巻号一覧
97 巻, 5 号
選択された号の論文の32件中1～32を表示しています
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Yasuki NONAKA, Shigeru FUJII, Toshio YAMANO1985 年 97 巻 5 号 p. 1263-1271
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe 31P NMR spectra of NADPH-adrenodoxin reductase and its complex with NADP+ are reported. The spectrum of adrenodoxin reductase showed two doublets arising from the phosphorus nuclei in the pyrophosphate group of FAD. Both doublets were shifted upfield to different extents in comparison with those of free FAD. Further, one of the doublets of phosphorus nuclei of the pyrophosphate group of bound NADP+ in the complex of adrenodoxin reductase and NADP+ was considerably shifted upfield in comparison with that of free NADP+. The spectrum of the complex of the reductase and NADP+ showed that the resonance of the 2'-phosphate group of NADP+ bound to the reductase was shifted downfield by 1.37 ppm compared with that of free NADP+ in the dianionic state. The 2'-phosphate resonance of bound NADP+ was independent of pH within the physiological range, whereas that of free NADP+ changed according to its ionization. The resonance of the 2'-phosphate group of NADP+ bound to the reductase also revealed that the ratio for the complex of NADP+ and the reductase was 1:1, and that this complex formation was inhibited by a high KCI concentration. These results were confirmed by electronic spectroscopic studies.抄録全体を表示PDF形式でダウンロード (741K) -
Hisashi OZASA, Shoko MIYAZAWA, Shuichi FURUTA, Takashi OSUMI, Takashi ...1985 年 97 巻 5 号 p. 1273-1278
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーRat hepatocytes were cultured for 72h with or without the addition of 0.5mM clofibric acid. The activities of individual enzymes of the peroxisomal β-oxidation pathway (acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydro-genase bifunctional protein, and 3-ketoacyl-CoA thiolase) decreased in the control culture, but markedly increased synchronously in the clofibric acid-treated culture. The levels of mRNAs coding for these enzymes and the rates of synthesis of the enzymes were also elevated in the clofibric acid-treated culture, although no proportional relationship was observed between the time-dependent changes of these parameters. The increase in mRNAs was much higher than the increase in the rate of synthesis of the enzymes. The activity of catalase, its mRNA level and the rate of its synthesis were slightly affected. The effects of clofibric acid on the peroxisomal β-oxidation enzymes and catalase in primary cultured hepatocytes were very similar to those observed in vivo. These results, therefore, suggest that primary culture of hepatocytes should provide a useful means for investigating the mechanism of induction of peroxisomal enzymes and the mechanism of action of peroxisome proliferators.抄録全体を表示PDF形式でダウンロード (1357K) -
Kenzo KOIKE, Tohru KOBAYASHI, Susumu ITO, Masahiro SAITOH1985 年 97 巻 5 号 p. 1279-1288
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーAn NADH oxidase was purified to homogeneity from Leuconostoc mesenteroides with a specific activity 100-fold higher than that of the crude extract.
1. The purified NADH oxidase was an acidic protein having an s020, w of 5.49 S and a molecular weight of 104, 000, consisting of a dimer with 53, 000 subunit size.
2. The enzyme could use O2, dichlorophenolindophenol and methylene blue as oxidants, but not H2O2, cytochrome c, or ferricyanide. The physiological substrate was β-NADH (Km=0.12mM) with O2 as the oxidant, probably forming H2O, rather than H2O2. Activity toward α-NADH was observed (Km=0.14mM), but the maximum velocity was 3 orders of magnitude lower than that with β-NADH. α-NADPH and β-NADPH were inert for the reaction.
3. The enzyme showed a flavoprotein absorption spectrum with maxima at 273, 379, and 450 nm with a shoulder at 465 nm: the absorption at 450-465 nm disappeared on adding excess NADH or hydrosulfite. One mol of the holoenzyme contained approximately 2 mol of FAD. The apoenzyme was obtained by treatment with EDTA-KBr solution and could be reconstituted partially by adding FAD, but not riboflavin or FMN.
4. The maximum activity of the reaction was observed at pH 6.5 in a temperature range of 35-45°C. The activation energy was estimated to be 3.77 kcal/mol.
5. The enzyme was inhibited by SH reagents, quinacrine, quinine, and Cue+, but not by EDTA. Adenine and its nucleoside 5'-di- and triphosphates showed competitive inhibitions, while various metabolites, such as H2O2, FDP, acetyl phosphate, lactate, ethanol, and acetate, did not affect the reaction.抄録全体を表示PDF形式でダウンロード (1613K) -
Haruo SUZUKI, Etsuko B. MUKOUYAMA1985 年 97 巻 5 号 p. 1289-1300
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe inhibition of globin synthesis in hemin-deficient rabbit reticulocyte lysates is due to the activation of a hemin-controlled translational inhibitor (HCI) that specifically phosphorylates eIF-2α.
High concentrations of cAMP (5-10mM) and GTP (1-2mM) stimulated the globin synthesis in heroin-deficient lysates when these compounds were added at the initial stage of incubation. The mechanism of the stimulation by cAMP and GTP was studied using hemin-deficient lysates, the N-ethylmaleimide(NEM)-treated HCI-supplemented lysates and a partially purified initiation factor, eIF-2. As the stimulation of globin synthesis by these compounds must be due to the prevention of the inhibition of globin synthesis, or due to the restoration of globin synthesis, or both, the preventive and restorative effects of these compounds were examined.
As for the preventive effect, it was observed that a) the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and b) cAMP and GTP inhibited the phosphorylation of eIF-2α in hemin-deficient lysates.
As for the restorative effect of cAMP and GTP, it was observed that c) these compounds restored the globin synthesis and the binding of [35S] Met-tRNAf to the 40 S ribosomal subunits, and promoted the dephosphorylation of eIF-2 (αP), d) the rates of the restored synthesis of globin were lower than the control, and e) cAMP promoted the release of [3H] GDP from the eIF-2 (αP)•[3H] GDP complex and the formation of eIF-2 (αP)•eIF-2B complex. Finding (d) indicates that steps involved in the restorative effect of these compounds may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates.
The data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. That is, cAMP inhibited the phosphorylation of eIF-2α and promoted both the release of GDP from eIF-2 and the formation of eIF-2 (αP)•eIF-2B complex, and GTP prevented both the activation of HCI and the phosphorylation of eIF-2α. Though cAMP and GTP affected multiple steps, it is suggested that cAMP stimulates the globin synthesis by inhibiting the phosphorylation of eIF-2α and that GTP stimulates the globin synthesis chiefly by preventing the activation of HCI in hemin-deficient lysates.抄録全体を表示PDF形式でダウンロード (2501K) -
Masato UMEDA, Shoshichi NOJIMA, Keizo INOUE1985 年 97 巻 5 号 p. 1301-1310
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーWe previously reported that liposomes containing glycophorin or gangliosides, both of which were isolated from human erythrocytes, are efficiently fused to erythrocyte membranes in the presence of HVJ (Umeda, M. et al., J. Biochein. 94, 1955-1966 (1983), and Virology 133, 172-182 (1984)). In the present work, the effect of lipid composition in glycophorin liposomes on their sensitivity to fusion with erythrocytes was studied.
Very little fusion occurred when glycophorin liposomes composed of dipalm itoylphosphatidylcholine-dicetylphosphate (9:1), dimyristoylphosphatidylcholine-dicetylphosphate (9:1), or egg yolk phosphatidylcholine-dicetylphosphate (9:1) were incubated with human erythrocytes in the presence of HVJ at 37°C. Addition of cholesterol into these liposomal membranes greatly enhanced the sensitivity of the liposomes to fusion. The presence of phosphatidic acid and phosphatidylethanolamine in liposomes also enhanced the sensitivity, whereas the presence of lysophosphatidylcholine had no significant effect on the ability of the liposomes to fuse. The fusion efficiency of liposomes was also enhanced by the presence of glucosylceramide. Change of lipid composition in liposomes had, however, no appreciable influence on the HVJ-mediated binding of liposomes to erythrocytes, suggesting that the interaction between HANA protein of HVJ and glycophorin in liposomes was not affected by the lipid composition of the liposomes.抄録全体を表示PDF形式でダウンロード (755K) -
Koei HAMANA, Shigeru MATSUZAKI1985 年 97 巻 5 号 p. 1311-1315
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe possible usefulness of polyamines as chemotaxonomic markers has been investigated in eukaryotic algae. Polyamines were analyzed in 12 species of primitive unicellular eukaryotic algae including some anomalous species. Norspermidine and norspermine in addition to putrescine and spermidine are widely distributed in most unicellular species of the algae. However, neither norspermidine nor norspermine was found in the taxonomically conflicting algae, Cyanophora and Glaucocystis, which contain cyanellae, or in a primitive red alga, Porphyridium. A thermoacidophilic eukaryotic alga, Cyanidium, is rich in both norspermidine and norspermine. Appreciable amounts of spermine and sym-homospermidine were detected only in the species belonging to the Rhodophyta (red algae).抄録全体を表示PDF形式でダウンロード (371K) -
Nobuo OKABE, Eiko TAKIMOTO1985 年 97 巻 5 号 p. 1317-1322
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe interaction between a thyroid hormone metabolite, 3-monoiodo-L-thyronine (3-T1) and bovine serum albumin (BSA) was investigated by using the CD method. An enhanced CD band was observed at the absorption wavelength region of 3-T1 around 293 nm suggesting the binding of 3-T1 to the BSA molecule. The ellipticity at 293 nm was measured at various molar ratios of 3-T1 to BSA, and the apparent binding constant and the maximum number of binding sites could be estimated as Kapp=8.85±1.07×104M-1 and n=23.8±0.9 respectively. The CD of a mixture of BSA, 3-T1 and thyroxine (T4) was also studied at various pH's. The pH profile of the two characteristic CD bands at 293 nm and 320 nm, attributed to bound 3-T1 and T4, suggested that the optimum binding condition of 3-T1 was attained at alkaline pH of around 9, while that of T4 was attained over a wide pH range between 5-10. A significant role of the ionized 4'-hydroxyl group of 3-T1 in the binding reaction with BSA is also suggested.抄録全体を表示PDF形式でダウンロード (337K) -
Kazuyo OHASHI, Makoto TSUNEOKA, Koscak MARUYAMA1985 年 97 巻 5 号 p. 1323-1328
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーFITC-labeled antibodies raised against chicken myofibrillar I-protein stained chicken myofibrils, which were fixed with formalin immediately after being cut from the sacrificed chicken breast muscle, at the junctional region of A-bands and I-bands. On the other hand, the antibodies stained the glycerinated myofibrils at the region around Z-bands. Aged glycerinated myofibrils stored in a cold room became stained with the same antibodies at the M-line and the A-band region except for the H-zone and the Z-band. I-Protein, which was originally localized at the A-I junctions, moved to the region around Z-bands and A-bands during the process of preparing myofibrils, paralleling the deterioration of myofibrils. Although I-protein is easily released from its original position, it is not a cytoplasmic protein of muscle but an intrinsic myofibrillar component, because immunoblotting tests showed that I-protein is contained in the myofibrillar fraction and not in the muscular cytoplasmic fraction.抄録全体を表示PDF形式でダウンロード (3371K) -
Masatoshi NAKANO, Bertram SACKTOR1985 年 97 巻 5 号 p. 1329-1335
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーFour forms of renal trehalase were isolated and purified to homogeneity. Hydrophobic interaction chromatography separated two forms; A-form and B-form. Both forms were subdivided further on Con A-Sepharose and were stained with periodic acid-Schiff reagent, indicating that they are glycoproteins.
The four forms of renal trehalase showed no significant difference in Km values for trehalose and K1 values for various inhibitors. The optimum pH of the four forms was pH 6.0 in phosphate buffer. Apparent molecular weights on gel filtration of the four forms were the same, 175, 000. Furthermore, the four forms showed the same antigenicity on double immunodiffusion. However, isoelectric point (pI), susceptibility to HgCl2, stability at - 80°C and Na+ activation behavior were different. Glycoprotein forms were more susceptible to HgCl2 and showed lower Na+ activation than nonglycoprotein forms. The pI of less hydrophobic forms (A1, A2) was more acidic than that of more hydrophobic forms (B1, B2). On the basis of these results, it is likely that four forms of renal trehalase are “isozymes.”抄録全体を表示PDF形式でダウンロード (1722K) -
Seiki KURAMITSU, Katsura INOUE, Kiyoshi KONDO, Kenji AKI1985 年 97 巻 5 号 p. 1337-1345
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーCytosolic and mitochondrial isozymes of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase [EC 2. 6. 1. 1]) were purified to homogeneity from rabbit liver. The rabbit liver isozymes were closely similar to the corresponding isozymes from other sources, including human heart, pig heart, chicken heart, and rat liver, in their molecular weights, absorption spectra, amino acid compositions, isoelectric points, and Michaelis constants for the substrates. The NH2-terminal amino acid sequences of rabbit liver isozymes were identified up to 30 residues, and showed some differences from those of the corresponding isozymes obtained from other animals so far studied.抄録全体を表示PDF形式でダウンロード (1306K) -
Nobukazu HASHIMOTO, Takashi MORITA, Sadaaki IWANAGA1985 年 97 巻 5 号 p. 1347-1355
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).抄録全体を表示PDF形式でダウンロード (3342K) -
Kazuo ITO, Masahiko KATAYAMA, Noshi MINAMIURA, Takehiko YAMAMOTO1985 年 97 巻 5 号 p. 1357-1362
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA mouse hybridoma cell line which produced an anti-human salivary α-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary α-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay.
The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary α-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic α-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with β-D-galacto-sidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary α-amylase and absolutely unreactive to pancreatic α-amylase.抄録全体を表示PDF形式でダウンロード (1237K) -
Masami HAYASHI, Mitsushi INOMATA, Megumi NAKAMURA, Kazutomo IMAHORI, S ...1985 年 97 巻 5 号 p. 1363-1370
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーTo determine the substrate recognition mechanism in calcium-activated neutral protease (CANP), the hydrolytic velocities for some possible substrates were compared. In general, succinylated polypeptides were poorer substrates than unmodified ones, suggesting that CANP interacts with positively charged amino groups and/or repels negatively charged succinyl groups in substrates. Among the substrates examined, protamine was degraded quite rapidly in a restricted manner. This degradation of protamine was remarkably accelerated by the addition of salt, and, in the absence of salt, protamine was inhibitory as to the degradation of vimentin by CANP. Protamine was separated into components and the sites cleaved by CANP were determined. CANP cleaved the clupeine YII and Z components at two sites, both being arginyl-arginine bonds, and the amino acid sequences around these sites were almost identical between YII and Z. No other arginyl-arginine bond was cleaved at all. These results showed that CANP prefers basic amino acid side chains but its specificity is very restricted.抄録全体を表示PDF形式でダウンロード (1039K) -
Takako HIROTA, Kenji HIROTA, Yoshikazu SANNO, Takehiko TANAKA1985 年 97 巻 5 号 p. 1371-1376
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーWhen rats were subjected to the stress of burns, tumors, or partial hepatectomy, a notable new peak of glucocorticoid binding protein appeared on DEAE-cellulose chromatography. This peak accompanied the original peak, which was the only dominant peak in intact rats. The appearance of the new binding protein was concomitant with a high rise in serum corticosterone levels. The new peak was eluted with 0.12-0.14M NaCl and another, small new peak with 0.02-0.03M NaCl, while the original peak of intact rats was eluted with 0.05-0.08M NaCl. In rats adrenalectomized prior to the stress, the new peaks did not appear. To mimic these stressful conditions which provoked a burst of endogenous glucocorticoid, rats were administered with an exogenous high dose of dexamethasone (100 μg/100g B. W.) in vivo. The new peak eluted with 0.12-0.14M NaCl was again observed and was more dominant in the hormone-treated rats than the stressed rats. These three peaks eluted with 0.02-0.03M, 0.05-0.08M, and 0.12-0.14M NaCl are called here Peak A, B, and C, respectively. This is the first demonstration of the effect of physiological changes in serum levels of glucocorticoid hormone on the nature of glucocorticoid binding protein by DEAE-cellulose chromatography.抄録全体を表示PDF形式でダウンロード (453K) -
Shuji TANAKA, Noriko MOHRI, Hiroshi KIHARA, Motonori OHNO1985 年 97 巻 5 号 p. 1377-1384
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーSelective separation of tryptophan-containing peptides has been attempted using 2-nitro-4-carboxyphenylsulfenyl chloride (NCps)-C1 as a reagent for hydrophobic modification of tryptophan. S-Carboxymethylated proteins were modified with NCps-C1 in 70% acetic acid and digested with TPCK-trypsin, and the digests were fractionated, directly or after partial fractionation on a Sephadex G-25 column, by high performance liquid chromatography using a reverse phase column. The tryptophan-containing peptides from the tryptic digests of S-carboxymethylated hen-egg white lysozyme and Trimeresurus flavoviridis phospholipase A2 were thus selectively separated and the amino acid sequences were determined, showing the validity of the method. The phenylthiohydantoin derivative of 2-(2'-nitro-4'-carboxyphenyl-thio)-L-tryptophan was synthesized and its spectroscopic and chromatographic properties determined, enabling us to identify the derivative on Edman sequencing.抄録全体を表示PDF形式でダウンロード (563K) -
Chikao NAKAYAMA, Mineo SANEYOSHI1985 年 97 巻 5 号 p. 1385-1389
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーIn order to determine the mode of action of cytostatic 9-β-D-xylofuranosyladenine (xylo-A), the inhibitory effects of 9-β-D-xylofuranosyladenine 5'-triphosphate (xylo-ATP) on DNA-dependent RNA polymerases I and II purified from cherry salmon (Oncorhynchus masou) liver nuclei were studied. This nucleotide showed strong inhibitory action on both RNA polymerases I and II. The Kl values are 14 μM for polymerase I and 5 μM for polymerase II (Km. values of ATP are 37 μm for polymerase I and 40 μM for polymerase II). The mode of xylo-ATP was competitive with respect to the incorporation of AMP into RNA and non-competitive to UTP and CTP.抄録全体を表示PDF形式でダウンロード (284K) -
Masanao TSUDA, Shigeru TAKETANI, Takaya SAWAMURA, Yasuko SHIOZAKI, Rik ...1985 年 97 巻 5 号 p. 1391-1399
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA single injection of D-galactosamine into rats caused acute liver cell injury, and the activity of ornithine transcarbamylase in the serum increased about 600-fold as compared with that in the normal serum. Some properties of the serum enzyme from galactosamine-treated rats have been studied together with those of the mitochondria) enzyme in liver. Both the enzyme activities gave similar pH profiles, showing an optimum of pH 8.5. Apparent Km values of the serum enzyme for ornithine under the standard conditions at pH 7.4 and pH 7.7 were 1.59mM and 0.94mM, respectively, and those of the mitochondria) enzyme were 1.69mM and 0.97mM, respectively. The Km, value of the serum enzyme for carbamyl phosphate was 0.34mM, which is similar to that of the mitochondrial enzyme. The mitochondria) enzyme was purified 78-fold to homogeneity with a 45% yield by ammonium sulfate fractionation, heat treatment, 2 nd ammonium sulfate fractionation, and phenyl-Sepharose CL-4B and CM-Sephadex C-50 column chromatographies. The specific activity of the purified enzyme was 282 μmol of citrulline formed per mg of protein per min at 37°C. The mitochondrial and serum enzymes have a molecular weight of 115, 000 as determined by Sephacryl S-300 gel filtration. Antibody specific for the mitochondrial enzyme was raised, and the immunological properties of the serum enzyme were examined. In immunoinhibition experiments, a decrease of the serum enzyme activity as well as the mitochondrial enzyme was observed on increasing the amount of the antibody. Analysis by sodium dodecyl sulfate-gel electrophoresis showed that the molecular weights of the serum and mitochondrial enzymes were both 39, 500. The serum enzyme activity after the injection of D-galactosamine varied in parallel with the enzyme amount. These results indicate that ornithine transcarbamylase in the serum is released as the intact form from the mitochondrial matrix during hepatic damage.抄録全体を表示PDF形式でダウンロード (2041K) -
Keiko TAKEDA, Junji MIKI, Hiroshi KANAZAWA, Tomofusa TSUCHIYA, Masamit ...1985 年 97 巻 5 号 p. 1401-1407
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe F1-ATPase from the uncD11 mutant of E. coli (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., & Futai, M. (1980) J. Biochem. 88, 695-703), showed different enzymological properties from the wild-type enzyme. The mutant F1-ATPase had biphasic kinetics and essentially the same Km values as the wild-type enzyme, although its Vmax values were lower. The mutant enzyme showed altered sensitivities to dicyclohexylcarbodiimide (DCCD), azide and quercetin; it was less sensitive than the wild-type to quercetin and DCCD, and its Mg2+-dependent ATPase activity was slightly more resistant to azide than that of the wild-type, whereas its Ca2+-dependent activity was more sensitive. On the other hand, the mutant and wild-type F1 were inhibited equally by 4-chloro-7-nitro-2, 1, 3-benzoxadiazole (NBD-Cl).
The fact that the Mg2+- and Ca2+-dependent F1-ATPase activities of the wild-type and mutant responded differently to quercetin and azide suggested that their mechanisms of action were different. Previous studies (Noumi, T., Mosher, M. E., Natori, S., Futai, M., & Kanazawa, H. (1984) J. Biol. Chem. 259, 10071-10075) indicated that Ser is replaced by Phe at residue 174 of the β subunit of the mutant. Thus the Ser residue or its neighboring area(s) may constitute the binding site of DCCD, quercetin and azide.抄録全体を表示PDF形式でダウンロード (538K) -
IV. Acylation of Enzyme-Bound L-2, 4-Diaminobutyric AcidSadaaki KOMURA, Kiyoshi KURAHASHI1985 年 97 巻 5 号 p. 1409-1417
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーAn enzyme fraction, which catalyzes the ATP-PP1 exchange reaction dependent on the three constituent amino acids of polymyxin E, was partially purified from crude extracts of Aerobacillus polyaerogenes. The approximate molecular weight was estimated to be 640, 000 by Sepharose 4 B gel filtration. Incubation of the enzyme with octanoyl coenzyme A and diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yielded octanoyldiaminobutyric acid thioesterified to the enzyme protein. On mild alkali treatment, octanoyldiaminobutyric acid, identified by paper chromatography, was released from the enzyme protein. From its acid hydrolyzate, diaminobutyric acid and octanoic acid were recovered in a molar ratio of 1 to 0.7. An ammonium sulfate fraction was required as the source of an acyltransferase for acylation of the enzyme-bound diaminobutyric acid. When [14C]-threonine was incubated with L-2, 4-diaminobutyric acid in the presence of octanoyl coenzyme A, octanoyldiaminobutyrylthreonine bound to the enzyme protein was formed. These results suggest that acyldiaminobutyric acid bound to the enzyme protein is a possible initiation complex in the biosynthesis of polymyxin E.抄録全体を表示PDF形式でダウンロード (1402K) -
Akira YAMANE, Tamio HIRABAYASHI1985 年 97 巻 5 号 p. 1419-1428
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA comparative study of vertebrate venticle tropomyosin has been carried out from the viewpoint of molecular evolution. The ventricles containing one-component tropomyosin were generally known, and in this paper those containing two components were also found in 8 species among mammals, reptiles, amphibia, and fish, but not among birds. The two components were concluded to be authentic tropomyosin and not artifacts since they showed lower electrophoretic mobilities in the presence of urea, and they were precipitated at pH 4.5 and bound to F-actin.
Studies on cysteine contents and cyanogen bromide cleavage peptide patterns revealed that the characteristics of the two tropomyosin components from pig, turtle, amphibia and carp ventricles varied increasingly in that order from typical α- and β-characteristics as seen in rabbit skeletal muscle tropomyosin.
The single component of chicken ventricle tropomyosin showed α component characteristics in its electrophoretic mobility and cysteine content, and β component characteristics in cyanogen bromide cleavage peptide pattern. The two components of carp ventricle tropomyosin seemed to be the most primitive, having two cysteine residues per molecule and a cyanogen bromide cleavage peptide pattern different from those of the two components of rabbit skeletal muscle.抄録全体を表示PDF形式でダウンロード (4529K) -
Tetsuo MIYAKE, Takanori OKA, Tsutomu NISHIZAWA, Fusakazu MISOKA, Toru ...1985 年 97 巻 5 号 p. 1429-1436
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーWe constructed a new vector containing the promoter and the signal sequence of E. coli phoA gene, the structural gene for the periplasmic alkaline phosphatase. One of the most useful characteristics of this vector is the unique HindIII restriction site located just at the end of the phoA signal sequence. This restriction site was generated by oligonucleotide-directed site-specific mutagenesis without changing the amino acid sequence of the signal peptide. Any kind of foreign structural gene can be easily inserted into the HindIII site by using synthetic oligonucleotides to construct a hybrid gene which has neither an extra sequence nor a deletion between the phoA signal sequence and the foreign structural gene. Human α-interferon gene was inserted into this HindIII site. When this hybrid gene was expressed under the control of the phoA promoter region, a low but significant activity was recovered in the cold water wash of the cells after an osmotic shock procedure.抄録全体を表示PDF形式でダウンロード (1060K) -
Masaharu EGUCHI, Ken KURIYAMA1985 年 97 巻 5 号 p. 1437-1445
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーMembrane-bound alkaline proteases from the midgut epithelia of the silkworm, Bombyx mori, were solubilized with 1% Lubrol-WX, at pH 11.2. They were purified by gel filtration on Sepharose 6 B and Ultrogel AcA-202 columns and a preparative polyacrylamide gel electrophoresis. Two proteases, caseinolytic (6 B 3-Tc) and benzoyl-arginine-p-nitroanilide-lytic (6 B 3-Tb) were obtained. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis. These enzymes showed high pH optima, 11.2, and pI values, above 11, and were extremely stable over a wide range of pH. The Km values for 6 B 3-Tb and Tc were 0.476mM and 2.5mg/ml respectively. Hammarsten casein and mulberry leaf protein were rapidly hydrolyzed by Tc, whereas the hydrolytic activity of Tb for Azocoll was higher than that of Tc. The protease Tb was strongly inhibited by diisopropylfluorophosphate, p-chloromercuribenzoate, benzamidine, leupeptin, and soybean trypsin inhibitor; Tc was inhibited by diisopropylfluorophosphate, tosyl phenylalanine chloromethylketone and chymostatin, but not by tosyl lysine chloromethylketone, p-chloromercuribenzoate, or iodoacetamide. The molecular weights of the proteases were estimated to be 12, 800 (Tb) and 13, 300 (Tc) by Sephacryl S-300 gel filtration. The amino acid analyses showed that both proteases contain a large number of acidic amino acids but a relatively small number of basic amino acids.抄録全体を表示PDF形式でダウンロード (1223K) -
Masaki TAKIGUCHI, Masataka MORI, Masamiti TATIBANA1985 年 97 巻 5 号 p. 1447-1459
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA procedure is described for the preparation of free and membrane-bound polysomes from rat liver. The procedure involves: (a) differential centrifugation of liver homogenate to separate free and membrane-bound polysomes; (b) treatment of the membrane-bound polysome fraction with a detergent to release bound polysomes from membranes; and (c) magnesium precipitation of both classes of polysomes. Free and bound polysomes prepared in this manner were essentially undegraded and highly active in cell-free protein synthesis. The recovery of polysomes was nearly quantitative and the distribution between the free and membrane-bound state was 41 and 59%, respectively. Polypeptides synthesized in vitro by the free and membrane-bound polysomes were quite different. The majority (81-84%) of mRNA activities of two secretory proteins (albumin and transferrin) were recovered in the membrane-bound polysomes, whereas the majority (81-85%) of mRNA activities of two cytosolic [aldolase B, EC 4. 1. 2. 13, and argininosuccinate synthetase, EC 6. 3. 4. 5], one mitochondrial [ornithine carbamoyltransferase, EC 2. 1. 3. 3] and one peroxisomal [catalase, EC 1. 11. 1. 6] proteins were recovered in the free polysomes. A polysome class synthesizing ornithine carbamoyltransferase was purified 42-fold from the free polysomes by immunoprecipitation. The procedure is rapid (4-5h) and reproducible, and provides a nearly quantitative means of separating the two classes of polysomes.抄録全体を表示PDF形式でダウンロード (3016K) -
Kazuyuki HIRANO, Yuichi IIIZUMI, Yukio MORI, Kazumi TOYOSHI, Mamoru SU ...1985 年 97 巻 5 号 p. 1461-1466
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーIn order to elucidate the physiological function of intestinal alkaline phosphatase, the characteristics of human intestinal alkaline phosphatase bound to brush border membrane vesicles were compared under optimal and physiological pHs. The Km value of this enzyme towards p-nitrophenylphosphate at the physiological pH was lower than that at the optimal pH. At the physiological pH, phosphate, arsenate and vanadate competitively inhibited the alkaline phosphatase activity, as they did at optimal pH, and the K1 values of these inhibitors at the physiological pH were also lower than those at the optimal pH. The effects of various inhibitors and antibody to human intestinal alkaline phosphatase on phosphate uptake into brush border membrane vesicles were investigated. The results indicated that phosphate uptake was affected by various inhibitors and the antibody to human intestinal alkaline phosphatase, but L-homoarginine, levamisole, and ouabain had no effect. From the above findings, it is strongly suggested that human intestinal alkaline phosphatase may function as a phosphate binding protein at low phosphate concentrations under physiological conditions.抄録全体を表示PDF形式でダウンロード (461K) -
Yukihiro FUSAUCHI, Koichi IWAI1985 年 97 巻 5 号 p. 1467-1476
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe ubiquitin-histone H2A conjugate, uH2A, of the protozoan Tetrahymena pyriformis was isolated by gel chromatography and octadecylsilyl-silica chromatography, from the fractions on the chromatographic purification of histone H2A [Fusauchi, Y. & Iwai, K. (1983) J. Biochem. 93, 1487-1497]. The uH2A showed an amino acid composition corresponding to the sum of an equimolar mixture of two protozoan H2A variants and protozoan free ubiquitin. N- and C-terminal sequencing of the uH2A, by Edman degradation and carboxypeptidase P digestion, showed a branched structure having two N-terminals, those of the H2A and ubiquitin components, and one C-terminal, that of each H2A variant component. Further structural analyses of the uH2A, by tryptic digestion of citraconylated uH2A and of a ubiquitinated BrCN fragment, showed that the ubiquitin C-terminal Gly-Gly is linked to the ε-amino group of either Lys-123, 125, or 126 in the H2A sequence, and that the ubiquitin sequence is similar to that of calf thymus but differs at least in the sequence of residues 12-27. The deduced structure was compared with the only known uH2A structure, that of calf thymus, with special reference to the branched site.抄録全体を表示PDF形式でダウンロード (1213K) -
Tadashi YOSHIMOTO, Daisuke TSURU1985 年 97 巻 5 号 p. 1477-1485
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーProline iminopeptidase [EC 3. 4. 11. 5] was purified about 2, 700-fold from cell-free extract of Bacillus coagulans by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose, and hydroxyapatite, and gel filtration on Sephadex G-150. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.3 with Pro-β-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X=amino acid including proline, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminus. Pro-D-amino acid bonds were also susceptible to the enzyme. The enzyme was completely inhibited by p-chloromercuribenzoate (PCMB) and partially by proline but not by metal chelators, diisopropylphosphorofluoridate (DFP), or phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by incubation with 2-mercaptoethanol. These results and the chromatographic profile on PCMB-T-Sepharose suggest that the enzyme is a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0, and the molecular weight of the enzyme was estimated to be 40, 000 by gel filtration on Sephadex G-100 and 35, 000 by sodium dodecyl sulfate (SDS) gel electrophoresis, indicating that the enzyme exists as a monomer.抄録全体を表示PDF形式でダウンロード (1128K) -
Shin NAKAMURA, Osamu TAKENAKA, Kenji TAKAHASHI1985 年 97 巻 5 号 p. 1487-1492
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーAmino acid sequences of fibrinopeptides A and B from the macaque, Macaca fuscata (Japanese monkey) and the guenon, Erythrocebus patas (patas monkey) were established. Fibrinopeptides A of the monkeys had a sequence identical with those of baboons:Ala-A_??_p-Thr-Gly-Glu-Gly-A_??_p-Phe-Leu-Ala-Glu-G_??_y-Gly-Gly-Val-A_??_g. Fibrinopeptides B were 9-residue, “short, ” peptides with the sequences Asn-Glu-Glu-S_??_r-Leu-Phe-S_??_r-Gly-Arg for M. fuscata and Asn-Glu-Glu-V_??_l-Leu-Phe-G_??_y-Gly-Arg for E. patas. The sequence of the B peptide of M. fuscata differed from that of a close-related species, M. mulatta (rhesus monkey), at a single site, L_??_u (M. f.)→P_??_o (M. m.). A single replacement between the B peptides of E. patas and Cercocebus aethiops (green monkey), V_??_l (E. p.)→G_??_y (C. a.), was detected. A phylogenic relationship of macaques, guenons, and baboons, named Cercopithecinae (Old World monkey), was deduced from the sequence data. A selective rather than random amino acid replacement was observed in the B peptides of these Old World monkeys, suggesting a restricted mutation of their fibrinopeptides during primate evolution.抄録全体を表示PDF形式でダウンロード (452K) -
Tetsuya SOMENO, Tetsushi SAINO, Kazuo KATOH, Hiroshi MIYAZAKI, Shin-ic ...1985 年 97 巻 5 号 p. 1493-1500
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーPyroglutamyl-lysyl-leucyl-argininal (Pyr-Lys-Leu-Argal) immobilized on gel matrix through the ε-amino group of its lysine residue was shown to be an efficient biospecific affinity adsorbent for purification of urokinase. Pyr-Lys-Leu-Argal dibutylacetal, a precursor of this immobilized ligand, was synthesized by a fragment condensation procedure, in which one of the thermolysin-digestion products of leupeptin dibutylacetal, H-Leu-Argal dibutylacetal, was used as a key intermediate. The precursor was coupled to CH-Sepharose 4 B with the aid of a water-soluble carbodiimide, and its acetal protecting group was then removed by mild acid treatment to free the essential aldehyde function. The Sepharose derivative thus prepared was shown to adsorb urokinase selectively and effectively from a crude human urine preparation at neutral pH and to release the bound enzyme under mild acidic conditions. The present technique afforded a highly purified urokinase preparation abundant in the high-molecular form with 90% recovery. The complex formed between urokinase and the immobilized ligand was found to have a dissociation constant of about 2×10-4M.抄録全体を表示PDF形式でダウンロード (609K) -
Noriko MATSUMOTO, Kazuhisa SEKIMIZU, Gen-Ichiro SOMA, Yoshihisa OHMURA ...1985 年 97 巻 5 号 p. 1501-1508
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーIn the previous paper, we described the identification of two abundant mRNAs of Sarcophaga peregrina (flesh-fly) which are selectively expressed in the fat body of middle third instar larvae. One of these mRNAs was found to encode a protein with a molecular mass of about 25, 000 (25-kDa protein) when translated in vitro (Tamura, H., et al. (1983) Dev. Biol. 99, 145-151).
Present paper reports the nucleotide sequence of a 2.3 kb DNA containing the entire gene for the 25-kDa protein. This gene consisted of four exons and contained an open reading frame for 184 amino acids. A CAT box and a TATA box were found in the 5'-flanking sequence. A poly A addition signal of AATAAA was assigned to the non-coding region in the fourth exon. A sequence having 75% homology with SV40 enhancer core sequence was identified in the non-coding region of the first exon.抄録全体を表示PDF形式でダウンロード (2216K) -
Shinji KAMIMURA, Masafumi YANO, Hiroshi SHIMIZU1985 年 97 巻 5 号 p. 1509-1515
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーUsing sea urchin (Hemicentrotus pulcherimus) sperm flagella, ATP hydrolysis coupled to sliding movement of microtubules was investigated. Flagellar axonemes were pretreated with trypsin and the microtubules induced to slide by addition of ATP (50-1, 000 μM) at 0-20°C. Motion-dependent hydrolysis of ATP was observed immediately after the addition of ATP, the rate of which was higher than that of steady state hydrolysis in axonemes without trypsin-treatment, or after complete disintegration. The rate of hydrolysis of ATP divided by the sliding velocity of microtubules reflects the ATP consumption necessary per unit distance of microtubule sliding. This parameter varied according to the experimental conditions in that it increased when the ATP concentration or temperature was decreased. Our results suggest that there is not a strict stoichiometric relationship between ATP hydrolysis and sliding distance in the dynein-tubulin system, indicating that the mechanochemical coupling is different from that in beating axonemes.抄録全体を表示PDF形式でダウンロード (578K) -
Koscak MARUYAMA, Kouichi MORIMOTO, Yoshiyuki KIJIMA, Kenji SOBUE, Shir ...1985 年 97 巻 5 号 p. 1517-1520
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーCaldesmon, calmodulin-, and actin-binding protein of chicken gizzard did not affect the process of polymerization of actin induced by 0.1M KCl. Caldesmon binds to F-actin, thus inhibiting the gelation action of actin binding protein (ABP; filamin). Low shear viscosity and flow birefringence measurements revealed that in a system of calmodulin, caldesmon, ABP, and F-actin, gelation occurs in the presence of micromolar Ca2+ concentrations, but not in the absence of Ca2+. Electron microscopic observations showed the Ca2+-dependent formation of actin bundles in this system. These results were interpreted by the flip-flop mechanism: in the presence of Ca2+, a calmodulin-caldesmon complex is released from actin filaments on which ABP exerts its gelating action. On the other hand, in the absence of Ca2+, caldesmon remains bound to actin filaments, thus preventing the action of ABP.抄録全体を表示PDF形式でダウンロード (1053K) -
Hiroshi YANAGAWA, Kiyotsugu KOJIMA1985 年 97 巻 5 号 p. 1521-1524
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーWe examined the possibility of chemical evolution in superheated hydrothermal environments and found the formation of microspheres at 250°C and above from a mixture of glycine, alanine, valine, and aspartic acid. The microspheres did not form at lower temperatures and consisted of silicates and peptide-like polymers that contained imide bonds and amino acid residues having an abundance of valine. The results show the possibility of thermophilic cellular structures, which might be adopted by the extremely thermophilic organisms, if they exist, reported by Baross and Deming.抄録全体を表示PDF形式でダウンロード (929K)
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