Mitsuo Tanaka

Effect of cupric ions on serum and liver cholesterol metabolism.

Cupric ions were administered subcutaneously to male Sprague-Dawley rats at a single dose of 200 μmol/kg. At 24 hr after administration, a remarkable increase of total and free cholesterol was seen in the rat serum. Also, when lecithin-cholesterol acyltransferase (LCAT) (E.C. 2.3.1.43) activity was expressed as the percentage of the total serum that free cholesterol esterified, the acyltransferase activity in rats treated with cupric ions showed a slight decrease while the triglyceride content in rat serum and liver decreased by 54% and 61%, respectively. However, the content of hepatic cholesterol in rats treated with cupric ions did not show such a marked change.On the other hand, acid cholesteryl ester hydrolase activity (Acid CEH) (E.C. 3.1.1.14) in liver lysosomes of rats treated with cupric ions showed a marked decrease with increasing cupric ion concentration both in vivo and in vitro. Furthermore, cupric ions caused a marked release of the lysosomal enzymes cathepsin D and β-glucuronidase into the cytosolic fraction. The changes in acid cholesteryl ester hydrolase activity induced by cupric ions appear to be a direct effect of cupric ions on the enzyme. These results suggest that excessive cupric ion concentrations could cause various disorders in lipid metabolism.

Characterization of a cytosolic protein inhibiting lysosomal acid cholesteryl ester hydrolase.

An inhibitor of lysosomal acid cholesteryl ester hydrolase (Acid CEH), (EC 3.1.1.13) was found in the cytosolic fraction of rat liver and various other tissues. The extent of the inhibitory effect was dependent on the concentration of the cytosolic protein. The Acid CEH inhibitor was heat-labile, nondialyzable, and its inhibitory activity significantly decreased by trypsin or chymotrypsin digestion, but not by lipase digestion. The inhibitor had no effect on the activity of cathepsin D, β-glucuronidase and acid phosphatase, which are other enzymes found in lysosomes. The present findings suggest that the inhibitor may be involved in the regulation of the hydrolysis of cholesteryl esters in lipoproteins that have been transferred into the liver.

Cupric ion-dependent inhibition of lysosomal acid cholesteryl ester hydrolase in the presence of hydroxylamine

In the presence of hydroxylamine or ascorbic acid, the inhibitory effects of Cu2+ on lysosomal acid cholesteryl ester hydrolase (acid CEH) partially purified from rat liver were studied.Hydroxylamine stimulated the inhibition of acid CEH activity by Cu2+ but not that by Zn2+, Fe2+, Co2+, Mn2+, Ca2+, Mg2+ and Hg2+. This Cu2+-dependent inhibition of acid cholesterol ester hydrolase (CEH) activity was completely prevented by ethylenediamine tetraacetic acid (EDTA), EGTA and o-phenanthroline, a chelator with a stability constant for Cu2+, and also by sulfhydryl agents and cytoplasmic reducing agents such as cysteine, glutathione and mercaptoethanol. In addition, the stimulative effects of hydroxylamine on Cu2+-dependent inhibition were maintained even after preincubation of Cu2+ with hydroxylamine.On the other hand, ascorbic acid was found to replace the stimulation by hydroxylamine of the Cu2+-dependent inhibition of acid CEH activity but the effects of ascorbic acid progressively became smaller with prolongation of the preincubation time. Moreover, addition of chemical radical scavengers to the reaction mixture did not prevent the Cu2+-dependent inhibition of acid CEH activity in the presence of ascorbic acid. These results suggest that Cu2+ causes inhibition of lysosomal acid CEH activity through the formation of Cu1+ in a reductive medium.

A diurnal variation of hepatic acid cholesteryl ester hydrolase activity in the rat

The diurnal variation in lysosomal acid cholesteryl ester hydrolase (Acid CEH), (EC 3.1.1.13) has been examined in fed, fasted and adrenalectomized rats. The Acid CEH activity of normal rat liver exhibits a diurnal rhythm with maxima at 06.00 hours and minima at 18.00 hours, but such a rhythm was not observed in spleen and brain. This rhythm was abolished after fasting for two days, and the resulting Acid CEH activity remained constant at the minimum level. However, adrenalectomy did not abolish the diurnal rhythm. These results indicate that the Acid CEH activity varies according to a diurnal rhythm with maxima and minima separated by approximately 12 hr. Further, it is evident that the appearance of this rhythm is dependent upon dietary, but not adrenal hormone influence.

Antioxidant activity of thiocholesterol on copper-induced oxidation of low-density lipoprotein.

The effect of thiocholesterol (SH-Chol) on the copper-inducedin vitro oxidation of low-density lipoprotein (LDL; 1.019<d<1.063) was investigated. Among the antioxidants tested, including cysteine, glutathione, 2-mercaptoethanol, dithiothreitol, probucol, thiopalmitic acid,. and SH-Chol, SH-Chol was the most effective antioxidant in copper-induced LDL oxidation. Also, SH-Chol completely inhibited the formation oxysterols i.e., 7-hydroxycholesterol and 7-ketocholesterol, in LDL particles and reduced 1,1-diphenyl-2-picrylhydrazyl used as stable free-radical model. Moreover, SH-Chol suppressed the degradation of endogenous α-tocopherol in LDL particles. These findings indicate that SH-Chol acts as antioxidant in the oxidative damage of LDLin vitro and as a free-radical scavenger in lipid peroxidation.

Properties of an acid cholesteryl ester hydrolase inhibitor from rat serum.

The inhibitory effect of a protein isolated from rat serum on lysosomal acid cholesteryl ester hydrolase (acid CEH; EC.3.1.1.13) activity was studied. An inhibitor was purified from rat serum following ultracentrifugation and heat treatment using column chromatography on Sephacryl S-200 and ultrafiltration. The purified inhibitor appeared as a single protein band in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight of the inhibitor was 28,000 Daltons as judged by gel filtration on Sephacryl S-200 and SDS-polyacrylamide gel electrophoresis. The purified inhibitor was shown to be apolipoprotein A-I (apo A-I), the major apolipoprotein of high-density lipoprotein (HDL), using immunoprecipitation with rat anti-apo A-I immunoglobulin (Ig)G. Inhibition of acid CEH activity by apo A-I was dependent on the concentration of apo A-I. The values of Vmax obtained were similar with or without apo A-I. Apo A-I of various other mammalian species, including human, bovine and rabbit, also inhibited acid CEH activity. Other apolipoproteins, such as apo A-II and apo B, also showed inhibiting activity. On the other hand, apo A-I had no effect on the activity of other enzymes found in lysosomes, such as cathepsin D, β-glucuronidase and acid phosphatase. The results suggest that apolipoproteins may play a role in the regulation of hydrolysis of cholesteryl esters in lipoproteins, that have been transferred to the liver, and that the inhibition of acid CEH activity by apo A-I may be a characteristic of the lipid-binding protein or be due to changes of the lipid/water interface.

The activity and properties of a hepatic acid cholesteryl ester hydrolase obtained from rats of different age groups

The activity of lysosomal acid cholesteryl ester hydrolase (acid CEH, EC 3.1.1.13) in rat liver was determined at 3, 5, 7, 10 and 20 wk following birth. The levels of acid CEH activity showed a marked decrease as rats grew older, whereas those of other lysosomal marker enzymes, such as acid phosphatase, β-glucuronidase and cathepsin B and D, showed only a slight decrease. On the other hand, acid CEH activity was detected in all subcellular fractions obtained from rat liver, but the enzyme activity in these fractions did not show the age-related decrease observed in the lysosomal fraction. The results presented here suggest that the marked alteration of lysosomal acid CEH activity that accompanies aging may be related to its possible involvement in the regulation of cholesterol concentration in rat liver.