巻号一覧
77 巻, 1 号
選択された号の論文の27件中1～27を表示しています
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Kiyoshi IKEDA, Kozo HAMAGUCHI1975 年 77 巻 1 号 p. 1-16
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーIn order to clarify the state of Trp 62 in hen egg-white lysozyme [EC 3.2.1.17] bound with N-acetylchitooligosaccharides in relation to the ionization states of side chains present in the substrate binding site, the binding of α- and β-N-acetylgluco-samine (GlcNAc), α- and β-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 was studied by use of the change in the tryptophyl circular dichroic (CD) band at 295 nm at various pH values. The effects of these saccharides on the charge-transfer binding of N1-methylnicotinamide to Trp 62 were also examined. The binding constants, estimated by use of N1-methylnicotinamide by assuming noncompetitive binding between the saccharides and N1-methylnicotinamide, were in good agreement with those determined from the CD change at 295nm.
The state of Trp 62 in saccharide-bound lysozyme was found to depend on the chemical structures of the saccharides and their binding orientations. The relation between the state of Trp 62 and the ionization of the catalytic Glu 35, which is far distant from Trp 62, was also confirmed for lysozyme complexes with various saccharides as well as for free lysozyme.抄録全体を表示PDF形式でダウンロード (1030K) -
Kenichi OHTSUBO, Hikoichi SAKAI, Hiromu MUROFUSHI, Ryoko KURIYAMA1975 年 77 巻 1 号 p. 17-21
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーThe modification of tubulin cysteine and cystine residues to S-sulfocysteines caused a distinct separation of the α and β subunits in a continuous sodium dodecyl sulfate polyacrylamide gel system. The well-separated subunit bands permitted investigation of the phosphorylation of α and βtubulin subunits. The incubation of tubulin frac-tion with [γ-32P]ATP demonstrated that both subunits were phosphorylated in vitro. The incorporation of 32PO4 into sea urchin eggs, however, failed to cause phospho-rylation of tubulin in vivo.抄録全体を表示PDF形式でダウンロード (1968K) -
Ryoko KURIYAMA1975 年 77 巻 1 号 p. 23-31
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーTubulin was polymerized to microtubules in a medium containing 4M glycerol-5mM2-(N-morpholino)ethanesulfonic acid-0.5mM MgSO4-1.0mMethyleneglycol-bis(2-amino-ethylether)-N, N, N', N'-tetraacetic acid-1.0mM ATP-50mM KCl at pH 6.5 (glycerol reassembly buffer) in vitro. Partially purified tubulin was obtained by sedimenting these reconstituted microtubules. There were two peaks in the partially purified tubulin fraction upon gel filtration through a Sephadex G-200 column. One was flow-through fraction and the other was purified tubulin dimer (molecular weight 105, 000±5, 000), having sedimentation coefficients of 20 S and 5.2 S, respectively. One mole of tubulin dimer bound 0.8 mole colchicine and contained α- and β-subunits whose amino acid compositions were determined.
Pure tubulin dimer obtained by Sephadex G-200 column chromatography failed to polymerize to microtubules by itself. Addition to the tubulin dimer of a small amount of partially purified tubulin fraction, flow-through fraction or fragmented outer fiber microtubules from sea urchin sperm flagella induced the polymerization of tubulin, as demonstrated by measurements of viscosity changes as well as by electron microscopic observations.抄録全体を表示PDF形式でダウンロード (1494K) -
II. Purification and Properties of Two CellulasesGentaro OKADA1975 年 77 巻 1 号 p. 33-42
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーTwo cellulase [EC 3.2.1.4] components derived from Meicelase, a commercial crude cellulase preparation from Trichoderma viride, were purified by consecutive column chromatography, and were designated as cellulase II-A and cellulase II-B. Cellulases II-A and II-B were each homogeneous on polyacrylamide gel electrophoresis. The molecular weights of cellulases II-A and II-B were 30, 000 and 43, 000, respectively, on the basis of Sephadex G-100 gel filtration. Both enzymes contained 12-14% carbohydrates (as glucose).
Some properties of the purified cellulases were investigated. The optimum pH and temperature for cellulases II-A and II-B were pH 4.5-5.0 and 60°, and pH 4.5-5.0 and 50°, respectively. Both enzymes were stable over the range of pH 5.0-7.0 at 4° for 24 hr. Cellulases II-A and II-B retained 27 and 41% of the original CM-cellulose-saccharifying activities, respectively, after heating at 100° for 10 min. Both enzymes were completely inhibited by some metal ions such as 1 mM Hg2+, and partially by 1mm Ag+ and Cu2+. However, Mg2+, Fe2+, and several other metal ions showed no inhibition at this concentration. The hydrolysis of CM-cellulose by cellulase II-A was more random than that by cellulase II-B.抄録全体を表示PDF形式でダウンロード (682K) -
Reassembly of Functionally Active 50S Ribosomal Particles from Proteins and RNAs of Escherichia coliDependency of 50S Ribosomal Reassembly on 30S SubunitsTomofusa TSUCHIYA, Hiroshi KANAZAWA, Hideo FUJIMOTO, Den'ichi MIZUNO1975 年 77 巻 1 号 p. 43-54
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーFurther studies were made on the reassembly of 50S ribosomal subunits from proteins and RNAs of E. coli. The reassembled particles had high activity in poly U-directed polyphenylalanine synthesis and their sucrose sedimentation properties were similar to those of the original intact particles.
Several factors affecting the reassembly were examined. The optimal pH for solubilization of ribosomal proteins was pH 9.5, and the optimal Tris concentration was 0.75 to 1.00M. In the reassembly mixture the pH was adjusted to 8.2. A sharp optimum magnesium ion concentration of 6 to 10mm was observed. The reassembly required 0.2 to 0.5M KCI, the optimum concentration being 0.40M. On incubation for 20 min a temperature of 34 and 40° was necessary, 37° being best, Oligonucleo-tides, which we previously added to the reassembly mixture were found not to be necessary for inhibition of RNase II [EC 3.1.4.20] activity remaining in the reaction mixture. The RNase II was inhibited by the high concentration of 1.0M Tris. It was found necessary to dialyze the reassembly mixture against a buffer containing 10 mm magnesium ion after the incubation.
Simultaneous reassembly of 30 and 50S subunits with time was observed, showing that 70S ribosomes were formed first and that they then dissociated into subunits.
Reassembly of 50S subunits from their component proteins and RNAs was com-pletely dependent on either 30S particles or the simultaneous reassembly of 30S subunits.
Other critical factors affecting the reassembly of 50S subunits must be examined, since the reproducibility of this reassembly is only about 60%, even under the above controlled conditions.抄録全体を表示PDF形式でダウンロード (910K) -
Amino Acid Sequence of a Fragment (“Histidine-Rich Peptide”) Released by Plasma KallikreinYong Nam HAN, Masanobu KOMIYA, Sadaaki IWANAGA, Tomoji SUZUKI1975 年 77 巻 1 号 p. 55-68
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーAn unknown peptide fragment, which was released from bovine high-molecular-weight kininogen by bovine plasma kallikrein [EC 3.4.21.8], was isolated and its chemical structure was established. The fragment consisted of 41 amino acids with serine and arginine at the NH2- and COOH-termini, respectively. The molecular weight was calculated to be 4, 584. It was very basic and contained eleven residues each of histidine and glycine and seven residues of lysine. Thus, the total number of these three amino acids accounted for about 70 percent of the total residues constituting the fragment.
The amino acid sequence of the fragment, designated tentatively as “His-rich peptide, ” was studied by Edman degradation and standard enzymatic and chemical techniques. These data made it possible to deduce the following sequence: H-S_??_r-His-G_??_y-Leu-Gly-His-Gly-His-Gln-L_??_s-Gln-His-G_??_y-Leu-Gly-
His-G1y-H_??_s-Lys-His-Gly-His-Gly-His-G_??_y-Lys-His-Lys-Asn-L_??_s-
Gly-Lys-Asn-Asn-G_??_y-Lys-H is-Tyr-Asp-T_??_p-Arg-OH
The fragment had an extremely interesting feature in that repeating sequences occur along the peptide chain. The repeats were of the type His-Gly-X or Gly-His-X and this sequence appeared six or seven times up to 26 residues from the N-terminal end. Moreover, three tetrapeptide sequences of Gly-His-Gly-His and two heptapeptide sequence consisting of His-Gly-Leu-Gly-His-Gly-His were found in the N-terminal portion.
It should be noted that plasma kallikrein liberates such a histidine-rich peptide from the kininogen in addition to a physiologically active peptide, bradykinin. The location of the “His-rich peptide” fragment in the precursor protein is also discussed.抄録全体を表示PDF形式でダウンロード (947K) -
I. Kinetic Parameters for the Hydrolysis of Synthetic SubstratesRikimaru HAYASHI, Yasuo BAI, Tadao HATA1975 年 77 巻 1 号 p. 69-79
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーKinetic parameters for carboxypeptidase Y [EC 3.4.12. 1], characterized as a non-specific enzyme, are given for the hydrolysis of a series of acylated peptides, acylated amino acid esters, and amides. We confirmed that the enzyme released COOH-terminal proline and β-alanine at an appreciable rate, as well as neutral amino acids with aromatic and aliphatic side chains at a very high speed. The rates of hydrolysis of ester and amide substrates were compatible with those produced by chymotrypsin [EC 3.4.21.1]. Stereospecificity was also demonstrated by the failure to hydrolyze peptide, ester, amide, and anilide substrates containing a D-amino acid.
The effects of pH, solvents, and salt concentrations on the kinetic parameters of hydrolysis of peptide and ester substrates are also described.抄録全体を表示PDF形式でダウンロード (755K) -
II. Effects of Substrate and Product Analogs on Peptidase and Esterase ActivitiesYasuo BAI, Rikimaru HAYASHI, Tadao HATA1975 年 77 巻 1 号 p. 81-88
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーReversible inhibition of the peptidase and esterase activities of CPase Y [EC 3.4.12.1] was investigated with substrate and product analogs known to be inhibitors or effectors of pancreatic carboxypeptidases A or B [EC 3.4.12.2 or 3]. L-Amino acids and NH2-blocked L-amino acids showed competitive-type inhibition, whereas their D-enantiomers caused less inhibition than the L-enantiomers, and showed non-com-petitive or mixed-type inhibition. Some phenylalanine analogs, e.g. β-phenylpropionic acid and t-cinnamic acid, were also reversible inhibitors of CPase Y. The type of these inhibitors and their Ki values were generally parallel for both the peptidase and esterase activities.抄録全体を表示PDF形式でダウンロード (547K) -
Susumu TSUNASAWA, Kozo NARITA, Kikuo OGATA1975 年 77 巻 1 号 p. 89-102
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーAn enzyme that releases acylamino acid from amino terminal acylated peptides and proteins has been isolated from rat liver in a highly purified form by a six-step procedure comprising extraction from liver homogenate, ammonium sulfate fraction-ation, heat treatment, chromatography on columns of DEAE-cellulose and hydrox-ylapatite and gel filtration on a Sepharose 6B column. About 1, 500-fold purification was achieved from the liver homogenate. The purified enzyme preparation showed a single band on polyacrylamide gel disc electrophoresis.
The enzyme specifically released acylamino acids from several amino terminal acylated peptides and proteins with different rates of hydrolysis depending on the acyl groups, terminal amino acid sequences and tertiary structure of the acyl protein substrates. The present enzyme may be useful for the removal of the N-terminal acylamino acid from some N-terminal blocked peptides and proteins in amino acid sequence analysis.
The molecular weight of the purified enzyme was estimated to be 360, 000- 420, 000 by gel filtration and sucrose density gradient ultracentrifugation. Disc electro-phoresis of the acylamino acid-releasing enzyme on SDS-polyacrylamide gel suggested that the enzyme consisted of five or six identical subunits having a subunit weight of about 75, 000. The N-terminal residue of the subunit, which consisted of a single polypeptide chain, was glycine. Other properties of the enzyme, inculding isoelectric point, the effects of metal ions and several chemical reagents on the enzyme activity, pH optimum, and amino acid composition were also examined.抄録全体を表示PDF形式でダウンロード (1051K) -
Yoshiki TAKESUE1975 年 77 巻 1 号 p. 103-115
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーLeucine β-naphthylamidase associated with the microvilli membranes of rabbit small intestine was solubilized with papain [EC 3.4.22.2] and purified by Sephadex G-200 gel filtration, DEAE-cellulose column chromatography, passage through a column of Sepharose 4 B coupled with anti-sucrase antibodies and preparative disc electrophoresis in polyacrylamide gel. The purified enzyme was homogeneous on ultracentrifugation and disc electrophoresis, but a double immunodiffusion test showed the presence of a minor component which was probably denatured enzyme. The molecular weight of the purified enzyme was estimated to be 225, 000 by Sephadex G-200 gel filtration and the sedimentation coefficient (S020, w) was found to be 6.90 S. Purified enzyme required bovine serum albumin for maximal activity, perhaps for its protection from autodigestion. It hydrolyzed, in addition to L-leucine β-naphthylamide, various L-amino acid β-naphthylamides and dipeptides with a free a-amino group, but did not hydrolyze benzoyl-L-arginine β-naphthylamide. Therefore, the purified enzyme is an aminopeptidase. Hg2+ and Cu2+ ions strongly inhibited the enzyme activity, but other metal ions and EDTA showed no or only slight effect. N-Ethylmaleimide exhibited a weak inhibition. Purified enzyme had an optimal pH and Km value for leucine β-naphthylamide similar to those of enzymes from other sources. Antibodies against the purified enzyme were raised in guinea pigs. The antibodies obtained were found by double immunodiffusion to be specific for the enzyme. They precipi-tated the enzyme quantitatively and partially inhibited the enzyme activity.抄録全体を表示PDF形式でダウンロード (4369K) -
Kaoru TSUJII, Toshio TAKAGI1975 年 77 巻 1 号 p. 117-123
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーSodium n-octylbenzene-p-sulfonate was successfully used in place of sodium dodecyl sulfate in SDS-polyacrylamide gel electrophoresis. This modification of the usual technique made it possible to scan the polyacrylamide gel for the distribution of the surfactant by measurement of UV absorption. It was found that a band electro-phoresed in advance of the complexes formed between protein polypeptides and the surfactant. The band could be observed even in the absence of protein polypeptide, and was ascribed to micelles derived from surfactant added in excess to the sample solution. The complexes were found to be detectable by scanning at 261 nm, usually with better sensitivity than by scanning at 280nm. Thus, this modification of SDS-polyacrylamide gel electrophoresis is useful both to investigate what is going on in the gel during electrophoresis and to improve the sensitivity of detection by UV scanning of protein complexes.抄録全体を表示PDF形式でダウンロード (1296K) -
Mutsumi SUGITA, Shigeki SHIRAI, Osamu ITASAKA, Taro HORI1975 年 77 巻 1 号 p. 125-130
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリー1. A unique subclass of ceramide oligosaccharides from whole tissue of the fresh-water bivalve Corbicula sandai has been isolated. Through the use of chemical, enzymatic, and physical techniques, two novel glycolipids were characterized as mannosyl-β (1→4)-glucosyl ceramide and mannosyl-α (1-4)-mannosyl-β(1→4)-glucosyl ceramide.
2. The components of fatty acids and long chain bases in the two glycolipids were analyzed by gas-liquid chromatography and were further identified by mass spectro-metry. The data show that, with respect to the major components, both lipids have similar caramide moieties.抄録全体を表示PDF形式でダウンロード (1436K) -
Koscak MARUYAMA, Shin-ichi ABE, Tetsuro ISHII1975 年 77 巻 1 号 p. 131-136
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーβ-Actinin, a regulatory protein of muscle, greatly decreased the dynamic rigidity modulus of an acto-heavy meromyosin solution. However, it was found that the mechanical mixing procedure used resulted in fragmentation of the decorated F-actin particles and β-actinin inhibited the reassociation of the fragmented particles. This was the reason for the decrease of the dynamic rigidity modulus of the acto-heavy meromyosin complex caused by β-actinin. When β-actinin was added to the acto-heavy meromyosin solution in the presence of ATP, it did not affect the visco-elasticity. F-actin particles dissociated by ATP were not fragmented by the mixing procedure, which was responsible for the apparent inactivation of β-actinin in the presence of ATP.抄録全体を表示PDF形式でダウンロード (419K) -
Dependence of the Rate on pH and Concentrations of SubstratesTaibo YAMAMOTO, Yuji TONOMURA1975 年 77 巻 1 号 p. 137-146
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーThe kinetic properties of pH jump-induced phosphorylation in thylakoidal membranes of spinach chloroplasts were investigated, and the following results were obtained. 1. The pH jump-induced P incorporation proceeded linearly with time for at least 2 sec after the start of the reaction. Phosphate was incorporated mainly into ATP. The amounts of incorporation into ADP during 0.1 and 2.0 sec were 0.02 and 0.1 mole/400 moles chl, respectively. The amounts of P incorporation into ADP and the β-position of ATP during a 2 sec reaction were less than 5 and 0.1% of the total amount of P incorporation, respectively. Even in the absence of added ADP, ATP was formed by the pH jump, but the amount was very small, i.e., less than 1% of that in the presence of a saturating amount of ADP. Formation of ATP was not enhanced by the addition of 0.1 mm AMP, instead of ADP.
2. The dependence of the rate of ATP formation, v, induced by a pH jump from 3.85 to 8.11 on the concentrations of ADP and Pi was given by
v=Vopt/{(1+Φ1/[ADP])(1+Φ2/[Pi])}
where the values of the constants, Vopt, Φ1, and Φ2 were 14-20 moles/106g chl/sec, 12.5-15μM and 11-20mM, respectively, at 0°.
3. The dependence of v on the concentration of protons was given by v=Va/{1+ (ΦH-a/[H+, a])2}, and v=V_??_/{1+([H+, b]/ΦHb)2},
in the acidic and basic phases, respectively. The values of the constants ΦHa and ΦHb, were 10-5.7 and 10-7.9M, respectively.
4. ATP formation was initiated by adding one of the substrates, ADP or Pi, at various times after the pH jump in the presence of the other substrate. The rate decreased logarithmically with increase in the time between the pH jump and the start of the reaction. When phosphorylation was initiated by adding Pi after the pH jump in the presence of ADP, the decay constant of v was about 0.08 sec-1, which was one-third of that observed when the order of addition of ADP and Pi was reversed.抄録全体を表示PDF形式でダウンロード (628K) -
II. Structure and FunctionNobuo TANAKA, Takashi YAMANE, Tomitake TSUKIHARA, Tamaichi ASHIDA, Mas ...1975 年 77 巻 1 号 p. 147-162
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーThe structure analysis of bonito heart ferrocytochrome c was carried out at 2.3 A resolution by X-ray diffraction, and a Kendrew-type skeletal model was built up. This molecule has an overall egg shape, 35 A in height, 30 A in width and 23 A in thickness; the 5th ligand of the heme iron atom is the Nε atom of the His-18 imidazole ring and the 6th is the Met-80 sulfur atom. Distinct α-helix regions are found between the N-terminus and residue 11, between 60 and 69, and between 90 and the C-terminus. The most distinct difference between the conformation of the present molecule and that of the horse oxidized molecule is the location of the Phe-82 phenyl ring. In the present reduced molecule, the phenyl ring is in closer contact with the iron atom and gives influences on the character of the iron atom. Inside the molecule, at the lower part of the heme pocket, there is an extended hydrogen bond network including the propionic acid residues of the heme group. Both Phe-82 and the hydrogen bond network may play a key role in the function of this molecule.抄録全体を表示PDF形式でダウンロード (986K) -
Noshi MINAMIURA, Yukio KIMURA, Kazuaki TSUJINO, Takehiko YAMAMOTO1975 年 77 巻 1 号 p. 163-169
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーSome physicochemical properties of crystalline α-amylase [EC 3.2.1.1] isolated from normal human urine were investigated.
A crystalline preparation of the enzyme was homogeneous on velocity sedimen-tation, gel filtration, and sodium dodecyl sulfate disc electrophoresis, and its molec-ular weight was estimated to be 4.5×104.
However, electrophoretic analyses revealed that the crystalline α-amylase consisted of at least five isozymes. The implications of the experimental results are discussed.抄録全体を表示PDF形式でダウンロード (3255K) -
Hachiro OZAKI, Isamu SHIIO1975 年 77 巻 1 号 p. 171-180
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーElastolytic enzyme was purified and crystallized from culture fluid of Flavobacterium immotum No. 9-35. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was determined by Sephadex G-100 gel filtra-tion to be 13, 000. The isoelectric point was between pH 8.3 and 8.9. The optimum pH of the enzyme was 7.2 for elastolytic activity. The purified enzyme showed not only elastolytic activity, but also non-specific proteolytic activity against various other proteins. Milk-clotting activity was also observed. The enzyme did not act on keratin, collagen, or fourteen amino acid esters, including N-benzoyl-L-alanine methyl ester, N-benzoyl-L-arginine ethyl ester, and N-acetyl-L-tyrosine ethyl ester, which were typical substrates of pancreatic elastase [EC 3.4.21.11], trypsin [EC 3.4.21.4], and chymotrypsin [EC 3.4.21.1], respectively. However, the enzyme selectively hydrolyzed elastin when both elastin and albumin were present in the reaction mixture.
The enzyme was inhibited by o-phenanthroline and various heavy metals such as cadmium, lead, zinc, and mercury. Various inhibitors, such as diisopropyl phos-phofluoridate, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, trypsin inhibitor, iodoacetamide, etc., had no effect on the elastolytic activity.抄録全体を表示PDF形式でダウンロード (1569K) -
Nobuo OKABE, Noriko MANABE, Ryo-zi TOKUOKA, Ken-ichi TOMITA1975 年 77 巻 1 号 p. 181-186
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーThe circular dichroism (CD) spectra of 3, 5, 3'-triiodothyronine-bovine serum albumin (T3-BSA) and thyroxine-bovine serum albumin (T4-BSA) complexes were measured at the various ratios of T3 or T4 to BSA (T/P) in the wavelength region of 200-350nm. No spectral change was observed in the wavelength region of 200-250nm, but induced Cotton effect was observed in the chromophoric wavelength region of 250-350nm. The value of [θ] at 319nm of the induced Cotton effect increased with increase of the T/P ratio in both complexes, and in T4-BSA complex, it increased remarkably. The number of thyroid hormone molecules bound to one BSA molecule was estimated to be one in the case of T4-BSA complex and four to five in T3-BSA.抄録全体を表示PDF形式でダウンロード (374K) -
Shuichi FUJIMOTO, Yoshiki OHBA1975 年 77 巻 1 号 p. 187-195
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーThe interaction of aflatoxin B1 with DNA was investigated. In the presence of native DNA the absorption spectrum of the toxin showed an obvious spectral shift in the region of 300-420nm with an isosbestic point at 376nm. DNA-bound aflatoxin is hydrolyzed in alkaline media more easily than the free toxin. The hydrolyzed form has a labile structure and can decompose further. The bound toxins are easily dissociated by heat as well as by salt, and all toxin molecules are released from DNA which remains double-stranded.
Aflatoxin can bind to thermally denatured DNA as well, with an accompanying spectral shift which depends on the particular preparation of denatured DNA, and there was an isosbestic point at 380nm. The complex of toxin and denatured DNA was stabilized by salt up to 0.1M. Thus it was concluded that aflatoxin was bound with denatured DNA in a different form from native DNA.
The number of binding sites of DNA was estimated by constructing Scatchard plots based on both spectral analysis and equilibrium dialysis. However, these show no definite value but fall in the region of 0.07 to 0.013, that is one toxin molecule per 80-140 nucleotide of native DNA. Several lines of evidence suggest the possibility that aflatoxin exists in aqueous solution as aggregates. The mechanism of the binding was discussed.
It is noteworthy that the number of binding sites of DNA doubles by the presence of histones.抄録全体を表示PDF形式でダウンロード (710K) -
I. Purification of Glucose-6-phosphate Dehydrogenase from the Vegetative Cell and Its Properties in Comparison with the Spore EnzymeSusumu UJITA, Kinuko KIMURA1975 年 77 巻 1 号 p. 197-206
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリー1. Glucose-6-phosphate dehydrogenase [D-glucose-6-phosphate: NADP+ 1-oxidoreduc-tase, EC 1.1.1.49] from vegetative cells of Bacillus subtilis was purified about 2, 400-fold. The purified enzyme was shown to be homogeneous as judged by chromato-graphic profile, polyacrylamide gel electrophoresis and ultracentrifugation. The corresponding enzyme from spores was also partially purified.
2. The molecular weight of the vegetative cell enzyme was estimated to be 350, 000 by Sepharose 6B chromatography and sedimentation equilibrium studies, and that of its subunit was 58, 000 as determined by SDS gel electrophoresis, indicating that the enzyme existed in a hexameric form. The molecular weight of the spore enzyme was found to be 240, 000.
3. The vegetative cell enzyme and spore enzyme have the same Km values and pH optima. The optimum p11 was about 9.2 for both enzymes and the Km values for NADP+ and glucose 6-phosphate were determined to be 6.7×10-6 and 7.5×10-5M, respectively.
4. The amino acid composition of the vegetative cell enzyme was examined and was compared with those of enzymes from other sources.抄録全体を表示PDF形式でダウンロード (1478K) -
Shin-ichi HAYASHI, Takehiko TANAKA, Junko NAITO, Masami SUDA1975 年 77 巻 1 号 p. 207-219
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーThe importance of the so-called phosphorylated pathway for the synthesis of serine in rat liver was confirmed by studies on in vitro serine synthesis and also by the existence of a correlation between the serine-synthesizing activity of liver extracts and the activities of the enzymes involved in the pathway under various dietary conditions. Serine-synthesizing activity was found to be distributed in various rat tissues such as kidney, brain, testis, spleen, pancreas, and fat pad. However, only in the liver was the synthesis regulated by dietary protein. In the liver, the three enzymes of the phosphorylated pathway were found to be repressed by high-protein diets or by starvation and induced by low-protein diets. The dietary induction of the enzymes required the presence of insulin and was suppressed by glucocorticoids. A suggestion is made that the effects of diet or hormones may be mediated by changes in the hepatic pool of essential amino acids.抄録全体を表示PDF形式でダウンロード (1021K) -
II. A Peptide-bound Flavin as the Prosthetic Group of L-Gulono-γ-lactone OxidaseHidehiko NAKAGAWA, Akira ASANO, Ryo SATO1975 年 77 巻 1 号 p. 221-232
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーL-Gulono-γ-lactone oxidase [EC 1. 1. 3. 8] was purified 80-fold from rat liver micro-somes. In confirmation of our previous finding with a cruder preparation, the purified enzyme was shown to contain an L-gulono-γ-lactone-reducible pigment as a prosthetic group. This pigment was not liberated from the protein by acid am-monium sulfate, 10% trichloroacetic acid or 2M urea, but was effectively released by proteolytic digestion. The pigment thus released showed a reduced-minus-oxidized difference spectrum characteristic of a flavin compound. The pigment was liberated from a trichloroacetic acid-treated preparation of the enzyme by pronase digestion and purified by Florisil column chromatography and paper chromatography. The absorption spectrum as well as the fluorescence emission and excitation spectra of the purified pigment indicated that it was actually a flavin peptide. It was, however, different not only from FMN but also from flavin peptides isolated from other sources such as succinate dehydrogenase [EC 1. 3. 99. 1] and monoamine oxidase [EC 1. 4. 3. 4] as regards the pH dependence of fluorescence intensity and the Rf value on thin-layer chromatography. A preliminary analysis showed that the purified flavin com-pound contained several amino acid residues. Alkaline photolysis of the purified flavin peptide suggested that the isoalloxazine ring of the flavin is involved in its binding to the peptide. The hypsochromic shift of the absorption peak in the near-ultraviolet region suggested further that the linkage between the flavin and the peptide may be mediated by the 8-methyl group of the isoalloxazine nucleus. It can be concluded that the prosthetic group of gulonolactone oxidase is a flavin which is covalently bound to the enzyme protein.抄録全体を表示PDF形式でダウンロード (974K) -
Studies on NADP+-specific Isocitrate Dehydrogenase from an Extreme Thermophile, Thermus flavus AT-62Partial Purification and PropertiesTakashi SAIKI, Kei ARIMA1975 年 77 巻 1 号 p. 233-240
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリー1. NADP+-specific isocitrate dehydrogenase [EC 1.1.1.42] was partially purified by about 440-fold from an extreme thermophile, Thermus flavus AT-62.
2. Remarkable thermostability of the enzyme was confirmed. The enzyme was not inactivated after 60 min at 70°, and the activity was lost only slowly at 80°. Above 90°, however, rapid inactivation was observed.
3. The dehydrogenase was susceptible to concerted inhibition by oxaloacetate plus glyoxylate. In the presence of oxaloacetate plus glyoxylate (each 1mM), 75% inhibi-tion was observed.
4. The degree of inhibition of the enzyme by oxaloacetate plus glyoxylate decreased markedly above 60°. The affinity of the enzyme for isocitrate and NADP+ was also reduced markedly above 60°. The activation energy calculated from Arrhenius plots below and above 60° were 14, 500 and 8, 000 cal per mole, respectively.
These observations suggest a possible conformation change of the enzyme protein at a transition temperature of 60°, and the physiological significance of this in the adaptation of thermophiles to elevated temperatures is discussed.抄録全体を表示PDF形式でダウンロード (543K) -
Yuji TANAKA, Akihiro KAGAMIISHI, Akira KIUCHI, Tadao HORIUCHI1975 年 77 巻 1 号 p. 241-247
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーβ-Galactosidase [EC 3.2.1.23] has been purified from a culture of Aspergillus oryzae by 2-propanol fractionation, column chromatography on DEAE-Sephadex A-50 and Sephadex G-200. The preparation was homogeneous on ultracentrifugation and disc electrophoresis.
The enzyme showed pH optima of 4.5 with ONPG1 as a substrate and 4.8 with lactose as a substrate. The stable pH range was from 4.0 to 9.0 and the optimum temperature was 46°.
The Michaelis constants were 7.2×10-4M with ONPG and 1.8×10-2M with lactose. Hg2+, Cu2+, N-bromosuccinimide, and sodium laurylsulfate caused marked inhibition.
The apparent molecular weight was calculated to be about 105, 000 by Sephadex gel filtration and sucrose density gradient centrifugation.抄録全体を表示PDF形式でダウンロード (1230K) -
Takahiko SHIOSAKA, Teruo ARIMA, Hideki TOIDE, Hiromichi OKUDA, Setsuro ...1975 年 77 巻 1 号 p. 249-256
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーDuring the fractionation of various enzymes concerned with DNA synthesis from the postmicrosomal supernatant fraction of various tissues, DNA polymerase [EC 2.7.7.7], thymidine kinase [EC 2.7.1.75], dTMP kinase [EC 2.7.4.9], deoxycytidine kinase [EC 2.7.1.74], and deoxycytidine monophosphokinase (dCMP kinase) [EC 2. 7. 4. 14] were found in the pellet fraction of postmicrosomal supernatant. Further, the uridine kinase [EC 2. 7. 1.48] and aspartate transcarbamylase [EC 2.1.3.2] activi-ties of postmicrosomal supernatant from various tissues were also present in this pellet fraction. The activities of DNA polymerase, thymidine kinase, uridine kinase, and aspartate transcarbamylase from normal and regenerating rat liver, and Yoshida sarcoma were higher in the pellet fraction than in the supernatant. On the other hand, the activities of dTMP kinase, dCMP kinase, and orotidine-5'-phosphate de-carboxylase [EC 4. 1. 1. 23] were lower in the pellet fraction than in the supernatant.
The pellet fractions of regenerating rat liver and Yoshida sarcoma showed a remarkable incorporation of various precursors (thymidine, dTMP, deoxycytidine, and dCMP) into DNA in the presence of a suitable DNA template, ATP and all four deoxynucleoside 5'-triphosphates for DNA synthesis. Normal adult rat liver catalyzed a much smaller incorporation of all these precursors, except for dCMP.抄録全体を表示PDF形式でダウンロード (607K) -
Homare KUWANA, Masao DATE1975 年 77 巻 1 号 p. 257-259
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーAcetohydroxy acid synthetase [EC 4.1.3.18] of Neurospora crassa has been solubilized from a mitochondria) pellet fraction by sonic treatment in 1.0M potassium phosphate buffer at pH 7.5 containing 10mM MgSO4 and centrifugation at 180, 000×g for 20 min. The soluble enzyme thus obtained has a high specific activity and a high sensitivity to valine comparable to the original mitochondria) pellet suspension when the activity was determined in high concentrations of potassium phosphate.抄録全体を表示PDF形式でダウンロード (181K) -
A New Technique for Investigation of Protein-ligand InteractionKen-ichi KASAI, Shin-ichi ISHII1975 年 77 巻 1 号 p. 261-264
発行日: 1975/01/25
公開日: 2008/11/18
ジャーナル フリーA new method for quantitative analysis of affinity chromatography which allows estimation of dissociation constants of a protein-ligand (either soluble or immobilized) complex is proposed. This method was successfully applied to a trypsin-glycylgly-cyl-L-arginine agarose system.抄録全体を表示PDF形式でダウンロード (248K)
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