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61.
62.
Y Ogawa 《Journal of biochemistry》1985,97(4):1011-1023
Calcium binding to troponin C and troponin was examined by a metallochromic indicator method under various conditions to obtain a further understanding of the regulatory roles of these proteins in muscle contraction. Troponin C has four Ca binding sites, of which 2 sites have a high affinity of 4.5 X 10(6) M-1 for Ca2+ and the other 2 sites have a low affinity of 6.4 X 10(4) M-1 in a reaction medium consisting of 100 mM KCl, 20 mM MOPS-KOH pH 6.80 and 0.13 mM tetramethylmurexide at 20 degrees C. Magnesium also binds competitively to both the high and low affinity sites: the apparent binding constants are 1,000 M-1 and 520 M-1, respectively. Contrary to the claim by Potter and Gergely (J. Biol. Chem. 250, 4628-4633, 1975), the low affinity sites are not specific only for Ca2+. The high and low affinity sites of troponin C showed different dependence on the ionic strength: the high affinity sites were similar to GEDTA, while the low affinity sites were similar to calmodulin, which has a steeper ionic strength dependence than GEDTA. Ca binding to troponin C was not affected by change of pH between 6.5 and 7.2. Troponin I enhanced the apparent affinity of troponin C for Ca2+ to a value similar to that for troponin. Trifluoperazine also increased Ca binding to troponin C. Troponin has four Ca binding sites as does troponin C, but the affinities are so high that the precise analysis was difficult by this method. The apparent binding constants for Ca2+ and Mg2+ were determined to be 3.5 X 10(6) M-1 and 440 M-1, respectively, for low affinity sites under the same conditions as for troponin C, being independent of change in pH between 6.5 and 7.2. The competitive binding of Mg2+ to the low affinity sites of troponin is consistent with the results of Kohama (J. Biochem. 88, 591-599, 1980). The estimate for the high affinity sites is compatible with the reported results. 相似文献
63.
Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca2+. To determine if Ca2+ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca2+, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca2+, was observed only in the perfusion solution containing Ca2+, indicating that myosin is responsible for the inhibitory effect of Ca2+ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca2+ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP
adenosine 5-triphosphate
- DTT
dithiothreitol
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid
- PIPES
piperazine-N,N-bis(2-ethanesulfonic acid) 相似文献
64.
Lambda DNA (125 micrograms/ml in Tris buffer, pH 7.4) was irradiated with 60Co gamma-rays and 3H beta-rays, respectively, and the number of strand breaks was determined by electrophoresis. Number of single-strand breaks increased linearly with radiation dose in both gamma- and beta-radiations and the relative effectiveness (beta/gamma) was found to be 1.82 in N2 and 1.16 in O2. Number of double-strand breaks increased with the square of the radiation dose in gamma-irradiation, but it increased linearly with radiation dose in beta-irradiation. Therefore, the relative effectiveness (beta/gamma) is higher at lower doses. O2 effects was observed by gamma-irradiation but was minimal after beta-irradiation. 相似文献
65.
Anti-O-phosphotyrosine antibodies in human sera 总被引:1,自引:0,他引:1
Antibodies reactive with O-phosphotyrosine (PTYR) were detected in 60 out of 621 inpatients, with high frequencies in hematologic and lung malignancies, hepatic diseases, cerebrovascular diseases, and autoimmune diseases. Affinity-purified antibodies proved capable of recognizing PTYR-containing proteins in a human carcinoma cell line, A431, both by immunofluorescent staining and by immunoaffinity chromatography, but had no detectable affinity for phosphorylated serine or threonine, or for the nucleotides tested. In these respects, the antibodies observed in human sera were indistinguishable from anti-PTYR antibodies raised experimentally in rabbits or mice. 相似文献
66.
M Imagawa S Hashida E Ishikawa Y Niitsu I Urushizaki R Kanazawa S Tachibana N Nakazawa H Ogawa 《Journal of biochemistry》1984,96(3):659-664
beta-D-Galactosidase from Escherichia coli and horseradish peroxidase were compared as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The anti-human ferritin Fab'-peroxidase conjugates gave lower nonspecific bindings and higher specific bindings than the corresponding Fab'-beta-D-galactosidase conjugates. As a result, the former provided more sensitive dose response curves for human ferritin than the latter. However, the peroxidase conjugates were required in a larger quantity, since peroxidase assay was much less sensitive than beta-D-galactosidase assay. 相似文献
67.
Interaction between the A2 and A19 amino acid residues is of critical importance for high biological activity in insulin: [19-leucine-A]insulin 总被引:1,自引:0,他引:1
The replacement of tyrosine at position A19 by leucine in the insulin molecule led to an analogue, [19-leucine-A]insulin [( Leu19-A]insulin), displaying insignificant receptor binding affinity and in vitro biological activity less than 0.1 and 0.05%, respectively, compared to the natural hormone. This analogue along with the previously reported [2-glycine-A]-, [2-alanine-A]-, and [2-norleucine-A]insulins is the least potent insulin analogue we have examined. Circular dichroic studies showed that all these analogues are monomeric at concentrations at which insulin is primarily dimeric. We conclude that an aromatic ring at position A19 and the presence of the side chain of isoleucine at position A2 are each of critical importance for high biological activity in insulin. It appears that the van der Waals interaction between the side chain of isoleucine A2 and tyrosine A19, present in crystalline insulin, is among the most important determinants for high biological activity in insulin. 相似文献
68.
Functional domains of Escherichia coli recA protein deduced from the mutational sites in the gene 总被引:20,自引:0,他引:20
Summary The sites of recA mutations of Escherichia coli, recA441 (tif-1), recA1, recA430 (lexB30) and recA44, were determined by analyses of the nucleotide sequences. All mutations are single point missense mutations within the coding region of the recA gene. In the recA441, recA1, recA430 and recA44 proteins, the 38th, 160th, 204th, and 246th amino acids, respectively, from the amino terminal ends are altered. Based on the properties of these mutant proteins and modified forms of recA protein, the locations of various regions of the recA protein that are involved in binding with ATP, binding with single-stranded DNA, hydrolysis of ATP, interaction between the recA protein molecules and interaction with the cI or lexA repressors are mapped on the primary structure of the protein. 相似文献
69.
Hideya Hayashi Seiji Ito Teruo Tanaka Manabu Negishi Hideo Kawabe Yokohama Hiromitsu Kikuko Watanabe Osamu Hayaishi 《Prostaglandins & other lipid mediators》1987,33(4)
In view of the recent finding that prostaglandin D2 is stereospecifically converted to 9α,11β-prostaglandin F2, an isomer of prostaglandin F2α, a highly specific and sensitive radioimmunoassay for 9α,11β-prostaglandin F2 was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9α-11β-prostaglandin F2 were raised in rabbits immunized with the bovine serum albumin conjugate, and [3H]9α,11β-prostaglandin F2 was enzymatically prepared from [3H]prostaglandin D2. The assay detected 9α,11β-prostaglandin F2 over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-section with prostaglandin F2α, prostaglandin F2β and 9β,11β-prostaglandin F2. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9α,11β-prostaglandin F2 was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 ± 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D2 (ca. 180 ng/g wet weight) and prostaglandin F2α (ca. 70 ng/g) were produced in the brain; however, 9α,11β-prostaglandin F2 was detected neither there nor in the blood. This result demonstrates that the conversion to 9α,11β-prostaglandin F2 is a minor pathway, if one at all, of prostaglandin D2 metabolism in the rat brain. 相似文献
70.