All issues
Volume 77 (1975)
- Issue 6 Pages 1127-
- Issue 5 Pages 909-
- Issue 4 Pages 689-
- Issue 3 Pages 473-
- Issue 2 Pages 265-
- Issue 1 Pages 1-
Volume 77, Issue 5
Displaying 1-26 of 26 articles from this issue
- |<
- <
- 1
- >
- >|
-
Hiroshi KIMURA, Toshihiro TSUDZUKI, Takashi MURACHI1975 Volume 77 Issue 5 Pages 909-912
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe catabolic degradation of hemoglobin and of its complex with haptoglobin by lysosomal enzymes from rat liver was studied with special emphasis on the action of cathepsins D and E. The digestion of free hemoglobin can be mainly attributed to the action of cathepsin D [EC 3.4.23.5], while the digestion of the complex in the pH range 2-3 is due more to the action of cathepsin E than that of cathepsin D. The enzymic activities of both cathepsins were strongly inhibited by pepstatin, and 4 M urea inactivated cathepsin E. Measurements of the peroxidase activity and optical rotatory dispersion of the hemoglobin-haptoglobin complex showed that the complex suffered rapid denaturation below pH 2.9.View full abstractDownload PDF (277K) -
Kuniko OKUMURA, Takashi MURACHI1975 Volume 77 Issue 5 Pages 913-918
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPapain [EC 3. 4. 22. 2] was photooxidized using methylene blue as a sensitizer. The photooxidzed enzyme lost its caseinolytic activity and had significantly decreased histidine and tryptophan contents. The tyrosine content was the same before and after the photooxidation. The SH content of the photooxidized enzyme, as deter-mined after reduction with dithiothreitol, was also unchanged. The loss of histidine was always slower than the loss of enzymatic activity, being less than one residue per molecule even when the enzymatic activity was completely lost. However, the inactivation and the oxidation of a histidine residue were pH-dependent in a similar fashion in the pH range of 5.0-8.0, the pH profiles conforming to theoretical titra-tion curves with apparent pKa values of 6.6 and 6.7, respectively. The fact that the ionization of a histidine residue in papain has a normal imidazole pKa value is entirely in accord with the finding for stem bromelain [EC 3. 4. 22. 4] (Murachi, T., Tsudzuki, T., & Okumura, K. (1975) Biochemistry 14, 249-255), and is of great significance in relation to the mechanism of catalysis by these enzymes.View full abstractDownload PDF (403K) -
Hiroshi SAITO, Zensaku YOSIZAWA1975 Volume 77 Issue 5 Pages 919-930
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIntima-media of bovine aorta was digested with pronase, after preliminary extraction of saline (1%)-soluble substances and fat. Crude glycopeptide fraction was then obtained from the resulting complex carbohydrate fraction by fractionation with CPC (cetylpyridinium chloride). Complete separation of sialoglycopeptides was achieved by chromatography on a DEAE-cellulose column at pH 7.2 followed by repeated chromatography on a DEAE-Sephadex A-25 column at pH 5.2.
Nine sialoglycopeptides (SGP 1-SGP 9) thus obtained were homogeneous on high-voltage paper electrophoresis at pH 3.5 and pH 5.2. The analytical data showed great heterogeneity of the carbohydrate chains of these preparations, although they consisted of the same monosaccharides (galactose, glucose, mannose, glucosamine, galactosamine, fucose, and sialic acid), except that SGP 1 lacked galactosamine. Heterogeneity was also observed in their peptide chains. It was noticed, however, that the contents of hexose, hexosamine, and aspartic acid of the fractions (SGP 3, SGP 4, and SGP 5) which eluted from the DEAE-Sephadex A-25 column at lower molarity of the eluting salt were higher than those of the fractions (SGP 7, SGP 8, and SGP 9) which eluted at higher molarity, while the contents of sialic acid and hydroxyamino acids were in an opposite relationship. Representative fractions (SGP 7 and SGP 9) of the latter contained many more alkali-sensitive linkages than those (SGP 3 and SGP 5) of the former, indicating the presence of many more 0-glycosidic linkages between hydroxyamino acid(s) and sugar(s) in the latter than in the former. The sialoglycopeptides contained significant amounts of sialic acid, ranging from 10% (SGP 1) to 32.4% (SGP 8). The highest contents were in SGP 8 and SGP 9, which contained equimolar amounts of sialic acid and hexosamine. Furthermore, infrared spectra indicated the presence of sulfate groups in most of the sialoglycopeptides.View full abstractDownload PDF (839K) -
Hiroshi SAITO, Noboru OTOTANI, Zensaku YOSIZAWA1975 Volume 77 Issue 5 Pages 931-938
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSclerotic and non-sclerotic tissues of intima-media of human aorta were digested with pronase, after preliminary extraction of saline (1%)-soluble substances and fat. Subsequently, sialoglycopeptide fractions were obtained from the pronase digests by fractionation with CPC (cetylpyridinium chloride). Complete separation of sialogly-copeptides was achieved by chromatography on a DEAE-Sephadex A-25 column at pH 7.2, followed by repeated chromatography on the same Sephadex column at pH 5.2.
The purified sialoglycopeptides thus obtained showed heterogeneity of their carbohydrate chains, although they contained the same monosaccharides (galactose, mannose, glucose, glucosamine, galactosamine, fucose, and sialic acid). Heterogeneity was also observed in their peptide chains. The contents of hydroxyamino acids and alkali-sensitive linkages of these preparations indicated that the fractions eluted from the DEAE-Sephadex A-25 column at higher molarity of the eluting salt at pH 5.2 contained many more O-glycosidic linkages between hydroxyamino acid(s) and sugar(s) than those eluted at lower molarity.
The present data indicate that the concentration of sialoglycoproteins in intima-media of human aorta increases significantly in arteriosclerosis in terms of defatted dry tissue. Moreover, the chemical nature of the sialoglycoproteins was modified slightly in the disease.View full abstractDownload PDF (541K) -
Toshio TAKAGI, Kaoru TSUJII, Keishiro SHIRAHAMA1975 Volume 77 Issue 5 Pages 939-947
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo clarify the mode of interaction between sodium dodecyl sulfate (SDS) and protein polypeptides with special reference to SDS-polyacrylamide gel electrophoresis, the binding of SDS to several protein polypeptides was investigated by the equilibrium dialysis technique. Each of the binding isotherms was characterized by the presence of two phases: an initial gradual increase in the amount of binding to 0.3-0.6g/g (first phase) and a subsequent steep increase to 1.2-1.5g/g (second phase). The binding was completed at a concentration of SDS below the critical micelle concentration. Throughout the first and second phases, the isotherms obtained were different for each kind of protein. On the basis of experiments with bovine serum albumin and ribonuclease [EC 3.1.4.22], the isotherms were profoundly affected by the method used for modification of the sulfhydryl groups. The claim of Reynolds and Tanford (Proc. Natl. Acad. Sci. U. S., 66, 1002 (1970)). that the isotherms are virtually identical for many kinds of proteins was not supported by the present data. Changes in the gross and local conformations were examined with reference to the isotherms by measurements of CD spectrum, free boundary electrophoresis, and gel filtration. The results obtained were collectively interpreted based on the model of SDS-protein polypeptide complexes proposed by the present authors (J. Biochem., 75, 309 (1974.)).View full abstractDownload PDF (696K) -
Norio KATOH, Shuichiro KUBO1975 Volume 77 Issue 5 Pages 949-955
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosins prepared from chicken and rabbit fast and slow muscles were treated with 5, 5'-dithiobis-(2-nitrobenzoic acid) (Nbs2). About half of the thiol groups of the fast muscle myosins reacted with Nbs2, but in slow muscle myosins, only about 10-20% of the thiol groups reacted.
This treatment removed 50-60% of L2 components, Nbs2 light chain, from fast muscle myosins, but did not result in specific dissociation of the light chains in slow muscle myosins. The treatment sometimes released L4 component from chicken muscle myosins instead of L2 component.
The changes of myosin ATPase [EC 3.6.1.3] activities caused by this treatment did not correlate with the release of Nbs2 light chain, but were dependent upon the species, chicken or rabbit.View full abstractDownload PDF (1176K) -
I. Liquefying α-Amylase from Bacillus subtilis1, 2Keitaro HIROMI, Masatake OHNISHI, Ken-ichiro KANAYA, Takashi MATSUMOTO1975 Volume 77 Issue 5 Pages 957-963
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRapid conformational changes due to pH jump were studied kinetically at 25° mainly by the stopped-flow method using liquefying α-amylase from Bacillus subtilis [EC 3. 2. 1. 1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump.
Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH.
Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation of phenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.View full abstractDownload PDF (1306K) -
Toshio SHIBAOKA, Takashi INATANI, Keitaro HIROMI, Takehiko WATANABE1975 Volume 77 Issue 5 Pages 965-968
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The action of saccharifying α-amylase from B. subtilis4 [EC 3. 2. 1. 1] on eq- (the equilibrium mixture of α- and β-forms) and β-maltose was studied at pH 5.4 and 25°.
2. The plots of initial velocity versus substrate concentration showed remarkable sigmoidal curves for both substrates.
3. The values of Hill's coefficient for eq- and β-maltose were determined to be 3.3 and 3.8, respectively.View full abstractDownload PDF (292K) -
Koscak MARUYAMA1975 Volume 77 Issue 5 Pages 969-972
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of three components of troponin (TN C, I, T) on the gelation of F-actin was investigated by measuring the increase in viscosity at a very low velocity gradient in a rotating viscometer. TN I or TN T greatly enhanced the generation of F-action. The effect of TN I-C or T-C complex became Ca-dependent: in the absence of Ca, the complex increased the rate of viscosity rise of F-actin, but in its presence this enhancing effect was almost absent. For these actions, the presence of tropomyosin or heat treatment at 45° was not required. These results can be explained in terms of strengthened interactions of F-actin particles bound with TN T or TN I and the release of TN I-C or TN T-C in the presence of Ca.View full abstractDownload PDF (260K) -
Naomi AZUMA, Akira ASAKURA, Koichi YAGI1975 Volume 77 Issue 5 Pages 973-981
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSActomyosin was extracted from smooth muscle of molluscan abalone with 0.1 M PPi at pH 6.4. Myosin was separated from the actomyosin by centrifugation at 100, 000×g in the presence of 5 mM ATP and 10 mM MgCl2. Myosin in the supernatant was further purified by gel filtration on a Sepharose 4B column. Paramyosin contamination of the actomyosin preparation interfered with the isolation of myosin and complete removal of actin and paramyosin from the myosin has not been accomplished.
The myosin appeared to consist of a single f-chain and a single g-chain, as examined by SDS-disc electrophoresis in 8 or 13.7% acrylamide gel. The ATPase [EC 3.6.1.3] activity of this myosin in 0.5m KCl at neutral pH and at 0° was rather unstable and decreased by 10-20% per day. The effects of p-chloromercuri-benzoate and EDTA on the ATPase activity were similar to those observed with other smooth muscle myosin but the dependence upon pH or KCl concentration was different.View full abstractDownload PDF (579K) -
Mikiharu YOSHIDA, Fumi MORITA1975 Volume 77 Issue 5 Pages 983-992
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of ADP to subfragment-1 was investigated by the gel filtration method. The amount of bound ADP was determined as a function of free ADP concentration. Linear Scatchard plots were obtained. The maximum binding number, 0.55 mole of ADP per 105g of protein, and the dissociation constant, 1.6×10-6 M, were obtained, using subfragment-1 prepared by tryptic digestion, in the presence of 0.083 M KCl-1G mM MgCl2-0.02 M Tris-HCl (pH 8), at 25°. Similar maximum numbers, about 0.5 mole per 106 g of protein, were obtained with subfragment-1 prepared by chymotryptic digestion of myosin or papain digestion of myofibrils. The maximum number did not depend on the KCl concentration or the temperature, while the dissociation constant decreased on decreasing either the KCl concentration or the temperature.
Adenylyl imidodiphosphate binding to subfragment-1 prepared by chymotryptic digestion was also measured by the gel filtration method. The maximum binding number, 0.41 mole per 105 g of subfragment-1, and the dissociation constant, less than 10-7 M, were obtained in the presence of 0.7 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 8°.
The difference absorbance at 288 nm of the difference absorption spectrum induced by ADP of subfragment-1 prepared by tryptic digestion was proportional to the amount of bound ADP.
The steady-state ATPase rate of subfragment-1 prepared by tryptic digestion was inhibited competitively by ADP in the presence of MgCl2. The extent of the initial burst of ATPase [EC 3.6.1.3] decreased from 0.46±0.06 to 0.30±0.09 mole of Pi per 105 g of subfragment-1 on adding ADP to a level of 0.6 mM.
Subfragment-1 prepared by tryptic digestion bound F-actin with a mole ratio of 1/0.96 of actin monomer. The binding was depressed by the addition of ADP.
On the basis of these results, subfragment-1 preparations were assumed to be a half-and-half mixture of two kinds of protein, and properties of each protein are discussed.View full abstractDownload PDF (694K) -
Yoshiko NAKAE, Kiyoshi IKEDA, Kozo HAMAGUCHI1975 Volume 77 Issue 5 Pages 993-1006
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe pH dependence of the extrinsic circular dichroic (CD) band at 375 nm of hen egg-white lysozyme [EC 3. 2. 1. 17] in which Trp 62 had been selectively 2-nitro-phenylsulfenylated (NPS-lysozyme) was studied. This pH dependence was interpreted in terms of the participation of Glu 35 (pK 6.2), Asp 101 (pK 4, 6), and Asp 66 (pK 1.5). The fact that the ionization of Glu 35 affects the extrinsic CD band of the NPS chromophore attached to Trp 62 confirms the presence of a relation between the state of Trp 62 and the ionization state of one of the catalytic groups, Glu 35, in hen lysozyme, as proposed by Ikeda and Hamaguchi (J. Biochem., 74, 221-230 (1973))..
The pH dependence of the binding constants of the dimer and trimer of N-acetylglucosamine (GlcNAc) and the p-methyl glycoside of GlcNAc (β-methyl-GlcNAc) to NPS-lysozyme were studied by measuring the changes in the extrinsic CD band. The changes in the CD spectrum on the binding of (G1cNAc)3 and (GIcNAc)2 were very similar to each other but were different from that on the binding of β-methyl-GIcNAc. However, β-methyl-G1cNAc competitively inhibited the binding of (GlcNAc)2 to NPS-lysozyme. The binding constants of the three saccharides to NPS-lysozyme were much smaller than those for intact lysozyme. The pH dependence of the binding constants of (GIcNAc)2 and (GlcNAc)3 were interpreted in terms of the participation of G1u 35 (pK 6.2), Asp 52 (pK 3.3), Asp 101 (pK4.6), and Asp 66 (pK 1.5). These pK values are very similar to those for intact lysozyme, as determined by Kuramitsu et al. (J. Biochem., 76, 671-683 (1974)); 77, 291-301 (1975). Comparison of the binding constants of Mn2+ and Co2+ ions to the catalytic carboxyls of NPS-lysozyme with those to intact lysozyme also indicated that the catalytic site of NPS-lysozyme is scarcely affected by this modification.
When (G1cNAc)2 or (G1cNAc)3 is bound to NPS-lysozyme, pK shifts of Glu 35, Asp 101, and Asp 66 occurred in the same directions as for intact lysozyme. In addition, a pK shift of Asp 52, which has not been observed for intact lysozyme, occurred. The participation of Asp 52 was also observed in the binding of β-methyl-GIcNAc. However, the binding of the monomer to NPS-lysozyme produced no significant pK shifts of Glu 35 and Asp 101, in contrast to the situation for intact lysozyme. These facts indicate a small difference in the binding orientation of the saccharides between the modified and intact lysozymes.View full abstractDownload PDF (1004K) -
Michiteru YOSHIDA, Aiko KIKUCHI, Kensuke SHIMURA1975 Volume 77 Issue 5 Pages 1007-1014
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1) A method is described for the separation and fractionation of nonhistone chromosomal proteins from salt-urea dissociated calf thymus chromatin. After precipitating DNA in the dissociated chromatin solution with LaCl3, the chromosomal proteins in the supernatant were fractionated by SP-Sephadex C-25 column chromatog-raphy using a combination of NaCl stepwise and linear gradient elutions. Much care was taken to prevent proteolytic degradation of the chromosomal proteins during the preparation.
2) Among the protein fractions separated by this chromatography, twenty subfrac-tions were found to be homogeneous on SDS-polyacrylamide gel electrophoresis. These purified proteins account for about 18% of the whole chromosomal protein. Eleven subfractions of these purified nonhistone proteins had ratios of acidic to basic amino acids above 1.0 and the nine remaining subfractions had ratios below 1.0, corresponding to nonhistone proteins of basic character.
3) The molecular weights of the purified nonhistone proteins ranged from 7, 400 to 19, 000.View full abstractDownload PDF (623K) -
Noshi MINAMIURA, Hiroshi CHIURA, Kazuaki TSUJINO, Takehiko YAMAMOTO1975 Volume 77 Issue 5 Pages 1015-1022
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis paper describes the isolation and study of glucose-forming amylase existing in human urine as a normal component. After removing a-amylase [EC 3. 2. 1. 1] by adsorption onto raw starch, urine was treated with DEAE-cellulose and Bio Gel P-150, and three fractionated proteins (F-l, F-2, and F-3), isolated in a homogeneous state by gel filtration, were shown to display glucose-forming amylase activity. They all hydrolyzed starch and glycogen, releasing glucose as the sole product, and also hydrolyzed maltose. However, their molecular weights, as estimated by gel filtration, isoelectric points, stabilities, and several enzymatic properties were differ-ent. The implications of the results are discussed.View full abstractDownload PDF (1072K) -
Isamu SHIMIZU, Hirohiko KATSUKI1975 Volume 77 Issue 5 Pages 1023-1027
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnaerobically grown Saccharomyces cerevisiae was aerated for 7 hr at 20°, 30°, or 40°, in a phosphate buffer containing 2% glucose. At elevated temperature (40°), de novo synthesis of squalene and sterols in the aerated yeast was only 32-35% of that at lower temperature (20° or 30°), and this decrease was attributed to the repression of the enzymes involved in the synthesis of mevalonate from acetyl-CoA. In addition, at elevated temperature, the metabolic flux from squalene to ergosterol was blocked at squalene epoxidation, lanosterol demethylation, and ergosta-5, 7, 22, 24 (28)-tetraene-3β-ol reduction.View full abstractDownload PDF (381K) -
Shuzo YAMAGATA, Kazuhito TAKESHIMA, Nobuo NAIKI1975 Volume 77 Issue 5 Pages 1029-1036
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nutritional requirements of three yeast mutants, previously shown to possess low O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) sulfhydrylase activities, were reinvestigated. It was thus found that one mutant (strain No. 16), previously identified as a homocysteine auxotroph, is in fact a double mutant requiring both cysteine and OAH. In agreement with the previous assignment, the other two strains (strains No. 13 and 17) were shown to be true cysteine auxotrophs. These results can best be explained by assuming the cystathionine pathway to be the main route of homocysteine synthesis in this organism.
It was further found that extracts of the three mutants contain genetically modified OAS-OAH sulfhydrylases with much reduced catalytic activities. Modified sulfhydrylase was partially purified from strain No. 16 by the same procedure as for the wild-type enzyme. Both OAS and OAH sulfhydrylase activities of the mutant enzyme were copurified and behaved identically on polyacrylamide gel elec-trophoresis. The enzymatic and physicochemical properties of the purified mutant enzyme were shown to be very similar to those of the wild-type enzyme, except that the catalytic activities of the former were only 3-5% of those of the latter, and that the ratio of OAH sulfhydrylase to OAS sulfhydrylase activity was some-what lower in the former than in the latter.View full abstractDownload PDF (1207K) -
I. Relationship of the Sulfhydryl Group Responsible for Mg2+-ATPase Activation to the S1 and S2 GroupsTatsuhisa YAMASHITA, Masashi KOBAYASHI, Tsuneyoshi HORIGOME1975 Volume 77 Issue 5 Pages 1037-1046
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reactivity of the sulfhydryl groups in myosin B to N-ethylmaleimide (NEM) was investigated under various conditions. Under the conditions where actin and myosin associate, i.e. at low ionic strength, only Mg2+-ATPase [EC 3. 6. 1. 3] activity was markedly activated by NEM treatment, whereas coupling of EDTA-ATPase inhibition with Ca2+-ATPase activation, which was seen on blocking S1 of myosin A with NEM, was observed under conditions at which the dissociation of actomyosin occurs, i.e. at high ionic strength, suggesting the covering with actin of the S1 region of myosin. Nevertheless, ATP accelerated the reactivity of Sl and S2 much more in the myosin B system than in myosin alone.
NEM-modified myosin B ATPase exhibited a shift of the KCl dependence curve to high concentration, a shift of the maximum activation of ATPase activity to high Mg ion concentration and a suppression of substrate inhibition at high substrate concentrations. These all indicate that the blocking by NEM of Sa, the sulfhydryl group related to the activation of Mg2+-ATPase of myosin B, brings about an increase in the association of myosin and actin in the myosin B system, resulting in an activation of Mg2+-ATPase activity. In addition, the relationship between Sa and a sulfhydryl group (s) essential for Ca2+ sensitivity was discussed.View full abstractDownload PDF (674K) -
Minoru HAMADA, Kichiko KOIKE, Yutaka NAKAULA, Tadayasu HIRAOKA, Masahi ...1975 Volume 77 Issue 5 Pages 1047-1056
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25°. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism.
Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM.
Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.View full abstractDownload PDF (589K) -
I. Purification from Ginseng Extract of the Active Component Stimulating Serum Protein BiosynthesisHikokichi OURA, Susumu HIAI, Yoshihiro ODAKA, Takako YOKOZAWA1975 Volume 77 Issue 5 Pages 1057-1065
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSystematic isolation and purification of the biologically active component of ginseng extract were followed by observing the incorporation of labeled leucine into serum protein at 6 hr after a single intraperitoneal injection in a mouse. Ginseng saponin mixture (fraction 5) exhibited high activity for such incorporation. Seven saponins were isolated from fraction 5 by means of preparative TLC, and assayed. Adminis-tration of all these saponins (ginsenoside-Rb2, Rc, Rc2, Rd, Re, and Rg1), except for ginsenoside-Rb1, caused an increase of leucine incorporation over that in control animals, The incorporation rate was directly proportional to the dose in the case of ginsenoside-Rd, which had the highest activity. The increased specific radio-activity of serum protein was not due to a decrease in the pool size of free amino acids in the liver. It was conclusively shown that the active component stimulating serum protein biosynthesis is saponin.View full abstractDownload PDF (582K) -
Koichiro TAKESHIGE, Shigeki MINAKAMI1975 Volume 77 Issue 5 Pages 1067-1073
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSElectron transport particles (ETP) prepared from beef heart mitochondria formed malondialdehyde by NADPH-dependent lipid peroixidation in the presence of ferric ions and ADP or ATP. The reaction was inhibited by MnCl2, EDTA, or radical scavengers, but was not inhibited by p-hydroxymercuribenzoate (PHMB) or respiratory chain inhibitors. The oxidation of NADPH and oxygen consumption by ETP were activated by the addition of ferric ions and ATP, and inhibited by inhibitors of lipid peroxidation. This peroxidation system was apparently different from those of liver microsomes and mitochondria as regards the effect of PHMB, optimal pH and the concentration of NADPH for half-maximal reaction velocity.View full abstractDownload PDF (493K) -
Masachika IRIE, Fumie SUITO1975 Volume 77 Issue 5 Pages 1075-1084
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to study the state of tyrosyl residues in a ribouuclease from bovine semina vesicles [EC 3. 1. 4. 22, RNase Vs1], several lines of experiments were carried out.
Spectrophotometric titration of RNase Vs1 indicated that two out of 8 tyrosine residues were titrated very easily and their apparent pKa, values were about 9.8. Next, about 4 residues were titrated at pH up to 13.5. The remaining 2 residues were titrated time-dependently at pH 13.5. In 8 M urea, about 6 tyrosine residues were titrated with apparent pKa values of about 11.2 and about 2 residues were titrated time-dependently at pH 13.5.
Acetylation of RNase Vs1 with N-acetylimidazole was studied at pH 7.5. In aqueous solution, about 1.1-3.5 tyrosine residues were acetylated, depending on the experimental conditions, and in 8 M urea, 5.3 tyrosine residues were modified.
RNase Vs1 was nitrated with tetranitromethane at pH 7.5. In aqueous solution, about 2.5 tyrosine residues were nitrated very easily; the enzymatic activity of the modified enzymes was 130-200% of that of the native enzyme. In 8 M urea, the reactivity of the tyrosine residues increased and about 4-5.5 residues were modified.
The results of chemical modification and spectrophotometric titration indicated that about two tyrosine residues in RNase Vs1 were exposed to the solvent and were more reactive to various reagents, and 3-4 tyrosine residues were less reactive. The final 2 residues were not accessible to the reagent even in the presence of urea, but were titraten at pH 13.5. The solvent perturbation difference spectrum using ethylene glycol as a perturbant indicated that about 4 tyrosine residues were perturbed.
When the pH of the enzyme solution was changed from 7.0 to 1.0, the change in optical density of RNase Vs1 due to denaturation blue shift was about 1, 600 at 287 nm. The optical density change at 287 nm of native RNase Vs1 on exposure to 8 M urea and 6 M guanidine-HCl indicated that the environments of 2-3 and 4 tyrosine residues were changed by the addition of the denaturants, urea and guanidine-HCl, respectively.
In RNase Vs1 having about four nitrotyrosine residues, the two most inaccessible tyrosine residues remained resistant to titration with alkali. On adding nucleotide, nitrated RNase Vs1 gave a difference spectrum in the ultraviolet region but not in 320-460 nm region, where nitrotyrosine residues absorb light. This may indicate that tyrosine residues located relatively near the surface of the molecule are not perturbed directly by nucleotide binding.View full abstractDownload PDF (724K) -
Kazuko OHGI, Masachika IRIE1975 Volume 77 Issue 5 Pages 1085-1094
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrom a commercial digestive produced from Aspergillus saitoi, a ribonuclease [EC 3.1.4.23] having a molecular weight of 12, 500 has been isolated in addition to the RNase reported previously, which had a molecular weight of 38, 000. The enzyme was found to be homogeneous by chromatography on DEAE-cellulose, disc electro-phoresis on polyacrylamide gel, and ultracentrifugation. The NH2-terminal amino acid was identified as glutamic acid. The amino acid composition indicated the presence of about 13 tyrosyl residues, 3 histidyl residues, and 2 half-cystine residues. The pH optimum of the RNase was 4.5, using RNA as a substrate. The enzyme was stable on heating at 70° for 5 min from pH 2 to 10. It hydrolysed RNA completely to mononucleotides via 2', 3'-cyclic nucleotides. The rates of release of nucleotides and 2', 3'-cyclic nucleotides were in the order: guanylic acid>adenylic acid>cytidylic acid>uridylic acid.View full abstractDownload PDF (2982K) -
I. Studies with Hydrophosbic ProbesKen-ichi ARAI, Takao TRAI, Masao KAWAKITA, Yoshito KAZIRO1975 Volume 77 Issue 5 Pages 1095-1106
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conformational difference between polypeptide chain elongation factor Tu (EF-Tu)•GTP and EF-Tu•GDP has been studied using hydrophobic and fluorescent probes. The interaction of EF-Tu.GDP with 1-anilino-8-naphthalenesulfonate (ANS) was measured in terms of the enhancement of the fluorescence intensity at the emission maximum of 475 nm. When EF-Tu• GDP•ANS complex was converted to EF-Tu.GTP•ANS complex by incubation with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40], there was a roughly 2-fold increase in fluorescence intensity and a blue shift of the emission maximum from 475 to 467 nm, indicating a con-formational transition of the protein. The conformational change was found to be reversible and the spectrum promptly returned to that of EF-Tu• GDP• ANS complex upon addition of excess GDP. A similar change in the spectrum was also observed when aminoacyl-tRNA, but not deacylated tRNA, was added to EF-Tu• GTP• ANS complex. Measurement of the number of binding sites by gel filtration indicated that EF-Tu•GTP and EF-Tu•GDP bind 2.9 and 1.7 molecules of ANS, respectively. These results suggest that in EF•Tu. GTP the conformation was altered and one additional binding site for ANS was created at or near the site interacting with aminoacyl-tRNA.
Another reagent, N-(1-anilinonaphthyl-4)maleimide (ANM) was covalently bound to the sulfhydryl group in EF-Tu•GDP which is essential for interaction with aminoacyl-tRNA. The binding could be determined spectrofluorometrically, since the reagent, which is nonfluorescent in aqueous solution, emitted a strong fluore-scence upon binding with the sulfhydryl group, indicating a marked hydrophobicity of the local environment. Measurements of the kinetics of the binding revealed that ANM reacted rapidly with the sulfhydryl group in EF-Tu•GTP, while the reaction with that in EF-Tu GDP proceeded more sluggishly. The difference in the reactivity of the sulfhydryl group essential for aminoacyl-tRNA binding between EF-Tu•GTP and EF-Tu•GDP probably reflects a conformational transition of the protein near the active site.
These results, together with those on spin-label studies previously published (Arai, Kawakita, Kaziro, Maeda, & Ohnishi (1974) J. Biol. Chem. 249, 3311), demon-strate that reversible conformational transition does occur in EF-Tu on changing the ligand from GDP to GTP.View full abstractDownload PDF (950K) -
Masahiro MASADA, Kazuo FUKUSHIMA, Goro TAMURA1975 Volume 77 Issue 5 Pages 1107-1115
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCysteine synthase [O-Acetyl-L-serine acetate-lyase (adding hydrogen-sulfide) EC 4.2.99.8] has been highly purified from the extract of rape, Brassica chinensis var. Komatsuna. The purified preparation appeared to be homogeneous on Sephadex G-100 gel filtration and dodecylsulfate-polyacrylamide gel electrophoresis, showing a molecular weight of about 62, 000. The latter method also suggested that this enzyme was composed of two identical subunits. The enzyme contained 2 moles of pyridoxal phosphate per mole of enzyme.View full abstractDownload PDF (1324K) -
Michihiko OGUCHI, Yoko MIYATAKE, Junko AYABE, Nobu AKAMATSU1975 Volume 77 Issue 5 Pages 1117-1121
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSD-Glucosamine was found to be phosphorylated by a rat liver extract in the presence of a high concentration of glucose, which was formerly believed to be a strong competitive inhibitor of this reaction. Results suggested that glucosamine may be phosphorylated by high Km hexokinase, i.e. glucokinase [EC 2.7.1.2]. The enzyme involved was separated from specific N-acetyl-D-glucosamine kinase [EC 2.7.1.59]. The phosphorylation was not inhibited by a physiological level of glucose or glucose 6-phosphate, which strongly inhibited low Km hexokinase. The apparent Km of glucokinase for glucosamine was estimated as 8 mM, which is ten times that of low Km hexokinase.View full abstractDownload PDF (333K) -
Yutaka ORII, Tetsutaro IIZUKA1975 Volume 77 Issue 5 Pages 1123-1126
Published: May 25, 1975
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAddition of NaHSO3 or HCN to the formyl group of heme a was greatly accelerated by freezing reaction mixtures prepared in aq. Na2CO3, and freezing resulted in characteristic color and spectral changes of the solutions. Similar changes were observed on decreasing the pH of alkaline reaction mixtures with HCl at room temperature, indicating that the effective pH of certain salt solutions is greatly lowered by freezing. The reactivity of the formyl group changed depending on the redox state of the heme iron and the species of ligand.View full abstractDownload PDF (224K)
- |<
- <
- 1
- >
- >|