巻号一覧
85 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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Retsu MIURA, Toshihiro SUGIYAMA, Toshio YAMANO1979 年 85 巻 5 号 p. 1107-1113
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーAdrenodoxin of bovine adrenocortical mitochondria was spin-labeled with two different spin-labeling reagents, N-(2, 2, 5, 5-tetramethyl-3-carbonylpyrroline-l-oxyl) imidazole (I) and N-(1-oxyl-2, 2, 6, 6-tetramethyl-4-piperidinyl) maleimide (II), without major loss of its activity for electron transport from NADPH to cytochrome c. The EPR spectrum of adrenodoxin spin-labeled with either of the reagents showed a pattern typical of a moderately immobilized spin label. When adrenodoxin was treated with (I), approximately two amino acid residues per molecule were spin-labeled, whereas a single residue was labeled by (II). While addition of NADPH to adrenodoxin spin-labeled with (I) did not diminish the EPR signal intensity, addition of the reductant to the labeled adrenodoxin in the presence of adrenodoxin reductase caused slow reduction of the spin label, the rate of which was dependent on the aerobicity. Addition of adrenodoxin reductase to adrenodoxin spin-labeled with (I) or (II) resulted in the appearance of a more immobilized component in the EPR spectrum. The ratio of the more immobilized component to the less immobilized component was saturated at a molar ratio of one to one. Addition of cytochrome P-450scc to adrenodoxin labeled with (I) had similar effects on the EPR spectrum.抄録全体を表示PDF形式でダウンロード (522K) -
A Chemical Modification StudyKuniyo INOUYE, Ben'ichiro TONOMURA, Keitaro HIROMI1979 年 85 巻 5 号 p. 1115-1126
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーAn ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7→>11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3. 4. 21. 14] at alkaline pH, ionic strength 0.1M, at 25°C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction.
Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitro-methane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' at alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule.
Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.抄録全体を表示PDF形式でダウンロード (977K) -
Kuniyo INOUYE, Ben'ichiro TONOMURA, Keitaro HIROMI, Kunio FUJIWARA, Da ...1979 年 85 巻 5 号 p. 1127-1134
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーThe interaction between Streptomyces subtilisin inhibitor (SSI) and thiolsubtilisin BPN', in which the catalytic residue Ser 221 of subtilisin BPN' [EC 3. 4. 21. 14] is converted chemically to Cys 221, was examined and compared with the interaction between SSI and native subtilisin BPN'.
SSI competitively inhibits the esterolytic activity of thiolsubtilisin BPN' toward p-nitro-phenyl acetate with an inhibitor constant, K1, of 13μM at pH 7.0, 25°C. An ultraviolet absorption difference spectrum having troughs around 245nm and 295nm was observed upon the binding of SSI and thiolsubtilisin BPN' at alkaline pH (pH 9.0-10.4), 25°C. This difference spectrum is similar in shape to that observed on the binding between SSI and subtilisin BPN', indicating the protonation of a tyrosyl residue upon the binding (Inouye, K., Tonomura, B., & Hiromi, K. (1975) abstracts of papers, 26th Forum on Protein Structure, Nagasaki pp. 117-120). Spectrophotometric titration of the difference spectra yielded a value for the dissociation constant of the SSI-thiolsubtilisin BPN' complex, Kd, of 40μM at pH 9.2-10.2, 25°C. These K1 and Kd values are more than 105 times greater than those for the interaction between SSI and subtilisin BPN' (Inouye, K., Tonomura, B., Hiromi, K., Sato, S., & Murao, S. (1977) J. Biochem. 82, 961-967; Fujiwara, K., Inouye, K., Tonomura, B., Murao, S., & Tsuru, D. (1977) J. Biochem. 82, 125-130; Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. 84, 1195-1202). This shows that a small change induced in the active site structure of subtilisin BPN' leads to a considerable decrease in its binding affinity (by about 30kJ/mol (7kcal/mol) in free energy units) to SSI.
The pH-dependence of the difference spectra observed on the binding of SSI and thiolsubtilisin BPN' at alkaline pH shows that a tyrosyl residue of the enzyme, which has been assigned to be either Tyr 104 or Tyr 217 (Inouye, K., Tonomura, B., & Hiromi, K. (1976) Seikagaku (in Japanese) 48, 542; Inouye, K., Tonomura, B., & Hiromi, K. (1979) J. Biochem. 85, 1115-1126), shifts its pKa value from 9.7 to 10.2-10.3 on complex formation with SSI. The extent of this upward pKa-shift (ΔpKa=0.5-0.6) is considerably smaller than that observed on the interaction of SSI and subtilisin BPN' (pKa 9.7→>11.5; ΔpKa>1.8) (Inouye, K., Tonomura, B., & Hiromi, K. (1975) abstracts of papers, 26th Forum on Protein Structure, Nagasaki pp. 117-120). The large difference in ΔpKa suggests that a change in the van der Waals radius as small as 0.45 A due to the conversion of -OH to -SH leads to a larger increase in the distance between the tyrosyl residue of the enzyme and the carboxyl group of SSI.抄録全体を表示PDF形式でダウンロード (652K) -
Seiya CHIBA, Keitaro HIROMI, Noshi MINAMIURA, Masatake OHNISHI, Tokuji ...1979 年 85 巻 5 号 p. 1135-1141
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーAnomeric forms of glucose produced from phenyl α-maltoside, maltose, or phenyl α-glucoside have been determined quantitatively by simultaneous measurements of optical rotation and reducing power, for eight kinds of glucose-producing 1, 4-α-glucosyl hydrolases, including glucose-forming amylase from human urine, and α-glucosidases from pig serum, honey bee, buckwheat seed, rice seed, sugar beet seed, flint corn seed, and brewer's yeast. All the eight enzymes studied were found to produce α-glucose exclusively.抄録全体を表示PDF形式でダウンロード (495K) -
Norio MUTO, Satoru TANI1979 年 85 巻 5 号 p. 1143-1149
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーTwo pepsinogens, the contents of which increase with developmental progress, were purified from the gastric mucosa of the adult rat by ammonium sulfate fractionation and chromatography on DEAE-cellulose and DEAE-Sepharose CL-6 B columns. The purified zymogens, designated as pepsinogens I and II, were each shown to be homogeneous by polyacrylamide gel disc electrophoresis. Pepsinogen II had a greater electrophoretic mobility toward the anode at pH 8.0 than pepsinogen I. The molecular weights of both zymogens were estimated to be 38, 000 by SDS-polyacrylamide gel electrophoresis. The activated enzymes, pepsins I and II, each had the same molecular weight of 32, 000. The pH optima for both enzymes were found to be 2.0. The enzymes showed high stabilities at pH 8.0, while they lost their activities within 60min at pH 10.0. The enzymes were inhibited by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN). The activities of the enzymes in hydrolyzing N-acetyl-L-phenylalanyl-3, 5-diiodo-L-tyrosine (APDT) were about 1/8 of that of porcine pepsin. These results suggest that pepsins I and II are very similar.抄録全体を表示PDF形式でダウンロード (1592K) -
Masako INUI(nee OUCHI), Sadahiko ISHIBASHI1979 年 85 巻 5 号 p. 1151-1156
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーThe function of mitochondria-bound hexokinase, the enzymatic form peculiar to the brain, in utilization of ATP generated inside the organelles, was examined by incubating rat brain mitochondria) fraction with [14C] glucose under various conditions. Addition of succinate and ADP to the incubation medium increased glucose 6-phosphate formation by the mitochondrial hexokinase and caused a smaller increase in ATP concentration in the mitochondria. The glucose phosphorylation was markedly inhibited by the addition of dinitrophenol, potassium cyanide, and oligomycin, and the ATP concentration was decreased. On the other hand, addition of atractyloside suppressed the glucose phosphorylation without affecting the mitochondrial hexokinase activity, whereas addition of antiserum against the mitochondrial hexokinase inhibited both glucose 6-phosphate formation and hexokinase activity. A part of both the glucose phosphorylation and hexokinase activities, however, remained even in the presence of the maximum dose of the anti-hexokinase serum and atractyloside. These results indicate the active utilization of intrinsically generated ATP by the mitochondria-bound hexokinase, a part of which may be located away from the surface of the mitochondrial membrane.抄録全体を表示PDF形式でダウンロード (512K) -
Eisuke FURUYA, Yukuo YOSHIDA, Kunio TAGAWA1979 年 85 巻 5 号 p. 1157-1163
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーSeveral kinds of hydrophilic proteins were examined to determine their interaction with artificial liposomes. Mitochondrial aspartate aminotransferase (m-GOT) [EC 2. 6. 1. 1], as well as cytochrome c, was found to interact strongly with negatively charged liposomes. In each case, an appreciable amount of the protein bound to liposomes remained unreleased after raising the salt concentration in the medium. The m-GOT tightly bound to the liposomes was also found to become latent in its enzymatic activity, and could be reversibly activated by solubilization of the liposomes with detergent. This is also the case for cytochrome c, which ceases to be reducible by external reductant, such as dithionite. Furthermore, the tightly bound m-GOT was not susceptible to the proteolytic action of trypsin, or that of Nagarse. From these observations it can be inferred that these basic proteins interact with acidic liposomes not only electrostatically but also hydrophobically. This kind of hydrophobic interaction was not observed in the combination of positively charged liposomes and acidic proteins, including s-GOT. Mitochondrial GOT was shown to be bound to isolated intact mito-chondria, but the bound enzyme was fully active, in contrast to the case of acidic liposomes. The hydrophobic interaction of water-soluble protein with liposomes is discussed in connection with the penetration of matrix enzyme through mitochondrial membranes.抄録全体を表示PDF形式でダウンロード (588K) -
Purification and Some PropertiesMakoto ANDO, Tadashi YOSHIMOTO, Susumu OGUSHI, Kaoru RIKITAKE, Shuji S ...1979 年 85 巻 5 号 p. 1165-1172
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーFormaldehyde dehydrogenase was isolated and purified in an overall yield of 12% from cellfree extract of Pseudonsonas putida C-83 by chromatographies on columns of DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite. The purified enzyme was homogeneous as judged by disc gel electrophoresis and was most active at pH 7.8 using formaldehyde as a substrate. The enzyme was also active toward acetaldehyde, propionaldehyde, glyoxal, and pyruvaldehyde, though the reaction rates were low. The enzyme was NAD+-linked but did not require the external addition of glutathione, in contrast with the usual formaldehyde dehydrogenase from liver mitochondria, baker's yeast, and some bacteria. The enzyme was markedly inhibited by Ni2+, Pd3+, Hg2+, p-chloromercuribenzoate, and phenylmethanesulfonyl fluoride. The molecular weight of the enzyme was estimated to be 150, 000 by the gel filtration method, and analysis by SDS-polyacrylamide gel electrophoresis indicated that the enzyme was composed of two subunit monomers. Kinetic analysis gave Km values of 67μm for formaldehyde and 56μM for NAD+, and suggested that the reaction proceeds by a “Ping-pong” mechanism. The enzyme catalyzed the oxidation of formaldehyde accompanied by the stoichiometric reduction of NAD+, but no reverse reaction was observed.抄録全体を表示PDF形式でダウンロード (1167K) -
Purification and Structure of Cytochrome-Binding FormKazunobu MATSUSHITA, Emiko SHINAGAWA, Osao ADACHI, Minoru AMEYAMA1979 年 85 巻 5 号 p. 1173-1181
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーA membrane-bound D-gluconate dehydrogenase [EC 1. 1. 99. 3] was solubilized from membranes of Pseudomonas aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a molecular weight of 138, 000 on polyacrylamide gel electrophoresis or 124, 000-131, 000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a molecular weight of 240, 000-260, 000 on sucrose density gradient centrifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100.
The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone.
In the presence of sodium dodecyl sulfate, the enzyme was dissociated into three components with molecular weights of 66, 000, 50, 000, and 22, 000. The components of 66, 000 and 50, 000 daltons corresponded to a flavoprotein and cytochrome cl, respectively, but that of 22, 000 dalton remained unclear as to its function.抄録全体を表示PDF形式でダウンロード (1210K) -
Miho FUKUYAMA, Yoshihiro MIYAKE1979 年 85 巻 5 号 p. 1183-1193
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーCholesterol oxidase [EC 1. 1. 3. 6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethyl-amido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53, 000 by SDS-gel electrophoresis and 46, 000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53, 000. The enzyme consumed 60μmol of O2/min per mg of protein with 1.3mM cholesterol at 37°C. The enzyme showed the highest activity with cholesterol; 3β-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33mM and the optimal pH was 5.0.
The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353nm and 455nm in 0.1M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid nor heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa, value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N(1)-histidyl FAD.抄録全体を表示PDF形式でダウンロード (793K) -
Yasuyuki SUZUKI, Yoshiji OKUMURA, Hyogo SINOHARA1979 年 85 巻 5 号 p. 1195-1203
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーTestosterone-binding globulin (TeBG) of canine serum was purified to apparent homogeneity by affinity chromatography on testosterone-17α-ethynylcarboxyaminoethyl-Sepharose 4 B followed by hydroxyapatite column chromatography. Canine TeBG was a glycoprotein containing 5.5% carbohydrates. Equilibrium sedimentation analysis in the presence and absence of 6M guanidine hydrochloride gave molecular weights of 40, 000 and 76, 000, respectively, suggesting that native TeBG consists of two subunits. Equilibrium dissociation constants at 0°C for testosterone and dihydrotestosterone were estimated to be 5.58×10-8M and 1.43×10-8M, respectively, and the number of binding site per native molecule was approximately unity for both androgens. Canine TeBG had virtually no affinity for estradiol, progesterone, or cortisol. Canine TeBG did not cross-react with a rabbit antiserum raised against bovine TeBG.抄録全体を表示PDF形式でダウンロード (1741K) -
IV. Sedimentation Coefficient, Diffusion Coefficient, Partial Specific Volume, and Particle Weight of the PhageYoshiyuki SAKAKI, Tadakazu MAEDA, Tairo OSHIMA1979 年 85 巻 5 号 p. 1205-1211
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーThe particle weight (molecular weight) of φNS11 was determined from the sedimentation coefficient, diffusion coefficient, and partial specific volume of the phage. The sedimentation coefficient of the phage (s020, W) is 416±2.7S. The diffusion coefficient (D020, W), which was determined by quasielastic light scattering measurement, is (0.57±0.03)×10-7cm2/s. The partial specific volume was determined by the mechanical oscillation technique to be 0.747±0.007cm3/g. Based on these values, the particle weight of the phage was calculated to be (70.3±4.3)×106 daltons, which agrees well with the particle weight (69-72×106 daltons) estimated from the molecular weight of phage DNA and the content of DNA. The Stokes radius of the phage particle was calculated to be 37.7±2nm and hydration of the phage was estimated to be 1.18cm3/g of dry phage. From the particle weight and the chemical composition of the phage, we estimated that one phage particle contains one doublestranded DNA molecule, 16, 000 residues of fatty acid, 72 protein I molecules, 920 protein II, 42 protein III, 48 protein IV, 290 protein V molecules, and 3, 700 molecules of polyamines.抄録全体を表示PDF形式でダウンロード (488K) -
II. Effect of cheB and cheZ Mutations on the Methylation of Methyl-Accepting Chemotaxis Protein of Escherichia coliHiroshi HAYASHI, Osamu KOIWAI, Masao KOZUKA1979 年 85 巻 5 号 p. 1213-1223
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーRadioactive proteins from chemotactic mutants of Escherichia coli with continuous tumbling phenotype (cheB and cheZ) and their otherwise isogenic parent were compared by two-dimen-sional gel electrophoresis. The system was capable of separating non-methylated methyl-accepting chemotaxis protein (MCP) from its methylated equivalent. The analysis of proteins from the envelope fraction of the bacteria showed that the cheB mutants contained a larger portion of methylated MCP than did the parent. However, the change of MCP methylation level was small, if any, in cheZ strains. The results suggest that the product of cheB gene and the product of cheZ gene are not functionally complementary. The product of cheB gene functions in controlling the level of methylation at the stationary state of the organisms.
In addition to known MCP species, a new MCP of about 43, 000 daltons was found. This MCP appears to be involved in transducing signals of some sugars.抄録全体を表示PDF形式でダウンロード (3452K) -
Masato OKADA, Seiichi KAWASHIMA, Kazutomo IMAHORI1979 年 85 巻 5 号 p. 1225-1233
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーPutrescine oxidase [EC 1. 4. 3. 4], putrescine: oxygen oxidoreductase (deaminating) (flavincontaining), from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens were adsorbed on amine-Sepharose 4B in which one of the terminal amino groups of diamine or triamine was covalently bound to Sepharose 4 B leaving the other terminal amino group(s) free. The affinities of these enzymes for the amine-Sepharose 4B increased on increasing the chain length of the methylene groups in the immobilized amines and fell upon addition of the substrate. The affinity of putrescine oxidase modified with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC) was reduced in comparison with that of the native enzyme so far as 1, 12-diaminododecane-Sepharose 4 B was concerned. From these results, it can be concluded that the interactions between the enzyme and the amine-Sepharose result from specific affinities mediated through the active sites of the enzymes. It is suggested that spermidine dehydrogenase as well as putrescine oxidase has as anionic point and a hydrophobic region in the active site.
On the basis of these results, the applicability of the enzyme affinities to purification procedures was examined. When partially purified enzymes were subjected to affinity chromatography, the following results were obtained. Putrescine oxidase gave a purification factor of 40-fold with about 100% recovery on a 1, 12-diaminododecane-Sepharose column. In the case of spermidine dehydrogenase, the purification factor and recovery on a 1, 8-diamino-octane-Sepharose column were about 1, 200-fold and 86%, respectively. By introducing affinity chromatography as a purification step, each enzyme could be purified more simply and with higher recovery.抄録全体を表示PDF形式でダウンロード (1441K) -
Masato OKADA, Seiichi KAWASHIMA, Kazutomo IMAHORI1979 年 85 巻 5 号 p. 1235-1243
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーThe substrate specificity of spermidine dehydrogenase from Serratia marcescens was studied, using many kinds of naturally occurring and synthetic polyamines. Diamines inhibited the enzyme competitively and their inhibitor constants tended to decrease with increasing methylene chain length in the diamines. All of the triamines and tetramines examined were active as substrates, and the amines containing a 4-aminobutylimino moiety (NH2(CH2)4NH-) in their structures were more active. N-Alkylputrescine was also oxidized by the enzyme. All of the amines containing a 4-aminobutylimino group were oxidized to form 1-pyrroline stoichiometrically as one of the products.
Tetramines containing a 3-aminopropylimino group (NH2(CH2)3NH-) were oxidized to form 1, 3-diaminopropane. However, in the case of an amine containing both 4-aminobutyl-imino and 3-aminopropylimino groups, the imino moiety of the former was preferentially oxidized by the enzyme.
On the basis of the substrate specificity, the binding characteristics of the enzyme are discussed and a subsite model for the binding site is proposed.抄録全体を表示PDF形式でダウンロード (707K) -
Hideto KUWAYAMA, Koichi YAGI1979 年 85 巻 5 号 p. 1245-1255
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーCa2+ bindings to pig cardiac myosin, subfragment-1 (S-1), and g2 light chain were investigated by the equilibrium dialysis method. Two different S-is, one of which can bind Ca2+ and another which cannot, were prepared. In order to calculate the free Ca2+ concentrations adequately, the amounts of Ca2+ included in various chemicals and proteins were measured by atomic absorption spectroscopy. Ca2+ contamination was greatest in KCl among the chemicals tested. In addition, the Ca2+ strongly bound to myosin and S-1 was released in the presence of Mg2+. When Mg2+ was not added, the Ca2+-binding constant of myosin was 4×105M-1 and the maximum binding number was 1.8 mol per mol of myosin. Cooperativity between the 2 Ca2+ bindings could not be demonstrated. Mg2+ strongly inhibited the Ca2+ binding: at a free Call concentration of 1×10-5M, 1.3 mol Ca2+ was bound to myosin in the absence of Mg2+, but 0.6 and 0.2 mol were bound in the presence of 0.3 and 4.5mM Mg2+, respectively. The Ca2+-binding constant of S-1, which contained a 15, 000 dalton component, was 8.6×105M-1, and the maximum binding number was 0.7 mol per mol of S-1. The 15, 000 dalton component could be exchanged with extraneous g2. S-1 which lacked the 15, 000 component could not bind Ca2+ at free Ca2+ concentrations less than 0.1 rum. The Ca2+ binding to free g2 light chain was about 100 times weaker than the binding to myosin, as indi-cated previously for skeletal myosin (Okamoto, Y. & Yagi, K. (1976) J. Biochem. 80, 111-120). The Ca2+-binding constant was obtained as 4.1×103M-1 in the absence of added Mg2+. Phosphorylation of g2 light chain did not affect the Ca2+ binding to the free g2 light chain or to myosin. Ca2+ binding to cardiac native tropomyosin was also measured.抄録全体を表示PDF形式でダウンロード (772K) -
I. Effect of Calcium-Dependent Regulator Protein on the Calcium Sensitivity of Microtubule AssemblyEisuke NISHIDA, Hiromichi KUMAGAI, Iwao OHTSUKI, Hikoichi SAKAI1979 年 85 巻 5 号 p. 1257-1266
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーWe examined the effect of porcine brain Ca2+-dependent regulator (CDR) protein on microtubule (MT) assembly from microtubular proteins isolated from porcine brain by temperature-dependent cycles of assembly-disassembly. CDR exhibited a potent inhibitory effect on MT assembly in the presence of Ca2+, whereas it had little or no effect on the extent of MT assembly in the absence of Ca2+ The increase in KCl concentration greatly potentiated the Ca2+-dependent inhibitory effect of CDR. The effect of CDR was reversible in a Ca2+ concentration-dependent manner, and the extent of inhibition by CDR at a fixed concentration of free Ca2+ was roughly proportional to the concentration of CDR. Moreover, the Ca2+ con-centration required for the half-maximal inhibition of MT assembly from a fixed concentration of purified microtubular proteins (PMP) decreased with increasing CDR concentration. On the basis of these results, together with data on the Ca2+-dependent association of CDR and tubulin (J. Biochem., accompanying paper), we propose the following model;
Ca2++CDR_??_Ca2+-CDR
Ca2+-CDR+tubulin_??_Ca2+-CDR-tubulin (nonpolymerizable)抄録全体を表示PDF形式でダウンロード (1302K) -
II. Association of Calcium-Dependent Regulator Protein with Tubulin DimerHiromichi KUMAGAI, Eisuke NISHIDA1979 年 85 巻 5 号 p. 1267-1274
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーThe Ca2+-dependent regulator protein (CDR) of cyclic nucleotide phosphodiesterase (PDE) was reported to be a Ca2+-dependent regulator of microtubule (MT) assembly in the preceding paper. In this paper, the binding of Ca2+-CDR complex to tubulin dimer was investigated in order to elucidate the Ca2+dependent inhibitory action of CDR on MT assembly. Purified microtubular proteins (PMPs) isolated from porcine brain did not affect the ability of CDR to activate Ca2+-activatable PDE, and did not include any inhibitory protein of Ca2+-activatable PDE. The binding of CDR to the tubulin dimer was observed on Sephadex G-200 gel filtration and ammonium sulfate fractionation in a Ca2+-dependent manner. CDR did not bind to microtubule associated proteins. We now assume that Ca2+ -dependent inhibition of MT assembly by CDR is due to the binding of CDR to tubulin dimer in a Ca2+-dependent manner.抄録全体を表示PDF形式でダウンロード (1112K) -
Masatoshi BEPPU, Tadao TERAO, Toshiaki OSAWA1979 年 85 巻 5 号 p. 1275-1287
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーDimeric succinyl-concanavalin A was cross-linked with ethylenediamine using I-ethyl-3-(3- dimethylaminopropyl)carbodiimide a condensing agent. Thus, a divalent dimer and a tetra- valent tetramer composed mostly of covalently cross-linked subunits bearing altered net charges were obtained.
Photoaffinity labeling of the cross-linked dimer with p-azidophenyl α-D-mannopyranoside resulted in a specific label for its saccharide-binding site and yielded a nonvalent dimer and a monovalent dimer (showing no subunit exchange). However, hemagglutination and glycogen precipitation data suggested that the labeled binding site is shielded but not destroyed by the label and can still bind weakly an external saccharide ligand possibly due to unsteadiness of the shielding label. Although nonvalent and monovalent derivatives were mitogenic as well as divalent and tetravalent derivatives for mouse splenic lymphocytes, binding and stimula- tion experiments indicated that their stimulating efficiencies after binding to the cells were far lower than those of the multivalent counterparts. Their activities were inhibited by methyl α-D-mannopyranoside, suggesting that the weak activities of nonvalent and monovalent derivatives were due to the labeled sites entirely and partly, respectively.
We suggest that the triggering of lymphocyte mitogenesis by concanavalin A may depend on cross-linkage of cell surface receptors.抄録全体を表示PDF形式でダウンロード (1542K) -
Yoshitaka HAMAGUCHI, Shinji YOSHITAKE, Eiji ISHIKAWA, Yuichi ENDO, Sac ...1979 年 85 巻 5 号 p. 1289-1300
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーThe procedures for the conjugation of rabbit IgG and Fab' antibodies with β-D-galactosidase from Escherichia coli using N, N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285-292, 1976; J. Immunol. 116, 1554-1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average numbers of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65-0.86) and 0.86 (0.80-0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4°C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab1'', and β-D-galactosidase in the conjugates were 23-29, 35-44, and 99Y., respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of P-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.抄録全体を表示PDF形式でダウンロード (915K) -
Yasuko SAWAI, Shigeharu UCHIDA, Junko SAITO, Nobuhiko SUGANO, Kinji TS ...1979 年 85 巻 5 号 p. 1301-1308
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーTwo forms of enzyme with ribonuclease H (RNase H) [EC 3. 1. 4. 34] activities, have been partially purified from cultured plant cells, strain GD-2, derived from carrot root. One is an Mn2+-dependent RNase H, and the second is an Mg2+-dependent RNase H. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at around 0.2M and 0.4M potassium chloride in phosphocellulose chromatography, and were further purified using blue Sepharose. Mg2+-dependent RNase H exhibits maximal activity at pH 9.0, and requires 10 to 15mM Mg2+ for maximal activity, whereas the Mn2+-dependent enzyme is most active at pH 8.0, is maximally active at an Mn2+ concentration of 0.4mM, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. The enzymes liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. The appar-ent molecular weight of the Mg2+-dependent RNase H was estimated to 18-20×104 and that of the Mn2+-dependent RNase H was estimated to be 14×104 by gel filtration.抄録全体を表示PDF形式でダウンロード (599K) -
Inhibition by Various Amino AcidsShigeo TAKADA, Takeyori SAHEKI, Yoshikazu IGARASHI, Tsunehiko KATSUNUM ...1979 年 85 巻 5 号 p. 1309-1314
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーInhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6. 3. 4. 5] are described.
1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates.
2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with K1 values of 1.3×10-4M, 2.5×10-4M, 6.7×10-4M, 6.3×10-4M, and 6.0×10-4M, respectively.
3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with K1 values of 9.5×10-4M and 1.2×10-3M, respectively.
4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively.
5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture.
6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.抄録全体を表示PDF形式でダウンロード (439K) -
Hideaki WATANABE, Kazuko OHGI, Masachika IRIE1979 年 85 巻 5 号 p. 1315-1320
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリー1. A base-nonspecific ribonuclease from Aspergillus saitoi [RNase Ms, EC 3. 1. 4. 23; molecular weight, 12, 500] was modified with phenylglyoxal (PG) and 1, 2-cyclohexanedione (CHD) in order to determine whether a single arginine residue was involved in the active site of the enzyme.
2. RNase Ms was inactivated by both PG and CHD with concomitant loss of one arginine residue. A competitive inhibitor of RNase Ms, 2', (3')-AMP, protected the enzyme from inactivation by PG. These findings strongly suggest that one arginine residue is involved in the active site of RNase Ms.
3. Difference CD spectra were measured at pH 5.5 for the binding of 2'-AMP and adenosine to native RNase Ms and the CHD- and PG-modified enzyme derivatives to determine the association constants. The arginine modification brought about a marked decrease in the binding affinity of 2'-AMP for the enzyme, but only a slight decrease for adenosine, suggesting that the arginine residue had interacted with the phosphate groups of the substrate.抄録全体を表示PDF形式でダウンロード (406K) -
Hiroyuki NAKAMURA, Takehiko WATANABE, Shiro KAKIUCHI, Hiroshi WADA1979 年 85 巻 5 号 p. 1321-1329
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーN6, O2'-Dibutyryl cyclic AMP and O2'-monobutyryl cyclic AMP were converted to N6-mono-butyryl cyclic AMP and cyclic AMP, respectively, in the supernatant fraction of rat liver. No further conversion of N6-monobutyryl cyclic AMP to cyclic AMP was observed. The enzyme responsible for these conversions (tentatively named O2'-esterase) was purified 1, 100-fold from rat liver supernatant by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Most of the nonspecific carboxylesterase activity was separated from this O2'-esterase at the DEAE-cellulose column step and only a distinct peak with 1/1, 000 of the total esterase activity eluted from the column hydrolyzed the O2'-butyryl ester bond of these cyclic AMP derivatives. This enzyme activity was found in a variety of rat tissues. The enzyme purified from liver, which was homogeneous upon poly-acrylamide gel electrophoresis, had the following properties: optimum pH, 7.0; Km for O2'-monobutyryl cyclic AMP, 7.7×10M; molecular weight, as determined by gel filtration, about 300, 000. This enzyme showed activity similar to that of nonspecific carboxylesterases with high affinity to butyryl esters such as α-naphthyl butyrate.
N6-Monobutyryl cyclic AMP, but not N6, O2'-dibutyryl cyclic AMP or O2'-monobutyryl cyclic AMP, bound to binding protein. The physiological actions of N6, O2'-dibutyryl cyclic AMP and O2'-monobutyryl cyclic AMP may be represented by the effects of their metabolites, N6-monobutyryl cyclic AMP and cyclic AMP, respectively. In this connection, the O2'-esterase plays a significant role in the biological actions of N6, O2'-dibutyryl cyclic AMP.抄録全体を表示PDF形式でダウンロード (1210K) -
III. Effect of Methyl Esters on the Chemotactic Response of Escherichia coliMasataka OHBA, Hiroshi HAYASHI1979 年 85 巻 5 号 p. 1331-1338
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーThe methylation-demethylation reaction of methyl-accepting chemotaxis protein (MCP) is tightly coupled to the appearance of the chemotactic response in Escherichia coli. The bacteria might therefore show a unique response upon the addition of a compound containing a methyl group. We selected methyl N-methyl anthranilate (NMMA) and its analogs for examination. When NMMA was added to a suspension of E. coli (wild type), the bacteria tumbled as it does in the presence of a repellent. NMMA caused tumbling of wild-type bacteria for at least 20min, while a conventional repellent makes the bacteria tumble for at most one min. The effect of NMMA requires functional MCP, cheA gene product, cheB gene product, and possibly cheX gene product. A positive signal of NMMA (i.e. sudden dilution) was detected by cheZ mutants with much higher sensitivity than that of a conventional repellent, indole, while both signals were rather poorly but equally detected by cheB mutants. These results suggest that the drug is related to the function of cheB gene product, a possible demethylating enzyme of MCP.抄録全体を表示PDF形式でダウンロード (544K) -
Toshihiko NAKANO, Atsushi IKAI, Masaaki NISHIGAI, Haruhiko NODA1979 年 85 巻 5 号 p. 1339-1346
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーRhodopsin from the bovine rod outer segment contains a covalently linked carbohydrate moiety (Heller, J. & Lawrence, M. A. (1970) Biochemistry 9, 864-868). We studied the location of this carbohydrate moiety on the disk membrane by using ferritin-conjugated concanavalin A and concanavalin A labelled with fluorescein isothiocyanate. Electron microscopic observation of sonicated disk membrane that was labelled with ferritin-concanavalin A revealed the electron-dense image of ferritin on the inner surface of the disk membrane and not on its outer surface. Intact disk membrane that was similarly treated with ferritin-concanavalin A showed a complete absence of ferritin molecules on its surface. In an independent series of experiments we confirmed that the sonicated disk membrane bound three to five times more fluorescein-labelled concanavalin A than the intact disk membrane did. From these experiments we conclude that the carbohydrate moiety of bovine rhodopsin is located on the inner surface of the disk membrane, in agreement with the report by Rohlich on the frog rod outer segment disk membrane (Rohlich, P. (1976) Nature 263, 789-791).抄録全体を表示PDF形式でダウンロード (2059K) -
Purification and PropertiesMichio TSUDA, Nobuhiko KATUNUMA, George WEBER1979 年 85 巻 5 号 p. 1347-1354
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーGlutamine 5-phosphoribosyl-l-pyrophosphate (PRPP) amidotransferase (amidophospho-ribosyltransferase), [EC 2. 4. 2. 14] was purified 1, 600-fold from rat liver. The preparation gave two protein bands on acrylamide gel electrophoresis, of which only the main band showed enzyme activity. The molecular weight of the enzyme was estimated to be 215, 000, 200, 000, and 195, 000 by Sephadex G-150 gel filtration, polyacrylamide gel electrophoresis, and sucrose density gradient ultracentrifugation, respectively. The apparent Km values for glutamine and PRPP were 1.24mM and 0.57mM, respectively. The concentration-activity curve for PRPP changed from a hyperbolic to a sigmoidal form on addition of AMP or GMP, and this inhibition by AMP was prevented by increasing the PRPP concentration. In the presence of high concentrations of inorganic phosphate, the catalytic activity was decreased and the sensitivity to AMP inhibition was slightly increased. The molecular size of liver amidotransferase was not changed by the addition of PRPP, AMP, or 2-mercaptoethanol. The purified rat liver enzyme has a broad pH-range of activity between 6.5 and 8.5.抄録全体を表示PDF形式でダウンロード (1080K) -
VI. Purification and Molecular Properties of NADP-linked Malic Enzyme from Escherichia coli WMasahiro IWAKURA, Jiro HATTORI, Yaeko ARITA, Masanobu TOKUSHIGE, Hiroh ...1979 年 85 巻 5 号 p. 1355-1365
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーNADP-linked malic enzyme [EC 1. 1. 1. 40] was highly purified from Escherichia coil W cells. The purified enzyme was homogeneous as judged by ultracentrifugation and gel electrophoresis. The apparent molecular weights obtained by sedimentation equilibrium analysis, from diffusion and sedimentation constants, and by disc electrophoresis at various gel concentrations were 471, 000, 438, 000, and 495, 000, respectively. The subunit molecular weights obtained by sedimentation equilibrium analysis in the presence of 6M guanidine hydrochloride and gel electrophoresis in the presence of sodium dodecyl sulfate were 76, 000 and 82, 000, respectively. The sedimentation coefficient (s020, W) was 13.8S, and the molecular activity was 44, 700min-1 at 30°C.
The amino acid composition of the enzyme was determined, and the results were compared with those of NAD-linked malic enzyme from the same organism and those of pigeon liver NADP-linked malic enzyme. The partial specific volume was calculated to be 0.738 ml/g.
The Km value for L-malate was 2.3mM at pH 7.4. Malonate, tartronate, glutarate, and DL-tartrate competitively inhibited the activity. The saturation profile for L-malate exhibited a marked cooperativity in the presence of both chloride ions and acetyl-CoA. However, acetyl-CoA alone did not show cooperativity or produce inhibition in the absence of chloride ions. Vmax and Km were determined as a function of pH. The optimum pH for the reaction was 7.8. Inspection of the Dixon plots suggested that three ionizable groups of the enzyme are essential for the enzyme activity. In addition to the oxidative decarboxylase activity, the enzyme preparation exhibited divalent metal ion-dependent oxaloacetate decarboxylase and α-keto acid reductase activities.
Based on the above results, the molecular properties of the enzymatic reaction are discussed抄録全体を表示PDF形式でダウンロード (1497K) -
Katsuyuki TANIZAWA, Kenji SODA1979 年 85 巻 5 号 p. 1367-1375
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーTwo types of kynureninase were isolated from Neurospora crassa IFO 6068. The formation of one of them, which was separated from the inducible kynureninase by DEAE-cellulose chromatography, was independent of the presence of tryptophan in the growth medium. Ouchterlony double-diffusion analysis and immunochemical titration indicated that the constitutive-type enzyme is immunologically different from the inducible enzyme. We confirmed by a selective assay method with antiserum that the addition of tryptophan to the medium does not affect the formation of one of the enzymes (constitutive-type). The constitutive kynureninase was purified approximately 650-fold and was free of the inducible enzyme as judged by analytical gel electrophoresis. The molecular weight and optimum pH values of both enzymes are very similar. However, the constitutive enzyme shows much higher activity and affinity for L-3-hydroxykynurenine than for L-kynurenine, suggesting that the enzyme functions biosynthetically as a 3-hydroxykynureninase. Constitutive kynureninase activities were widely found in all the fungi tested, whereas the inducible enzyme activity was not present in Mucor or Rhizopus species. The inducible enzymes of all the Neurospora strains examined were shown to be immunologically identical.抄録全体を表示PDF形式でダウンロード (1407K) -
Iwao OHTSUKI1979 年 85 巻 5 号 p. 1377-1378
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーAnti-troponin formed 25 to 29 striations with a period of 38nm along the whole length of thin filaments in chick embryonic breast muscle, in contrast with the uniform formation of 24 striations in adult muscle. This indicates that the thin filament in embryonic breast muscle is longer than that in the adult muscle.抄録全体を表示PDF形式でダウンロード (757K) -
Eisaku KATAYAMA1979 年 85 巻 5 号 p. 1379-1381
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーThe interaction of troponin-I (TN-I) and troponin-T (TN-T) was examined using immobilized TN-I as an affinity adsorbent. TN-T dissolved in 0.4M NaCl bound strongly to immobilized TN-I and required the application of 5M urea for its elution. A chymotryptic fragment of TN-T (Ohtsuki, 1978), perhaps the C-terminal fragment, retained the ability of the original TN-T to bind to immobilized TN-I.抄録全体を表示PDF形式でダウンロード (847K) -
Noboru OTOTANI, Narikazu SATO, Zensaku YOSIZAWA1979 年 85 巻 5 号 p. 1383-1385
発行日: 1979/05/01
公開日: 2008/11/18
ジャーナル フリーHigh performance liquid chromatography was performed by the ion pair method on unsaturated disaccharides produced from chondroitin sulfates by the action of chondroitinase. Completely separated peaks corresponding to ΔDi-OS, ΔDi-4S, and ΔDi-6S were obtained on a column of μ-bondapak-C18 with 0.035M tetra-butylammonium phosphate (pH 7.54) as a mobile phase. There was a linear relationship between the ratio of the peak area and the amount of each standard tested.抄録全体を表示PDF形式でダウンロード (226K)
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