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Volume 82 (1977)
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- Issue 5 Pages 1189-
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Volume 82, Issue 5
Displaying 1-39 of 39 articles from this issue
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Yoshiaki NAKAMARU1977 Volume 82 Issue 5 Pages 1189-1195
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of spectral shift and absorption intensity change of the divalent bromocresol purple (BCP) anion was further investigated and it was characterized as a spectrophotometric membrane probe.
At high concentrations (1-40mM), the absorption intensity of the BCP anion at 590nm (monomer band) decreased markedly with increase of the dye concentration, while another absorption band appeared at 554nm. Analysis of the change of absorption intensity showed that the marked decrease resulted from dimer formation of BCP (polymer formation at concentrations higher than 20mM).
Wavelengths of maximum absorption (λmax) of the BCP anion were determined in various solvents and comparison of these λmax's with λmax of the BCP anion bound to SR showed that the hydrophobicity of the area of BCP anion binding to SR corresponded to a refractive index of 1.429.
While the BCP anion bound to SR showed a monomer spectrum, a dimer band appeared for the BCP anion bound to SR-P1 (phosphorylated protein) with a marked decrease in the absorption intensity at the monomer band, indicating that two polar groups, binding sites for the BCP anions, closely approached each other in the SR-P1 configuration.View full abstractDownload PDF (555K) -
IV. Purification and Properties of Hexose-Binding ProteinsHanwant C. BHANDARI, Masaya HAYASHIBE1977 Volume 82 Issue 5 Pages 1197-1205
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo glucose-binding (G1 and G2) proteins and one mannose-binding (M) protein were isolated and purified from the fraction containing plasmalemma of Schizosaccharomyces pombe. The purification was performed by a sequence comprising of extraction with nonionic detergent Tween 80, ammonium sulfate precipitation, DEAE-cellulose, Biogel P150, and hydroxyapatite column chromatographies. The presence of only two glucose- and one mannose-binding proteins was confirmed by isoelectric focusing. The proteins were homogeneous as shown by polyacrylamide gel electrophoresis.
The purified proteins contained appreciable amounts of carbohydrate, but did not contain detectable amounts of lipid. Approximate molecular weights determined by gel filtration were 1.9×105, 2.2×105, and 1.8×105, and isoelectric points were pH 8.3, 6.2, and 7.3 for G1, G2, and M proteins, respectively. pH optima for hexose binding were 5.3-5.5 for G1 and G2 proteins and 5.3-5.8 for M protein.
Binding activities of G1 and G2 proteins were strongly inhibited by amino group-modifying reagents such as dinitrofluorobenzene, phenylisothiocyanate and dansyl chloride. Copper ions inhibited glucose binding of G1 and G2 proteins and this inhibition was reversible on the addition of L-histidine. These results suggest that amino groups, especially histidine in some form, play an important role in glucose binding of these proteins. However, mannose-binding activity was inhibited by sulfhydryl reagents.View full abstractDownload PDF (1061K) -
Kuniyo INOUYE, Ben'ichiro TONOMURA, Keitaro HIROMI, Sakae SATO, Sawao ...1977 Volume 82 Issue 5 Pages 1207-1215
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S. & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studied by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.View full abstractDownload PDF (581K) -
II. Factors Influencing the Enzyme ActivityRyo TAGUCHI, Hiroh IKEZAWA1977 Volume 82 Issue 5 Pages 1217-1223
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe apparent activity of phospholipase C [EC 3. 1. 4. 3] of Clostridium novyi type A toward phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine increased in the presence of sodium deoxycholate (SDC). The effects of divalent cations on phospholipase C activity were examined in detail at various concentrations of these cations. These effects varied with substrate. Hydrolysis of phosphatidylcholine by this enzyme significantly increased in the presence of Mg2+ or Ca2+. Hydrolysis of sphingomyelin was inhibited by Ca2+, but increased in the presence of Mg2+. Phosphatidylethanolamine-hydrolyzing activity increased only slightly in the presence of Mg2+ and Ca2+. Zn2+ rather inhibited hydrolysis of these substrates. The effects of divalent cations and detergent appear to be directly related to the physical state of the phospholipid micelles used as substrates.
When phosphatidylcholine, sphingomyelin, or phosphatidylethanolamine was used as a substrate, phospholipase C activity was completely inhibited by 2.5mM EDTA or o-phenanthroline (concentration in the final incubation mixture: 0.5mM), and was fully restored by Zn2+ alone. Both Ca2+ and Mg2+ were ineffective for reactivation.
The isoelectric point of the enzyme was 7.1±0.1.View full abstractDownload PDF (470K) -
Ryo TAGUCHI, Hiroh IKEZAWA1977 Volume 82 Issue 5 Pages 1225-1230
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubstrate specificities of phospholipases C [EC 3. 1. 4. 3] from Clostridium novyi, Clostridium perfringens, Bacillus cereus, and Pseudomonas aureofaciens were studied under the same conditions. Phospholipases C from Clostridium novyi and Bacillus cereus show wide substrate specificities while those of Clostridium perfringens and Pseudomonas aureofaciens show relatively narrow specificities.
On the basis of these results, the hydrolytic actions of these phospholipases on membrane lipids of Escherichia coli, Bacillus cereus, and Clostridium novyi were examined under the same conditions. The enzymes of Clostridium novyi and Bacillus cereus attacked all the membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine, phosphatidylglycerol, lysophosphatidylethanolamine, and o-aminoacylphosphatidylglycerol. Phospholipase C from Pseudomonas aureofaciens attacked these three membranes and their lipid extracts, hydrolyzing phosphatidylethanolamine. Phospholipase C from Clostridium perfringens hardly attacked the phospholipids of these bacterial membranes. However, phospholipase C from Clostridium perfringens hydrolyzed phosphatidylethanolamine in a mixture containing lipid extract from Escherichia coli membrane and purified phosphatidylcholine from egg yolk.View full abstractDownload PDF (474K) -
Keizo SHIMADA, Norio MURATA1977 Volume 82 Issue 5 Pages 1231-1236
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy means of a chemical modification technique, the surface structure of the intracytoplasmic membrane (chromatophores) of the photosynthetic bacterium Chromatium vinosum was investigated. Trinitrobenzenesulfonate was employed to modify the primary amino groups of phosphatidylethanolamine and proteins.
The experimental results indicate that upon the chemical modification of intact cells, the amino groups on the inner surface of the chromatophores are trinitrophenylated, while upon the modification of isolated chromatophores, the amino groups of the outer surface are trinitrophenylated. Phosphatidylethanolamine is essentially equally distributed on the inner and outer surfaces of the chromatophores, while most proteins exist at the outer surface. In particular, a protein fraction, which is soluble in a mixture of chloroform/methanol, is located at the outer surface exposing a part of the amino groups in the aqueous phase.View full abstractDownload PDF (413K) -
Yoshio IMAI, Ryo SATO, Takashi IYANAGI1977 Volume 82 Issue 5 Pages 1237-1246
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStopped-flow spectrophotometric measurements were carried out by the rate of cytochrome P-450 reduction by NADPH in a reconstituted system consisting of NADPH-cytochrome c (P-450) reductase and cytochrome P-450 purified from phenobarbital-induced rat and rabbit liver microsomes, respectively. The reduction rate was increased by the addition of hydroxylatable substrates and the apparent first-order rate constant of the reduction in the presence of 1mM benzphetamine was determined to be 4-12s-1, a value which corresponds to a potential rate of 240-720 nmol per min per nmol of cytochrome P-450. The rate constant of the binding of benzphetamine to cytochrome P-450 was also measured by stopped-flow spectrophotometry and found to be of the order of 50s-1. The rate constants determined for the two partial reactions are much higher than the overall rate of benzphetamine-dependent NADPH oxidation of 50-70nmol per min per nmol of cytochrome P-450. A difference spectrum attributable to an oxygenated form of ferrous cytochrome P-450 was observed during the steady state of the benzphetamine-dependent NADPH oxidation. It is concluded from these and other lines of evidence that the rate-limiting step of benzphetamine N-demethylation by the reconstituted system is at or after the introduction of the second electron. Although the reduction of cytochrome P-450 is not rate-limiting, it was affected in parallel with the overall NADPH oxidation in response to various substrates, pH, and benzphetamine concentration, suggesting that substrate binding to cytochrome P-450 causes parallel enhancement of the cytochrome reduction and the rate-limiting step of the overall process.View full abstractDownload PDF (813K) -
Determination of the Monomer-Dimer Equilibrium Constant and the Energetics of Subunit AssociationKihachiro HORIIKE, Kiyoshi SHIGA, Yasuzo NISHINA, Akio ISOMOTO, Toshio ...1977 Volume 82 Issue 5 Pages 1247-1255
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe enzyme concentration dependence of spectrophotometric titrations of hog kidney D-amino acid oxidase [EC 1. 4. 3. 3] with p-aminobenzoate was studied. The monomer-dimer equilibrium constant of the oxidized holoenzyme at 25°C was estimated to be 7×105M-1 at pH 7.5 and 4×106M-1 at pH 8.3. The energetics of subunit association are discussed.View full abstractDownload PDF (566K) -
Yukio NISIMOTO, Fumio TAKEUCHI, Yukio SHIBATA1977 Volume 82 Issue 5 Pages 1257-1266
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUpon solubilization with sodium cholate, the cytochrome b5-like hemeprotein associated with the outer membranes of rat liver mitochondria (abbreviated here as OM-cytochrome b5) could be preferentially released from sonicated mitochondrial outer membrane preparations. The OM-cytochrome b5 thus solubilized was highly purified by DEAE-cellulose, DEAF-Sepharose CL-6B, hydroxylapatite column chromatography in the presence of 0.5% sodium cholate and the isoelectric focusing method. Sepharose 6B gel chromatography in the presence of 6M guanidine hydrochloride and Sephadex G-75 gel chromatography in the presence of 0.5% sodium dodecyl sulfate (SDS) indicated a monomeric molecular weight of about 16, 000 for the detergent-solubilized OM-cytochrome b5. However, in aqueous solutions, this cytochrome occurs as a stable polymeric species since its molecular weight has been found to be approximately 60, 000 even in 0.5% sodium cholate. The absorption spectrum of reduced OM-cytochrome b5 in the visible region closely resembled that of microsomal cytochrome b5, however, the OM-cytochrome b5 showed a broad, symmetrical α-band with a maximum around 557nm. A high level of NADH-cytochrome c reductive activity was detected by the addition of both the purified L-kynurenine 3-hydroxylase [EC 1. 14. 13. 9] and OM-cytochrome b5 preparation to a sonicated phospholipids liposome bilayer, although only the purified monooxygenase or OM-cytochrome b5 could not catalyze the reduction of cytochrome c by NADH. The rate of NADH-cytochrome c reduction catalyzed by the monooxygenase and this hemeprotein in the presence of PC liposomes was affected by high concentrations of rotenone or dicumarol.View full abstractDownload PDF (796K) -
Nozomu NISHI, Kumiko SAKATA-SOGAWA, Gilbu SOE, Jinpei YAMASHITA1977 Volume 82 Issue 5 Pages 1267-1279
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The light-induced pH change of chromatophore suspensions from Rhodospirillum rubrum was stimulated significantly and similarly by KCl, NaCl, LiCl, RbCl, CsCl, MgCl2, MnCl2, and CaCl2. In the dark, the pH of chromatophore suspensions decreased immediately and markedly on adding these salts.
2. The light-induced pH change stimulated by KCl plus valinomycin was inhibited by LiCl and NaCl, but not by RbCl.
3. The optimum pH values for light-induced pH change and photosynthetic ATP formation were around 5 and 8, respectively. The amount of chromatophore-bound ubiquinone-10 reduced in the light was independent of pH from 5 to 9. At pH 8, the number of protons incorporated into chromatophores in the light Was one-half of the number of ubiquinone-10 molecules reduced in the light.
4. Among several pH indicators tested, bromothymol blue (BTB) and neutral red (NR) showed absorbance changes on illumination of chromatophores. Although the pH change indicated by the absorbance change was opposite to the light-induced pH change of the medium, the effect of KCl on the absorbance changes of BTB and NR, and the effect of valinomycin on that of NR, but not on that of BTB, were similar to those on the light-induced pH change.
5. The light-induced absorbance change of BTB was significantly inhibited by NR, whereas that of NR was hardly influenced by BTB.
6. Oligomycin stimulated the light-induced absorbance change of BTB under either nonphosphorylating or phosphorylating conditions. On the other hand, that of NR under phosphorylating conditions was 50% of that under non-phosphorylating conditions, and was increased by oligomycin.View full abstractDownload PDF (939K) -
IV. Structure of a Branched Globoside Containing Mannose from Spermatozoa of the Fresh-Water Bivalve, Hyriopsis schlegeliiTaro HORI, Hitoshi TAKEDA, Mutsumi SUGITA, Osamu ITASAKA1977 Volume 82 Issue 5 Pages 1281-1285
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. A second novel globoside, provisionally named Lipid II in the previous study, was obtained from spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii. The structure of this globoside was established by the results of partial acid hydrolysis, methylation studies, and oxidation with chromium trioxide.
2. The structure was shown to be GlcNAcβ(1→2)Manβ(1→3)[Xylβ(1→2)]Mangβ(1→4)Glcβ-(1→1)-ceramide. It is structurally related to the previously described globoside (Lipid I), except that a branched xylose is linked to the heterooligo saccharide chain of the latter lipid.
3. The predominant fatty acids were palmitic and stearic acids, and octadeca-4-sphingenine was the principal base, amounting to 70% of the total. The fatty acid and long-chain base compositions show nearly the same distribution in both of these globosides.View full abstractDownload PDF (841K) -
I. The Mode of Contribution of Hyaluronidase, β-Glucuronidase, and β-N-Acetylhexosaminidase on Hyaluronic AcidShiro HAYASHI1977 Volume 82 Issue 5 Pages 1287-1295
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHyaluronic acid and its oligosaccharides (tetra-, hexa-, and octasaccharides) were digested by canine liver lysosomes at pH 4.0. The degradation products were separated by gel filtration and ion exchange chromatography. The contribution of hyaluronidase and exoglycosidases (β-glucuronidase and β-N-acetylhexosaminidase) on the degradation of hyaluronic acid was inferred from the distribution of the oligosaccharides, glucuronic acid, and N-acetylglucosamine in the digestion mixture.
Hyaluronic acid was degraded into a large amount of even-numbered oligosaccharides and to a trace amount of free glucuronic acid in the initial stage of the digestion. On the other hand, the prolonged digestion study showed an increase of odd-numbered oligosaccharides such as the tri- and pentasaccharides in proportion to an increase of free glucuronic acid.
On the degradation of the hyaluronic acid oligosaccharides, tetra- and hexasaccharides gave tri- and pentasaccharides as the main oligosaccharides in the degradation products respectively. Octasaccharide was not degraded into heptasaccharide by the action of β-glucuronidase but into a mixture of di-, tetra-, and hexasaccharides by hyaluronidase. The activity of β-glucuronidase for tetra- and hexasaccharides was higher than that of β-N-acetylhexosaminidase for tri- and pentasaccharides.
On the basis of these findings, hyaluronic acid is first depolymerized by hyaluronidase alone and octasaccharide is the minimum substrate size in the degradation by hyaluronidase. Hexasaccharide and other smaller oligosaccharides are degraded by the action of exoglycosidases. The level of β-N-acetylhexosaminidase activity is a limiting factor in the degradation by the concerted action of exoglycosidases.View full abstractDownload PDF (670K) -
Takako KATO, Yukio KATO, Hisataka KASAI, Tsuneo OKUYAMA1977 Volume 82 Issue 5 Pages 1297-1305
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHighly acidic proteins were extracted from bovine liver with 55% saturated ammonium sulfate and purified by DEAE-Sephadex A-50 column chromatography and Sephadex G-75 column chromatography. Four acidic protein fractions (BLA I-IV) were obtained in yields of 150, 100, 50, and 450mg, respectively, from 6kg of bovine liver. When analyzed by 7.5% and 15% polyacrylamide gel disc electrophoresis, BLA I and BLA II fractions were still heterogeneous, and BLA III fraction was composed of two main and one minor protein components. On the other hand, BLA IV fraction was homogeneous after rechromatography on DEAE-Sephadex A-50. Gel electrophoresis also showed that the four acidic protein fractions were clearly different each other. The molecular weights were estimated to be 12, 500 and 14, 500 for the two main protein components of BLA III fraction, and 16, 000 for BLA IV, as determined by SDS-polyacrylamide gel electrophoresis. Three N-terminal amino acids for BLA III fraction were identified by a dansylation method, but none was found for BLA IV. BLA IV showed an unusual UV spectrum, while BLA I-III fractions gave usual protein spectra.
BLA III and BLA IV fractions were compared with highly acidic proteins of bovine brain (PAP I and PAP II). BLA III fraction was different from PAP I (bovine brain S-100 protein) and BLA IV fraction was virtually identical with PAP II, on the basis of amino acid compositions and peptide maps of tryptic digests. Immunodiffusion tests with anti-S-100 serum demonstrated that S-100 protein could not be detected in bovine liver; the content is less than one-thousandth of that in bovine brain. Thus, S-100 protein was not detected in bovine liver by chemical analysis or by immunochemical tests. However, it appears that almost identical acidic proteins are present in bovine liver and brain (BLA IV and PAP II) as well as in chick brain (CBA II).View full abstractDownload PDF (1940K) -
I. The Isolation and Characterization of Ceramide Mono- and Di-HexosidesMutsumi SUGITA1977 Volume 82 Issue 5 Pages 1307-1312
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCeramide mono- and di-hexosides were isolated from the starfish, Asterina pectinifera. These glycolipids had both a novel ceramide which contained almost entirely phytosphingosines (long-chain bases) and a high content of 2-hydroxy fatty acids. The long-chain base composition of the glycolipids shows an unusual pattern with both branched bases (iso-C16, iso-C17 iso-C18, anteiso-C17, and anteiso-C18) and normal ones (C16, C17, and C18). Of these, iso-C16- and anteiso-C18-phytosphingosines had been undetected up to now.
The glycolipids were identified as glucosyl and lactosyl ceramides. Lactosyl ceramide had not been previously reported in starfish, although the wide occurrence of this glycolipid has been demonstrated in other organisms.View full abstractDownload PDF (1812K) -
II. Properties of the Major Enzyme Purified from the LiverKazuo ASAOKA, Kenji TAKAHASHI1977 Volume 82 Issue 5 Pages 1313-1323
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The molecular and enzymatic properties of the major component (Fraction IV) of glutathione S-aryltransferases [EC 2. 5. 1. 13] purified from the liver of monkey (mainly rhesus monkey) have been investigated. The enzyme had a molecular weight of about 48, 000 and was composed of two subunits of apparently identical molecular weight (ca. 24, 000) bound to each other non-covalently. Each subunit contained one SH group. The amino acid composition showed characteristic high contents of leucine and glutamic acid residues. No amino-terminal residue was detected by the dansyl method.
2. The enzyme showed a rather broad optimum pH range from 7.5 to 9 with 1, 2-dichloro-4-nitrobenzene as a substrate. It was moderately stable below 40°C at pH 7.5. However, it showed an anomalous instability at pH around 4.2. It was reversibly denatured at least partially by urea or guanidine hydrochloride and irreversibly denatured by sodium dodecylsulfate. It was significantly inhibited by Zn2+, Cd2+, and Hg2+, and also by benzene hexachloride. It was extensively inactivated by reaction with phenylglyoxal or 2, 4, 6-trinitrobenzene sulfonate, whereas several SH reagents were without marked effect on the activity under the reaction conditions employed.View full abstractDownload PDF (1178K) -
Yousuke SEYAMA, Takeshi KASAMA, Tamio YAMAKAWA, Akihiko KAWAGUCHI, Kaz ...1977 Volume 82 Issue 5 Pages 1325-1329
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of hydrogen incorporation into fatty acids was investigated with an enzyme preparation from baker's yeast. Fatty acids synthesized from malonyl-CoA and acetyl-CoA in the presence of D2O or stereospecifically deuterium-labeled NADPH were isolated and analyzed by mass chromatography to examine the localization of deuterium atoms in the molecule. The following results were obtained:
1. Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom). The second hydrogen atom was incorporated as the result of hydrogen exchange phenomenon between the methylene group of malonyl CoA and water.
2. HB hydrogen of NADPH was used for β-ketoacyl reductase.
3. HB hydrogen of NADPH was also used for enoyl reductase.
4. Hydrogen atoms from HB position of NADPH were found on the odd-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom).View full abstractDownload PDF (302K) -
Yosinobu NAGAO, Tosifusa TODA, Kaoru MIYAZAKI, Takekazu HORIO1977 Volume 82 Issue 5 Pages 1331-1345
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Spleen-type (M2-type) pyruvate kinase was purified and crystallized from Rhodamine sarcoma of rats by a procedure involving affinity chromatography on a P-cellulose column and isoelectric separations in the presence and absence of fructose 1, 6-diphosphate (FDP).
2. In sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, the purified enzyme migrated, forming a single band corresponding to a molecular weight of approximately 60, 000.
3. In molecular-sieve chromatographies of 0.16-0.79mg/ml of the enzyme in the absence of FDP on a Sephadex G-200 column, the enzyme could exist as monomer, dimer and tetramer, all of which were enzymically active. It existed as a mixture of monomer and dimer at 0.16mg/ml and as a mixture of dimer and tetramer at 0.79mg/ml. In the presence of FDP, all the enzyme existed as tetramer at all the concentrations tested, having a molecular weight of approximately 240, 000.
4. It was previously reported that the enzyme possessed two different kinds of FDP-binding sites (1st and 2nd sites). It was found that the rate of binding of FDP to the 1st site was remarkably slow, whereas that to the 2nd site was rapid provided that the 1st site had been bound with FDP. The Km values for FDP in its binding to the 1st and 2nd sites were nearly the same, 3_??_4×10-7M.
5. The FDP bound to the 2nd site was rapidly dissociable, but the FDP bound to the 1st site hardly so. The amount of FDP bound to the 1st site was approximately 2 mol per mol of tetramer. Perhaps, a maximum of 4 mol of FDP per mol of tetramer could be bound.
6. Of the several salts, with which the enzyme was preincubated in the presence and absence of FDP, 0.3M LiCl, NaCl, KCl, RbCl, NH4Cl, (NH4)2SO4, KI, KHCO3, and Tris-HCl increased the Vmax significantly to nearly the same extent.View full abstractDownload PDF (1558K) -
Norio IWATSUKI1977 Volume 82 Issue 5 Pages 1347-1359
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDeoxythymidine kinase [EC 2. 7. 1. 21] activity in Escherichia coli increased in response to infection with T even phages but not with T odd phages. This was due to the formation of a new enzyme distinct from the deoxythymidine kinase of E. coli. Among the phages tested, T4 am N82 (gene 44) induced the enzyme most strikingly. Phage-induced deoxythymidine kinase was purified 440-fold from a crude extract of T4 am N82-infected E. coli KY896 (tdk-: deoxythymidine kinase deficient) by streptomycin treatment, ammonium sulfate fractionation, and DEAE- and phosphocellulose chromatographies. The molecular weight of the enzyme was estimated to be about 86, 000 by Sephadex G-200 gel filtration. The enzyme seemed to be composed of subunits with a molecular weight of about 28, 000.
The partially purified enzyme had a pH optimum at 6.8 and was stimulated several-fold by Mg2+, Mn2+, and Co2+. Deoxythymidine served as a good phosphate acceptor but other deoxynucleosides and 5-halogenated deoxyuridines did not. As phosphate donors, dATP and dGTP were about 50% as active as rATP. dTTP was a potent inhibitor of the enzyme, and the inhibition was highly dependent on pH. No remarkable activation was found with various nucleotides. The saturation curve for ATP was sigmoidal. Only one Km value for deoxythymidine (7.0×10-5M) was obtained from Lineweaver-Burk plots with an ATP concentration of 1.43mM, whereas two Km values (3.3×10-5M and 2.4×10-4M) were found with 0.46mM ATP. When dTTP was present, two Km values (7.0×10-5M and 4.2×10-4M) were found even at 1.43mM ATP. Unlike the E. coli enzyme, T4 deoxythymidine kinase was inactivated quickly above 50°C but was stable at moderate temperatures, and no drastic conformational change was detected even though in the presence of regulatory nucleotides.View full abstractDownload PDF (897K) -
Shinya YOSHIKAWA, Tetsuji UENO, Tanhaku SAI1977 Volume 82 Issue 5 Pages 1361-1367
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reaction of an oxygenated form of cytochrome oxidase [EC 1. 9. 3. 1] with cyanide was examined under conditions where spontaneous decay was prevented. The equilibrium and kinetic constants for the reaction agreed well with those for the normally operating enzyme, indicating that the oxygenated form is one of the active intermediates of the cytochrome oxidase reaction.View full abstractDownload PDF (477K) -
VIII. Correlation between Drug Accumulation and Acidic PhospholipidsYuji MATSUZAWA, Akira YAMAMOTO, Susumu ADACHI, Mitsuo NISHIKAWA1977 Volume 82 Issue 5 Pages 1369-1377
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of 4, 4'-bis (β-diethylaminoethoxy) α, β-diethyldiphenylethane (DH) on lipid metabolism in the liver differed considerably in different animal species, humans, monkeys, and rats, because of differences in drug-metabolizing ability.
Monkeys retain considerable drug-metabolizing ability as compared with humans, but the DH-hydroxylating activity in monkeys seems to be much lower than in rats. The hydroxyl derivative was the major substance which accumulated in rat liver following the administration of DH, while DH itself and its N-dealkylated substances accumulated in monkey liver. N-Dealkylated substances were also observed in human liver, but the amount was much smaller than in monkeys.
Bis (monoacylglyceryl) phosphate (BMGP), which is characteristic of this kind of druginduced lipidosis, did not increase as much in monkey liver as in human liver, but a marked increase in phosphatidyl inositol (PI) was observed in monkey liver during administration of DH.
The concentration of acidic phospholipids (BMGP+PI) in liver showed a close correlation with the accumulation of the drug (DH+its metabolites), irrespective of species differences.
Among subcellular particles isolated from a monkey liver following administration of DH, the crude mitochondrial fraction, including lysosomes, was richest in BMGP.View full abstractDownload PDF (584K) -
Yoshikazu AYAKI1977 Volume 82 Issue 5 Pages 1379-1388
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of estradiol-17β on the metabolism of bile acids has been studied in male rats.
1. A 3-day acute treatment with 100μg/100g body weight of the hormone caused a decrease in serum cholesterol and an increase in hepatic cholesterol. A chronic treatment with the same quantity twice a week for 3 weeks induced hypercholesteremia, which continued for 10 days after the final dosage, but there was no change in hepatic total cholesterol.
2. Fistula bile of both groups of treated rats collected during 30min was analyzed for bile acid components. Hyodeoxycholic acid and/or chenodeoxycholic acid increased relative to cholic acid in both groups, as compared with the controls.
3. Kinetic study of the metabolism of bile acids in the chronically treated rats revealed a more than twofold enlargement of the pool size but an almost unchanged half-life of chenodeoxycholic acid. As for cholic acid, essentially no change in pool size or half-life was observed.
4. These results indicate that chronic treatment of male rats with estradiol-17β causes an increase in elimination of hepatic cholesterol via the bile acid pathway.View full abstractDownload PDF (723K) -
Tsutomu UNEMOTO, Maki HAYASHI, Makoto HAYASHI1977 Volume 82 Issue 5 Pages 1389-1395
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane-bound NADH oxidase activities from slightly halophilic marine Vibrio alginolyticus and moderately halophilic V. costicolus required 0.3 and 0.5M Na+, respectively, for maximum activity; other cations such as Li+, K+, Rb+, Cs+, Mg2+, and Ca2+ were relatively ineffective as replacements for Na+. The concentration of Na+ required to give half-maximum activity with the NADH oxidase from V. alginolyticus was 82mM. This value was reduced to 6.4 and 13.8mM in the presence of 400mM K+ and 10mM Mg2+, respectively, indicating that K+ and Mg2+ cooperated with Na+ for activation. The same pattern of cation dependence was observed with the NADH oxidase from V. costicolus. The NADH oxidase from nonhalophilic Escherichia coli, however, had no specific requirement for Na+. Thus, Na+-dependent activation of NADH oxidase appeared to be a characteristic feature of these halophilic bacteria.
All NADH oxidases examined were influenced by the species of anion present and the order of activating effect followed the lyotropic series: SO42-, CH3COO->Cl->NO3->SCN-. Chaotropic anions such as NO3- and SCN- were inhibitory to the NADH oxidases, irrespective of the halophilic nature of the bacterial source.View full abstractDownload PDF (506K) -
Kenji ENDO, Hiroyuki KONDO, Makoto ISHIMOTO1977 Volume 82 Issue 5 Pages 1397-1402
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSulfite formation from benzenesulfonate was studied in extracts from a bacterium grown on this compound as a main carbon source. The activity of sulfite formation depended on the presence of NADH and oxygen as well as magnesium, suggesting an oxygenation-type reaction. The activity was found in a fraction precipitated by ammonium sulfate at 35-50% saturation; the specific activity was 15 times higher than that of crude extract, probably due to the elimination of inhibitory substances of low molecular weight from the preparation. In a distillate of the reaction mixture, phenol was found. Pyrocatechol as well as benzenesulfonate was oxidized in the crude extract, but phenol was not.View full abstractDownload PDF (448K) -
I. Subcellular Localization, Biosynthesis, and Intracellular Translocation of Glutamate DehydrogenaseKaname KAWAJIRI, Tomoyuki HARANO, Tsuneo OMURA1977 Volume 82 Issue 5 Pages 1403-1416
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The presence of glutamate dehydrogenase in the microsomal fraction of rat liver was confirmed. The identities of mitochondrial and microsomal glutamate dehydrogenases were proved by immunochemical methods and by SDS polyacrylamide gel electrophoresis of purified enzymes.
2. Synthesis of glutamate dehydrogenase by the membrane-bound ribosomes of rough endoplasmic reticulum was determined. Newly synthesized enzyme molecules were discharged on the cytoplasmic surface of endoplasmic reticulum membranes.
3. A precursor-product relationship was found between microsomal and mitochondrial glutamate dehydrogenases. About six hours were needed for the transport of glutamate dehydrogenase from the site of synthesis to mitochondria.
4. The half-life of glutamate dehydrogenase was about 5.5 days, which was somewhat longer than that of mitochondrial total protein determined in the same experiment.
5. Mitochondrial-type malate dehydrogenase was also present in the microsomal fraction. Subfractionation of smooth microsomes revealed the existence of particular light microsomal vesicles in which both glutamate dehydrogenase and malate dehydrogenase were concentrated. These vesicles may participate in intracellular transport of matrix enzymes from microsomes to mitochondria.View full abstractDownload PDF (1843K) -
II. Significance of Binding of Glutamate Dehydrogenase to Microsomal MembraneKaname KAWAJIRI, Tomoyuki HARANO, Tsuneo OMURA1977 Volume 82 Issue 5 Pages 1417-1423
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Glutamate dehydrogenase and malate dehydrogenase solubilized from liver microsomes were able to rebind to microsomal vesicles while the corresponding dehydrogenases extracted from mitochondria showed no affinity for microsomes.
2. Competition was noticed between microsomal glutamate dehydrogenase and microsomal malate dehydrogenase in the binding to microsomal membranes. Mitochondrial malate dehydrogenase or bovine serum albumin did not inhibit the binding of microsomal glutamate dehydrogenase to microsomes.
3. Binding of microsomal glutamate dehydrogenase to microsomal membranes decreased when microsomes was preincubated with trypsin.
4. Rough microsomal glutamate dehydrogenase was more efficiently bound to rough microsomes than smooth microsomes. Conversely, smooth microsomal glutamate dehydrogenase had higher affinity for smooth microsomes than for rough microsomes.
5. A difference was noticed among the glutamate dehydrogenases isolated from rough and smooth microsomes, and from mitochondria, which suggested the possibility of minor posttranslational modification of enzyme molecules in the transport from the site of synthesis to mitochondria.View full abstractDownload PDF (527K) -
Yoshihiro UCHIDA, Yoji TSUKADA, Tsunetake SUGIMORI1977 Volume 82 Issue 5 Pages 1425-1433
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNeuraminidase [sialidase, EC 3. 2. 1. 18] was found to be widely distributed in bacteria belonging to Arthrobacter. Among these bacteria, Arthrobacter ureafaciens, A. oxydans, and A. aurescens produced relatively potent neuraminidase activities. For the production of this enzyme, not only colominic acid, a homopolymer of N-acetylneuraminic acid, but also N-acetylneuraminic acid, the reaction product of this enzyme, are effective as sources of carbon.
An affinity adsorbent specific for neuraminidase was prepared by cross-linking colominic acid with soluble starch by means of epichlorohydrin. Neuraminidase from A. ureafaciens could be purified on this affinity column. The purified neuraminidase was shown to be free from protease, N-acetylneuraminic acid aldolase, phospholipase C, and glycosidases. Aminoff's assay procedure for sialic acid was modified to avoid the centrifugation step. The modified procedure gave a higher molecular extinction coefficient.View full abstractDownload PDF (1232K) -
II. Effect of the Interaction with Actin on the Reactivities of Light Chain SH GroupsKeiichi YAMAMOTO, Takamitsu SEKINE1977 Volume 82 Issue 5 Pages 1435-1441
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the role of light chains in the actin-myosin interaction, we measured the changes in reactivities of the SH groups of light chains with a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)maleimide, under various conditions differing in the manner of actin-myosin interaction and in the activity of actin-activated Mg2+-ATPase at 20°C. The reactivity was not greatly influenced by changes in the manner of protein aggregation. An apparent difference observed between the states before and after superprecipitation was mainly attributed to the resulting ADP. The changes in reactivities of alkali light chains apparently depended on the actin-activated Mg2+-ATPase activity of myosin during the reaction with the thiol reagent. Since it can be considered that the population of actin-myosin complex in the reaction solution is larger at high ATPase activity of myosin than at low ATPase activity, alkali light chains are supposed to participate in the actin-myosin interaction. The change in alkali light chain-2 was more marked than that in alkali light chain-1. However, the finding that alkali light chains also showed large changes at 0°C, where the actin-activated Mg2+-ATPase activity of myosin was very low, suggests much more complicated mechanisms for such reactivity changes. Although the mechanisms of the reactivity changes in light chains and actin are not clear, these results agree well with our previously reported observations using glycerinated muscle.View full abstractDownload PDF (482K) -
Toshio KAYA, Masumi AIKAWA, Tomoko MATSUMOTO1977 Volume 82 Issue 5 Pages 1443-1449
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe yeast α-mannosidase [EC 3. 2. 1. 24] was purified 1160-fold from the crude extract of the autolysate. The purified preparation was practically free from α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, β-mannosidase, and β-N-acetylhexosaminidase activities. After the separation of yeast mannan during the purification procedures the enzyme became unstable but could be stored at 5°C for three weeks with 50% loss of activity. The purified enzyme hydrolyzed both aryl and alkyl mannosides, but hydrolysis of yeast mannan proceeded slowly. Yeast mannan and Zn2+ increased the enzyme catalyzed hydrolysis of p-nitrophenyl mannoside, whereas NaN3, monoiodoacetate and methyl α-D-mannoside acted as inhibitors. The molecular weight was estimated to be 450, 000 by gel filtration.View full abstractDownload PDF (470K) -
II. Purification and Some Properties of the PhageYoshiyuki SAKAKI, Kazuko YAMADA, Mieko OSHIMA, Tairo OSHIMA1977 Volume 82 Issue 5 Pages 1451-1456
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn improved procedure for the purification of a lipid-containing acidophilic thermophilic bacteriophage, φNS 11, is described. The procedure involves Cs2SO4 discontinuous density gradient, Cs2SO4 equilibrium density gradient and sucrose gradient centrifugations. The phage has an icosahedral shape, 75nm in diameter, with spike-like structures radiating from the vertices. It contained about 12% by weight of lipid, 13-14% of DNA and 74-75% of protein. The phage was stable between pH 2 and 5, but rapidly inactivated above pH 6. It was highly sensitive to organic solvents and detergents. Some other basic properties such as the buoyant density and UV spectrum of the purified phage are also described.View full abstractDownload PDF (886K) -
III. Characterization of Viral ComponentsYoshiyuki SAKAKI, Mieko OSHIMA, Kazuko YAMADA, Tairo OSHIMA1977 Volume 82 Issue 5 Pages 1457-1461
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComponents of a lipid-containing phage φNS 11 were characterized. The phage had five protein components, the molecular weights of which were 59, 000, 44, 000, 33, 000, 23, 000, and 18, 000. Viral lipid consisted of six components, which were also found in the host bacterial lipid. The relative amounts of these viral lipid components were very similar to those of the bacterial lipid. The phage contained ω-cyclohexyl fatty acids characteristic of Bacillus acidocaldarius as the main fatty acids. The phage nucleic acid was a linear double-stranded DNA, the molecular weight of which was 9.3-9.4×106 daltons. The guanine plus cytosine content of the DNA was determined to be about 52% from chemical analysis, buoyant density (1.711g/cm3 in CsCl) and melting temperature (90.6°C in 0.15M NaCl plus 0.015M sodium citrate). The phage contained two kinds of polyamine; spermidine and spermine.View full abstractDownload PDF (1479K) -
Evidence for the Presence of Myosin Isozymes in Rabbit Skeletal MuscleKoichi YAGI, Sadayuki MATSUDA, Hideto KUWAYAMA1977 Volume 82 Issue 5 Pages 1463-1467
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHeavy meromyosin prepared from rabbit skeletal myosin by chymotryptic digestion was separated into two different heavy meromyosins by Sepharose 4B-6 aminohexyl PP1 column chromatography. SDS-gel electrophoresis of one fraction of heavy meromyosin, which was eluted with 75mM ammonium acetate, showed that it contained the small polypeptide chains, g3 and g2, as well as the large chains. The other fraction of heavy meromyosin, which was eluted with 85mM ammonium acetate, contained g1 and g2. We concluded that the two heavy meromyosins arose from two different populations (isozymes) of myosin. No significant difference in Ca2+-ATPase activity was detected between the two heavy meromyosins.View full abstractDownload PDF (781K) -
A Photochemically Stable Electron Mediator between NADH and Various Electron AcceptorsRyuki HISADA, Tatsuhiko YAGI1977 Volume 82 Issue 5 Pages 1469-1473
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis paper describes the properties and application of 1-methoxy-5-methylphenazinium methyl sulfate (1-methoxyPMS), which is a photochemically stable, versatile electron carrier. Like 5-methylphenazinium methyl sulfate (PMS), it mediates electron transfer between NADH and various electron acceptors such as tetrazolium dyes or the electrode of an enzymic electric cell, and yet it does not deteriorate upon storage under scattered light in normal laboratories. The rate of reduction of 1-methoxyPMS coupled to the reoxidation of NADH produced by the lactate dehydrogenase reaction, was even faster than that of PMS. It was also successfully employed as an electron mediator in the enzymic electric cell method for the assay of NAD-linked dehydrogenases. 1-MethoxyPMS solution is rosy pink, and its standard redox potential (Eo') is approximately +0.063V. The use of 1-methoxyPMS will be beneficial in biochemistry as well as medical technology, where PMS has been used as an electron mediator in various electron transfer systems.View full abstractDownload PDF (339K) -
IV. Quantitative Comparison of Affinity Adsorbents Containing Various Arginine PeptidesMakoto NISHIKATA, Ken-ichi KASAI, Shin-ichi ISHII1977 Volume 82 Issue 5 Pages 1475-1484
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to study the mechanism of substrate binding of trypsin by affinity chromatography, we synthesized various L-arginine-terminated oligopeptides having different chain length and amino acid sequences, and immobilized them on agarose gel. The interaction of β-trypsin with these adsorbents was studied by a quantitative affinity chromatographic procedure which gave the dissociation constant (Kd) of the trypsin-immobilized ligand complex. This procedure proved to be very useful and to give information equivalent to that obtained by kinetic procedures. The contribution of the amino acid residue at P2 of the ligands to the affinity was studied by using tripeptide (Gly-X-Arg) Sepharoses, and alanine was found to be more effective than glycine or valine. This conclusion was supported by a kinetic experiment in which K1 values of the corresponding soluble tripeptides (Ac-Gly-X-Arg) were determined. A significant decrease in Kd was observed when the ligand was elongated from dipeptide to tripeptide. However, Kd decreased only slightly when the ligand was elongated further. This suggests that a tripeptide is sufficiently long as a ligand. On the basis of these results, the mode of subtsrate binding of trypsin is discussed.View full abstractDownload PDF (817K) -
Takashi YOKOTA, Ken-ichi ARAI, Yoshito KAZIRO1977 Volume 82 Issue 5 Pages 1485-1489
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of 30S ribosomal protein S1 on poly (U)-directed polyphenylalanine synthesis was studied using a highly purified cell-free system which was devoid of endogenous S1. The system consisted of homogeneous preparations of EF-Tu, EF-Ts, and EF-G, and 70S ribosomes from which protein S1 had been removed by poly (U)-cellulose column chromatography. It was found that protein S1 was indispensable for translation of poly (U) by an S1-depleted system at low concentrations of poly (U). On the other hand, at higher concentrations of poly (U), a considerable amount of polyphenylalanine was synthesized in the absence of added S1. The stimulatory effect of S1 was observed at all Mg2+ concentrations examined but was most pronounced at 10mM Mg2+. Some physicochemical properties of the protein were also studied. It was demonstrated that the protein has an elongated shape with an axial ratio of approximately 8.5.View full abstractDownload PDF (353K) -
II. Fluorescence of the Tryptophanyl GroupsTakashi SHIMIZU1977 Volume 82 Issue 5 Pages 1491-1494
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe intrinsic fluorescence of bovine platelet myosin was found to change upon addition of the substrate or its analogs in a manner similar to that of rabbit skeletal muscle myosin. The result suggests that the mechanism of ATP cleavage is analogous for myosins from diverse origins.View full abstractDownload PDF (269K) -
Takashi MORITA, Hisao KATO, Sadaaki IWANAGA, Katsumi TAKADA, Terutoshi ...1977 Volume 82 Issue 5 Pages 1495-1498
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwenty peptide-4-methylcoumarin amides (MCA) were newly synthesized and tested as possible substrates for α-thrombin, factor Xa, kallikreins, urokinase, and plasmin. These fluorogenic peptides contained arginine-MCA as the carboxyl-terminus. Release of 7-amino-4-methylcoumarin was determined fluorometrically. Of these peptides, the following were found to be specific substrates for individual enzymes: Boc-Val-Pro-Arg-MCA for α-thrombin, Boc-Ile-Glu-Gly-Arg-MCA, and Boc-Ser-Gly-Arg-MCA for factor Xa, Z-Phe-Arg-MCA for plasma kallikrein, Pro-Phe-Arg-MCA for pancreatic and urinary kallikreins, and glutaryl-Gly-Arg-MCA for urokinase. Moreover, these peptide-MCA substrates were resistant to plasmin.View full abstractDownload PDF (253K) -
Kazuyuki SUGAHARA, Moriaki AKASAKI, Ikuo FUNAKOSHI, Pertti AULA, Ikuo ...1977 Volume 82 Issue 5 Pages 1499-1501
Published: November 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structures of two glycoasparagines composed of one mole each of N-acetylneuraminic acid, galactose, N-acetylglucosamine, and asparagine were determined by periodate oxidation, enzymatic degradation, and methylation analysis. The structures were NANAα2→3Galβ1→4GlcNAcβ→Asn and NANAα2→6Galβ1→4GlcNAcβ→Asn, respectively.View full abstractDownload PDF (183K) -
1977 Volume 82 Issue 5 Pages 1503
Published: 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (43K) -
1977 Volume 82 Issue 5 Pages 1504
Published: 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (16K)
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