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71.
A gene coding for lipase-solubilized bovine liver microsomal cytochrome b5 has been synthesized, expressed in Escherichia coli, and mutated at functionally critical residues. Characterization of the recombinant protein revealed that it has a reduction potential that is approximately 17 mV lower than that of authentic wild-type protein at pH 7 (25 degrees C). Structural studies determined that the recombinant protein differed in sequence from authentic wild-type cytochrome b5 owing to three errors in amidation status in the published sequence for the protein on which the gene synthesis was based. The structural origin of the lower reduction potential exhibited by the triple mutant has been investigated through X-ray crystallographic determination of the three-dimensional structure of this protein and is attributed to the presence of Asp-57 within 3.3 A of heme vinyl-4 in the mutant. In addition, the model developed by Argos and Mathews [Argos, P., & Mathews, F.S. (1975) J. Biol. Chem. 250, 747] for the change in cytochrome b5 oxidation state has been studied through mutation of Ser-64 to Ala. In this model, Ser-64 is postulated to stabilize the oxidized protein through H-bonding interactions with heme propionate-7 that orients this propionate group 6.2 A from the heme iron. Spectroelectrochemical studies of a mutant in which Ser-64 has been changed to an alanyl residue demonstrate that this protein has a reduction potential that is 7 mV lower than that of the wild-type protein; moreover, conversion of the heme propionate groups to the corresponding methyl esters increases the potential by 67 mV.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
72.
A rapid fluorogenic method for the demonstration of 5′-nucleotide phosphodiesterase in human serum has been developed. This method uses the substrate 4-methylumbelliferyl 5′-thymidylate impregnated in agarose gels or filter paper strips. Zymograms are developed in less than 30 min at 25°C, and the sensitivity of this method has been compared with that of the indigogenic method. 相似文献
73.
Light-induced pigment granule migration in the retinular cells of Drosophila melanogaster. Comparison of wild type with ERG-defective mutants 总被引:3,自引:3,他引:0 下载免费PDF全文
The dependence of pigment granule migration (PGM) upon the receptor potential was examined using several strains of electroretinogram (ERG)- defective mutants of Drosophila melanogaster. The mutants that have a defective lamina component but a normal receptor component of the ERG (no on-transient A [nonA] and tan) exhibited normal pigment granule migration. The mutants that have very small or no receptor potentials (certain no receptor potential A [norpA] alleles), on the other hand, exhibited no PGM. In the case of the temperature-sensitive norpA mutant, norpAH52, normal PGM was present at 17 degrees but not at 32 degrees C or above, corresponding to its electrophysiological phenotype. In the transient receptor potential (trp) mutant, whose receptor potential decays to the baseline within a few seconds during a sustained light stimulus, the pigment granules initially moved close to the rhabdomere when light was turned on but moved away after about 5 s during a sustained light stimulus. All these results lend strong support to the notion that PGM is initiated by a light-evoked depolarization of the receptor membrane, i.e., the receptor potential. However, under certain experimental conditions, the receptor potentials failed to induce PGM in the trp mutant. The depolarization of the receptor, thus, appears to be closely associated with PGM but is not a sufficient condition for PGM. 相似文献
74.
Block and inactivation of sodium channels in nerve by amino acid derivatives. II. Dependence on temperature and drug concentration. 总被引:2,自引:0,他引:2 下载免费PDF全文
The interaction of n-propylguanidinium (nPG) with sodium channels has been further characterized. From experiments at varying temperatures, the Q10 for the sodium current decay time constant in the two [Na+] gradients is 2.6-2.9 independent of drug. Testing several nPG concentrations we find that peak sodium current declines sharply with [nPG] at all levels, but the decay time constant approaches an asymptote above 4 mM. No "hooks" in sodium tail currents are seen. If the sodium current is allowed to decay completely before repolarization no tail current is observed. We have developed a kinetic model in which nPG acts at a single site within the sodium channel. Reaction of nPG with its receptor requires two steps. Fitting the temperature data shows that the first step involves diffusion of the drug to the site and close association with it. The second step may include molecular reorganization of the complex. The rate constants for the reaction are all simple exponential functions of voltage. Using them, the model successfully predicts decay time constants and peak currents, and their dependence on potential, [Na+] gradient, temperature, and nPG concentration. The results are consistent with the idea that an arginine residue may be closely associated with inactivation. 相似文献
75.
Relationship between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in the action of thyroid hormone on rat jejunal mucosa. 下载免费PDF全文
Administration of three successive doses of triiodothyronine (T3) (50 micrograms/100 g body wt), given on alternate days to thyroidectomized and euthyroid rats, stimulated oxygen consumption (QO2) and Na+ transport-dependent respiration (QO2 [5]) in the stripped jejunal mucosa, a preparation that consisted mostly of epithelial cells. The increase in QO2(t) accounted for 57% of the increment in QO2 in the transition from the hypothyroid to the euthyroid state and for 29% of the increment in the transition from the euthyroid to the hyperthyroid state. Administration of T3 to hypothyroid rats also increased the yield of epithelial cells. Injection of T3 into thyroidectomized and euthyroid rats increased the specific activity (at Vmax) of the (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase) in jejunal crude membrane preparations. No significant change was recorded in the activity of Mg-ATPase in the same preparation. The ratio of QO2/NaK-ATPase and QO2(t)/NaK-ATPase in the various thyroid states remained constant, indicating proportionate increased in the respiratory and enzymatic indices. The effect of administration of T3 to thyroidectomized rats on the number of NaK-ATPase units (recovered in the crude membrane preparation) was estimated by: (a) Na+ + Mg++ + ATP-dependent binding of [3H]-ouabain to crude membrane fractions, and (b) the amount of the phosphorylated intermediate formed in the NaK-ATPase reaction from AT32P(gamma). Estimates were obtained of the maximal number of [3H]ouabain binding sites (Nm) and dissociation constants (Kd). Nm for [3H]ouabain and Nak-ATPase specific activity increased to about the same extent after T3 administration to thyroidectomized rats, with no change in the apparent Kd values. The amount of phosphorylated intermediate formed in jejunal crude membrane preparations also increased significantly. Thus, thyroid hormone administration may increase the number of active Na+pump sites in the plasma membrane. The apparent increase in the number of Na+ pump sites also correlated with the hormone dependent increases in QO2 and QO2(t). 相似文献
76.
Antibodies to staphylococcal alpha toxin were separated into two distinct populations. One population prevented binding of alpha toxin onto erythrocyte membranes. The other population neutralized after the toxin was bound onto erythrocytes and thereby brought about an indirect hemagglutination reaction. The data suggest that alpha toxin has a membrane-binding region. 相似文献
77.
我国北方异色瓢虫越冬集群的调查研究:越冬场所的调查初报 总被引:2,自引:0,他引:2
在毛主席革命路线指引下,我国农林害虫的生物防治得到了迅速发展。为了在本世纪内,全面实现农业、工业、国防和科学技术现代化,在植物保护工作上,我们要积极贯彻“预防为主,综合防治”的方针,采取综合防治消灭害虫。“以虫治虫”是其中的重要措施。研究益虫越冬集群的规律,扩大益虫来源,给利用天敌提供丰富的物质基础,为此开展本题的调查研究。 相似文献
78.
79.
Everted hamster jejunum was loaded with D-galactose and then escape into an initially galactose-free mucosal solution was followed. Mucosal anaerobiosis greatly increased the rate of escape, an effect which might have been caused by inhibiting reuptake from the unstirred layer and/or by augmenting the ease of unidirectional efflux across the brush border membrane. The former effect was expected because of our previous results from influx studies, and the main object here was to find out if the ease of efflux is affected by anaerobiosis. With phlorizin present in the mucosal solution during escape, information about unidirectional efflux was obtainable. We estimated that 10(-4) M phlorizin inhibited the ease of efflux via the phlorizin-sensitive pathway by about 65%. Apparently the reason why mucosal phlorizin accelerates escape of sugar from loaded mucosa, an effect which has been reported previously by others, is that it inhibits unidirectional efflux less effectively than it inhibits reuptake from the unstirred layer. Residual efflux via the phlorizin-sensitive pathway was markedly increased by mucosal anaerobiosis. This increase did not require an elevation of intracellular Na+ concentration. These results, together with those of our previous study, show that mucosal anaerobiosis abolishes uphill transport of galactose across the brush border of hamster jejunum by inhibiting unidirectional influx and by increasing the ease of unidirectional efflux. Neither of these effects requires a rise in intracellular Na+ concentration. 相似文献
80.