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41.
Mitsuro Nakano Toshihiro Hidaka Takashi Ueta Ryohei Ogura 《Prostaglandins & other lipid mediators》1983,25(4):503-510
Incubation of [1-14C]arachidonic acid (AA) with homogenates of bovine gallbladder muscle generated a large amount of radioactive material having the chromatographic mobility of 6-keto-PGF1α (stable product of PGI2) and smaller amounts of products that comigrated with PGF2α and PGE2. Formation of these products was inhibited by the cyclooxygenase inhibitor indomethacin. The major radioactive product identified by thin-layer chromatographic mobility and by gas chromatography - mass spectrometric analysis was found to be 6-keto-PGF1α. The quantitative metabolic pattern of [1-14C]PGH2 was virtually identical to that of [1-14C]AA. Incubation of arachidonic acid with slices of bovine gallbladder muscle released labile anti-aggregatory material in the medium, which was inhibited by aspirin or 15-hydroperoxy-AA.These results indicate that bovine gallbladder muscle has a considerable enzymatic capacity to produce PGI2 from arachidonic acid. 相似文献
42.
Masato Hirata Takafumi Hamachi Eiichi Suematsu Toshitaka Koga 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,763(4)
Effects of N-formyl chemotactic peptides on the Ca2+ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of 45Ca2+ into macrophages, whereas it stimulated the efflux of 45Ca2+ from macrophages at concentrations ranging from 10−10 M to 10−7 M. fMet-Met-Met and fMet-Leu also stimulated the 45Ca2+ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN3, did not modify the 45Ca2+ efflux induced by the chemoattractants, yet they did induce the release of 45Ca2+ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of 45Ca2+ from the NaN3-insensitive Ca2+ store site and mimicked the enhancement of the 45Ca2+ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca2+ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca2+ from the NaN3-insensitive Ca2+ store site. 相似文献
43.
Masato Hirata Takafumi Hamachi Eiichi Suematsu Toshitaka Koga 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,763(4):339-345
Effects of N-formyl chemotactic peptides on the Ca2+ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of 45Ca2+ into macrophages, whereas it stimulated the efflux of 45Ca2+ from macrophages at concentrations ranging from 10?10 M to 10?7 M. fMet-Met-Met and fMet-Leu also stimulated the 45Ca2+ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN3, did not modify the 45Ca2+ efflux induced by the chemoattractants, yet they did induce the release of 45Ca2+ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of 45Ca2+ from the NaN3-insensitive Ca2+ store site and mimicked the enhancement of the 45Ca2+ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca2+ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca2+ from the NaN3-insensitive Ca2+ store site. 相似文献
44.
The lipase production of a plant pathogenic fungus, Fusarium oxysporum f. sp. lini SUF 402, was induced by fat as the carbon source, and its release was stimulated by the infusion of intracellular free calcium ion with a calcium ionophore, A23187. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, a calmodulin inhibitor) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl- L-tyrosyl]-4-phenylpiperazine (KN-62, a Ca2+/calmodulin dependent protein kinase II inhibitor) reduced the extracellular release of lipase in vivo. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7, a protein kinase C inhibitor) did not have this ability. After K2H32PO4 had been incorporated into the cells, they were treated with W-7 or KN-62 and stimulated by Ca2+ ionophore. On SDS-PAGE of intracellular proteins followed by autoradiography, W-7- and KN-62-treated cells showed inhibition of the incorporation of 32Pi into the 20 kDa protein resulting from Ca2+ stimulation. F. oxysporum had calmodulin (CaM)-dependent protein kinase activity in the cytoplasmic fraction and had the ability to phosphorylate of syntide 2, a specific substrate of CaM kinase II. The partially purified CaM-dependent protein kinase was inhibited by 10 microM KN-62 in vitro. Increase of the intracellular Ca2+ concentration of F. oxysporum activated CaM and CaM-dependent protein kinase, resulting in the extracellular lipase release. These results suggest the existence of a Ca2+ signalling system in F. oxysporum like those observed in higher eucaryotes. 相似文献
45.
Purification and characterization of phosphoenolpyruvate phosphomutase from Pseudomonas gladioli B-1.
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Phosphoenolpyruvate phosphomutase (PEPPM) catalyzes C-P bond formation by intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvate (PnPy). We purified PEPPM from a gram-negative bacterium, Pseudomonas gladioli B-1 isolated as a C-P compound producer. The equilibrium of this reaction favors the formation of the phosphate ester by cleaving the C-P bond of PnPy, but the C-P bond-forming reaction is physiologically significant. The C-P bond-forming activity of PEPPM was confirmed with a purified protein. The molecular mass of the native enzyme was estimated to be 263 and 220 kDa by gel filtration and polyacrylamide gel electrophoresis, respectively. A subunit molecular mass of 61 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the native protein was a tetramer. The optimum pH and temperature were 7.5 to 8.0 and 40 degrees C, respectively. The Km value for PnPy was 19 +/- 3.5 microM, and the maximum initial velocity of the conversion of PnPy to phosphoenolpyruvate was 200 microM/s/mg. PEPPM was activated by the presence of the divalent metal ion, and the Km values were 3.5 +/- 1.4 microM for Mg2+, 16 +/- 5 nM for Mn2+, 3.0 +/- 1.5 microM for Zn2+, and 1.2 +/- 0.2 microM for Co2+. 相似文献
46.
H Tokumitsu A Mizutani H Minami R Kobayashi H Hidaka 《The Journal of biological chemistry》1992,267(13):8919-8924
A calcyclin-associated protein with an apparent molecular weight of 50,000 (CAP-50) was purified from rabbit lung. The procedure included ammonium sulfate precipitation, anion and cation ion-exchange, and calcyclin affinity chromatographies. Interestingly, partial amino acid sequences of lysyl-endpeptidase-digested fragments indicated that CAP-50 was a member of the Ca2+/phospholipid-binding proteins, the annexin family. The sequence of a proteolytic peptide with Staphylococcus aureus V8 protease on NH2-terminal region is not homologous with any other annexin family proteins. Phospholipid binding studies showed that CAP-50 bound to phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid-containing vesicles, in a Ca(2+)-dependent manner. In the presence of Ca2+/calcyclin, CAP-50 formed a complex with calcyclin and bound to the PS-containing vesicles. The apparent Kd value of calcyclin for CAP-50 was calculated to be 1.61 x 10(-6) M. Zero-length cross-linking studies indicated that 1 mol of CAP-50 bound to an equimolar unit of calcyclin. CAP-50 inhibited the phospholipase A2 activity, dose-dependently (IC50 = 0.2 microM), however, calcyclin did not alter the inhibitory effect. With the 125I-calcyclin gel overlay method, calcyclin bound tightly to CAP-50 in a Ca(2+)-dependent manner after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that rabbit lung CAP-50 is a newly identified member of the annexin family. Ca2+/calcyclin apparently regulates the function of CAP-50 on cytosolic face of the plasma membrane. 相似文献
47.
M Terasawa A Nakano R Kobayashi H Hidaka 《The Journal of biological chemistry》1992,267(27):19596-19599
A novel calcium-binding protein (molecular weight 23,000-24,000, pI 5.3-5.5), which we term neurocalcin, was identified in bovine brain. Using calcium-dependent drug affinity chromatography ((S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxycarbonylpiperazinyl++ +)-1-(P- methoxybenzyl)ethyl]-N-methylbenzene-sulfonamide dihydrochloride, W-77, -coupled Sepharose 6B), we purified neurocalcin from bovine brain. The partial amino acid sequence of neurocalcin revealed it to be an as yet unidentified protein with three putative calcium binding sites (EF-hands). Further purification and sequence analysis demonstrated the presence of four isoprotein forms designated alpha, beta, gamma 1, and gamma 2. When the 165 sequenced residues of neurocalcin beta are compared with sequences of other proteins, neurocalcin beta has a 38.2% sequence homology with visinin and 45.5% with recoverin (Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H., and Miki, N. (1990) Neuron 2, 469-476; Dizhoor, A. M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A., Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918). Both visinin and recoverin are expressed specifically in retinal photoreceptors and are not found in brain. Unlike visinin and recoverin, neurocalcin is purified not only from retina but also from bovine brain. Our results suggest that neurocalcin is a recoverin-like protein expressed in bovine brain. 相似文献
48.
M Tozuka H Hidaka M Miyachi K Furihata T Katsuyama M Kanai 《Biochimica et biophysica acta》1992,1165(1):61-67
A new apolipoprotein complex designated as the apo(AII-E2-AII) complex was identified in the lipoprotein fractions of human plasma with apoE phenotypes containing apoE2 (E4/E2, E3/E2, and E2/E2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by an immunoblotting assay using anti-apoE or anti-apoAII antibodies, established that the apo(AII-E2-AII) complex, with a molecular weight of 58,000, was identical to the complex consisting of apoE and apoAII, and that it also dissociated following reduction with beta-mercaptoethanol. This new complex was also demonstrated to be distinct from the apo(E-AII) complex and apoE monomer by isoelectric focusing, in the samples that were not treated with beta-mercaptoethanol. In apoE phenotype E3/E2, the apo(AII-E2-AII) complex was primarily included in the high-density lipoprotein (HDL, 1.063 < d < 1.21 g/ml) fraction, but was also observed in a small quantity in the very-low-density lipoprotein (VLDL, d < 1.006 g/ml) fraction. For further characterization, the apo(AII-E2-AII) complex was isolated by preparative SDS-PAGE, and no contamination of apo(E-AII) complex and apoE monomer was detected by immunoblotting assay using an anti-apoE antibody. It was confirmed by an enzyme-linked immunosorbent assay (ELISA) system that a molecular ratio between apoAII monomer and apoE in the isolated apo(AII-E2-AII) complex was approx. 2, when the apo(E-AII) complex was used as a standard with the ratio of 1:1. It indicates that the apo(AII-E2-AII) complex is formed from two molecules of apoAII monomer and one molecule of apoE.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
49.
M Hidaka T Kobayashi Y Ishimi M Seki T Enomoto M Abdel-Monem T Horiuchi 《The Journal of biological chemistry》1992,267(8):5361-5365
DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases. 相似文献
50.
M G Traber G W Burton L Hughes K U Ingold H Hidaka M Malloy J Kane J Hyams H J Kayden 《Journal of lipid research》1992,33(8):1171-1182
To study the mechanisms of discrimination between various forms of vitamin E, four normal subjects, one patient with lipoprotein lipase deficiency, and three patients with abnormal apolipoprotein B-100 production were given an oral dose containing three tocopherols labeled with differing amounts of deuterium (2R,4'R,8'R-alpha-(5,7-(C2H3)2)tocopheryl acetate (d6-RRR-alpha-tocopheryl acetate), 2S,4'R,8'R-alpha-5-(C2H3)tocopheryl acetate (d3-SRR-alpha-tocopheryl acetate), and 2R,4'R,8'R-gamma-(3,4-2H)tocopherol (d2-RRR-gamma-tocopherol). The tocopherol contents of plasma, red cells, and lipoproteins were measured up to 76 h after the dose. In normal subjects all three tocopherols were absorbed and secreted in chylomicrons with equal efficiencies. Both d2-gamma- and d3-SRR-alpha-tocopherols peaked at similar concentrations in the other lipoprotein fractions, then decreased similarly, but 2-4 times more rapidly than did d6-RRR-alpha-tocopherol. A lipoprotein lipase-deficient patient and a patient with prolonged production of chylomicrons with absent apolipoprotein B-100 also demonstrated the lack of discrimination between tocopherols during absorption. Despite abnormal apolipoprotein B-100 production in two patients, the "VLDL" was preferentially enriched in d6-RRR-alpha-tocopherol. Our results show that there is no discrimination between the three tocopherols during absorption and secretion in chylomicrons, but subsequently there is a preferential enrichment of very low density lipoprotein (VLDL) with RRR-alpha-tocopherol. Catabolism of this VLDL results in the maintenance of plasma RRR-alpha-tocopherol concentrations. 相似文献