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31.
Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I. 相似文献
32.
Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range 总被引:1,自引:0,他引:1
H Yoshimura T Nishio K Mihashi K Kinosita A Ikegami 《Journal of molecular biology》1984,179(3):453-467
The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin. 相似文献
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T Yoshimura T Miyoshi M Imaki T Tamura M Fujii K Matsumoto T Toumiya Y Hori T Nakamura M Tanada 《Acta biologica Hungarica》1986,37(3-4):235-241
Changes in the activity of serum gamma-glutamyl transpeptidase (gamma-GTP) and the percentage of the gamma-GTP fraction in healthy young men given a high carbohydrate diet (480-636 g/day, 80% of the total energy) for 21 days were examined. Serum total gamma-GTP activity showed no significant change in four healthy young volunteers who received high carbohydrate diet for 21 days. However, the percentage of the gamma-GTP (1) fraction increased significantly (P less than 0.01) from the basal level of 55.6 +/- 4.0% to 67.6 +/- 0.9% on day 10, and then decreased to 58.4 +/- 1.4% on day 21. When the experimental diet was replaced by usual diet, the percentage of the gamma-GTP (1) fraction returned to the same level as before the experiment. It is concluded from the results that the nutrient intake affects the percentage of gamma-GTP (1), but not the total serum gamma-GTP activity. 相似文献
35.
K Nakahama K Yoshimura R Marumoto M Kikuchi I S Lee T Hase H Matsubara 《Nucleic acids research》1986,14(14):5843-5855
The gene encoding an extracellular metalloproteinase from Serratia sp. E-15 has been cloned, and its complete nucleotide sequence determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature protein of the Serratia protease consists of 470 amino acids with a molecular weight of 50,632. The G+C content of the coding region for the mature protein is 58%; this high G+C content is due to a marked preference for G+C bases at the third position of the codons. The gene codes for a short pro-peptide preceding the mature protein. The Serratia protease gene was expressed in Escherichia coli and Serratia marcescens; the former produced the Serratia protease in the cells and the latter in the culture medium. Three zinc ligands and an active site of the Serratia protease were predicted by comparing the structure of the enzyme with those of thermolysin and Bacillus subtilis neutral protease. 相似文献
36.
T Yoshimura 《Archives of biochemistry and biophysics》1983,220(1):167-178
Infrared and electron paramagnetic resonance spectra of nitrosyl(protoporphyrin IX dimethyl ester)iron(II)(Fe(PPDME)(NO)) and its complexes with nitrogenous bases (N bases) such as imidazoles, pyridines, aliphatic amines, and anilines have been measured in various solvents. At room temperature, giso, Aiso, and nu NO values of five-coordinate Fe(PPDME)(NO) decreased with an increase in solvent polarity parameter ET, indicating the interaction between the solvent and the vacant axial coordination position. It has been found that the nu NO value of six-coordinate species is very sensitive to the solvent polarity, while the giso value is less sensitive. The solvent effect on the equilibrium constants, which are evaluated from the intensity change of the NO stretching band for five- and six-coordinate species, is discussed. 相似文献
37.
38.
Summary Zymogramic analysis of esterase in the mouse, rat and guinea-pig salivary glands was undertaken and demonstrated species and organ specificities of esterase with electrophoretic method. Salivary glands esterase was classified into A, B and C types based on the electrophoretic mobility. Mouse submandibular gland had the most complicated pattern, while guinea-pig showed the simpliest patterns which was devoid of B type of esterase. Rat salivary glands exhibited rather regular patterns. Similar zymogram patterns were obtained with many kinds of ester compounds, that is simple and substituted naphthol esters and indoxyl derivatives. The tests of inhibition and activation for esterase activity was obtained. Histochemical properties applied to inhibitor test in the esterase zymogram patterns showed no marked differences between ducts and acini. 相似文献
39.
40.