Biomedical subjects
Publications and source records attributed to S Horie.
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When 14C-labeled N-(alpha-methylbenzyl)azelaamic acid (C9), which is an intermediate in the metabolism of N-(alpha-methylbenzyl)linoleamide, a potent hypocholesterolemic agent, was administered to rats, 84% of the radioactivity was recovered in the urine in 24 hr, which contained 66.1% of N-(alpha-methylbenzyl)glutaramic acid (C5) and 8.6% of N-(alpha-methylbenzyl)pimelamic acid (C7) as major metabolites. While 14C-labeled C9 was incubated with isolated hepatocytes, similar metabolites were found, whereas none of the metabolites with an even number of carbon atoms in the acyl side chain was detected. The activity of the chain-shortening of C9 by hepatocytes isolated from clofibrate-treated rats was stimulated to about twice that of untreated hepatocytes, in parallel with the elevation of C9-dependent H2O2-generation. A subcellular fractionation study of the liver revealed that the subcellular distribution of cyanide-insensitive C9-oxidation activity was coincident with that of catalase and of cyanide-insensitive palmitoyl-CoA oxidation. In this reaction, C7 and C5 were produced. For this reaction, the formation of C9-CoA thioester was essential as an intermediary step. These results indicate that peroxisomes are capable of shortening the acyl side-chains of drugs by beta-oxidation and, thus, suggest an additional metabolic role for peroxisomes.
The oscillator strengths of hemoproteins in the light frequency range of 1.11 X 10(4) to 3.23 X 10(4) cm-1 (wavelength range of 900 to 310 nm) were measured by means of computer-assisted spectrophotometry. The obtained values of oscillator strength per molar heme ranged from about 1.4 to 2.2. By comparing the oscillator strength values of the ferric and ferric cyanide-bound forms of hemoproteins and also the values of low molecular weight ferric heme complexes, it was found that the oscillator strength was lower for those hemoproteins whose heme was coordinated with strong field ligands. It was also found that the hemoproteins showing a smaller pH-dependent change in the carbon monoxide-difference spectrum had lower oscillator strengths. The following linear relation was observed, with various ligand complexes of bovine methemoglobin, horse metmyoglobin, and ferric horseradish peroxidase, between the oscillator strength (f) determined in the present study and the respective magnetic susceptibility (10(6) X chi 20 degrees M) values in the literature: f = A (10(6) X chi 20 degrees M) + B. The values of constants A and B in the equation were estimated for horseradish peroxidase, methemoglobin, and metmyoglobin. On varying the temperature in the range of 0 to 40 degrees C, the oscillator strength of the metmyoglobin-azide complex changed in parallel with the change in the spin state. Taking advantage of the fact that fluoride complexes of many hemoproteins show 10(6) X chi 20 degrees M values close to 14,500 and also that the values of intersection B are around 86.4% of the respective values of the fluoride complexes of ferric horseradish peroxidase, methemoglobin, and metmyoglobin, an empirical equation was evolved for the calculation of an approximate 10(6) X chi 20 degrees M value from the f value of a given complex (fobs) and that of the fluoride complex (fF) of a hemoprotein. The approximate magnetic susceptibilities of various ligand complexes of bovine lactoperoxidase could be thus calculated with the equation. The oscillator strengths of ferrous hemoproteins were also investigated and ligand-dependent regular changes were found.
Changes in peroxisomal CoA pools in the liver of fasted, diabetic, high-fat diet-fed and clofibrate-treated rats were studied. Total-CoA increased slightly in the fasted group and markedly in the diabetic, high-fat and clofibrate-treated groups. Fractionation studies showed that changes in free CoA levels were much greater in peroxisomes than in mitochondria. The concentrations of CoAs were calculated from the contents of CoAs in organelles and the changes in volume of organelles under these conditions; the concentration of total CoA in peroxisomes was higher than that in cytosol, but lower than that in mitochondria. These changes were accompanied by an increase in the activity of peroxisomal beta-oxidation. The results obtained from these experiments indicate that the peroxisomal beta-oxidation system is controlled not only at the enzyme level but also at the substrate or co-factor level.
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A purified NADPH-cytochrome c reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was prepared from swine testis microsomes by detergent solubilization followed by a procedure including chromatofocusing. The reductase was eluted at an isoelectric point of 4.8 from the chromatofocusing column. 730-fold purification was achieved with an overall yield of 1.2%. The preparation was found to be homogeneous upon polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (SDS). Upon SDS-polyacrylamide gel electrophoresis, however, the purified preparation resolved into one major band (Mr 78 000) and two minor bands (Mr 60 000 and 15 000). The enzyme contained about 1 mol each of FMN and FAD, which were both extractable with trichloroacetic acid and also boiling water. The oxidized form of the enzyme showed the absorption spectrum of a typical flavoprotein. Aerobic reduction with NADPH resulted in conversion of the spectrum into one of an air-stable semiquinone form. The activity of the purified preparation was 26 mumol cytochrome c reduced/min per mg protein under the standard assay conditions at 22 degrees C. The enzyme catalyzed the reaction through a ping-pong mechanism.
The effects of valproic acid on peroxisomal beta-oxidation and on lipid levels of liver and serum in the rat and mouse were studied. When the animals were fed diet containing 1% valproic acid for 2 weeks, the activity of peroxisomal beta-oxidation increased 4-fold in the rat liver and 2-fold in the mouse liver. Other peroxisomal enzymes such as catalase and urate oxidase also increased by the treatment though to a lesser extent than beta-oxidation. The contents of triglyceride and cholesterol in the serum decreased significantly in the rat but not in the mouse. The time course curves of the activities of cyanide-insensitive palmitoyl-CoA oxidation and carnitine-dependent palmitoyltransferase indicated that peroxisomal beta-oxidation was enhanced more rapidly than that of mitochondrial. The distributions of these enzymes were not changed by the treatment with valproic acid, though increases in liver weight and protein content were observed. These results indicate that the action of valproic acid in enhancing hepatic beta-oxidation is similar to that of clofibrate and other hypolipidemic drugs.
The effect of a 0.25% clofibrate diet for 2 weeks on peroxisomal and mitochondrial beta-oxidation in chicken liver was studied. The activities of antimycin antimycin A-insensitive palmitoyl-CoA oxidation (peroxisomal beta-oxidation) and carnitine acetyltransferase increased about two-fold. The activities of palmitoyl-CoA-dependent O2 consumption (mitochondrial beta-oxidation) and carnitine palmitoyltransferase were also slightly activated by the administration of clofibrate, but not significant. Thus, clofibrate may be a typical drug which activates the peroxisomal beta-oxidation more than the mitochondrial one in various species. The effect of clofibrate on peroxisomal carnitine acetyltransferase was the same as that on the mitochondrial one in chicken liver. Serum lipids were not lowered, but hepatomegaly was observed in the present experiment with chicken.
Spectral changes of hemoproteins in the near ultraviolet region on binding to a ligand and on oxidation-reduction of the heme-iron were studied by computer-controlled spectrophotometry. Near ultraviolet difference spectra between the low spin and high spin forms of ferric hemoproteins were classified into three groups: Those showing two absorption peaks having maxima at around 285 and 295 nm, those showing a peak at around 275 nm, and those showing a peak at around 300 nm. No corresponding absorption peak was observed with model heme complexes of low molecular weight. The intensity of the peak in cyanide difference spectra of catalase and horseradish peroxidase in the near ultraviolet region was dependent on the concentration of added cyanide and paralleled the intensity of the spectral changes in the Soret region. The spectral changes in both the near ultraviolet and Soret regions developed within 6 ms after the addition of cyanide. Difference spectra between the reduced and oxidized forms of cytochrome c, cytochrome oxidase-cyanide complex, hemoglobin, and lactoperoxidase-cyanide complex showed a characteristic peak at around 285-290 nm. Various difference spectra of hemoglobin in the near ultraviolet region were also measured. The observed positions, shapes, combinations, and relative intensities of the peaks were compared with those of solvent perturbation difference spectra and pH difference spectra of proteins and aromatic amino acids and also with the diacetylchitobiose-induced difference spectrum of lysozyme. The kinds of aromatic amino acid residues possibly responsible for the observed difference peaks were discussed on the basis of the results of the comparison. Based on the results obtained, the common occurrence of a heme-linked functional response of the hemoprotein conformation was suggested.
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A drug, (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid, was metabolized to 4-(1-imidazolylmethyl)benzoic acid in isolated hepatocytes of rats, which was enhanced markedly by the pretreatment of rats with clofibrate. With liver homogenates, the formation of the CoA-ester of this drug and its subsequent chain-shortening were demonstrated. In the series of these reactions, acyl-CoA synthetase, CoA, ATP and NAD were required, whereas cyanide did not inhibit the reaction. These results indicate that peroxisomes are capable of shortening the acyl side-chains of drugs by the beta-oxidation, giving an additional suggestion on the functions of peroxisomes.
Effects of vitamin E deficiency and its restoration on biochemical characteristics of hepatic peroxisomes were studied. Rats were maintained on the vitamin E-deficient diet for 25 weeks and then on a diet supplemented with vitamin E for 5 weeks. Blood hemolysis by hydrogen peroxide and lipid peroxidation in the liver increased markedly in vitamin E-deficient rats. The former returned to the control level after the resupplying of vitamin E, but the latter did not. Of liver peroxisomal enzymes, the activities of catalase, D-amino-acid oxidase and urate oxidase decreased in vitamin E-deficient rats. On the other hand, activities of fatty acyl-CoA oxidase and carnitine acetyltransferase increased significantly in vitamin E-deficient rats. All activities of these peroxisomal enzymes were restored to the control levels in vitamin E-supplemented rats. The activities of the mitochondrial, lysosomal and microsomal enzymes tested showed no apparent change except that the change of mitochondrial palmitoyltransferase was shown to be similar to that of peroxisomal fatty acid oxidation. These results were also supported by cell fractionation techniques. Following the methods of aqueous polymer two-phase systems, the characteristics of peroxisomal surface membranes altered in respect of their hydrophobicity, but not in respect of the surface charge of peroxisomal membranes. These results indicate that peroxisomal functions, especially those of the fatty acid oxidation system, change their activities more sensitively than other intracellular organelles in response to the condition of vitamin E deficiency.
Subcellular localization of malate dehydrogenase and glycerol-3-phosphate dehydrogenase in rat liver was studied by sucrose density gradient centrifugation. The specific adsorption of cytosolic malate dehydrogenase to the peroxisomes was observed. This phenomenon was eliminated by washing peroxisome-rich fraction with 100 mM potassium chloride. It is suggested that the malate shuttle between the cytosol and the mitochondria is more dominant than the glycerophosphate shuttle with respect to the transfer of reducing equivalents, while NADH produced by fatty acid oxidation in peroxisomes can not be transferred into the cytosol via the malate shuttle in the rat liver.