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41.
42.
Yuichiro Hiraizumi 《American journal of human genetics》1964,16(3):375-379
43.
44.
The effect of a carboxylic ionophore (lasalocid) on the sarcoplasmic reticulum Ca2(+)-ATPase was investigated. The purified enzyme was preincubated with lasalocid in the presence of Ca2+ and the absence of K+ at pH 7.0 and 0 degrees C for 2 h. The Ca2(+)-dependent ATPase activity was strongly inhibited by this preincubation, whereas the activity of the contaminant Mg2(+)-ATPase was unaffected. The steady-state level of the phosphoenzyme (EP) intermediate remained constant over the wide range of lasalocid concentrations. The Ca2(+)-induced enzyme activation was unaffected. The kinetics of phosphorylation of the Ca2(+)-activated enzyme by ATP as well as the rate of conversion of ADP-sensitive EP to ADP-insensitive EP were also unaffected. Accumulation of ADP-insensitive EP was greatly enhanced, and almost all of the EP accumulating at steady state was ADP-insensitive. Hydrolysis of ADP-insensitive EP was strongly inhibited. A similar strong inhibition of the Ca2(+)-dependent ATPase activity by lasalocid was found with sarcoplasmic reticulum vesicles. To examine the effect of lasalocid on the conformational change in each reaction step, the Ca2(+)-ATPase of sarcoplasmic reticulum vesicles was labeled with a fluorescent probe (N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine) without a loss of catalytic activity and then preincubated with lasalocid as described above. The conformational changes involved in hydrolysis of ADP-insensitive EP and in the reversal of this hydrolysis were appreciably retarded by lasalocid. The conformational changes involved in other reaction steps were unaffected. These results demonstrate that hydrolysis of ADP-insensitive EP in the catalytic cycle of this enzyme is selectively inhibited by lasalocid. 相似文献
45.
K Oshimi Y Oshimi O Yamada M Wada T Hara H Mizoguchi 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(9):3312-3317
The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering. 相似文献
46.
Both 25-epimers of (22E)-22-dehydro-1 alpha,25-dihydroxy-26-methylvitamin D3 [22-dehydro-26-methyl-1,25-(OH)2D3] were synthesized. The biological activity of these compounds was tested in binding affinity to chick intestinal receptor protein of 1 alpha,25-dihydroxy-vitamin D3 [1,25-(OH)2D3] and in stimulating for intestinal calcium transport and bone calcium mobilization with vitamin D-deficient rats. The relative potency of (25R)- and (25S)-22-dehydro-26-homo-1,25-(OH)2D3 and 1,25-(OH)2D3 in competing for the intestinal cytosolic binding was 1.7:1.5:1. A similar order of activity was observed on intestinal calcium transport and bone calcium mobilization. In the ability for stimulation of intestinal calcium transport, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were about 3.6 and 2.1 times as active as 1,25-(OH)2D3, respectively. In bone calcium mobilization tests, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were estimated to be 2.2 and 1.6 times as potent as 1,25-(OH)2D3, respectively. 相似文献
47.
Measurement of the cytosolic free calcium ion concentration of individual lymphocytes by microfluorometry using quin 2 or fura-2 总被引:3,自引:0,他引:3
H Komada H Nakabayashi H Nakano M Hara T Yoshida H Takanari K Izutsu 《Cell structure and function》1989,14(2):141-150
The cytosolic free calcium ion concentration ([Ca2+]i) of individual lymphocytes was measured by microfluorometry with dual excitation wavelengths using quin 2 for fura-2. Fura-2 was a more suitable fluorescent Ca2+ indicator than quin 2 for measurements of single cells because of the standard curve calibrated for fura-2 had a good linearity, and the standard deviation (SD) of the value of the intensity ratio of fura-2-loaded cells was much smaller than that of quin 2-loaded cells. The [Ca2+]i in quiescent lymphocytes was about 1 x 10(-7) M, and an increase in the [Ca2+]i was observed within a few minutes of ionomycin, protein A, phorbol myristate acetate (PMA) or concanavalin A (Con A) stimulation. Ionomycin-induced proliferation occurred when the initial [Ca2+]i was approximately 3 x 10(-7) M or greater. The increase in the [Ca2+]i induced by Con A occurred transiently, and another rise in the [Ca2+]i was observed in the stage prior to the S-phase. These results indicate that Ca2+ is necessary for stimulated lymphocytes to enter the cell cycle and S-phase. 相似文献
48.
Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conversion of 3 alpha- or 20 alpha-hydroxy groups of several bile acids and 5 beta-pregnanes to the corresponding 3- or 20-ketosteroids. The kcat values for the steroids determined at pH 7.4 were low, but the kcat/Km values for the 3-ketosteroids were comparable to or exceeded those for 1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3 beta- or 20 beta-face of the ketosteroid substrate. Competitive inhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyprogesterone acetate, hexestrol, and 1,10-phenanthroline suggests that both 1-indanol and hydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor of the enzyme, 1,10-phenanthroline, suppressed the 3 alpha-hydroxysteroid dehydrogenase activity in the crude extract of monkey liver by 50%. The results strongly suggest that indanol dehydrogenase acts as a 3(20)alpha-hydroxysteroid dehydrogenase in the metabolism of certain steroid hormones and bile acids. 相似文献
49.
A stable covalent complex was prepared by cross-linking adrenodoxin reductase with adrenodoxin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was purified extensively until free components were removed completely. The major component of the complex had a molecular weight of 63 kDa, which corresponds to a 1:1 stoichiometric complex between adrenodoxin reductase and adrenodoxin. NADPH-cytochrome c reduction activity of the covalent complex was comparable to that of an equimolar mixture of adrenodoxin reductase and adrenodoxin (native complex), and the NADPH-ferricyanide reduction activity of the complex was equal to that of the native one. In contrast to the native complex, the covalent complex produced much less superoxide upon NADPH-oxidation, and the covalent complex was found to be more stable than the native complex, suggesting that the complex state is more favorable for catalysis. From these results, we conclude that the adrenodoxin molecule does not need to dissociate from the complex during electron transfer from NADPH to cytochrome c. 相似文献
50.
An ATP-driven Cl- pump in the brain 总被引:2,自引:0,他引:2
T Shiroya R Fukunaga K Akashi N Shimada Y Takagi T Nishino M Hara C Inagaki 《The Journal of biological chemistry》1989,264(29):17416-17421
EDTA-treated microsomes prepared from rat brain mainly consisted of sealed membrane vesicles 200-500 nm in diameter and were rich in both Cl- -ATPase and Na+,K+-ATPase activities. Such Cl- -ATPase-rich membrane vesicles accumulated Cl- in an ATP-dependent and osmotically reactive manner in the presence of 1 nM ouabain. The Cl- uptake was maximally stimulated by ATP with a Km value of 1.5 mM; GTP, ITP, and UTP partially stimulated Cl- uptake, but CTP, beta, gamma-methylene ATP, ADP, and AMP did not. The ATP-dependent Cl- uptake was accelerated by an increase in the medium Cl- concentration with a Km value of 7.4 mM. Such stimulation of Cl- uptake by ATP was dependent on the pH of the medium, with an optimal pH of 7.4, and also on the temperature of the medium, with an optimal range of 37-42 degrees C. Ethacrynic acid dose dependently inhibited the ATP-dependent Cl- uptake with a concentration for half-maximal inhibition at 57 microM. N-ethylmaleimide (0.1 mM) completely inhibited and sodium vanadate (1 mM) partially inhibited the ATP-dependent Cl- uptake. The membrane vesicles did not accumulate H+ in the Cl- uptake assay medium. The ATP-dependent Cl- uptake profile agreed with that of Cl- -ATPase activity reported previously (Inagaki, C., Tanaka, T., Hara, M., and Ishiko, J. (1985) Biochem. Pharmacol. 34, 1705-1712), and this strongly supports the idea that Cl- -ATPase in the brain actively transports Cl-. 相似文献