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Biomedical subjects

K Shirai

Publications and source records attributed to K Shirai.

At least 199 records · Page 11Linked to original sources
PubMed1984

Methyl butyrate-hydrolyzing activity of hepatic triglyceride lipase from rat post-heparin plasma.

Hepatic triglyceride lipase was obtained from post-heparin plasma of rats in an electrophoretically homogeneous form. The enzyme had an isoelectric point at pH 4.9 and molecular weight of 65,000. The relation between the "lipase" and "esterase" activities of the enzyme was studied using emulsified triolein and water-soluble methyl butyrate (80 mM) as substrates. The same enzyme protein catalyzed the hydrolyses of both emulsified triolein and water-soluble methyl butyrate. The relation of activity to the methyl butyrate concentration differed from those for pancreatic lipase and liver esterase. During purification, the ratio of methyl butyrate to triolein-hydrolyzing activity of the enzyme increased. On digestion of the enzyme with trypsin, the "lipase" activity was retained. However, the trypsin-treated enzyme was adsorbed to a heparin-Sepharose column and eluted with 0.75 M NaCl, like the untreated enzyme. These results suggest that rat hepatic triglyceride lipase contains a so-called "hydrophobic recognition site" that is destroyed by trypsin treatment and is distinct from the heparin-binding and catalytic sites.

Animals↗
PubMed1984

Lipoprotein lipase- and phospholipase A2-catalyzed hydrolysis of phospholipid vesicles with an encapsulated fluorescent dye. Effects of apolipoproteins.

The self-quenching dye, 6-carboxyfluorescein, has been encapsulated into sonicated vesicles of egg phosphatidylcholine. Porcine pancreatic phospholipase A2 and bovine milk lipoprotein lipase catalyze the hydrolysis of the phosphatidylcholine resulting in the release of the encapsulated dye and a large increase in 6-carboxyfluorescein fluorescence. The fluorescence increase occurs in parallel with the formation of lysophosphatidylcholine and is strongly dependent on Ca2+ for phospholipase A2 catalysis and on apolipoprotein C-II for hydrolysis by lipoprotein lipase. Other apolipoproteins, including apolipoproteins C-III, C-I, and A-I, do not enhance lipoprotein lipase activity towards this substrate. We conclude that the enhancement of lipoprotein lipase activity by apolipoprotein C-II is a specific property of the activator protein due to its interaction with lipoprotein lipase or an enzyme/lipid interface and not a characteristic of lipid-binding proteins in general.

Animals↗
PubMed1984

Post-heparin plasma hepatic triacylglycerol lipase-catalyzed hydrolysis of tributyrin. Effect of lipid interface.

The mechanism of action of hepatic triacylglycerol lipase (EC 3.1.1.3) was examined by comparing the hydrolysis of a water-soluble substrate, tributyrin, with that of triolein by hepatic triacylglycerol lipase purified from human post-heparin plasma. The hydrolyzing activities toward tributyrin and triolein were coeluted from heparin-Sepharose at an NaCl concentration of 0.7 M. The maximal velocity of hepatic triacylglycerol lipase (Vmax) for tributyrin was 17.9 mumol/mg protein per h and the Michaelis constant (Km) value was 0.12 mM, whereas the Vmax for triolein was 76 mumol/mg per h and the Km value was 2.5 mM. The hydrolyses of tributyrin and triolein by hepatic triacylglycerol lipase were inhibited to similar extends by procainamide, NaF, Zn2+, Cu2+, Mn2+, SDS and sodium deoxycholate. Triolein hydrolysis was inhibited by the addition of tributyrin. Triolein hydrolysis was also inhibited by the addition of dipalmitoylphosphaidylcholine vesicles. In contrast, the additions of triolein emulsified with Triton X-100 and dipalmitoylphosphatidylcholine vesicles enhanced the rate of tributyrin hydrolysis by hepatic triacylglycerol lipase. In the presence of dipalmitoylphosphatidylcholine, the Vmax and Km values of hepatic triacylglycerol lipase for tributyrin were 41 mumol/mg protein per h and 0.12 mM, respectively, indicating that the enhancement of hepatic triacylglycerol lipase activity for tributyrin by dipalmitoylphosphatidycholine vesicles was mainly due to increase in the Vmax. The enhancement of hepatic triacylglycerol lipase activity for tributyrin by phospholipid was not correlated with the amount of tributyrin associated with the phospholipid vesicles. On Bio-Gel A5m column chromatography, glycerol tri[1-14C]butyrate was not coeluted with triolein emulsion, and hepatic triacylglycerol lipase activity was associated with triolein emulsion even in the presence of 2 mM tributyrin. These results suggest that hepatic triacylglycerol lipase has a catalytic site for esterase activity and a separate site for lipid interface recognition, and that on binding to a lipid interface the conformation of the enzyme changes, resulting in enhancement of the esterase activity.

Heparin↗
PubMed1984

Post-heparin plasma hepatic triacylglycerol lipase-catalyzed tributyrin hydrolysis. Effect of trypsin treatment.

Hepatic triacylglycerol lipase (EC 3.1.1.3) hydrolyzes water-insoluble fatty acid esters, e.g., trioleoylglycerol (lipase activity) and water-soluble fatty acid esters, e.g., tributyrin (esterase activity). Esterase activity of hepatic triacylglycerol lipase is enhanced by triolein emulsion and phospholipid vesicles [1]. The catalytic mechanism and structure of human hepatic triacylglycerol lipase isolated from human post-heparin plasma and the effect of trypsin treatment on the lipase and esterase activities of the enzyme were examined. Treatment of hepatic triacylglycerol lipase with trypsin resulted in loss of its lipase activity, but had no effect on its esterase activity. Chromatography of hepatic triacylglycerol lipase on Bio-Gel A5m showed that hepatic triacylglycerol lipase binds to dipalmitoylphosphatidylcholine vesicles. However, on chromatography of the trypsin-treated enzyme after incubation with dipalmitoylphosphatidylcholine vesicles, a part of hepatic triacylglycerol lipase that retained esterase activity was eluted separately from the dipalmitoylphosphatidylcholine vesicles. Addition of vesicles of dipalmitoylphosphatidylcholine to the trypsin-treated enzyme did not enhance its esterase activity. These results are consistent with the hypothesis that hepatic triacylglycerol lipase has a catalytic site that hydrolyzes tributyrin and a lipid interface recognition site, and that these sites are different: trypsin modified the lipid interface recognition site of the hepatic triacylglycerol lipase but not the catalytic site.

Binding Sites↗
PubMed1984

Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase.

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4 -yl) amino]-caproyl] phosphatidylcholine (C6-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A2 (PA2) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C6-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca++ was required for PA2 catalysis. Identification of the products of hydrolysis showed cleavage at the sn-1 and sn-2 positions for LpL and PA2, respectively. For PA2, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.

Apolipoprotein C-II↗
PubMed1984

Hydrolysis of cholesterol ester in artificial lipid mixtures by cholesterol esterase released from particulate fractions of rat arterial wall.

Acid and neutral cholesterol esterase activities in rat arterial wall were released from the lysosomal fraction and microsomal fraction respectively into the 105,000 X g supernatant fraction by treatment with Triton X-100, heparin and dextran sulfate. The percentage releases of acid cholesterol esterase by Triton X-100 (0.1%), heparin (50 micrograms/ml) and dextran sulfate (1 mg/ml) were 21%, 18% and 4%, respectively, while those of neutral cholesterol esterase were 66%, 56% and 39%, respectively. The cholesterol esterase released by dextran sulfate, especially that from the microsomal fraction, hydrolyzed cholesterol ester in artificial lipid mixtures with similar lipid compositions to those of the deposits in fatty streaks and fibrous plaques of atheromatous lesions.

Animals↗
PubMed1984

Effect of niceritrol on lipid metabolism of aorta in atherosclerotic rats.

Atherosclerotic lesions were formed in the aorta of rats given a high cholesterol diet containing propylthiouracil (PTU) and vitamin D2 (atherogenic diet) for 8 weeks. The effects of niceritrol (pentaerythritol tetranicotinate), which lower the plasma lipid level, on lipid metabolism in the arterial wall of the atherosclerotic rats were studied. Niceritrol significantly decreased the plasma cholesterol level of atherosclerotic rats, which was 823 mg/100 ml, or about ten times that of control rats. On treatment with niceritrol, the cholesterol level was reduced most in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Heparin-releasable lipoprotein lipase activity in epididymal adipose tissue, lipoprotein lipase activity in post-heparin plasma, and VLDL-triolein hydrolyzing activity in adipose tissue stromal vessels were all higher in niceritrol-treated atherosclerotic rats. Of the enzymes in the arterial wall concerned with cholesterol ester metabolism, acid cholesterol esterase activity was decreased in atherosclerotic rats, while niceritrol treatment increased this activity. The ratio of acyl-CoA cholesterol acyltransferase activity (ACAT) to neutral cholesterol esterase activity was higher in atherosclerotic rats than in control rats, but was lower in niceritrol-treated rats than in atherosclerotic rats. From these results, it is concluded that niceritrol modifies enzyme activities in such a way as to reduce the cholesterol ester content of the arterial wall and lower plasma VLDL and LDL cholesterol levels.

Adipose Tissue↗
PubMed1983

Effect of pantethine on fatty acid oxidation in microvessels of rat brain.

Fatty acid oxidation in brain microvessels decreased greatly when persistent hypertension developed in spontaneously hypertensive rats (SHR). Treatment of SHR with pantethine [D-bis-(N-pantothenyl-beta-aminoethyl) disulfide] in vivo for 4 weeks restored their fatty acid oxidation activity to the control level. The mechanism of the activating effect of pantethine on fatty acid oxidation was investigated in brain microvessels. Pantethine and its metabolites (pantetheine and 4'-phosphopantetheine) activated three steps of fatty acid oxidation, i.e., acyl-CoA synthetase, carnitine acyltransferase and intramitochondrial oxidation. The relation between changes in fatty acid oxidation activities and injuries of brain microvessels and the protective effect of pantethine against such injuries is discussed.

Animals↗
PubMed1983

Effect of clinofibrate on lipid metabolism of aorta in atherosclerotic rats.

Atherosclerotic lesions formed in the aorta of rats given diet containing propylthiouracil (PTU), vitamin D2 and high cholesterol diet (atherogenic) for 8 weeks. The effect of clinofibrate, which lowers the plasma lipid level, on lipid metabolism in the arterial wall of the atherosclerotic rats was studied. Clinofibrate significantly decreased the high plasma cholesterol level of atherosclerotic rats, which was 823 +/- 256 (mean +/- SD) mg/dl, or about ten times that of control rats (85 +/- 11 mg/dl). On treatment with clinofibrate, the cholesterol level was reduced most in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Heparin-releasable lipoprotein lipase activity in epididymal adipose tissue, lipoprotein lipase activity in post heparin plasma, and VLDL-triolein hydrolizing activity in adipose tissue stromal vessels were higher in clinofibrate-treated rats than in atherosclerotic rats. Of the enzymes in the arterial wall concerned with cholesterol ester metabolism, acid cholesterol esterase activity was decreased in atherosclerotic rats, and clinofibrate treatment increased this activity. The ratio of acyl-CoA cholesterol acyltransferase activity (ACAT) to neutral cholesterol esterase activity was higher in atherosclerotic rats than in control rats and was lower in clinofibrate-treated rats than in atherosclerotic rats. From these results, it is concluded that clinofibrate modifies enzyme activities in such a way as to cause a reduction of cholesterol accumulation in the arterial wall and lowers the plasma VLDL and LDL cholesterol levels.

Animals↗
PubMed1983

Lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholine of guinea pig very low density lipoproteins and discoidal complexes of phospholipid and apolipoprotein: effect of apolipoprotein C-II on the catalytic mechanism.

To elucidate the mechanism by which apolipoprotein C-II (apoC-II) enhances the activity of lipoprotein lipase (LpL), discoidal phospholipid complexes were prepared with apoC-III and di[(14)C]palmitoyl phosphatidylcholine (DPPC) and containing various amounts of apoC-II. The rate of DPPC hydrolysis catalyzed by purified bovine milk LpL was determined on the isolated complexes. The rate of hydrolysis was optimal at pH 8.0. Analysis of enzyme kinetic data over a range of phospholipid concentrations revealed that the major effect of apoC-II was to increase the maximal velocity (V(max)) some 50-fold with a limited effect on the Michaelis constant (K(m)). V(max) of the apoC-III complex containing no apoC-II was 9.2 nmol/min per mg LpL vs. 482 nmol/min per mg LpL for the complex containing only apoC-II. The effect of apoC-II on enzyme kinetic parameters for LpL-catalyzed hydrolysis of DPPC complexes was compared to that on the parameters for hydrolysis of DPPC and trioleoylglycerol incorporated into guinea pig very low density lipoproteins (VLDL(p)) which lack the equivalent of human apoC-II. Tri[(3)H]oleoylglycerol-labeled VLDL(p) were obtained by perfusion of guinea pig liver with [(3)H]oleic acid. Di[(14)C]palmitoyl phosphatidylcholine was incorporated into the VLDL(p) by incubation of VLDL(p) with sonicated vesicles of di[(14)C]palmitoyl phosphatidylcholine and purified bovine liver phosphatidylcholine exchange protein. The rates of LpL-catalyzed hydrolysis of trioleoylglycerol and DPPC were determined at pH 7.4 and 8.5 in the presence and absence of apoC-II. In the presence of apoC-II, the V(max) for DPPC hydrolysis in guinea pig VLDL(p) increased at both pH 7.4 and pH 8.5 (2.4- and 3.2-fold, respectively); the value of K(m) did not change at either pH (0.23 mm). On the other hand, the kinetic value of K(m) for triacylglycerol hydrolysis in the presence of apoC-II decreased at both pH 7.4 (3.05 vs. 0.54 mm) and pH 8.5 (2.73 vs. 0.62 mm). These kinetic studies suggest that apoC-II enhances phospholipid hydrolysis by LpL in apoC-III-DPPC discoidal complexes and VLDL(p) mainly by increasing the V(max) of the enzyme for the substrates, whereas the activator protein primarily causes a decrease in the apparent K(m) for triacylglycerol hydrolysis.-Shirai, K., T. J. Fitzharris, M. Shinomiya, H. G. Muntz, J. A. K. Harmony, R. L. Jackson and D. M. Quinn. Lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholine of guinea pig very low density lipoproteins and discoidal complexes of phospholipid and apolipoprotein: effect of apolipoprotein C-II on the catalytic mechanism.

Animals↗
PubMed1982

Lipoprotein lipase catalyzed hydrolysis of water-soluble p-nitrophenyl esters. Inhibition by apolipoprotein C-II.

Bovine milk lipoprotein lipase (LpL) catalyzes the hydrolysis of the water-soluble esters p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB). The same protein and same active site are involved in hydrolysis of water-soluble p-nitrophenyl esters and emulsified trioleoylglycerol since (a) trioleoylglycerol hydrolysis and PNPB hydrolysis activities coelute from the heparin-Sepharose affinity column used to purify LpL and (b) LpL-catalyzed hydrolyses of trioleoylglycerol and PNPB are inhibited to equal extents by phenylmethanesulfonyl fluoride. The effect of apolipoprotein C-II (apoC-II) on the LpL-catalyzed hydrolysis of PNPA and PNPB has been determined. ApoC-II inhibits hydrolysis of both esters, with a maximum extent of inhibition of 70-90%. Inhibition of the LpL-catalyzed hydrolysis of PNPB is specific for apoC-II, since apolipoproteins A-I, C-I, and C-III-2 have little effect on this reaction, and is partial noncompetitive in form. KI values for apoC-II inhibition of the LpL-catalyzed hydrolysis of PNPA and PNPB are in the range 0.26-0.83 microM. The effect of apoC-II on the temperature dependences of LpL-catalyzed hydrolysis of both esters and on NaCl inhibition of LpL-catalyzed PNPB hydrolysis is consistent with a change in rate-determining step with LpL and apoC-II interact. These results indicate not only that there is an interaction between apoC-II and LpL in aqueous solution in the absence of a lipid interface but also that this interaction conformationally modulates the active site of the enzyme.

Animals↗