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S Ohkuma

Publications and source records attributed to S Ohkuma.

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

Acridine orange as a fluorescent probe for lysosomal proton pump.

Acridine orange was found to accumulate in pure lysosomal particles (tritosomes) in vitro, and the quenching of its fluorescence correlated well with the delta pH (inside acid) across the lysosomal membrane. Use of this dye showed that Mg-ATP caused lysosomal acidification. This acidification was sensitive to N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, and azide, but not to oligomycin, ouabain or vanadate. These results supported the idea of the existence of a lysosomal H+-pump, suggested in a previous paper (Ohkuma et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762).

Acridine Orange↗
PubMed1981

Purification and properties of rabbit liver acid-lipase (4-methylumbelliferyl oleate hydrolase).

An acid lipase was purified from rabbit liver lysosomes by, in sequence, osmotic treatment of the lysosomal fraction, Sephadex LH-20, DEAE-Sephadex A-50, Bio-Gel A-5m, hydroxyapatite and, finally, Sephadex G-200 column chromatography. The substrate was 4-methylumbelliferyl oleate. The enzyme was solubilized by Sephadex LH-20 column chromatography instead of detergents and organic solvents, to obtain an intrinsic macromolecule. 4-Methylumbelliferyl oleate hydrolase, osmotically released from lysosomal particles, had a very high molecular weight (greater than 800 000) which was reduced by gel filtration on a Sephadex LH-20 column; the final molecular weight of the purified enzyme was 58 000. The specific activity of 4-methylumbelliferyl oleate hydrolase increased at almost the same rate as acid cholesterol esterase and triacylglycerol lipase after Sephadex LH-20 column chromatography; the thermal stability of the activity of the three enzymes was almost identical. We also discuss the properties of the enzyme molecule and the interaction between the enzyme and the lysosomal membrane.

Animals↗
PubMed1981

Differential effects of morphine withdrawal on cerebral beta 1- and beta 2-adrenergic receptors.

Effect of morphine dependence and its withdrawal on the 3H-dihydroalprenolol (3H-DHA) binding for beta -adrenergic receptors, beta 1 and beta 2, was examined by a computerized analysis of biphasic Hofstee plots. The relative density of beta 1 and beta 2 receptors in the rat cerebral cortex was found to be approximately 70% and 30%, respectively. In rats rendered dependent on morphine by a subcutaneous implantation of a morphine pellet, the 3H-DHA binding to beta 1 and beta 2 receptors was not altered. During the stage of withdrawal induced by administration of naloxone, however, the 3H-DHA binding to the cerebral particulate fractions was increased, and this increase was due to the increased binding sites in beta 1 and beta 2 receptors. On the other hand, the apparent affinities of beta and beta 2 for atenolol and salbutamol, selective antagonists for beta 1- and beta 2-adrenergic receptors, respectively, were not altered under these experimental conditions. These results suggest that an abrupt increase in cerebral beta 1-receptor binding sites occurs at morphine withdrawal, and the occurrence of such a super-sensitivity in cerebral beta 1 receptor may be involved in the exhibition and/or maintenance of the abstinence syndrome in morphine-dependent subjects.

Albuterol↗
PubMed1981

Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances.

With few exceptions, weakly basic compounds that are sufficiently lipophilic in their neutral forms and sufficiently hydrophilic in their protonated forms accumulate in lysosomes. When the concentration within the lysosomes becomes sufficiently high, osmotic swelling occurs. The cells than take on a vacuolated appearance. The concentrations at which different weak bases cause lysosomal vacuolation vary over almost three orders of magnitude. For any particular weak base, it is the concentration of the neutral form that determines the extent of uptake and the degree of vacuolation. Chloroquine is anomalous in that concentrations greater than approximately 30 microM cause less uptake and less vacuolation than do lower concentrations.

Amines↗
PubMed1981

Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages.

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.

Amines↗
PubMed1981

Characteristics of taurine transport in freshly isolated rat hepatocytes.

Characteristics of taurine (2-aminoethanesulfonic acid) transport were studied in freshly isolated rat hepatocytes prepared by a collagenase perfusion technique. The uptake of taurine at 37 degrees C was linear up to 30 min of incubation, but gradually decreased thereafter and reached a plateau at 90 min after initiation of the incubation. Taurine uptake at 4 degrees C by isolated hepatocytes was not saturable, whereas that at 37 degrees C was saturable with the following parameters: Km, 37 microM; Vmax, 0.043 nmoles/mg prot./min; and EA, 5.6 Kcal/mol. The taurine uptake at 37 degrees C was found to be sodium dependent, and this was inhibited competitively by guanidinoethyl sulfonate and beta-alanine with the Ki values of 1.75 mM and 285 microM, respectively. Conjugated cholate, conjugated chenodeoxycholate, alanine, isethionate and leucine had no effect on the taurine uptake. The present results indicate that taurine uptake by isolated hepatocytes consists of unsaturable and energy independent, and carrier-mediated and energy dependent transport processes.

Amino Acids↗
PubMed1981

Hepatic lipogenesis and mobilization of peripheral fats in the formation of alcoholic fatty liver.

Biochemical mechanisms underlying the development of alcoholic fatty liver was investigated. Acute ethanol (EtOH) administration for 3 days by an inhalation method, and continuous EtOH treatments by feeding with liquid diet or drinking water containing EtOH induced a significant increase of hepatic triglycerides (TG). A small but significant increase of TG was also observed in the blood serum. Although hepatic acetyl CoA carboxylase activity, measured in the presence and absence of citrate, was not altered by either acute or chronic EtOH administrations, fatty acid synthetase and malic enzyme activities in the liver were increased by continuous EtOH administration, but not in the acutely EtOH-treated animals. The incorporations of [14C]palmitate and [14C]acetate into hepatic RG were also increased significantly in animals treated continuously with EtOH. The lipoprotein lipase activity in adipose tissues was activated by both acute and continuous EtOH treatments, whereas lipase activity in adipose tissues and the epinephrine-stimulated and cyclic AMP-mediated release of free fatty acid (FFA) from this tissue were not altered by these treatments. These results indicate that acute alcoholic fatty liver is caused mostly by the increased mobilization of FFA from peripheral adipose tissues via the activation of lipoprotein lipase, whereas alcoholic fatty liver induced by continuous EtOH administration involves the increased synthesis of FFA due to the activation of fatty acid synthetase and malic enzyme in the liver in addition to the increased mobilization of peripheral FFA.

Acetyl-CoA Carboxylase↗
PubMed1980

[Intravenous chronic toxicity of lentinan in rats: 6-month treatment and 3-month recovery (author's transl)].

Chronic toxicity of lentinan was studied in male and female JCL : SD rats. Lentinan was given intravenously into tail vein. Dosage levels employed were 0 (5% mannitol), 0.01, 0.1, 1 (with or without dextran), and 10 mg/kg/day for 6 months in a volume of 1 ml/100 g body weight. After 6 months, the treatment was discontinued and a recovery study was performed for 3 months. Rats receiving 10 mg/kg had redness and necrosis of the tail, the treatment was stopped at week 5, and the rats were sacrificed. Rats receiving 1 mg/kg showed redness of the ear, tail, and scrotum, which was remarkable in the 2nd and 3rd months. Body weight gains were not adversely affected. Laboratory examinations revealed an increase in leukocyte count, decreases in differential eosinophil count and platelet count, and an increase in serum beta-globulin level in drug-treated rats. At autopsy after 6 months, rats from the drug-treated groups had pulmonary hemorrhage and enlargements of the spleen and mesenteric lymph nodes. Histologic changes attributable to treatment included (1) activation of reticulo-endothelial system such as small epithelioid cell nodule in the liver, spleen, and mesenteric lymph nodes, and mobilization of Kupffer cells; (2) arteritis in various organs, especially notable in the spleen, testis, and epididymis ; (3) hemorrhage in the lung; and (4) hypospermatogenesis. All these changes described above had a propensity to recover. The maximum no effect level was estimated to be less than 0.01 mg/kg in the present study in male and female rats.

Animals↗
PubMed1978

Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.

A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.7-4.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (2-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping.

Animals↗
PubMed1976

Synthesis of apoferritin in mouse peritoneal macrophages. Characterization of 20 S particles.

Apoferritin particles were found in mouse peritoneal macrophages cultured in vitro. They were found as 20S particles in the "ribosomal fraction" of macrophages labeled with L-[14C]glutamic acid. Possibilities that they were breakdown products of ribosomes or of other well-known contaminants of the ribosomal fraction were excluded because they did not incorporate [5-3H]uridine. They were resistant to RNase and were relatively resistant to detergent. The antibody against horse spleen apoferritin precipitated about 70% of the particles in the 20S region, judging by measurement of radioactivity. On in vitro incubation with Fe2+ and suitable oxidizing agents the sedimentation coefficient of 80% of the 20S particles changed to about 60S, which corresponds to that of ferritin. SDS-polyacrylamide gel electrophoresis revealed the presence of subunit structures with the same molecular size as that of mouse liver apoferritin. Under the electron microscope, the particles appeared spherical with a relatively uniform diameter of about 130 A.

Animals↗