巻号一覧
86 巻, 5 号
選択された号の論文の52件中1～50を表示しています
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Hiroshi NAKAJIMA, Koichi SUZUKI, Kazutomo IMAHORI1979 年 86 巻 5 号 p. 1169-1177
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーFructose 1, 6-bisphosphate (FBP) stimulates the reaction of Bacillus stearothermophilus acetate kinase (AK). FBP changes the reaction curve for ATP from a sigmoidal type to a Michaelis-Menten one. The binding of FBP to AK was studied by an equilibrium dialysis method and by measuring changes in fluorescence. The extent of binding of FBP to the enzyme paralleled its activation. In addition, the binding constant for FBP increased in the presence of sub-strate, ATP. These results suggest that FBP is an allosteric activator of B. stearothermophilus AK.
Only two moles of FBP bound to this tetrameric enzyme. No cooperativity was found for the binding of FBP. These observations support the previous conclusion, that a set of two subunits in the tetramer is a unit of the enzymatic function.
A model is presented to interpret the sigmoidal kinetics for ATP, the absence of cooperativity for FBP binding, and the allosteric activation by FBP of this enzyme. The kinetic properties of the enzyme can be explained quantitatively by this model.抄録全体を表示PDF形式でダウンロード (611K) -
Kazuhiko NAGATA, Koichi SUZUKI, Kazutomo IMAHORI1979 年 86 巻 5 号 p. 1179-1189
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーA reactive ATP analog, N6-(6-bromoacetamidohexyl)-AMP•PCP, reactedspecifically with the ATP inhibitory site of rabbit skeletal muscle phosphofructokinase without affecting the active site. Modification resulted in the incorporation of 1.01 mol of the reagent per mol of enzyme subunit. The modified enzyme was insensitive to allosteric inhibition by ATP and to activa-tion by AMP at pH 7.2, where the native enzyme exhibits allosteric kinetic behavior. These observations demonstrate that we had succeeded in obtaining PFK fixed in the T state. Using the kinetic parameters of this modified enzyme, the kinetic properties of native enzyme can be quantitatively accounted for by the allosteric model of Monod-Wyman-Changeux. Fur-ther, the reagent was shown to have reacted with a specific cysteine residue near or at the ATP inhibitory site, and the sequence around the cysteine was determined as Cys-Lys-Asp-Phe-Arg.抄録全体を表示PDF形式でダウンロード (874K) -
Takashi SHIMAKATA, Yoko FUJITA, Takashi KUSAKA1979 年 86 巻 5 号 p. 1191-1198
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリー3-Hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35] was purified 100-fold to homogeneity from crude extracts of Mycobacterium smegmatis, using ammonium sulfate fractionation, gel fil-tration, and chromatography on DEAE-cellulose, hydroxyapatite, and NAD-Sepharose 4B columns. Its molecular weight was estimated to be 50, 300 by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. NADH acted twelve times more efficiently than NADPH as an electron donor for the reduction of 3-ketoacyl-CoA, and there was strict substrate stereo-specificity (L form) in the oxidation of 3-hydroxyacyl-CoA. The pH optimum depended upon the direction of reaction, i. e., 6.0 for the oxidation of NADH and 9-10 for the reduction of NAD. The Km values for different thioesters of acetoacetate, i. e., esters of CoA, pante-theine, and acetyl-cysteamine were determined to be 0.036, 1.19, and 44.4mm, respectively.
Antibodies raised against the dehydrogenase of M. smegmatis strongly inhibited the enzyme activity, but did not affect the corresponding dehydrogenase of pig heart. The anti-bodies were found to inhibit the acetyl-CoA dependent elongation of fatty acids by the crude extract of M. smegmatis. These findings, together with those on the reconstitution of the elongation activity reported previously (Shimakata, T., Fujita, Y., & Kusaka, T. (1977) J. Biochem. 82, 725-732) indicate that 3-hydroxyacyl-CoA dehydrogenase is involved in the acetyl-CoA dependent elongation of fatty acids in M. smegmatis.抄録全体を表示PDF形式でダウンロード (1202K) -
Katsuyuki TANIZAWA, Kenji SODA1979 年 86 巻 5 号 p. 1199-1209
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーSeveral kynurenine analogs have been prepared and examined for thei susceptibility to hydrolytic cleavage by bacterial kynureninase. In addition to L-kynurenine, 4-fluoro-and 5-fluoro-L-kynurenines were hydrolyzed rapidly. 3-Hydroxy-, 5-hydroxy-, 5-methyl-, and N'-formyl-L-kynurenines, and β-benzoyl-DL-alanine were hydrolyzed slowly, whereas D-kynurenine, S-benzyl-L-cysteine, and L-asparagine were not hydrolyzed. Kinetic parameters for these kynurenine analogs indicate that a substituent on the benzene ring of kynurenine does not greatly affect the affinity of the enzyme for the substrate but does markedly affect the rate of hydrolysis. γ-(ο-Aminophenyl)-L-homoserine was converted into L-alanine and ο-amino-benzaldehyde, suggesting that the σ-bond electrons between the β- and γ-carbon atoms of this kynurenine analog remain in the alanyl moiety during the enzyme reaction. Aromatic compounds such as ο-aminobenzaldehyde and ο-aminoacetophenone strongly inhibited the kynurenine hydrolysis. It was shown that kynurenic acid is not produced by kynureninase by the use of isotopically labeled substrate. A small amount of pyruvate was definitely formed in the kynureninase reaction. On the basis of these results, a reaction mechanism is proposed for the enzymatic kynurenine cleavage, involving hydrolysis of the α, γ-diketone intermediate to give anthranilic acid and the pyruvate-pyridoxamine 5'-phosphate Schiff base, which is further converted into the alanine-pyridoxal 5'-phosphate Schiff base, or directly hydrolyzed to give pyruvate and the pyridoxamine 5'-phosphate form of the enzyme.抄録全体を表示PDF形式でダウンロード (840K) -
Nozomu NISHI, Mikio KATAOKA, Gilbu SOE, Tomisaburo KAKUNO, Tatzuo UEKI ...1979 年 86 巻 5 号 p. 1211-1224
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリー1. The membrane of Rhodospirillum rubrum chromatophores was disintegrated with mild detergents (cholate and deoxycholate) in order to study the spatial arrangement of the functional proteins in the photochemical apparatus and the electron transport system in the membrane.
2. The components solubilized from the membrane by a mixture of cholate and deoxycholate (C-DOC) were separated into four fractions by molecular-sieve chromatography in the presence of C-DOC; they were designated as F1, F2, F3, and F4 in the order of elution. The fractions were further purified by repeated molecular-sieve chromatography in the presence of C-DOC until each fraction was chromatographically homogeneous.
3. F1 appeared to be conjugated forms of F2.
4. The purified F2 was composed of a rigid complex having a weight of 7 × 105 daltons, containing approximately 10 different kinds of protein species with molecular weights of 3.8 × 104, 3.6 × 104, 3.5 × 104, 2.8 × 104, 2.7 × 104, 2.6 × 104, 1.3 × 104, 1.2 × 104, 1.1 × 104, and 1.0 × 104. The complex contained 33 bacteriochlorophylls, 4 iron atoms, and 90 phosphates, but no cytochrome, ubiquinone, or phospholipid. It showed the same reaction center activity as chromatophores, indicating that the complex was a unit of the photochemical apparatus (photoreaction unit). Each chromatophore of average size was estimated to possess about 24 photoreaction units.
5. The purified F3 showed an absorbance spectrum characteristic of reaction centers, and contained 3.4 bacteriochlorophylls, 2.0 bacteriopheophytins, and 1.9 acid-labile iron atoms, but no cytochrome or ubiquinone (C-DOC reaction center). It had a weight of 1.2 × 105 daltons, and the main components were 4 protein species with molecular weights of 2.8 × 104, 2.7 × 104, 2.6 × 104, and 1.0 × 104.
6. The purified F4 showed a molecular weight of about 11, 000, and contained one mole of ubiquinone-10 per mole (ubiquinone-10 protein).
7. The reaction center activity of C-DOC reaction centers was stimulated by ubiquinone-10 protein. In addition, the reaction center oxidized reduced cytochrome c2 in the light, provided that ubiquinone-10 protein was present (photo-oxidase activity).抄録全体を表示PDF形式でダウンロード (847K) -
Hisao NAKASAKI, Taijiro MATSUSHIMA, Shigeaki SATO, Takashi KAWACHI1979 年 86 巻 5 号 p. 1225-1231
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーAlkaline phosphatase [orthophosphoric monoester phosphohydrolase, EC 3.1.3.1] was purified from the mucosa of rat small intestine by butanol extraction, ethanol fractionation, gel filtra-tion, with controlled-pore glass-10 and DEAE-cellulose column chromatography. On the gel filtration, the enzyme activity was separated into three peaks; A in the void volume, B and C at lower molecular weight positions. Enzyme A was purified to homogeneity. The activity of enzymes A, B, and C was detected even on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at the position of the protein of enzyme A, which had a molecular weight of 110, 000 daltons. Enzymatic properties such as pH optimum, Km value for the substrate, heat inactivation and inhibition by amino acids were the same in all three enzymes. Based on these findings, together with the elution positions on gel filtration, enzyme A was regarded as an aggregate, and enzymes B and C as dimer and monomer molecules, respectively.抄録全体を表示PDF形式でダウンロード (883K) -
Masataka OHBA, Osamu KOIWAI, Shozo TANADA, Hiroshi HAYASHI1979 年 86 巻 5 号 p. 1233-1238
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーA protein existing mainly in the supernatant fraction of Escherichia coli was found to be methylated by accepting the methyl moiety originating from methionine. The protein was identified as peptide synthesis elongation factor Tu (EF-Tu) by the following criteria. 1) The methylatable protein separated at the same position as purified EF-Tu on two-dimensional gel electrophoresis. 2) The methylatable protein interacted with antiserum specific for EF-Tu.
Amino acid analysis of the methyl-labeled protein suggested that the site of methylation was an ε-amino group of lysine.抄録全体を表示PDF形式でダウンロード (1884K) -
VII. Structural and Functional Characteristics of Sulfhydryl Groups in NADP-Linked Malic Enzyme from Escherichia coli WMasahiro IWAKURA, Masanobu TOKUSHIGE, Hirohiko KATSUKI1979 年 86 巻 5 号 p. 1239-1249
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーNADP-linked malic enzyme from Escherichia coli W contains 7 cysteinyl residues per enzyme subunit. The reactivity of sulfhydryl (SH) groups of the enzyme was examined using several SH reagents, including 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM).
1. Two SH groups in the native enzyme subunit reacted with DTNB (or NEM) with different reaction rates, accompanied by a complete loss of the enzyme activity. The second-order modification rate constant of the “fast SH group” with DTNB coincided with the second-order inactivation rate constant of the enzyme by the reagent, suggesting that modification of the “fast SH group” is responsible for the inactivation. When the enzyme was denatured in 4 m guanidine-HCl, all the SH groups reacted with the two reagents.
2. Although the inactivation rate constant was increased by the addition of Mg2+, an essential cofactor in the enzyme reaction, the modification rate constant of the “fast SH group” was unaffected. The relationship between the number of SH groups modified with DTNB or NEM and the residual enzyme activity in the absence of Mg2+ was linear, whereas that in the presence of Mg2+ was concave-upwards. These results suggest that the Mg2+-dependent increase in the inactivation rate constant is not the result of an increase in the rate constant of the “fast SH group” modification.
3. The absorption spectrum of the enzyme in the ultraviolet region was changed by addition of Mg2+. The dissociation constant of the Mg2+-enzyme complex obtained from the Mg2+-dependent increment of the difference absorption coincided with that obtained from the Mg2+-dependent enhancement of NEM inactivation.
4. Both the inactivation rate constant and the modification rate constant of the “fast SH group” were decreased by the addition of NADP+. The protective effect of NADP+ was increased by the addition of Mg2+.
Based on the above results, the effects of Mg2+ on the SH-group modification are discussed from the viewpoint of conformational alteration of the enzyme.抄録全体を表示PDF形式でダウンロード (757K) -
Affinity Labeling with Bromopyruvate1Isamu KAMESHITA, Masanobu TOKUSHIGE, Katsura IZUI, Hirohiko KATSUKI1979 年 86 巻 5 号 p. 1251-1257
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーPhosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli W was alkylated by incubation with bromopyruvate, a substrate analog, leading to irreversible inactivation. The reaction followed pseudo-first-order kinetics. Mg2+, an essential cofactor for catalysis, enhanced the inactivation, and the enhancing effect increased as the pH increased. The inactivation rate showed a tendency to saturate with increasing concentrations of bromo- pyruvate, indicating that an enzyme-bromopyruvate complex was formed prior to the alkyla- tion. DL-Phospholactate, a potent competitive inhibitor with respect to phosphoenolpyr-uvate, protected the enzyme from inactivation in a competitive manner. Examination of the acid hydrolysate of the enzyme modified with [14C]bromopyruvate by paper chromatography showed that radioactivity was solely incorporated into carboxyhydroxyethyl cysteine. In addition, determination of sulfhydryl groups of the native and modified enzymes with 5, 5'- dithiobis(2-nitrobenzoate) showed that inactivation occurred concomitant with the modification of one cysteinyl residue per subunit. These results indicate that bromopyruvate reacted with the enzyme as an active-site-directed reagent.抄録全体を表示PDF形式でダウンロード (516K) -
Tatsuo SENSHU, Mochihiko OHASHI1979 年 86 巻 5 号 p. 1259-1267
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe synthesis and association of histones with chromatin were studied using MH-134SC cells in suspension culture. Cultures containing approximately equal numbers of cells were pulse-labeled with [3H]lysine at various times after the interruption of DNA synthesis with hydroxyurea. Each culture was mixed with a fixed volume of a culture generally labeled with [14C]lysine at the time of harvesting. Acid-soluble proteins extracted from different subcellular fractions of cells labeled under various conditions were compared by electrophoresis on poly-acrylamide gels containing acetic acid and urea. All types of chromatin histones were labeled nearly equally as [14C] marker histones by a 15min pulse under normal conditions, except that a considerable portion of pulse-labeled H4 was in highly acetylated forms. Addition of hy-droxyurea at the start of the pulse markedly reduced the labeling of H3 and H4, but affected the labeling of the other histones only slightly. When DNA synthesis was inhibited before the start of the pulse, labeling of all histones decreased significantly. The addition of hy-droxyurea was found to cause transient accumulation of newly synthesized proteins in the cytoplasmic soluble fraction; these were characterized as H3 and H4 from their metabolic properties and their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The results suggest that association of newly synthesized H3 and H4 histones is closely coupled with ongoing DNA replication. The implications of the results for the mechanism of formation of new nucleosomes are discussed.抄録全体を表示PDF形式でダウンロード (621K) -
Motonori OHNO, Yuji KARASAKI1979 年 86 巻 5 号 p. 1269-1274
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーPresteady state and steady state analyses of the α-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of three specific ester substrates and three ring-substituted derivatives were carried out to elucidate the effect of hydrophobic interactions due to the different side chains of the substrates on the individual steps of the reaction. Hydrolysis of all the substrates except for Nα-acetyl-Nin-formyltryptophan methyl ester (Ac-Trp (CHO)-OMe) was controlled by the deacylation rate. In spite of their comparable Ks values, the substrates with small kcat, such as Nα-acetyltryptophan methyl ester and Nα-acety1-2-(2-nitro-4-carboxyphenylsulfeny1)-tryptophan methyl ester, characteristically gave Km values one order of magnitude smaller than the others. For the reaction of Ac-Trp (CHO)-OMe, it was ascertained that the deacylation step was not rate-controlling. It is suggested that the acylation step controls the rate in this case.抄録全体を表示PDF形式でダウンロード (382K) -
Toshimitsu AIBA, Issei OHTSUKA, Hirofumi ONISHI, Shizuo WATANABE1979 年 86 巻 5 号 p. 1275-1282
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーIkebe et al. ((1977) J. Biochem. 82, 299-302, and (1978) J. Biochem. 83, 1643-1655) proposed thatcalcium is required only for the activation of myosin light-chain kinase, and that phos-phorylation and dephosphorylation of a specific light-chain component of gizzard myosin are required for superprecipitation (increase in turbidity) and for its reversal (decrease in turbidity), respectively. The following results support their proposals:
1. Gizzard myosin was freed from myosin light-chain kinase by chromatography on a DEAE-Sephadex A-50 column or by washing with 10mM EDTA solution.
2. Phosphorylated myosin was chromatographed on a DEAE-Sephadex A-50 column, and combined with actin to obtain acto-phosphorylated myosin. The Mg-ATPase activity of the acto-phosphorylated myosin was always insensitive to calcium. On the other hand, the superprecipitation activity of acto-phosphorylated myosin in the absence of calcium (i. e., in the presence of EGTA) was lower than that in the presence of 0.1mM CaCl2 (showing an apparent calcium sensitivity). However, some results were obtained suggesting that a trace amount of heavy-metal contaminant in the buffer used may have been responsible for the apparent calcium sensitivity. For example, the superprecipitation activity in the presence of 0.2mM CaC12 plus 0.1mM EGTA was equal to that in the presence of 1mM EGTA.
3. With a combined system of acto-phosphorylated myosin and gizzard native tropomyosin, both the ATPase reaction and superprecipitation proceeded in a manner independent of calcium in the early stages of the reactions, but both reactions in the absence of calcium some-times slowed down in the later stages. Urea-gel electrophoresis revealed that myosin light-chain phosphatase, which is contained in native tropomyosin, caused dephosphorylation of phosphorylated myosin in the absence of calcium.抄録全体を表示PDF形式でダウンロード (1715K) -
Hirofumi ONISHI, Sadatoshi IIJIMA, Hirotaka ANZAI, Shizuo WATANABE1979 年 86 巻 5 号 p. 1283-1290
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーIsolation of myosin light-chain phosphatase (MLCP) from chicken gizzard muscle was at-temped, using three different methods of fractionation successively, in the following order: ammonium sulfate salting-out, DEAE-cellulose chromatography, and Sephadex G-200 gel filtration. Three different preparations of MLCP were obtained with increasing degrees of purity: the ammonium sulfate fraction, the chromatography fraction, and the gel filtration fraction. The following findings were obtained with these preparations.
1. The ATPase reaction catalyzed by skeletal acto-gizzard phosphorylated myosin proceeded at the same rate in the presence or absence of calcium ions, and it was inhibited by addition of the MLCP preparations.
2. Superprecipitation of acto-phosphorylated myosin was induced by adding ATP, and it was reversed by a subsequent addition of the MLCP preparations.
3. Urea-gel electrophoresis showed that inhibition of the ATPase reaction or reversal of the superprecipitation was always accompanied by the dephosphorylation of phosphorylated light chains.
4. The concentration of MLCP preparations required to produce the same extent (at a limited incubation time) of dephosphorylation of phosphorylated light chains decreased as the frac-tionation progressed: the ammonium sulfate fraction > the chromatography fraction > the gel filtration fraction. The same order was found for inducing the same rate of reversal of superprecipitation. It is thus strongly suggested that dephosphorylation of light chains by MLCP is essential for the relaxation of gizzard muscle.抄録全体を表示PDF形式でダウンロード (2038K) -
Nobuyuki YAMASAKI, Takahiro TSUJITA, Tatsuo ETO, Shinichi MASUDA, Kens ...1979 年 86 巻 5 号 p. 1291-1300
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーA novel method for the preparation of Kyn 62-lysozyme, in which tryptophan 62 is replaced by kynurenine, is reported. Hen egg-white lysozyme was ozonized in aqueous solution to yield one N'-formylkynurenine residue and deformylated with dilute hydrochloric acid in frozen solution at-10°C. Crude Kyn 62-lysozyme was purified by affinity and Bio Rex 70 chromatography successively. Kyn 62-lysozyme retains affinity for chitin and is essentially an active enzyme with a slightly weakened but distinct catalytic activity. After this modifi-cation, the enzyme activity was changed differently depending on the kind of substrate. At the individual optimum pH's, lytic activity was largely retained (80% active), but the catalytic efficiency for hydrolyzing glycol chitin was relatively low (30% active)
Lysis of M. lysodeikticus cell suspensions was optimally catalyzed by Kyn 62-lysozyme at pH 6.2 and at 0.088 ionic strength. These values are lower by 1.3 pH unit and 0.04 ionic strength, respectively, than those of intact lysozyme. The optimum pH and ionic strength for the hydrolysis of neutral substrates were scarcely affected. These results suggest the significance of electrostatic interaction in the lysis of lysozyme.
Relatively limited loss of activity induced by modification of the 62nd residue, which is thought to participate directly in the binding of the substrate at subsite C, is discussed on the basis of the similarity of side chain structure in tryptophan and kynurenine.抄録全体を表示PDF形式でダウンロード (762K) -
Hisao UEYAMA1979 年 86 巻 5 号 p. 1301-1311
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーPoly(A) polymerase [polyadenylate nucleotidyltransferase, EC 2.7.7.19] was extracted from Tetrahymena pyriformis The enzyme was demonstrated to be present in three forms by column chromatography on DEAE-cellulose, and they were termed poly(A) polymerase Ia, Ib, and H in order of increasing affinity to the column. The properties of enzymes Ia and Ib were similar except that Ia utilizes poly(A) as a primer rather efficiently. Enzyme II differed from enzymes Ia and Ib not only in elution profile on DEAE-cellulose column chromatography but also in pH and temperature preferences, molecular weight, requirement for divalent cations, sensitivity to salts at high ionic strength, optimal primer concentration, and subcellular localization. The molecular weights of enzymes Ia and Ib measured by gel filtration were both 43, 000, and that of enzyme II was 95, 000. All three enzymes required Mn2+ for maximal activity; Mg2+ could replace Mn2+ in the reaction of enzyme II, but only partially. In the presence of 0.1M ammonium sulfate the activities of enzymes Ia and lb were both completely inhibited, whereas enzyme II still showed 42% of its original activity. These findings suggest that there are two distinct types of poly(A) polymerase in Tetrahymena pyriformis.抄録全体を表示PDF形式でダウンロード (798K) -
Solubilization, Partial Purification, and PropertiesKazuhiro SOGAWA, Kenji TAKAHASHI1979 年 86 巻 5 号 p. 1313-1322
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーConditions for the solubilization of membrane-bound neutral proteinase associated with monkey liver microsomes were investigated. Among the reagents tested, deoxycholate, cholate, and some nonionic detergents, including Triton X-100, with hydrophilic-lipophilic balance values of around 13, were effective. The solubilization profile indicated that the enzyme is bound to the microsomal membranes by strong hydrophobic interaction.
The enzyme was partially purified from monkey liver microsomal fraction, previously washed with 1 M KC1 and 0.05% sodium dodecyl sulfate, by Triton X-100 extraction, followed by chromatography on columns of hydroxylapatite and Sepharose CL-6B. The apparent molecular weight of the enzyme was estimated to be about 88, 000 from the elution position on Sepharose CL-6B column chromatography in the presence of 0.5% sodium cholate. It was optimally active at pH 8.0 with heat-denatured casein as a substrate. It was strongly inhibited by diisopropyl phosphorofluoridate and phenylmethanesulfonyl fluoride, indicating that the enzyme is a serine proteinase. EDTA, EGTA, and chymostatin also inhibited the enzyme strongly. Among urea-denatured protein substrates tested, calf thymus histone was hydrolyzed most rapidly, followed by casein, hemoglobin, and bovine serum albumin, whereas practically no hydrolysis occurred with denatured ovalbumin, fibrinogen, and γ-globulin as substrates.抄録全体を表示PDF形式でダウンロード (716K) -
Kinzo NAGASAWA, Yoko INOUE, Tsugumi TOKUYASU1979 年 86 巻 5 号 p. 1323-1329
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーBy heating the pyridinium salts of chondroitin 4- and 6-sulfates in dimethyl sulfoxide con-taining 10% of water or methanol at 80°C for 1-5 h, several chondroitin preparations with sulfur contents of 0.02-1.05% were prepared in 83-96% yields, respectively. Chemical properties of the preparations, such as degrees of desulfation and of depolymerization, were compared with those of the products obtained by the previously described methods.抄録全体を表示PDF形式でダウンロード (1044K) -
Kinetic and Molecular PropertiesKeiko MURAKAMI1979 年 86 巻 5 号 p. 1331-1336
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe kinetic and molecular properties of AMP deaminase [AMP aminohydrolase, EC 3.5.4.6] purified from baker's yeast (Saccharornyces cerevisiae) were investigated. The enzyme was activated by ATP and dATP, but inhibited by P1 and GTP in an allosteric manner. Alkali metal ions and alkaline earth metal ions activated the enzyme to various extents. Kinetic negative cooperativity was observed in the binding of nucleoside triphosphates. Kinetic analysis showed that the number of interaction sites for AMP (substrate) and P1 (inhibitor) is two each per enzyme molecule.
The molecular weight of the native enzyme was estimated to be 360, 000 by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme gave a single polypeptide band with a molecular weight of 83, 000, suggesting that the native enzyme has a tetrameric structure.
Baker's yeast AMP deaminase was concluded to consist of two “protomer” units which each consist of two polypeptide chains with identical molecular weight.抄録全体を表示PDF形式でダウンロード (435K) -
Hirokazu KATAYAMA, Yasuo KITAGAWA, Etsuro SUGIMOTO1979 年 86 巻 5 号 p. 1337-1343
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe distribution of glycerate kinase [ATP: D-glycerate 2-phosphotransferase, EC 2.7.1.31] in kidney was studied. This enzyme was found to be present in the renal cortex. By differential centrifugation of the homogenate and sucrose density gradient analysis, it was found that 42% and 60% of the renal glycerate kinase were localized in the cytosol and mitochondria, respectively. The mitochondrial enzyme appeared to be present in the inner membrane and/or matrix. No difference was found between the solubilized-mitochondrial and cytosolic gly-cerate kinase as regards kinetic properties, thermal stability, electrochemical properties, and molecular size. Immunochemical identity of these enzymes was demonstrated using a rabbit antibody against mitochondrial glycerate kinase purified from rat liver. Although the hepatic enzyme was induced by dietary protein (Kitagawa, Y., Katayama, H., & Sugimoto, E. [1979] Biochim. Biophys. Acta 582, 260-275), the renal enzyme in mitochondria and cytosol was not affected by dietary protein. These results on renal glycerate kinase are compared with those for the hepatic enzyme, and the regulatory mechanism for intracellular distribution of the enzyme is discussed.抄録全体を表示PDF形式でダウンロード (505K) -
Tatsuzo FUJII, Akira TAMURA1979 年 86 巻 5 号 p. 1345-1352
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーChanges in the membrane morphology and phospholipid content of human erythrocytes were determined after incubation of intact cells with each of various exogeneous phospho-lipases (PLases). PLase A2 from Naja naja or bee venom induced crenation of the cells in parallel with hydrolysis of the membrane phosphatidylcholine (PC). This crenated cell shape was reversed to a biconcave disc or cup-like form by a further treatment with lysophospho-lipase. In contrast, bacterial PLase C from Clostridium perfringens and Pseudomonas aureofaciens or fungal PLase D from Streptomyces chromofuscus induced invagination of the cells in parallel with hydrolysis of the PC. The action of the latter group of PLases on the mem-brane morphology was counteracted by PLase A2, and vice versa. Thus, participation of the membrane lipid bilayer in the induction of membrane conformational change and hence cell shape change was demonstrated.抄録全体を表示PDF形式でダウンロード (1125K) -
The Presence of Three Forms, and Their Physicochemical, Catalytic, and Immunochemical PropertiesShigeo TAKADA, Takashi KUSUMI, Takeyori SAHEKI, Michio TSUDA, Tsunehik ...1979 年 86 巻 5 号 p. 1353-1359
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe physicochemical, catalytic, and immunochemical properties of three forms of purified rat liver argininosuccinate synthetase (ASS) [EC 6.3.4.5] are described.
1. Three forms of rat liver ASS were separated by column chromatography on DEAE-Sephadex A-50, and named ASS I, II, and III in the order of elution.
2. Their molecular weights were identical as determined by ultracentrifugation and by electrophoresis at pH 6.6 in various concentrations of polyacrylamide gel. The S20, w values of ASS I and II were 8.9S and 8.8S, respectively. The subunit molecular weights of the three forms of ASS were also identical in sodium dodecyl sulfate (SDS)-polyacrylamide gel elec-trophoresis.
3. Each form of ASS gave a single band with complete fusion in Ouchterlony double-diffusion analysis with antisera to the purified ASS II.
4. On titration of ASS I, II, and III with 5, 5'-dithiobis(2-nitrobenzoic acid), they had 16 free sulfhydryl groups per mole of the enzyme in the presence of SDS and 4 free sulfhydryl groups per mole of the enzyme in the absence of SDS.
5. The amino acid compositions of ASS I and II were practically the same.
6. Catalytic constants, such as Km values and pH optima, of ASS I and II were identical, and the specific activities of the three forms were nearly equal.
7. ASS I and II contained 4 and 8 moles of argininosuccinate (ASA) per mole of the enzyme, respectively, and ASS III contained no ASA.
8. ASA bound to ASS I and II was released from the enzymes by incubation in 500 mM potassium phosphate, pH 6.2, containing 10 mM AMP, 10 mM Na4P2O7, and 10 mM MgCl2.
9. ASS I and II that were made free of ASA regained 2 and 4 moles of ASA per mole of the enzyme, respectively, when incubated in 50 mM Tris-HCl, pH7.5, containing 10 mM ATP, 10 mM L-aspartate, 10 mM L-citrulline, and 10 mM MgCl2.
10. When native ASS I and II (with 4 and 8 moles of ASA/mole of the enzyme, respec-
tively) were incubated with L-[14C]aspartate under conditions which allow the synthesis of ASA, half of the ASA molecules bound to ASS were replaced by newly formed [14C]ASA.
11. Native ASS I and II were less sensitive to chymotrypsin than the ASA-free ASS I and II, and ASS III.抄録全体を表示PDF形式でダウンロード (981K) -
Masayuki OZAWA, Akira ASANO, Yoshio OKADA1979 年 86 巻 5 号 p. 1361-1369
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe presence and nature of interpeptide disulfide bonds in HANA (hemagglutinin and neuraminidase) glycoprotein and F (fusion) glycoprotein of HVJ (Sendai virus) are described.
In the case of HANA, subunits of the same or very similar molecular weight were interconnected with a disulfide bond(s). Cleavage of the bond(s) can easily be achieved by the addition of 1mM dithiothreitol with concomitant loss of the biological activities of the glyco-protein. After splitting of the interconnecting bonds, all the HANA protein subunits remainedbound on the viral membrane.
To observe the cleavage of the interpeptide disulfide bond between the F1 and F2 subunits of F glycoprotein, higher concentrations of sulfhydryl compounds were required than were necessary for HANA protein. Splitting of the disulfide bond under either denaturing or nondenaturing conditions failed to release both segments of F protein from the virion. Therefore, F glycoprotein seems to have at least two membrane binding sites, one on F1 and the other on F2.
On the other hand, the disulfide bond which connects the HA1 and HA2 subunits of influenza virus is hardly cleaved under nondenaturing conditions. Addition of 8M urea or 6M guanidine HCl, which completely inactivates HA activity, was necessary for the splitting of this disulfide bond by thiol compounds. Interestingly, the HA1 subunit was released from the virion after the cleavage. Thus, unlike F1 and F2 of HVJ, the HA1 subunit seems to have no hydrophobic binding site to the membrane. A model for the arrangement of these subunits on the viral membrane is proposed.抄録全体を表示PDF形式でダウンロード (1839K) -
Osamu URAYAMA, Makoto NAKAO1979 年 86 巻 5 号 p. 1371-1381
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーWe compared several Na, K-ATPase preparations from various organs of the rat. The brain Na, K-ATPase differed from the enzymes of other organs in its pH dependence and responses to ouabain and N-ethyImaleimide in spite of similarities in the kinetic parameters of activation by Na+, K+, Mg2+, and ATP. The optimum pH of the brain NaI-enzyme was at 7.4 to 7.5 at 37°C. The Lubrol extract of this brain enzyme preparation showed a lower optimum pH of 6.6. When the Lubrol extract of the brain was fractionated with (NH4)2SO4, the activity of the precipitate in the neutral pH region was restored. On the other hand, the optimum pH of the kidney NaI-enzyme was slightly affected by Lubrol and ammonium sulfate treatments (pH7.5→7.3). The brain enzyme (K1/2=0.9 μM) showed about 100-fold higher sensitivity to ouabain than the enzymes from other organs (K1/2=100μM) in the presence of 120mM Na+ and 10mM K+ In a Hill plot of the ouabain inhibition, the former failed to give a linear relationship, while the latter gave a straight line with a Hill coefficient of 1.0. The effect of K+ on the brain enzyme-ouabain interaction led us to consider that the brain enzyme might have two components as regards ouabain affinity, high and low affinity components. The time course of N-ethylmaleimide inhibition of the brain enzyme was rapid and biphasic, while the kidney enzyme showed only a slow phase following pseudo-first order kinetics. ATP protected the kidney enzyme activity completely against N-ethylmaleimide inhibition, but the protection of the brain enzyme activity by ATP was only partial. We
divided rat Na, K-ATPases into two groups, the brain type, which is restricted to the central nervous system, and the kidney type, which is found in most organs.抄録全体を表示PDF形式でダウンロード (835K) -
Rie MASUDA-MIKAWA, Yoshiaki FUJII-KURIYAMA, Masahiko NEGISHI, Yutaka T ...1979 年 86 巻 5 号 p. 1383-1394
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーHepatic microsomal cytochrome P-450 and P-448 have been purified from Phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rats, by modifications of Imai and Sato's procedures (1974). The purified preparations of cytochrome P-450 and P-448 were homogeneous judging from their specific contents (17 and 16nmol per mg protein, respectively) and the results of SDS-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusion analyses. These two cytochromes are different in their physico-chemical and immunological properties, and their substrate specificities.
In reconstituted systems containing the purified cytochrome and NADPH-cytochrome P-450 reductase, ethoxycoumarin deethylation and benzo(a)pyrene hydroxylation catalyzed by cytochrome P-450 and P-448 were completely inhibited by the homologous antibody, while essentially no effect was observed with heterologous combinations of antigen and antibody. In contrast, the benzphetamine demethylation activities of cytochrome P-450 and P-448 were markedly inhibited by the heterologous antibody as well as by the homologous one. These results suggest that the two cytochromes are immunologically different but have some antigenic determinants in common.
Drug metabolizing activities of microsomes from PB- and MC-treated rats were inhibited by the antibodies, essentially as expected from the results with the reconstituted systems. The remaining activities in the presence of excess concentrations' of the antibody, however, were higher in MC-microsomes treated with anti P-448 antibody than in PB microsomes treated with anti P-450 antibody. These results suggest that cytochrome P-448 molecules may be so localized in the microsomal membrane that the membrane structure may hinder the access of the antibody to the antigenic determinant.抄録全体を表示PDF形式でダウンロード (1946K) -
Takao TAKI, Yoshio HIRABAYASHI, Rvoichi KONDO, Makoto MATSUMOTO, Kiyoh ...1979 年 86 巻 5 号 p. 1395-1402
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe effect of butyrate on glycolipid metabolism and morphological differentiation in the cell culture system of rat ascites hepatomas, AH 7974 of island-forming type and AH 7974F of free type, was studied. Both cell lines adhered to the substratum in the presence of 1 mm butyrate. In the case of AH 7974, the addition of butyrate induced a distinct morphological change but the other cell line showed no such conspicuous change. Butyrate-treated AH 7974 cells showed a 2 to 3-fold elevation of CMP-N-acetylneuraminic acid: lactosylceramide sialyl-transferase activity to form N-acetylneuraminylgalactosylglucosylceramide (GM3). On the other hand, no enzyme activity could be detected in AH 7974F cells. Four glycosyltransferase activities involved in glycolipid synthesis, including sialyltransferase in AH 7974F cells, were reduced by butyrate. From these observations we concluded that sialyltransferase to form GM3, or GM3 itself, is prerequisite for the morphological alteration induced by butyrate.抄録全体を表示PDF形式でダウンロード (1143K) -
Hiroshi KURAMOCHI, Hiroyasu NAKATA, Shin-ichi ISHII1979 年 86 巻 5 号 p. 1403-1410
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーLeupeptin (acyl peptidyl-L-argininal) is a potent inhibitor of trypsin and related proteases. We analyzed the association of leupeptin with bovine trypsin kinetically, assuming that it proceeds by a pathway which involves two steps:
E + I K1_??_ Complex I K+2 K-2 _??_ Complex II
The observed dissociation constant (K1) for the first step was 1.24 × 10-3 M (at pH 8.2, 15°C) and the two first-order rate constants (k+2 and k-2) were 166s-1 and 1.75 × 10-3•s-1, respectively (at pH 8.2, 15°C). The dissociation constant (Kd) for the whole process was calculated from these parameters to be 1.34 × 10-8 M. This value is compatible with that determined directly by an independent static method (2.36 × 10-8 M). We also measured Kd for the leupeptin complex of anhydrotrypsin, a trypsin derivative in which the active-site hydroxyl group is missing. The observed value was about 5 orders of magnitude larger than Kd and was rather similar to K1 in native trypsin. A leupeptin isomer which contains a D-argininal residue did not show strong affinity towards trypsin. These findings suggest that complex II consists of a covalent hemiacetal adduct formed between the serine hydroxyl group in the enzyme active site and the aldehyde group in the inhibitor. The pH dependencies of the dissociation constant and other parameters show that deprotonation of the charge-relay system in the active site is important for the formation and stabilization of complex II.抄録全体を表示PDF形式でダウンロード (593K) -
Tatsuo MIYAZAKI, Sigehiro MORITA, Masahiro HATANO, Tsunenori NOZAWA1979 年 86 巻 5 号 p. 1411-1417
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーComparison of absorption and circular dichroism (CD) spectra in the near infrared region was made with chromatophore and subchromatophore preparations obtained from Rhodopseudomonas sphaeroides. The 850nm absorption band had a positive correlation with the 850nm and 870nm CD bands. The 800nm and 870nm absorption bands seemed not to correlate with any CD Lands. Lipid contents in chromatophores and subchromatophores were measured. Lipids in membranes seemed to contribute to the appearance of the 870nm absorption band, but not to that of the 800nm and 850nm absorption bands. The time courses of absorbance changes were compared at 800, 850, and 870nm in detergent-treated chromatophores. Relative changes of absorbances differed from one another. The present results suggest that the three absorption bands are due to three different bacteriochlorophyll a-types and the 850nm absorption band originates from exciton-coupling of bacteriochlo-rophyll a.抄録全体を表示PDF形式でダウンロード (447K) -
III. Changes of Enzyme Activities on Cell Membranes during CultureSatoshi KATO, Kazushi AOYAMA, Toshikazu NAKAMURA, Akira ICHIHARA1979 年 86 巻 5 号 p. 1419-1425
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーWhen liver cells were dispersed with collagenase, their 5'-nucleotidase activity decreased to half the initial level, but it increased to the original level again on culture of the cells for a few days. The activity of another membrane enzyme, alkaline phosphatase, did not decrease on dispersion of the cells, but it increased about 10-fold on culture of the cells. These inductions did not require any hormone, but the effects were greater at a high cell density. These enzymes are located in both the plasma membranes and the cytoplasm, but the enzymes in these two locations can be distinguished by differences in their pH optima, substrate specificities, and susceptibilities to inhibitors. The increases were found to be due to increases in the activity of only the enzymes in the plasma membranes. The increases in enzyme activities were inhibited by actinomycin D, cycloheximide, and puromycin. The activities of leucine aminopeptidase and aminopeptidase B, other membrane enzymes, remained constant during dispersion and culture of the cells.
These results show that enzymes in the cell membranes are affected in different ways by cell dispersion with collagenase and subsequent culture of the cells.抄録全体を表示PDF形式でダウンロード (437K) -
Its Application to the Synthesis of Oligonucleotides Containing Guanylyl ResiduesYo KIKUCHI, Keiko HIRAI, Kenji SAKAGUCHI1979 年 86 巻 5 号 p. 1427-1432
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーPolynucleotide phosphorylase from Thermus thermophilus catalyzed the addition of short guanylyl blocks from GDP to the 3'-hydroxyl termini of oligonucleotide primers at low temperature in a simple reaction mixture. Polyguanylic acid formation was inhibited at 37°C, but the addition of one or two guanylyl residues to oligonucleotide primers proceeded in high yields. The reaction was applied to the synthesis of oligonucleotides containing guanylyl residues at the 3'-end. Using (Ap)2A and (Up)2U as primers, (Ap)3G, (Ap)3GpG, and (Up)3G were synthesized in yields of 25-52%. (Ap)2GpG was synthesized from ApA and GDP in a yield of 13%.抄録全体を表示PDF形式でダウンロード (381K) -
Yuji GOTO, Kozo HAMAGUCHI1979 年 86 巻 5 号 p. 1433-1441
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe conformation and stabilities of the CL fragment isolated from a type λ Bence Jones protein and the fragment in which the intrachain disulfide bond had been reduced were studied by measuring CD, fluorescence, and ultraviolet absorption. The results indicated that no great conformational change occurs on reduction of the disulfide, unless the SH groups are alkylated. Intact CL was more resistant than reduced CL to guanidine hydrochloride. The denaturation curves were analyzed using an equation based on the binding of guanidine hydrochloride and the free energy changes of denaturation in the absence of the denaturant were estimated as about 6kcal•mol-1 for intact CL and about 1.8 kcal•mol-1 for reduced CL. The difference in stability between intact CL and reduced CL was explained to a great extent in terms of the entropy change associated with reduction of the intrachain disulfide bond of the fragment in the denatured state.抄録全体を表示PDF形式でダウンロード (638K) -
Satoshi YOSHIDA, Yutaka ORII, Suguru KAWATO, Akira IKEGAMI1979 年 86 巻 5 号 p. 1443-1450
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーIsolated mammalian cytochrome oxidase gave an Arrhenius plot with a break (Tb) at about 20°C when assayed in a medium containing Emasol. The activation energies above and below 20°C were 9.3 (EH) and 18.9 kcal/mol (EL), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature Tt=40°C), dimyristoyl phosphatidylcholine (DMPC, Tt =23°C) and dioleoyl phosphatidylcholine (DOPC, Tt=-22°C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36°C with E=22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with Tb=23°C. EH and EL were 6.6 and 15.8 kcal/mol, respectively. The plot for the oxidase-DMPC also showed a break (Tb=26°C) with EH=6.6 and EL=26.6 kcal/mol.
These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the “hot” and “cold” conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.抄録全体を表示PDF形式でダウンロード (1321K) -
Masao TSUDA1979 年 86 巻 5 号 p. 1451-1461
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーA rice bran lectin was purified to homogeneity by precipitation with ammonium sulfate and chromatography on ovomucoid-Sepharose and CM-cellulose. The molecular weight of the dimer lectin was estimated to be around 37, 000 by ultracentrifugation studies. The sedimentation coefficient was 3.8S. On Sepharose 6B gel filtration in the presence of 6M guanidine-HCl, the lectin showed a molecular weight of 19, 000. On reduction and carboxymethylation, the lectin further dissociated into two nonidentical subunits, with molecular weights of about 11, 000 and 8, 000. These subunits did not show hemagglutinating activity.
Equilibrium dialysis experiments using N-acetyl-[1-14C]glucosamine indicated that about 1.8mol of the sugar was bound to 19, 000g of the lectin. The lectin was mitogenic against mouse splenic lymphocytes and human peripheral lymphocytes. The lectin enhanced the rate of glucose oxidation and inhibited epinephrine-stimulated lipolysis in mouse adipocytes. Some characteristics of the lectin are compared with those of wheat germ agglutinin.抄録全体を表示PDF形式でダウンロード (2275K) -
Masao TSUDA1979 年 86 巻 5 号 p. 1463-1468
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーA lectin was purified to homogeneity from the mushroom, Flammulina veltipes, by zinc acetate treatment and CM-cellulose column chromatography. Its molecular weight was estimated to be 20, 000 by gel filtration and polyacrylamide gel electrophoresis. The lectin does not contain carbohydrate, half-cystine, methionine, or histidine.
On gel filtration with Sepharose 6B in the presence of 6M guanidine-HCl, the purified lectin dissociated into two nonidentical subunits, FVA-L (molecular weight, 12, 000) and FVA-S (8, 000). The hemagglutinating activity was retained only in the FVA-L subunit. The lectin is mitogenic with respect to mouse spleen lymphocytes.抄録全体を表示PDF形式でダウンロード (1206K) -
Akira WAKIZAKA, Eiji OKUHARA1979 年 86 巻 5 号 p. 1469-1478
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe conformational changes of double-stranded DNA induced during irradiation with ultravio-let, were immunologically investigated. These studies revealed that at least two distinct anti-genic sites were induced in the irradiated DNA molecule, giving rise to two different antibodies specific for ultraviolet-irradiated (uv) DNA and thermally denatured DNA-like structure, and these were demonstrable using radioimmunoassay and double diffusion tests. A series of experiments, including melting profile, fluorescence intensity of the ethidium bromide complex and chromatographic behavior on hydroxyapatite, performed on the antigenically active uvDNA indicated that the duplex structure of DNA separated irregularly during irradiation. Furthermore, the data showed that the conformational determinants of uvDNA are located on the exposed single-stranded regions.抄録全体を表示PDF形式でダウンロード (1537K) -
Takao OHYASHIKI, Kenzo CHIBA, Tetsuro MOHRI1979 年 86 巻 5 号 p. 1479-1485
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリー1. The addition of Tb3+ to rat intestinal mucosal membranes resulted in a marked enhance-ment of Tb3+ fluorescence at 550 nm and some decrease in the native fluorescence of the membranes at 330nm.
2. The pH dependence profile of Tb3+ fluorescence indicates that the development of Tb3+ fluorescence upon its addition to the membranes is related to changes in the states of tryptophan and/or tyrosine residues of the membrane proteins.
3. More than one type of Tb3+-binding site appears to be present in the membranes; the apparent dissociation constant of one type of Tb3+-membrane complex is 10.2 μM at pH 7.4.
4. Tb3+ fluorescence increased in a sigmoidal fashion with increasing concentration of Tb3+ in the presence of Ca2+, but the Hill coefficient (n_??_2) was almost the same regardless of the presence of Ca2+.
5. Addition of Ca2+ or Mg2+ to the medium induced marked decreases in both Tb3+ fluorescence and native fluorescence of the membranes. The apparent dissociation constants of Ca2+- and Mg2+-membrane complexes were estimated from the changes in Tb3+ fluorescence to be 2.89 and 5.35mM, respectively. Mg2+ markedly reduced the binding affinity of Ca2+ for the membranes.
6. The Ca2+-induced reduction of the fluorescence of Tb3+-membrane complex was attenuated at high ionic strength.
On the basis of these results, the nature of the Ca2+-binding sites of rat intestinal mucosal membranes is discussed.抄録全体を表示PDF形式でダウンロード (495K) -
Satoshi MARUYAMA, Taro TATSUKI, Shintaro SUGAI1979 年 86 巻 5 号 p. 1487-1494
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe equilibrium and kinetics of thermal unfolding of yeast 5S ribosomal RNA have been studied by optical methods, in a low ionic strength environment without Mg2+, to follow the disruption of the secondary structure base pairs in the molecule. The equilibrium results demonstrated that all of the helical regions melted simultaneously, and the kinetics of the thermal unfolding were first order. These findings suggest the validity of the two-state approximation for the unfolding reaction under the present conditions. The total number of secondary structure base pairs estimated from our experiment was consistent with that contained in the conformational model based on the Raman spectrum rather than that in the one derived by the enzymic digestion method. Taking our results on the kinetic behavior of the thermal unfolding overall, we propose that the 5S RNA has a partly melted secondary structure under the solvent conditions used.抄録全体を表示PDF形式でダウンロード (496K) -
Eisaku KATAYAMA, Yoshiaki NONOMURA1979 年 86 巻 5 号 p. 1495-1509
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe mechanism of formation of the negatively stained image in electron microscopy was investigated with native collagen fibrils as a model. The negatively stained image was simulated from the primary structure by using the values of volume or bulkiness of each amino acid residue as a parameter for stain-excluding capacity. The pattern simulated from the bulkiness values gave an excellent fit with the negatively stained image. Since some contribution of positive staining components to negative staining has been suggested, positive staining with uranyl acetate was tested with various washing solutions of different pH. While acidic conditions did not produce any stained image, a positively stained image was easily obtained at alkaline pH. On the other hand, negatively stained images with stains of different charge character remained essentially the same as those obtained with acidic uranyl stains. It was concluded that the contribution of positive components to the negatively stained image is negligible under the conventional conditions for negative staining with uranyl acetate.
In order to demonstrate the utility of the analytical method employing the values of “bulkiness, ” we studied the unknown molecular packing in the polar lead paracrystal of rabbit skeletal tropomyosin. Utilizing the primary sequence data for α-tropomyosin we successfully showed the polar paracrystal to be an array of molecules which are parallel and in register. Further, our analysis made it possible to deduce the position of a given residue in the negatively stained pattern of the polar paracrystal.抄録全体を表示PDF形式でダウンロード (3620K) -
Eisaku KATAYAMA, Yoshiaki NONOMURA1979 年 86 巻 5 号 p. 1511-1522
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーNegatively stained images of divalent cation-induced tropomyosin paracrystals show polymorphic patterns which are almost bipolar. Although these bipolar forms are naturally due to antiparallel arrays of molecules, the precise molecular arrangements have not been clarified yet except in the case of one type of these polymorphic paracrystals by Stewart and McLachlan [(1976) J. Mol. Biol. 103, 251-269].
In the previous paper we showed that the lead-induced polar paracrystal is a parallel and in-register array of tropomyosin molecules. Moreover, we have made it possible to locate a given residue on the staining pattern. By overlapping two photographic transparencies of the polar paracrystal antiparallel, directly observed images of polymorphic bipolar paracrystals could be synthesized photographically with fidelity. The overlap length between N-terminals of antiparallel pairs of molecules could be easily determined without any assumptions.
Next, we considered the stabilizing forces involved in the morphogenesis of such polymorphic paracrystals. The cation-bridged attractive forces already proposed by some groups were insufficient to account for the stability of some specific forms of tropomyosin paracrystals. From the primary amino acid sequence of tropomyosin, we calculated the changes of repulsive forces between basic residues with changes of molecular overlap length between the N-terminals of antiparallel pairs. By setting the values of charge appropriately, we could account well for the stability of the polymorphic structures observed by electron microscopy.抄録全体を表示PDF形式でダウンロード (3082K) -
II. Assignment of a New SubgroupNobuhiro TAKAHASHI, Tatunori TAKAYASU, Toshiaki ISOBE, Tomotaka SHINOD ...1979 年 86 巻 5 号 p. 1523-1535
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe primary structure of the variable region of the human λtype Bence Jones protein NIG-48 was determined by analysis of the N-terminal sequence of the completely reduced and amino-ethylated protein, as well as of five cyanogen bromide fragments. The variable region of NIG-48 contains 112 amino acid residues.
The protein NIG-48, having a unique sequence of the variable region, a low degree of homology (about 50%) with λchains of the five other subgroups and the addition of two residues around 65, may represent a new subgroup, namely VλVI.抄録全体を表示PDF形式でダウンロード (830K) -
Its Application to Purification of Porcine Pancreas EnzymesIkuharu SASAKI, Hirosi GOTOH, Ryohei YAMAMOTO, Hideyuki HASEGAWA, Jinp ...1979 年 86 巻 5 号 p. 1537-1548
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリー1. All the porcine pancreas enzymes tested, regardless of their pI's were adsorbed on Amber- lite CG-50 (a weakly acidic cation exchange resin) at pH 4, where the ion-exchange group (carboxyl group) is not dissociated. The adsorption is hardly influenced by ionic strength.
2. At pH 4, the adsorbed enzymes were partially eluted by organic solvents such as 50% propanol.
3. The adsorbed enzymes were effectively eluted by increasing the pH from 4 to 6. Trypsin (pI 10.5) was eluted before carboxypeptidase A (pI 4.5 and 5.3) with 0.5 ivt acetate buffer, whereas the former enzyme was eluted after the latter enzyme with 0.2 NI 3, 3-dimethyl glu-tarate buffer. However, with either buffer, the elution order of enzymes was not always the same as the order of their pI's.
4. By a single Amberlite CG-50 column chromatography of porcine pancreas extracts, kallikrein, carboxypeptidase B, deoxyribonuclease, carboxypeptidase A, and trypsin were purified 100-fold, 16-fold, 17-fold, 8-fold, and 32-fold, respectively.
5. The adsorption-elution mechanism of proteins on Amberlite CG-50 may be as follows. At pH_??_4.5, proteins are adsorbed by hydrophobic affinity on loci of the resin, each of which includes at least one carboxyl group. As the pH is increased, the carboxyl groups dissociate, resulting in a decrease of the hydrophobicity and an increase of the negative charge of the loci; thus, the proteins are now retained by the remaining hydrophobic affinity plus the increased electrostatic affinity or minus the increased electrostatic repulsion.抄録全体を表示PDF形式でダウンロード (1030K) -
Wataru SAKAMOTO, Osamu NISHIKAZE1979 年 86 巻 5 号 p. 1549-1557
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーA low molecular weight (LMW) kininogen was isolated from pooled human serum by chro-matography on DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-150, and Sephadex G-100. It was shown to be homogeneous by ultracentrifugation, polyacrylamide gel elec-trophoresis, and immunoelectrophoresis. The sedimentation coefficient, S020, w, of purified LMW kininogen was 3.85 s, and its molecular weight was determined to be 78, 000 by Se-phadex G-100 gel-filtration. The LMW kininogen contained 79.3% protein, 8.0% hexose, 3.9% hexosamine, and 4.9% sialic acid.
In order to determine the immunochemical properties of LMW kininogen, specific anti-serum was prepared in rabbits. The antigenic determinant of LMW kininogen was not related to the sialic acid and kinin moieties in the kininogen molecule, but could not be distinguished from that of high molecular weight (HMW) kininogen. In the quantitative single radial im-munodiffusion test, a sialic acid-free LMW kininogen reacted to a greater extent with the antiserum than the native LMW kininogen. The kininogen level in human serum was esti-mated by single radial immunodiffusion. The antiserum cross-reacted with monkey serum, but not with sera from dogs, rats, mice, horses, pigs, guinea pigs, oxen, and rabbits.抄録全体を表示PDF形式でダウンロード (3223K) -
Masahiko ENDO, Noboru HIYAMA1979 年 86 巻 5 号 p. 1559-1565
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーL-Fucose dehydrogenase [EC 1.1.1.122] was isolated from a rabbit liver extract and purified about 390-fold with a yield of approximately 13 %. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 cellulose column chromatography, gel filtration on Sephadex G-100, preparative polyacryl-amide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sepharose 4B, was useful for the removal of other dehydrogenases.
The enzyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92, 000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mm, 1.4 mm, and 0.07 mm for L-fucose, D-ara-binose, and NAD+, respectively.
A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.抄録全体を表示PDF形式でダウンロード (451K) -
Shuhei KONDO, Tetsuya ASAKAWA, Fumi MORITA1979 年 86 巻 5 号 p. 1567-1571
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーA difference UV-absorption spectrum induced by ATP was observed with scallop myosins purified from both striated and smooth adductor muscles. The difference spectra showed a positive peak at 289 nm with a shoulder around 295 nm, and two small negative troughs around 280 nm. Tryptophanyl movement similar to that in rabbit skeletal myosin is indicated. Some tyrosyl movements in scallop myosins, however, may be in the opposite direction.抄録全体を表示PDF形式でダウンロード (276K) -
Yoshihiro UCHIDA, Yoji TSUKADA, Tsunetake SUGIMORI1979 年 86 巻 5 号 p. 1573-1585
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーNeuraminidase I and neuraminidase II from Arthrobacter ureafaciens were characterized. As determined by gel filtration on Ultrogel AcA 44, the molecular weights of neuraminidases I and II were 51, 000 and 39, 000, respectively. Neuraminidases I and II were similar to each other in their enzymatic properties except for the substrate specificities towards gangliosides and erythrocyte stroma. Their optimal pHs were between 5.0 and 5.5 with N-acetylneur-aminosyl-lactose or bovine submaxillary mucin as substrates, but with colominic acid as a substrate, the pH optimum was between 4.3 and 4.5. They were most active around 53°C, were stable between pH 6.0 and 9.0, and were thermostable up to 50°C. They did not require Ca2+ for activity and were not inhibited by EDTA. They were inhibited only slightly or not at all by p-chloromercuribenzoic acid or Hg2+.
Both neuraminidases I and II were able to hydrolyze the α-ketosidic linkage of N-glycolyl-neuraminic acid as well as that of N-acetylneuraminic acid, and were able to liberate sub-stantially all of the sialic acid from various kinds of substrates. However, they cleaved only about 50% of the sialic acid from bovine submaxillary mucin. The saponification of bovine submaxillary mucin by mild alkali treatment, on the other hand, resulted in an increased susceptibility to the neuraminidases and brought about the complete liberation of sialic acid.
Remarkable differences were observed between neuraminidases I and II as regards sub-strate specificities on gangliosides; the initial rate of hydrolysis by neuraminidase I was 74 times, and its maximum velocity constant was 91 times those of neuraminidase II. The addition of sodium cholate markedly stimulated the enzymatic hydrolysis of gangliosides, and increased the maximum velocity constant of neuraminidase I twofold and that of neu-raminidase II 143-fold.
Although neuraminidases I and II were able to hydrolyze (α, 2-3), (α, 2-6), and (α, 2-8) linkages, the initial rate of hydrolysis of N-acetylneuraminosyl-α, 2-6-lactose was greater than that of the α, 2-3-isomer.抄録全体を表示PDF形式でダウンロード (1605K) -
Hidehiro TANAKA, Miyako TANAKA, Haruo SUGI1979 年 86 巻 5 号 p. 1587-1593
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe effect of sarcomere length and stretching on the tension and the rate of ATP splitting was studied using small fiber bundles from glycerinated rabbit psoas muscle. The rate of ATP splitting was determined by measuring ADP production, while the tension development in response to a contracting solution (at pCa 5.3) was recorded in the same preparation.
The isometric tension developed by the preparation decreased when the sarcomere length was increased. The decrease of tension development was accompanied by a decrease in the rate of ATP splitting. If a preparation exerting steady isometric tension was stretched by 5-10% at a velocity of 0.1 mm/s, the rate of ATP splitting was increased after stretching, while the steady isometric tension attained after stretching was also higher than the initial value. The extent of the excess ATP splitting caused by stretching decreased with increasing sarcomere length. These results suggest that the rate of the interaction cycle between actin and myosin molecules may increase as a result of stretching.抄録全体を表示PDF形式でダウンロード (471K) -
Peter GRÖBNER1979 年 86 巻 5 号 p. 1595-1605
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーIsoelectric focusing of plasmodial extracts of Physarum polycephalum demonstrated the presence of several multiple enzyme variants of thymidine kinase, which appear sequentially during the nuclear division cycle. Variants (A)+(A1) are the only enzyme variants found in the late G2-phase, whereas the variants (C)+(C1) are only present at the time of mitosis and S-phase (1, 2). Evidence is presented that multiple forms of thymidine kinase (A)+(A1) with high pI arise by dephosphorylation of a primary translation product with low pI (C and/or C1). The thymidine kinase fractions (A)+(A1) and (C)+(C1) were separated and partially purified by DEAE-cellulose chromatography. The enzyme variants (C)+(C1) are converted in vitro by an endo-genous enzymatic factor as well as by bacterial alkaline phosphatase into the variants (A)+(A1).抄録全体を表示PDF形式でダウンロード (821K) -
Kinetics and Properties of the Enzyme VariantsPeter GRÖBNER1979 年 86 巻 5 号 p. 1607-1614
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThymidine kinase variants of Physarum polycephalum separated by repeated DEAE-cellulose chromatography have been characterized. The enzyme variants show similar catalytic properties (e.g., substrate specificity, pH optimum) and molecular weights, as judged by their sedimentation in sucrose gradients. However, they differ significantly with respect to pI, inhibition by dTTP and thermostability, and they have slightly different Km values for deoxythymidine as a substrate.抄録全体を表示PDF形式でダウンロード (487K) -
Hideo IGARASHI, Takashi MORITA, Sadaaki IWANAGA1979 年 86 巻 5 号 p. 1615-1618
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーStaphylocoagulase was isolated from a culture filtrate of Staphylococcus aureus, strain st-213, by a two step purification procedure of chromatography on a bovine prothrombin-Sepharose 4B affinity column and gel filtration on Sephadex G-25. The yield of the coagulase activity ranged from 75-83% and the purified preparation gave a single precipitin line in im-munodiffusion tests against anti-crude and anti-purified staphylocoagulase sera. However, the final product was shown to contain one major and two minor components by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chemical analysis of the material indi-cated that it does not contain any cystine residues and that its NH2-terminal residue is a single isoleucine.抄録全体を表示PDF形式でダウンロード (232K) -
Mori SETAKA, Masafumi YANO, Takao KWAN, Hiroshi SHIMIZU1979 年 86 巻 5 号 p. 1619-1622
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーMultilayer planar membranes were constructed between a pair of cellulose sheets from fragmented sarcoplasmic reticulum (FSR) as well as a mixture of egg yolk lecithin and the Ca2+- ATPase purified from FSR. Since sodium deoxycholate was used instead of organic solvents in order to dissolve phospholipids in the process of the membrane preparation, the total activity of the Ca2+-ATPase was still preserved in the planar membrane of FSR. It was also indicated using a spin label technique that the orientation of phospholipids in the planar membrane of FSR was considerably disturbed by the presence of proteins such as the Ca2+- ATPase included in FSR.抄録全体を表示PDF形式でダウンロード (304K) -
Sumiko HAMANAKA(née YASUE), Shizuo HANDA, Tamio YAMAKAWA1979 年 86 巻 5 号 p. 1623-1626
発行日: 1979/11/01
公開日: 2008/11/18
ジャーナル フリーThe gangliosides from the erythrocytes of various strains of inbred mice were analyzed. The ganglioside compositions differed in different strains and the strains could be divided into 4 types on the basis of this difference. The main ganglioside is GM4 or sialosyl galactosyl ceramide in Type 1, an unidentified ganglioside in Type 2, GM4 and GM2 in almost equal amounts in Type 3, and GM2 in Type 4.抄録全体を表示PDF形式でダウンロード (1427K)