied in rat arterial wall. The optimal pH of CPT of the arterial wall was about 8.5. On subcellular fractionation of the arterial wall, the highest activity was found in the microsome-rich fraction; the cytosolic fraction showed only a trace of activity. The Michaelis constant (KM) for CDP-choline was 0.019 mM. The CPT activity of a homogenate of arterial wall increased linearly with increase in concentration of diolein up to 3.2 mM. 20 mM magnesium and 0.2 mM manganese ions caused marked activation respectively and essential for the activity. Calcium, barium, cobalt, copper, and ferrous ions were inhibitory. 0.5 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 mM glycoletherdiamine-N,N,N'N'-tetraacetic acid (GEDTA) increased the activity in the presence of 10 mM magnesium ion. Sonication of the enzyme solution and addition of high concentration of detergent, such as Triton X-100 and Tween 20, markedly decreased the activity. Porcine liver phosphatidylcholine, phosphatidylethanolamine, and especially polyenephosphatidylcholine increased CPT activity of the arterial wall, while lysophosphatidylcholine was strongly inhibitory. The properties of arterial CPT activity under various conditions are discussed.

Animals↗
PubMed1982

Lipid metabolism in arteriosclerotic arterial wall of rats.

Arteriosclerotic lesions were formed in rat aorta by the administration of vitamin D2, a high-fat diet and a thyroid suppressing agent. This treatment increased the serum total cholesterol level to 12 times the control level. In the arteriosclerotic lesions that were induced the activities of lysosomal enzymes, such as acid phosphatase and acid lipase, were higher than in controls, that of acid cholesterol esterase was decreased, those of microsomal lipid-synthesizing enzymes--such as acyl-CoA synthetase and cholesterol ester synthesizing activity--were increased and that of neutral cholesterol esterase was decreased. These data suggest that lipid metabolism in arteriosclerotic lesions was changed, resulting in the accumulation of cholesterol esters in the aorta. Administration of high-fat diet and thyroid suppressing agent also increased the serum cholesterol levels to 12-fold the control level, but did not induce arteriosclerotic lesions. After this treatment the activities of hydrolyzing enzymes, such as acid and neutral cholesterol esterase and lipase, in the aorta increased, but the activities of lipid synthesizing enzymes also increased. These data suggest that lipid metabolism in the aorta in this condition changed to compensate for the large influx of serum lipids and to prevent arteriosclerosis. The roles of the serum lipid level, cell injury and lipid metabolism in the aorta in forming arteriosclerotic lesions are discussed on the basis of these results.

Animals↗
PubMed1982

Effect of tocopherol deficiency on lipid metabolism in arterial wall of spontaneously hypertensive rats on normal and high cholesterol diets.

Lipid metabolism in the arterial wall of spontaneously hypertensive rats (SHR) fed on tocopherol-deficient diet, high-cholesterol diet or both was studied. Serum tocopherol was greatly decreased in tocopherol-deficient SHR. Lipoperoxide, determined as thiobarbituric acid-reactive substances, was higher in tocopherol-deficient SHR than in normal SHR. Tocopherol-deficient SHR showed a decrease in acid cholesterol esterase activity, but no change in neutral cholesterol esterase, acid and neutral lipase, acyl CoA synthetase, cytidine-diphosphate choline-1-2-diacyl glycerol choline phosphotransferase (CPT) or triglyceride synthesizing activity.

Animals↗
PubMed1982

Effect of phospholipids on lipase activity in rat arterial wall homogenate.

Triolein hydrolyzing activity in homogenates of the rat arterial wall was studied. Two optimal pH values were observed around 5.0 (acid lipase) and 7.0 (neutral lipase). Neutral lipase activity was enhanced by serum and was inhibited by 1 M NaCl but was not inhibited by protamine. Heparin decreased the acid lipase activity. A 1-mM solution of Cu2+ ion and Zn+2 ion decreased both lipase activities. The activities of both acid and neutral lipases were increased by the addition of rat serum (2 micrograms protein serum/ml) and were decreased by the addition of more than 5 micrograms protein serum/ml. The activities of neutral and acid lipases were increased by the addition of 0.25 mM phosphatidylcholine and phosphatidylserine. Both lipase activities were decreased by the addition of 0.25 mM phosphatidylethanolamine. The addition of 0.25 mM sphingomyelin increased neutral lipase activity and decreased acid lipase activity. At concentrations higher than 2 mM, these phospholipids decreased the activities of both lipases. These results suggest that at least acid and neutral lipases exist in the arterial wall cells, and their activities might be modified by phospholipids.

Animals↗
PubMed1982

Lipid metabolism in the aorta and the brain microvessels of rabbits on high cholesterol diet.

The characteristics and properties of lipid metabolism in the aorta and the brain microvessels of rabbits were investigated to clarify the role of lipid metabolism in formation of atherosclerosis. In rabbit aorta, cholesterol esterase and lipase each had an acidic and a neutral optimum pH, whereas acyl-CoA synthetase, acyl-CoA: cholesterol acyltransferase, triglyceride synthesizing activity and choline phosphotransferase each had one neutral optimum pH. These pH optima were similar to those in rats. High cholesterol diet induced atheromatous lesions in the aorta, but not in the brain microvessels. In atheromatous aorta, the acid lipase and acid phosphatase activities were higher than in controls, but not the acid cholesterol esterase activity. Moreover the activities of neutral lipase, acyl-CoA synthetase, acyl-CoA:cholesterol acyltransferase, triglyceride synthesis and choline phosphotransferase were increased, but neutral cholesterol esterase activity was normal. These data suggest that lipis metabolism in the atheromatous aorta is changed in a manner favoring accumulation of lipids, especially cholesterol esters. In controls, most of the above enzyme activities in the brain microvessels were higher than those in the aorta. However, these enzyme activities in the brain microvessels were not changed by cholesterol feeding. Thus it is suggested that the properties of lipid metabolism in the aorta and brain microvessels, including permeability of lipoproteins into the vessel walls, are important in formation of atherosclerosis in addition to the serum factors.

Animals↗
PubMed1981

Interaction of lipoprotein lipase with phospholipid vesicles. Role of apolipoprotein C-II and heparin.

Lipoprotein lipase is bound to heparin-like molecules at the surface of capillary endothelial cells. For maximal activity, the enzyme requires apolipoprotein C-II, a protein constituent of triacylglycerol-rich lipoproteins. In this report, the interactions of apolipoprotein C-II, heparin and sonicated vesicles of dipalmitoylphosphatidylcholine with purified bovine milk lipoprotein lipase were studied by gel filtration on Bio-Gel A5m. In the presence of vesicles of dipalmitoylphosphatidylcholine (1 mg), lipoprotein lipase (25 micrograms) associated with phospholipids even in the absence of apolipoprotein C-II. With limited phospholipid (40 micrograms), the amount of enzyme which associated with lipid decreased in the presence of apolipoprotein C-II (20 micrograms). Human plasma apolipoprotein C-III, another protein constituent of triacylglycerol-rich lipoproteins, also caused a decrease in the amount of enzyme associated with phospholipid. These results suggest that apolipoprotein C-II does not increase the activity of the enzyme by facilitating its interaction with a lipid interface. In the absence of lipid, lipoprotein lipase and apolipoprotein C-II (molar ratio, 1 : 1) eluted from Bio-Gel A5m as two separate components. The interaction of heparin with lipoprotein lipase was studied using a specific [3H]heparin, which was isolated by affinity chromatography on immobilized lipoprotein lipase; the [3H]heparin eluted with 0.6 M NaCl. Specific [3H]heparin coeluted with lipoprotein lipase when the enzyme was associated with phospholipid; the [3H]heparin was released from the enzyme by 0.75 M NaCl.

Animals↗
PubMed1981

Studies on lipase in rat brain.

Lipase activity was measured in homogenates of rat cerebral hemispheres using radioactive glycerol trioleate emulsified with Triton X-100 as substrate. The labeled oleic acid was separated from the ester with a methanol-chloroform-heptane mixture. Under these assay conditions, the activity showed pH optima at about 5.5 and 7.5. The final products of these lipase activities were suggested to be free fatty acid and glycerol.

Animals↗
PubMed1981

Regulation of lipase activities in rat brain in vitro.

Regulation of acid and neutral lipase activities in rat brain was examined in vitro. Both activities were decreased by SDS, CuCl2 and ZnCl2 and by delipidation. The neutral lipase activity was also markedly reduced by N-ethylmaleimide and PbCl2. The activity of delipidated preparation was increased by addition of phosphatidyl choline at both pH 5.5 and 7.5 and by phosphatidyl serine at acidic pH value. Pretreatments of the enzyme preparation with phospholipase A and trypsin reduced the lipase activities at both pH values. It is suggested that phospholipids play an important role on lipase activity in brain.

Animals↗
PubMed1981

Catabolism of human very low density lipoproteins in vitro: a fluorescent phospholipid method for monitoring lipolysis.

The catabolism of human plasma very low density lipoproteins (VLDL) by purified bovine milk lipoprotein lipase has been measured in vitro using a fluorescent phospholipid as a method to monitor lipolysis. Dansyl phosphatidylethanolamine (DPE) was incorporated into VLDL to form DPE-VLDL, and the rate of catabolism was followed by measuring the increase in fluorescence at 490 nm after the addition of the enzyme. The studies were performed with VLDL isolated from 20 normal individuals. In addition, the VLDL from 8 mildly obese subjects with primary hypertriglyceridemia (Type IV phenotype) was studied. With this in vitro system and with a constant amount of lipoprotein lipase, the rate of lipolysis did not differ in normal and in these hypertriglyceridemic subjects. Furthermore, there was no correlation between the rates of hydrolysis and the plasma levels of triglyceride or high density lipoprotein cholesterol.

Adult↗