巻号一覧
87 巻, 3 号
選択された号の論文の38件中1～38を表示しています
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Tohru TAKASHI, Keizo INOUE, Shoshichi NOJIMA1980 年 87 巻 3 号 p. 679-685
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe relation between the immune reactions of phosphatidylcholine liposomes containing cardiolipin with or without cholesterol and the physical state of the liposomes was studied. In egg yolk phosphatidylcholine liposomes, both immune agglutination and complement-mediated immune damage occurred irrespective of the presence of cholesterol. Immune agglutination also occurred at all temperatures tested (0-40°C). On the other hand, in dipalmitoylphosphatidylcholine liposomes, both reactions depended on the cholesterol content, but immune agglutination occurred even in the absence of cholesterol above the phase transition temperature at which liposomal membranes became fluid. The binding of antibodies to liposomes was found to be independent of the physical state of the liposomal membranes.
The requirement of the fluid state of liposomal membranes for immune reactions is discussed.抄録全体を表示PDF形式でダウンロード (544K) -
Kayoko FUKUDA, Motowo TOMITA, Akira HAMADA1980 年 87 巻 3 号 p. 687-693
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe O-glycosidically linked carbohydrate units of glycophorin derived from horse erythrocyte membrane were released as reduced oligosaccharides by alkaline borohydride treatment. One tetrasaccharide, two trisaccharides, and two disaccharides were purified by gel filtration and ion-exchange chromatography.
Studies employing periodate oxidation, methylation analysis and gas-liquid chromatog-raphy-mass spectrometry revealed the following structures for these oligosaccharides; a tetra-saccharide; N- glycolylneuraminyl-(2→3)-D-galactopyranosyl-(1→3)-[N-glycolylneuraminyl-(2→6)]-D-N-acetylgalactosaminitol, trisaccharides; N-glycolylneuraminyl-(2→3)-D-galacto-pyranosyl-(1→3)-D-N-acetylgalactosaminitol and D-galactopyranosyl-(1→3)-[N-glycolyl-neuraminyl-(2→6)]-D-N-acetylgalactosaminitol, disaccharides; D-galactopyranosyl-(1→3)-D-N-acetylgalactosaminitol and N- glycolylneuraminyl-(2→3)-D-galactitol.抄録全体を表示PDF形式でダウンロード (506K) -
II. Affinity Chromatography and Cooperative LigationsYoshikazu SUKENAGA, Hiroshi AKANUMA, Chizuko SUEKANE, Makoto YAMASAKI1980 年 87 巻 3 号 p. 695-707
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーAffinity chromatography was used to characterize the binding properties of carboxypeptidase B with its ligands. The affinity adsorbents employed included arginine directly attached to agarose beads, arginine attached to the same support through hydrophilic and hydrophobic spacers, and immobilized caproylphenylalanine. The enzyme showed marked retardation on all of the arginine columns but only slight retardation on the phenylalanine column. Several hydrophobic ligands in solution enhanced to some extent the enzyme retardation on columns having arginine directly attached to the solid support, while several amino- and guanidino-alkylcarboxylic acids (lysine and arginine analogs) greatly enhanced the enzyme retardation on the phenylalanine column and somewhat enhanced it on the other columns having hydrophobic spacer arms. These observations confirmed that the enzyme has two binding sites for the soluble and immobilized ligands and that these two sites exhibit cooperative ligations. Binding constants of the enzyme for various soluble ligands were also calculated from their chromatographic effects and the resulting values were interpreted in terms of the cooperative action of the two binding sites, i.e., one for the primary binding of basic amino acid analogs (Site I) and the other for hydrophobic compounds (Site II). In this chromato-graphic study, however, such cooperation of the two sites was obscure when arginine, acylarginine, or alkylarginine was the ligand directing to Site I.抄録全体を表示PDF形式でダウンロード (980K) -
Kazuo NAGASAKI, Michiki KASAI1980 年 87 巻 3 号 p. 709-716
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe Ca2+ and Mg2+ permeabilities of sarcoplasmic reticulum vesicles were measured by using a fluorescence chelate probe, chlortetracycline. The Mg2+ permeability was studied exten-sively. The data were analyzed under the assumption that the increment of fluorescence intensity is proportional to the amount of the divalent cations inside the vesicles. The validity of this assumption was confirmed by the tracer method with Ca2+ The presence of Ca2+ or Mg2+ had little effect on the Mg2+ efflux, but greatly affected the Ca2+ efflux. In the absence of extravesicular divalent cations, the permeabilities for Ca2+ and Mg2+ were almost the same, but the Ca2+ permeability was slightly reduced by the presence of Mg2+ and greatly reduced by Ca2+. In the absence of extravesicular divalent cations, the Ca2+ and Mg2+ permeabilities increased in parallel with increasing pH. For example, the permeation times for Mg2+ and Ca2+ were about 3 min at pH 6.5 and 20 s at pH 8.1 at room temperature. Kinetic analysis of the fluorescence change suggested the existence of two kinds of vesicles with different permeabilities.抄録全体を表示PDF形式でダウンロード (549K) -
Norio MUTO, Kunio MURAYAMA, Kiso AKAHANE, Satoru TANI1980 年 87 巻 3 号 p. 717-723
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーBiochemical and immunological properties of two kinds of pepsinogens isolated from the gastric mucosal extracts of adult Wistar rats were studied. Their activated enzymes were prepared from the zymogens using a DEAE-Sepharose CL-6B column. The isoelectric points of pepsinogens I and II were estimated to be 3.90 and 3.75, respectively, by isoelectric focusing, and those of pepsins I and II to be 3.60 and 3.45, respectively. Amino acid compositions of the two pepsinogens or pepsins were strikingly similar to each other and neither pepsinogen I nor II contained organic phosphate. The biochemical properties of rat preparations compared with porcine pepsinogens A and C and pepsins A [EC 3. 4. 23. 1] and C [EC 3. 4. 23. 3] showed that rat pepsinogens and pepsins resembled porcine pepsinogen C and pepsin C, respectively. Pepsinogens I and II were demonstrated to share a similar immunogenic molecular structure by double diffusion analysis and Laurell immunoelectrophoresis. Rabbit antipepsinogen I serum cross-reacted with the mouse preparation but did not with the rabbit and porcine preparations. The possibility of the genetically controlled occurrence of pepsinogens I and II in the rat is discussed.抄録全体を表示PDF形式でダウンロード (931K) -
Purification and CharacterizationTakashi KAGEYAMA, Kenji TAKAHASHI1980 年 87 巻 3 号 p. 725-735
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーA cathepsin D-like acid proteinase from human gastric mucosa was purified for the first time to homogeneity as examined by polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 85, 000 by gel filtration on Sephadex G-150 and, after treatment with 2-mercaptoethanol, about 38, 000 by sodium dodecyl sulfate-poly-acrylamide disc gel electrophoresis. These results indicate that the native enzyme is composed of two apparently identical monomeric units. The protein band could also be stained with the Schiff reagent after periodate oxidation, indicating that the proteinase is a glycoprotein. Analyses showed that the enzyme contains approximately 4 glucosamine and 16 mannose residues per molecule (M.W. 85, 000). The amino acid composition generally resembled
those of cathepsins D except for the lower contents of basic amino acid residues, especially lysine. It also showed some similarity to pepsinogens. The optimal pH was 2.0-3.5 with hemoglobin as a substrate. The enzyme was strongly inhibited, like pepsin, by 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as well as by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. The proteinase was stable in alkaline solution up to pH 9.0, and was rather stable to sequential acidification and neutralization. The acidification resulted in an increase of electrophoretic mobility towards the anode.抄録全体を表示PDF形式でダウンロード (1438K) -
Akihiko MORIYAMA, Kenji TAKAHASHI1980 年 87 巻 3 号 p. 737-743
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe distribution of acid proteases in primate lungs and some other tissues was investigated with bovine hemoglobin as a substrate at pH 3.0. The activity in lung per tissue weight as well as per protein weight varied about 10-fold among different animals, but there was not much difference between primates and non-primates. The total acid protease activity was highest in the human lung. These activities were generally inhibited over 90% by pepstatin. Upon chromatography on a DEAE-cellulose column, the activity was fractionated into one or two major peaks. The activity due to ordinary cathepsin D (cathepsin D-I type protease) was eluted near the break-through position and showed pH-activity profiles with an optimum at pH 3.0 to 4.0. This protease fraction was present in all the animals examined. In addition, a second major peak of activity was observed in most primate lungs examined, and was thought to correspond to cathepsin D-II of Japanese monkey lung, as reported previously (Moriyama, A. and Takahashi, K. (1978) J. Biochem. 83, 441-451). This fraction gave a typical pH-activity profile with an optimum at pH 2.0-3.0; at pH 1.0 it showed about 50 to 90%. of the maximum activity. This protease fraction was not found in the lungs of non-primates nor in various tissues other than the lungs of Japanese monkey. Therefore this cathepsin D-II type protease appears to be specifically present in some primate lungs.抄録全体を表示PDF形式でダウンロード (501K) -
Temperature Dependence of Acitivity and StabilityYuzuru SUZUKI, Norihisa NAKAMURA, Takashi KISHIGAMI, Shigeo ABE1980 年 87 巻 3 号 p. 745-751
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーA p-nitrophenyl-α-D-glucopyranoside-hydrolyzing α-glucosidase from an obligate thermophile, Bacillus thermoglucosidius KP 1006, gave a triphasic relationship at pH 6.8 in the van't Hoff plot of Km, in the Arrhenius plot of the first order rate constant of inactivation with 0.1% sodium dodecyl sulfate, and in the logarithmic plot of the maximal fluorescence intensity at 346 nm versus reciprocal of temperature. The respective plots exhibited two breaks at 40 and 61°C, 43 and 62°C, and 40 and 61°C. However, the Arrhenius plot of the molecular activity at pH 6.8 had a single discontinuity at 64°C. These findings, together with thermodynamic quantities for the enzyme, suggest that the thermal conformational changes in the enzyme protein occur around 40-43°C and 61-64°C. The Arrhenius plot of the rate constant of heat inactivation at pH 6.8 was bent at 73°C. Thermodynamic data indicate that the enzyme is transformed from a heat stable form into a heat unstable form at 73°C with temperature elevation. The critical points localized near the minimum, optimum, and maximum temperatures (40, 60, and 72°C) of the cell growth, respectively.抄録全体を表示PDF形式でダウンロード (495K) -
Kenji KITAMURA, Akira ITO, Yo MORI1980 年 87 巻 3 号 p. 753-760
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe existing forms of collagenase [EC 3. 4. 24. 7] in the human uterine cervix were examined. The latent collagenase extracted by homogenization in 0.25% Triton X-100 containing 0.01M CaCl2 was indicated to be a complex of collagenase with α2-macroglobulin by the behavior of the fraction of this enzyme before and after treatment with NaSCN on Sephadex G-150 column chromatography and an immunodiffusion method. The active collagenase was extracted by rehomogenization in 50mM Tris-HCl buffer, pH 7.4, containing 0.1M CaCl2 from the insoluble residue at 0°C. Another latent collagenase was extracted from the insoluble fraction in the same buffer by heating at 60°C for 4min and this enzyme was activated by 4-aminophenylmercuric acetate or trypsin. The molecular weights of the active and the latent forms were approximately 7.3×104 and 9.4×104, respectively. This indicates that the latency is due to the formation of a low molecular weight inhibitor enzyme complex.
These results clarified that the human uterine cervix contains three existing forms (α2-macroglobulin complex, active form and low molecular weight inhibitor complex) of col-lagenase under these experimental conditions.抄録全体を表示PDF形式でダウンロード (1081K) -
Koichi SUZUKI, Shigeo OHNO, Kazutomo IMAHORI1980 年 87 巻 3 号 p. 761-769
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe physicochemical nature of an active fragment (T2A) of colicin E3 was examined together with those of its specific inhibitor, B, and the T2A-B complex under various conditions by means of CD and fluorescence spectroscopy.
1. T2A was mainly composed of unordered structure, although the molecule was folded into a rather compact structure, probably by β-turns and ionic interactions.
2. The structure of T2A was not stable, and unfolding of the molecule was observed on treatments with acid, alkali, heat, guanidine hydrochloride, and detergents. However, once denaturing conditions were removed, the unfolded T2A molecule quickly regained the native conformation, recovering its full activity.
3. Formation of a one-to-one complex of T2A and B was confirmed. No gross confor-mational changes in T2A and B were found upon formation of the T2A-B complex.
4. A mechanism of dissociation of colicin E3 into A and B, or of T2A-B into T2A and B, during passage through the membrane of colicin E3-infected cells is proposed, based mainly on the results obtained here.抄録全体を表示PDF形式でダウンロード (604K) -
Seiki KURAMITSU, Kozo HAMAGUCHI, Shiro MIWA, Koji NAKASHIMA1980 年 87 巻 3 号 p. 771-778
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe pH difference absorption spectra of human lysozyme [EC 3. 2. 1. 17] were measured. The difference spectra in the acidic region had a peak at 300nm, as observed for hen and turkey lysozymes. The pH dependence curve of the extinction difference at 300nm was well interpreted in terms of the pK values of the catalytic groups (3.4 for Asp 52 and 6.8 for Glu 35 at 0.1 ionic strength and 25°C) determined from the pH dependence of the circular dichroism at 303.5nm (Kuramitsu et al. (1974) J. Biochem. 76, 671-683) and the fluorescence excited at 305nm (Kuramitsu et al. (1978) J. Biochem. 83, 159-170).
The difference spectra of human lysozyme in the alkaline pH region were characteristic of tyrosyl ionization. The perturbation of tryptophyl residues, which had been observed for hen and turkey lysozymes (Kuramitsu & Hamaguchi (1979) J. Biochem. 85, 443-456), was not observed for human lysozyme. On the basis of the pH dependence curves of the extinction difference at 245 and 295 nm, we roughly estimated the apparent pK values of the six tyrosyl residues as 9.2, 9.2, 10.5, 10.9, 12.4, and 12.5. A time-dependent spectral change observed above pH 11 was not due to the exposure of buried tyrosyl residues on alkali denaturation but was due mainly to disulfide cleavage and exposure of buried tryptophyl residues.抄録全体を表示PDF形式でダウンロード (503K) -
Michio NIINOBE, Yuji HITOMI, Setsuro FUJII1980 年 87 巻 3 号 p. 779-783
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーA convenient and highly sensitive colorimetric assay for various proteases, such as trypsin, chymotrypsin, plasmin, thrombin, and urokinase is described. The substrates used are α-naphthyl ester derivatives of Nα-tosyl-L-lysine, Nα-acetylglycyl-L-lysine, and Nα-acetyl-L-tyrosine, and activity is assayed by colorimetric determination of α-naphthol released from them. Use of these α-naphthyl ester derivatives made the method more sensitive than the use of the corresponding methyl or ethyl ester derivatives.
The minimum detectable concentrations of trypsin, chymotrypsin, plasmin, thrombin and urokinase in this method were about 0.002μg, 0.01μg, 0.002 CU, 0.01 IU, and 2 IU, respectively.
The Km values of trypsin and thrombin for TLNE were 0.11mM and 0.15mM, while those for TLME were 2.5mM and 6.7mM, respectively; the Km values of chymotrypsin for ATNE and ATEE were 0.18mM and 0.7mM, respectively; and the Km values of urokinase for AGLNE and AGLME were 0.17mM and 4mM, respectively.
Zymograms of various proteases were easy to prepare using these α-naphthyl ester substrates, and zymograms of trypsin and chymotrypsin were made with TLNE and ATNE, respectively, as substrates.抄録全体を表示PDF形式でダウンロード (880K) -
Issei MABUCHI, James A. SPUDICH1980 年 87 巻 3 号 p. 785-802
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーUnfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were homogenized in a buffer containing 0.1M KCl and 2mM MgCl2 at pH 6.85. About 50% of the actin was recovered in the high-speed supernate of the homogenate. More than 80% of the actin in this supernate was found to be monomeric upon gel filtration chromatography through a Sephadex G-150 column or by a DNase I inhibition assay. The critical concentration for polymerization of this actin prior to further purification was 0.3-0.9mg/ml under various conditions. Actin was purified to near homogeneity from the Sephadex G-150 pool with a high yield. The purified actin had a critical concentration for polymerization of 0.02-0.03mg/ml. The isoelectric point of the crude actin and the purified actin was the same. Indeed, we found that there is only one isoelectric focusing species of actin in the sea urchin egg, and it has an isoelectric point more basic than rabbit skeletal muscle actin. The discrepancy between the polymerizability of the crude and purified actin may be due to the presence of factors in the crude fraction which inhibit the polymerization of actin.抄録全体を表示PDF形式でダウンロード (3840K) -
Toshihiko HAYASHI, Yutaka NAGAI1980 年 87 巻 3 号 p. 803-808
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe anomalously low mobility of collagen peptides on SDS-gel electrophoresis was investi-gated, using tadpole skin collagen and bovine Type I, H, and III collagens.
The free electrophoretic mobility of al chains of collagen was found to be smaller than those of α2 chain and common proteins of similar size, which migrate on SDS-gel according to their molecular weights. The retardation coefficient of collagen peptides was normal. Therefore, the overall SDS-collagen complex may be comparable in size with SDS complexes of common proteins with similar molecular weights. One characteristic difference of collagen in comparison with common proteins is its low content of hydrophobic amino acid residues. This may account for the low free electrophoretic mobility in SDS of collagen al chains, if the SDS-protein complex is of a necklace type, not a rod-like type.抄録全体を表示PDF形式でダウンロード (916K) -
Toshihiko HAYASHI, Toshitaka NAKAMURA, Hisae HORI, Yutaka NAGAI1980 年 87 巻 3 号 p. 809-815
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe degradation rates of type I, II, and III collagens by tadpole collagenase were studied by measuring the viscosity of the solution and the contents of α chains and α chains of collagen, using SDS-polyacrylamide gel electrophoresis followed by densitometric analysis of the separated peptide bands. An empirical parameter was derived from the viscosity, and was shown to change in parallel with the content of α chains upon incubation with tadpole collagenase almost to the stage of complete digestion of collagen. Linear plots of parameters reflecting the concentration of intact collagen molecules against time were obtained, indicating the degradation to be pseudo-first order. The first-order rate constants for the degradation of Type I, II, and III collagens with tadpole collagenase at 30, 25, and 20°C gave activation energies of 60 kcal/mol for Type III collagen and 40 kcal/mol for Type I and II collagen. There appeared to be a dependency of the degradation rates on the conformation of the collagen molecules (which is affected by temperature).抄録全体を表示PDF形式でダウンロード (1616K) -
Contribution of Three-Dimensional Structure and Individual Amino Acid ResiduesMakoto SASAKI, Shinya TAKEDA, Taiji KATO, Kazuhisa MATSUBA1980 年 87 巻 3 号 p. 817-824
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe contribution of three-dimensional structure and individual amino acid residues to the antigenicities of macromolecular protein was investigated for a thiol protease stem bromelain as antigen. The extent of the participation was demonstrated by a decrease in antigenicity when the enzyme was denatured in 8 M urea before and after reductive cleavage of intrapeptide disulfide bonds or modified in particular amino acid residues. The results showed that the enzyme treated with 8M urea without reductive cleavage of disulfide bonds preserved about 90% of antigenicity to antibodies against native stem bromelain, while the enzyme denatured after the reductive cleavage of disulfide bonds brought about almost 80% disappearance of the antigenicity. Modification of individual amino acid side chains revealed that lysine was the most immunodominant amino acid, showing 2.5% contribution per residue, and tyrosine followed with 1.2%. However, acidic amino acids such as glutamic and aspartic acids were found to be as low as 0.3%, and tryptophan was 0.2%. These data suggest that most of the antigenic determinants of stem bromelain are of the steric conformation in which lysine and/or tyrosine are most frequently involved as immunodominant amino acids.抄録全体を表示PDF形式でダウンロード (607K) -
Akira ISHIHAMA, Masayoshi ENAMI, Yoshikazu NISHIJIMA, Toshikazu FUKUI, ...1980 年 87 巻 3 号 p. 825-830
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe effects of 2'-substitutions of ATP on the substrate and inhibitor properties for RNA synthesis were studied in the poly(dAT)-dependent reaction of Escherichia coli RNA polymerase. In the presence of UTP, 2'-deoxy-2'-azidoadenosine 5'-triphosphate (AzTP) was incorporated into an acid-insoluble fraction at one-tenth of the rate of ATP incorporation; it thus acts as a competitive inhibitor for poly(AU) synthesis. On the other hand, another ATP analog, 2'-deoxy-2'-fluoroadenosine 5'-triphosphate (AfTP), was co-polymerized with UTP into acid-insoluble materials at a rate less than 1% of that of ATP incorporation; in addition, it exerted a strong but mixed-type inhibition on poly(AU) synthesis. Different modes of action of the two ATP analogs are discussed in connection with the specificity of substrate recognition by RNA polymerase.抄録全体を表示PDF形式でダウンロード (481K) -
Old Yellow Enzyme-Phenol ComplexesYasuzo NISHINA, Teizo KITAGAWA, Kiyoshi SHIGA, Hiroshi WATARI, Toshio ...1980 年 87 巻 3 号 p. 831-839
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーResonance Raman scattering from the old yellow enzyme-phenol complexes was observed on exciting them at the characteristic charge transfer band of the complexes. An excitation profile of Raman intensities was obtained for the enzyme-pentafluoro phenol complex and was compared with that of riboflavin bound to riboflavin binding protein with no charge transfer band. The pentahalogenated and para-substituted phenols in the complex form commonly exhibited an intense Raman line of ring deformation mode (vυ6a) upon excitation at 568.2 nm. The pentahalogenated phenols displayed the Raman line of ring breathing mode (υl) but the para-substituted phenols did not. Other intensity-enhanced Raman lines of bound phenols were limited to in-plane modes including υ3, υ8a, υ12, υ14, and υ19a. Several Raman lines of FMN were also resonance-enhanced by the charge transfer band. The intensity enhance-ment was remarkable for the Raman lines at 1585 and 1550cm-1, which are known to involve the vibrational displacements of the N (5) and C (4a) atoms of isoalloxazine. The Raman line at 1628cm-1, a ring I mode, was little intensified while a few ring III modes were moder-ately intensified. These features of resonance enhancement of Raman intensity suggest that π electrons of the benzene ring interact as a whole with ring III of isoalloxazine in a geometry of parallel arrangement of their molecular planes with phenolate oxygen above the N (5)-C (4a) bond. A relatively localized interaction between oxygen of phenolate and a particular atom of FMN appears unlikely.抄録全体を表示PDF形式でダウンロード (682K) -
Hirosuke FUKUDA, Mitsuaki MORIGUCHI, Tatsurokuro TOCHIKURA1980 年 87 巻 3 号 p. 841-846
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーGlyoxylate reductase II was purified about 2, 400-fold from a cell extract of baker's yeast by protamine sulfate treatment, and column chromatographies on DEAE-cellulose, hydroxyl-apatite, Sephadex G-150, and phosphocellulose. The purified enzyme was electropho-retically homogeneous. The molecular weight was determined to be approximately 65, 000 by gel filtration. The enzyme was greatly stabilized by the addition of 20% (v/v) glycerol. It catalyzed the reduction of glyoxylate and hydroxypyruvate and was specific for NADPH as an electron donor, but showed slight affinity towards NADH. The Michaelis constants for glyoxylate, hydroxypyruvate, NADPH, and NADH were found to be 16mM, 1.4mM, 5.7μM, and 0.43mM, respectively. The enzyme was inhibited by p-chloromercuribenzoate (PCMB) and iodoacetate, but inhibition was prevented by dithiothreitol (DTT) or L-cysteine. The reduction of glyoxylate and hydroxypyruvate was not stimulated by anions.抄録全体を表示PDF形式でダウンロード (851K) -
Studies by Crossed Immunoelectrophoresis and Crossed Immuno-AffinoelectrophoresisAnne M. GOLOVTCHENKO-MATSUMOTO, Toshiaki OSAWA1980 年 87 巻 3 号 p. 847-854
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe binding of the human erythrocyte membrane major intrinsic protein (Band 3) to con-canavalin A (Con A), Ricinus communis agglutinin (RCA), and pokeweed mitogens (PWM) has been studied by crossed immunoelectrophoresis and crossed immuno-affinoelectrophoresis.
Although pure by the criterion of polyacrylamide gel electrophoresis, Band 3 was resolved by crossed immunoelectrophoresis into three peaks. A major peak was split on both the anodic and cathodic sides, and consisted of at least two components. A smaller additional peak was also observed. By introducing Con A in the first dimension of the run, the main peak was dissociated into 2 peaks of different heights. Partial binding to Con A was confirmed by introducing Con A Sepharose 4 B in an intermediate gel in the second dimension of the electrophoresis. When the first dimension gel of the crossed immunoelectrophoresis contained RCA, or when RCA Sepharose 4 B was present in an intermediate gel in the second dimension of the run, the major peak was far smaller because of binding to that lectin. In experiments with a first dimension gel containing pokeweed mitogens, the major peak was higher but narrower than in the control experiments, presumably due to selective binding of some constituents of Band 3.
Immuno-affinoelectrophoretic methods therefore show that Band 3 is heterogeneous and that its components bind differently to the above lectins.抄録全体を表示PDF形式でダウンロード (2822K) -
Hiroshi YANAGAWA, Yukiko KOBAYASHI, Fujio EGAMI1980 年 87 巻 3 号 p. 855-869
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーMarigranules, highly organized particles of 0.3 to 2.5μm in diameter, were produced from a reaction of glycine and acidic, basic, and aromatic amino acids at 105°C for 8 weeks under a N2 atmosphere in a modified sea medium enriched with six essential transition elements. The resulting marigranules were purified by centrifugation in ascending concentrations of Ficoll 400 (5 to 20%). The marigranules (C, 58.22; H, 3.76; N, 14.23; ash, 7.62%) were ununiformly packed with KOH-soluble polymers and they also had membrane-like structures. Their infrared spectrum suggested the presence of peptide bonds. The interior structure of marigranules was solubilized by treatment with KOH solution. The KOH-solubilized component was characterized by gel filtration and polyacrylamide gel electrophoresis. The component consisted of polymers with molecular weights of 1, 800, 6, 800, 15, 000, and 82, 000 daltons, and 34% of the total nitrogen was revealed as primary amino groups after treatment with elastase. This suggests that the polymers have at least one peptide bond per three amino acid residues. Thus, it appears that the interior of marigranules consists of polymers with an elastin-like structure. The surface structure of marigranules was solubilized with SDS and Triton X-100. The SDS-solubilized components were 3, 400, 350, and 150 daltons in size.
We found that organized particles were phase-separated from a concentrated aqueous solution of freeze-dried powder prepared from the reaction mixture mentioned above, and we called the particles marisomes. Marisomes (C, 63.67; H, 6.71; N, 10.56; ash, 6.54%) had a spherical structure of 2 to 3μm in diameter. They were found to be empty particles with a soft envelope by scanning electron microscopic observation. Marisomes were completely solubilized by SDS, ethanol, and Triton X-100. The solubilized marisomes were phase-separated again by dialysis against water. Marisomes were also solubilized in boiling water and were phase-separated again on cooling. Thus the system was reversible. Marisomes mainly consisted of lipophilic polymers with molecular weights of approx. 3, 400 daltons. The molecular size and lipophilic properties of marisomes were quite similar to those of the surface structure of marigranules.
Taking into consideration the characteristics of both the particles, we were led to the working hypothesis that a marisome may be the precursor of a marigranule, that is, the marisome was first phase-separated from the reaction mixture and then amino acids and their oligomers were absorbed into the marisome, in which they were polymerized to give elastin-like polymers with cross-links. Thus, both the organized particles, marisomes and marigranules, were good models for the course of evolution of protocells. In addition, the modified sea medium proved to be a good simulative marine environment for studies of chemical evolution in the primeval sea.抄録全体を表示PDF形式でダウンロード (4878K) -
Acylation Kinetics at Low pH'sKazuhiko IKEDA, Shigeru KUNUGI, Hideo HIROHARA1980 年 87 巻 3 号 p. 871-880
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーDimeric α-chymotrypsin (D) formed over a range of low pH (3.7-5.2) showed a different specificity from monomeric α-chymotrypsin (M) for the acylation step, though the catalytic system functions as well in the dimeric enzyme as in the monomeric enzyme. For aliphatic substituted phenyl esters, a bulky substituent at the ortho-position of the phenyl ring reduced the acylation rate of the dimeric enzyme compared with the monomeric enzyme. Especially for aliphatic p-nitrophenyl esters, the dissociation constants of the DS complexes were always smaller than those of the MS complexes, independent of the aliphatic chain length of the substrate. The intrinsic acylation rate constant of the dimeric enzyme increased with the chain length of the substrate. These catalytic properties are discussed with reference to the structures of the catalytic and binding sites of the dimeric enzyme.抄録全体を表示PDF形式でダウンロード (642K) -
Shin-ichi SUGIMOTO, Isamu SHIIO1980 年 87 巻 3 号 p. 881-890
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリー3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase of Brevibacterium flavum, which is the first enzyme in the aromatic amino acid biosynthetic pathway, was purified 540-fold to a disc-gel-electrophoretically homogeneous state. During the purification processes, component A of chorismate mutase, a common enzyme for phenylalanine and tyrosine biosynthesis, was purified together with DAHP synthetase in a constant ratio. It was concluded that DAHP synthetase and chorismate mutase component A form a bifunctional enzyme. The molecular weight of bifunctional DAHP synthetase-chorismate mutase component A was estimated to be 250, 000 by gel-filtration. On SDS-polyacrylamide-gel electrophoresis, a single protein band was obtained and the molecular weight was estimated to be 55, 000, suggesting that DAHP synthetase-chorismate mutase component A is an oligomer of identical subunits. 5, 5'-Dithio-bis(2-nitrobenzoic acid) (DTNB) reacted with 3 mol of sulfhydryl groups per mol of the subunit. One of the sulfhydryl groups was essential for the DAHP synthetase activity, whereas the chorismate mutase activity was not affected by DTNB at all. The optimum pH of the DAHP synthetase reaction was 6.5 with the purified preparation. The DAHP synthetase activity was stimulated 30% by chorismate mutase component B but not by chorismate, the substrate of chorismate mutase. Double-reciprocal plots of the reaction rate against erythrose-4-phosphate concentration gave straight lines with Km of 0.4mm for erythrose-4-phosphate. Plots of the reaction rate against phosphoenolpyruvate concentration curved upwards. The Hill coefficient and S0.5 for phosphoenolpyruvate were estimated to be 4.1 and 0.4mM, respectively. Inhibition by inorganic phosphate, one of the products, was noncompetitive for erythrose-4-phosphate and was not competitive for phosphoenol-pyruvate. Phenylalanine and tyrosine were competitive inhibitors for erythrose-4-phosphate (apparent K1, 0.066mM) and mixed-type inhibitors for phosphoenolpyruvate. Tryptophan did not affect DAHP synthetase activity at all.抄録全体を表示PDF形式でダウンロード (1311K) -
Mineko SAKAI, Shoji ODANI, Tokuji IKENAKA1980 年 87 巻 3 号 p. 891-898
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe carboxyl-terminal amino acid sequence of Streptomyces subtilisin inhibitor (SSI) was reinvestigated by analysis of the amino acid sequences of the thermolysin peptides from the C-terminal decapeptide, and the sequence -Val110-Ala-Phe-Phe113, which was reported in J. Biochem. 76, 1191-1209 (1974), was revised to -Val110-Phe-Ala-Phe113. Carboxypeptidase A digestion of SSI resulted in loss of the inhibitory activity in parallel with the release of the carboxyl-terminal four amino acid residues. The resulting modified inhibitor, des (Val110-Phe113)-SSI, possessed almost full inhibitory activity against subtilisin BPN' when the inhibitor was incubated with the enzyme in amounts less than one mol of enzyme per mol of the inhibitor. However, no inhibitory activity was observed when the molar ratio of the inhibitor to the enzyme was less than one. This phenomenon suggests that the carboxyl-terminal four amino acid residues might play an important role in the maintenance of the three-dimensional structure of SSI, which resists the action of the proteinase. The addition of more than 30-fold molar excess of SSI-(104-113)-decapeptide (C-terminal decapeptide of SSI) to the modi-fied inhibitor resulted in refolding of the polypeptide chain, rendering it immune from proteolytic digestion.抄録全体を表示PDF形式でダウンロード (595K) -
Hiroshi NAKASHIMA, Shio MAKINO1980 年 87 巻 3 号 p. 899-910
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーBand 3 from bovine erythrocyte membranes was isolated in a state of high purity by the following steps in the presence of a nonionic detergent, nonaethyleneglycol n-dodecyl ether (C12E9): (1) selective removal of Band 2.6 from ghosts by solubilization with 2% C12E9, (2) extraction of Band 3-rich fraction with 4% C12E9 from 2% C12E9-treated membrane residues, and (3) purification of Band 3 by aminoethyl-conjugated Sepharose 4 B column chromatography. Human Band 3 was also purified in good yield by aminoethyl-conjugated Sepharose 4 B column chromatography of erythrocyte membrane proteins solubilized with 1% C12E9 and treated with 2, 3-dimethylmaleic anhydride. There were no significant differences in CD spectra in C12E9, amino acid compositions, and migration mobilities in sodium dodecyl sulfate-gel electrophoresis between bovine and human Band 3. Calculations of average hydrophobicity and discriminant function demonstrated that bovine Band 3 could be categorized as a typical integral membrane protein. Bovine Band 3 showed a tendency to form a dimer and higher aggregates in 0.1% C12E9; these were resistant to dissociation into monomers in sodium dodecyl sulfate solution and, further, the protein retained residual secondary structure in highly concentrated guanidine hydrochloride solution, indicating the possible presence of an extended sequence of hydrophobic amino acid residues.抄録全体を表示PDF形式でダウンロード (2941K) -
Masao TANAKA, Shonen YOSHIDA1980 年 87 巻 3 号 p. 911-918
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe anthracycline antibiotics, daunomycin and adriamycin, strongly inhibited the reactions of both DNA polymerase α and β from calf thymus by competing with the template primers, i.e., activated DNA or initiated deoxyhomopolymers. DNA polymerase α was more sensitive to both drugs than DNA polymerase β with all the template-primers tested. With poly (dT)• oligo (dA), the activity of the α-enzyme was extremely sensitive to these drugs (K1, 0.9μm for daunomycin), while that of the β-enzyme was relatively resistant (K1, 25μm for dauno-mycin).
Much stronger inhibition was produced by preincubating these drugs with the enzymes than with the template-primers, and the inhibition of DNA polymerase activity was reversed by the addition of excess template-primers. These results indicate that the inhibition was produced mainly by direct interaction of the drugs with DNA polymerases rather than by impairing the template activity of DNA due to intercalation of the drugs.
Although adriamycin inhibited DNA polymerases α and β to slightly higher extents than daunomycin, the modes of inhibition by these two drugs were quite similar.抄録全体を表示PDF形式でダウンロード (482K) -
Isolation of Cysteinyl Peptides by Covalent Chromatography from the Potato Enzyme and Their Amino Acid SequencesKenichi NAKANO, Toshio FUKUI, Hiroshi MATSUBARA1980 年 87 巻 3 号 p. 919-927
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーTo elucidate the structural similarity between α-glucan phosphorylases from different sources, the amino acid sequences of the cysteinyl regions in potato phosphorylase were determined, and compared with the complete sequence of the rabbit muscle enzyme. Cysteinyl peptides were purified by covalent chromatography based on thiol-disulfide exchange. Potato phosphorylase was coupled to Thiopropyl-Sepharose 6 B in the presence of urea, and, after tryptic digestion, 10 distinct cysteinyl peptides which accounted for all the cysteine present in the enzyme were finally isolated in high yields. From the sequence comparison, all of the peptides surrounding the cysteinyl residues in the potato enzyme are homologous with widely distributed specific regions in the rabbit muscle enzyme, although only 4 of 10 cysteinyl residues are conserved between the two enzymes. These observations, in addition to the previously established homology in the cofactor site, show that potato and rabbit muscle phosphorylases are similar in terms of the primary structure. The different properties of cysteinyl residues in the two enzymes are discussed based on the present sequence comparison and the recent X-ray crystallographic data for the rabbit muscle enzyme.抄録全体を表示PDF形式でダウンロード (663K) -
Shigemi KATO, Masahiro TSUJI, Yasuo NAKANISHI, Sakaru SUZUKI1980 年 87 巻 3 号 p. 929-939
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーIncubation of rat liver microsomes with GDP-[14C] mannose and phenyl phosphate for 30min, followed by paper-chromatographic analysis, revealed the presence of a new radioactive compound that was isolated and characterized as phenyl β-D-mannosyl phosphate. The enzyme catalyzing this reaction was very closely related to a dolichyl β-D-mannosyl phosphate-synthesizing activity found in the same microsomes. The two activities were affected in a parallel fashion by mild heating and were found in the same proportions in whole microsomes, in rough-surfaced microsomes, and in smooth-surfaced microsomes. Increasing the con-centration of phenyl phosphate from 0 to 15mM resulted in a progressive reduction in the rate of mannose incorporation into endogenous dolichyl phosphate. It is concluded that the two reactions are catalyzed by a single enzyme. The labeled product from phenyl phos-phate was rapidly excluded from the microsomes, while the product from dolichyl phosphate was retained in the microsomes, suggesting that phenyl phosphate may act as a mannose-trapping agent, thereby inhibiting mannosyltransferase reactions involving dolichyl phosphate as a glycosyl carrier.抄録全体を表示PDF形式でダウンロード (904K) -
Hisako FUSHIMI, Masao SHIBATA, Seiichiro TARUI1980 年 87 巻 3 号 p. 941-949
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーHereditary diabetic mice (NSY) were inbred from original streptozotocin diabetic ICR mice for 8-9 generations using hyperglycemia as an index. The normoglycemic ICR mice were used as controls for the NSY line. The nonfasting blood sugar level of the NSY mice was 305±14mg/100ml, while their immunoreactive insulin level was 30±4μU/ml (the values of the controls were 165±12mg, /100ml and 79±14μU/ml, respectively). β-N-Acetylglu-cosaminidase [EC 3. 2. 1. 29], β-galactosidase [EC 3. 2. 1. 23], α-glucosidase [EC 3. 2. 1. 21], and α-mannosidase [EC 3. 2. 1. 24] activities were determined in the 1, 000×g supernatant of the liver and the kidney of control and streptozotocin diabetic ICR mice and their NSY line.
In the kidneys of the insulinopenic NSY mice, the β-galactosidase and α-mannosidase activities were significantly decreased. No significant changes were found in liver enzyme activities. Insulin treatment increased the kidney β-galactosidase activity significantly. The insulinopenic state, which caused a decrease in the glycosidase activities in the kidney, could induce retarded breakdown of glycoprotein.抄録全体を表示PDF形式でダウンロード (3168K) -
III. The Absolute Configuration at the C-6 Chiral Center of Tetrahydrobiopterin Cofactor and Related CompoundsSadao MATSUURA, Takashi SUGIMOTO, Hiroyuki HASEGAWA, Shunsuke IMAIZUMI ...1980 年 87 巻 3 号 p. 951-957
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーTetrahydrobiopterin, the natural pteridine cofactor for aromatic amino acid hydroxylases, was produced stereospecifically with reference to the C-6 chiral center from 7, 8-dihydrobiopterin by the action of dihydrofolate reductase. Similarly, 7, 8-dihydro-6-methylpterin was reduced to tetrahydro-6-methylpterin having the same 6-configuration by the same enzyme. The absolute configuration of these tetrahydropterins at the C-6 chiral center was determined to be L, as in (2 S)-1, 2, 3, 4-tetrahydro-2-methylquinoxaline, which was derived from L-alanine, by comparison of the CD spectra.抄録全体を表示PDF形式でダウンロード (458K) -
Yasuhiro NATORI, Masahiro NISHIJIMA, Shoshichi NOJIMA, Hiroshi SATOH1980 年 87 巻 3 号 p. 959-967
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーA phospholipase A2 bound tightly to the particulate fractions of rat ascites hepatoma cells was purified approximately 13, 000-fold with a reasonably high yield (34%) by extraction with sodium cholate, ammonium sulfate fractionation, solubilization with sodium dodecyl sulfate, column chromatographies on Sephadex G-150 in the presence of sodium dodecyl sulfate, and on DEAE-cellulose and CM-cellulose in the presence of Triton X-100.
The enzyme has a unique substrate specificity; namely, it preferentially hydrolyzes phosphatidylethanolamine and, to a lesser degree, phosphatidylglycerol. However, it does not attack phosphatidylcholine, phosphatidic acid or cardiolipin in the present experimental conditions. The final preparation shows both phospholipase A2 and lysophospholipase L2
activities, but neither lysophospholipase L1 nor lipase activity. The purified enzyme has a rather broad pH optimum ranging from 7 to 9, requires Ca2+, and is resistant to heat-treatment at 95°C for 5min.抄録全体を表示PDF形式でダウンロード (678K) -
Haruki YAMAGUCHI1980 年 87 巻 3 号 p. 969-977
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーChondroitin sulfates were quantitatively extracted from bovine and whale nasal septum without indiscriminate depolymerization by treatment with 0.5M sodium hydroxide containing 0.26M sodium borohydride at 3-5°C for 10 days. The reducing-end xylitol of the glyco-saminoglycans was accurately determined by a microchemical method which had been developed for the separation and determination of the reducing-end alditols of reduced polysaccharides with high molecular weight (Yamaguchi, H., Inamura, S., & Makino, K. (1976) J. Biochem. 79, 299-303; Yamaguchi, H. & Makino, K. (1977) J. Biochem. 81, 563-569). The xylitol proportion of each glycosaminoglycan was in fair agreement with the xylose proportion of the corresponding chondroitin sulfate prepared by proteolytic digestion, suggesting that the xylose residues of the chondroitin sulfate chains are all situated in the reducing-end position and are quantitatively converted to xylitol by reductive β-elimination during the extraction with alkaline sodium borohydride. Accordingly, the molecular weight of the glycosaminoglycans was readily derived from the ratio of xylitol content to polysaccharide weight. The combined use of this method for molecular weight estimation and gel chromatography made it feasible to analyze the molecular weight range of chondroitin sulfates on relatively small amounts of materials. The chondroitin sulfates prepared by alkali extraction were fractionated on Sephadex G-200 into 12 fractions. The molecular weight determination of the glycosamino-glycan in each fraction revealed that the molecular weight ranges of the chondroitin sulfates are appreciably wider than those previously reported. Linear relationships were found for every chondroitin sulfate preparation when the partition coefficient Kav and the elution volume of each fraction were plotted against the log of the corresponding molecular weight determined
by the present method.抄録全体を表示PDF形式でダウンロード (692K) -
Hiromu MUROFUSHI1980 年 87 巻 3 号 p. 979-984
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーA tubulin-tyrosine ligase was purified from porcine brains using DEAF-cellulose chromatog-raphy, Sepharose-sebacic acid hydrazide-ATP affinity chromatography and Sepharose-tubulin affinity chromatography. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 46, 000. The apparent molecular weight was 37, 000 on a high speed liquid chromatograph equipped with gel filtration columns. The pH optimum for the activity was around 8 and a second peak was observed at around 6.5. 8.5μM ATP or 30μM tyrosine gave half-maximal activity. The purified enzyme catalyzed the tyrosination of the α subunit of tubulin in vitro.抄録全体を表示PDF形式でダウンロード (446K) -
Hideo SAWADA, Masakazu HAYASHIBARA, Akira HARA, Toshihiro NAKAYAMA1980 年 87 巻 3 号 p. 985-988
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーAromatic ketone reductase activity of microsomes showed a unique cofactor requirement: Addition of NADP and glucose-6-phosphate was as effective as that of an artificial NADPH generating system, whereas NADPH alone served as a cofactor less efficiently. Microsomal aromatic ketone reductase, purified partially from guinea pig liver microsomes after solubilization with Triton X-100, reduced 5β-dihydrotestosterone, aromatic aldehydes, and ketones with NADPH as a cofactor. However, addition of hexose-6-phosphate dehydrogenase, purified from the same source, as an NADPH generator produced about 2 times higher activity than that of yeast glucose-6-phosphate dehydrogenase or NADPH alone.抄録全体を表示PDF形式でダウンロード (289K) -
Shigeo OHNO, Kimiko SAITO, Koichi SUZUKI, Kazutomo IMAHORI1980 年 87 巻 3 号 p. 989-992
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーThe effects of carboxypeptidase digestion on the function of colicin E3 which had been demon-strated to be a complex of proteins A and B was investigated. It was indicated that removal of 7 amino acids from the C-terminal region of protein A has no significant effect on the inter-action with the inhibitor, protein B, or with the specific cell surface receptor but does have an effect on the efficient interaction with the final target, ribosomes.抄録全体を表示PDF形式でダウンロード (302K) -
Toshihiko HAYASHI, Toshitaka NAKAMURA, Hisae HORI, Yutaka NAGAI1980 年 87 巻 3 号 p. 993-995
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーDegradation of type II and III collagens by tadpole collagenase in a mixture of both substrates was monitored by SDS-polyacrylamide gel electrophoresis in 3M urea, followed by densitometric quantitation. The degradation rates of type II and III collagens were increased and decreased, respectively, by the mutual presence, compared with those of type II and III collagens alone. The results suggest that the degradation of type II collagen in vivo may be regulated by the presence of other type (s) of collagen, particularly in such a case as resorption of cartilage in the region of newly forming osteoid tissue.抄録全体を表示PDF形式でダウンロード (656K) -
Kazuhiro KOHAMA1980 年 87 巻 3 号 p. 997-999
発行日: 1980/03/01
公開日: 2008/11/18
ジャーナル フリーAn actin preparation extracted at 25°C for 2h from the residue which had previously been subjected to the routine extraction procedure, i.e. at 4°C for 15min, showed markedly increased rates of amino acid incorporation, i.e. 48% higher than routinely extracted actin. The nature of the actin having higher amino acid incorporation rates was discussed in relation to 10 S-actinin.抄録全体を表示PDF形式でダウンロード (229K) -
1980 年 87 巻 3 号 p. 1001
発行日: 1980年
公開日: 2008/11/18
ジャーナル フリーPDF形式でダウンロード (14K)
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