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Volume 84 (1978)
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- Issue 5 Pages 1021-
- Issue 4 Pages 737-
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Volume 84, Issue 5
Displaying 1-37 of 37 articles from this issue
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Koichi SUZUKI, Kazutomo IMAHORI1978 Volume 84 Issue 5 Pages 1021-1029
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conditions of digestion of colicin E3 with trypsin were examined to obtain an active fragment (T2A) of colicin E3, and a method suitable for large-scale preparation of T2A was developed. The T2A preparation thus obtained was homogeneous on SDS-polyacrylamide gel electrophoresis and the molecular weight was estimated to be about 11, 000. T2A was composed of 97 amino acid residues and was rich in basic amino acids; methionine, valine, cysteine, and cystine were absent. The N-terminal residue was lysine and the structure near the C-terminus was -Lys-Lys-Tyr-Leu. Since T2A had no lysine or arginine residue at the C-terminus and since the C-terminal structure was identical to that of protein A, it was concluded that T2A was derived from the C-terminal region of protein A.
No clear differences were detected among T2A preparations obtained from 3 different fractions of colicin E3, suggesting that the apparent homogeneity of colicin E3 does not involve the T2A region.View full abstractDownload PDF (2331K) -
Koichi SUZUKI, Kazutomo IMAHORI1978 Volume 84 Issue 5 Pages 1031-1039
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of an active fragment (T2A) of colicin E3 composed of 97 amino acid residues was determined. The structures of 6 tryptic peptides obtained from citracony-lated T2A were analyzed, mainly by Edman degradation. These tryptic peptides were aligned by examining overlapping peptides obtained from T2A with a Staphylococcal protease. These results, together with sequence analyses of whole T2A from both termini, established the complete amino acid sequence of T2A. The structure and function of colicin E3 are discussed based on the determined primary structure.View full abstractDownload PDF (639K) -
Yoh SANO, Yuzo NOZU, Hideo INOUE1978 Volume 84 Issue 5 Pages 1041-1049
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular weight of the coat protein subunit of cucumber green mottle mosaic virus was determined by various methods and compared with that of tobacco mosaic virus. The molecular weights of both proteins were found to be the same; 17, 100 as determined by gel chro-matography in 6M guanidinium chloride and 14, 000 by gel chromatography in 0.1% sodium dodecyl sulfate. Sedimentation equilibrium experiments in 6M guanidinium chloride previously indicated molecular weights of 17, 700 and 17, 200 for the tobacco virus and the cucumber virus proteins, respectively. However, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis the cucumber virus protein migrated faster than the tobacco virus protein. This behavior was confirmed at various gel concentrations. Plotting the logarithm of the relative mobility against the gel concentration yielded a “Ferguson plot, ” and the slopes indicated molecular weights of 18, 000 and 16, 200 for the tobacco virus and the cucumber virus proteins, respectively. When gel electrophoresis was carried out in the presence of 8M urea, both coat proteins migrated at the same rate. The Ferguson plots of both proteins were obtained under these conditions and indicated a molecular weight of 17, 700 for both proteins.
We have examined the anomalously fast migration rate of the cucumber virus protein in the absence of 8M urea. Investigations of the amounts of binding of dodecyl sulfate to both proteins with or without 8M urea, the conformation of both proteins under various denaturing conditions, and the difference in the intrinsic viscosity of the proteins confirmed our proposal that the anomalous mobility of the cucumber virus protein is attributable to a higher intrinsic negative charge density, smaller hydrodynamic volume and a slightly higher α-helix content as compared with the tobacco virus protein.View full abstractDownload PDF (629K) -
V. Basic EnzymesStudies of Quantitative Affinity ChromatographyKen-ichi KASAI, Shin-ichi ISHII1978 Volume 84 Issue 5 Pages 1051-1060
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The K1, values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.View full abstractDownload PDF (695K) -
Ken-ichi KASAI, Shin-ichi ISHII1978 Volume 84 Issue 5 Pages 1061-1069
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of β-trypsin [EC 3.4.21.4] immobilized on Sepharose with competitive inhibitors was quantitatively studied by frontal affinity chromatography. Analysis of the dependence of the elution volume of an inhibitor on its initial concentration gave two im-portant parameters. One is the dissociation constant (Kd) of the immobilized trypsin-inhibitor complex, and the other is the total amount of trypsin retaining binding ability that is present in the column (Bt). Experiments using benzamidine showed that frontal affinity chromatography is a theoretically simple system and can be treated in the same way as enzyme kinetics. The Kd value obtained for benzamidine was very similar to the K1 value. The Bt value corresponded to 73% of the immobilized trypsin. These results suggest that the function of trypsin was hardly affected by the immobilization. Results obtained with β-naphthami-dine were somewhat less satisfactory, probably due to a considerable non-specific interaction. In the case of proflavine, analysis was almost impossible due to extremely strong non-specific interaction. This procedure is very useful to analyze specific interactions if non-specific interactions are not significant. The basic features of frontal affinity chromatography are discussed.View full abstractDownload PDF (668K) -
Essential Histidyl and Cysteinyl Residues of the NADPH-Binding Site and Environment of the Adrenodoxin-Binding SiteAtsuo HIWATASHI, Yoshiyuki ICHIKAWA1978 Volume 84 Issue 5 Pages 1071-1086
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPig NADPH-adrenodoxin reductase was crystallized from pig adrenocortical mitochondria and its physicochemical properties were investigated. Pig NADPH-adrenodoxin reductase is a typical flavoprotein. Its optical absorption spectrum showed peaks at 272, 377, and 450 nm in the oxidized form. The adrenodoxin reductase contained one FAD per mol. The molecular weight was 49, 000. The isoelectric points of the adrenodoxin reductase and its complex with adrenodoxin were 5.3 and 4.6, respectively. Pig NADPH-adrenodoxin reductase, unlike bovine NADPH-adrenodoxin reductase, was found to be free of carbohydrate. The fluorescences of tryptophanyl residues and FAD of the adrenodoxin reductase were quenched by holo- and apo-adrenodoxins. The NADPH-binding site of the adrenodoxin reductase was examined by photooxidation and selective chemical modifications with diethyl pyrocarbonate and sulfhydryl reagents. The results indicate that a histidyl and a cysteinyl residue of the adrenodoxin reductase are essential for the NADPH-binding site. The circular dichroism spectrum of the adrenodoxin reductase showed negative ellipticity in the visible region. Spur formation was observed between pig and bovine NADPH-adrenodoxin reductases against the antibody to bovine NADPH-adrenodoxin reductase in Ouchterlony double-diffusion agar plates. The antibody did not interact with spinach ferredoxin-NADP+ reductase.View full abstractDownload PDF (2273K) -
II. An Order-Disorder Phase Transition in the Chemo-Mechanical ConversionMasafumi YANO, Hiroshi SHIMIZU1978 Volume 84 Issue 5 Pages 1087-1092
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe dependence of the acto-heavy meromyosin (acto-HMM) ATPase activity on the velocity of streaming of the HMM solution was measured in our stream systems constructed from rabbit skeletal muscle. The acto-HMM ATPase activity was an increasing function of the streaming velocity. It was verified that this increase of the ATPase activity is caused by a mechanism such that the rate of decomposition of AM*P into A+M+P is increased by the streaming. Both the streaming velocity and the ATPase activity showed biphasic behavior with respect to the inverse of the temperature: below the critical temperature Tc, the temperature dependence of the acto-HMM ATPase activity was essentially the same as that in homogeneous systems randomly dispersed in a test tube, while the streaming was observed as a linear function of l/T only in the phase appearing above Tc, and the acto-HMM ATPase activity there shows a remarkable increase against -1/T. The critical temperature Tc is not attributable to a transition of the molecular state inside acto-HMM but to the dynamic state of the stream system, which is controlled by changing the amount of F-actin fixed onto the stream cell. It can be concluded that the phase appearing below Tc is a disordered phase where the molecular dynamics of acto-HMM are essentially those of random and independent motion, while the phase appearing above Tc is an ordered one and the molecular dynamics of acto-HMM show some cooperativity (the dynamic cooperativity).View full abstractDownload PDF (447K) -
III. Dynamic Cooperativity - A New Cooperativity in Actomyosin Systems with a Polarized Arrangement of F-actinHiroshi SHIMIZU, Masafumi YANO1978 Volume 84 Issue 5 Pages 1093-1102
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe order-disorder phase transition in the chemo-mechanical process in our streaming system, which was reported in the preceding paper [Yano, M. & Shimizu, H., J. Biochem. 84, 1087-1092], was studied in detail. Starting from the hypothesis that a new type of cooperativity, dynamic cooperativity, is present in the elementary cycles of the chemo-mechanical conversion, quantitative and consistent agreement was obtained between the theoretical and experimental data on the temperature dependences of the streaming velocity and the ATPase activity, including the presence of the phase transition. The hypothesis was also supported by observations of the temperature dependence of the ATPase activity in resisted streaming. The physical and physiological significance of dynamic cooperativity is considered. It is shown that dynamic cooperativity arises in actomyosin system which have a polarized arrangement of F-actin by directed streaming of the solution, because the rate of the elementary cycle can be accelerated by the streaming. Furthermore, dynamic cooperativity is the key mechanism of self-organization of a dynamical order such that in ordered motions, and is one of the essential conditions for the direct conversion of the chemical energy of ATP to the mechanical energy of ordered and directed motion.View full abstractDownload PDF (796K) -
Tomohiko MORI, Yoshie WAKABAYASHI, Sadaichi TAKAGI1978 Volume 84 Issue 5 Pages 1103-1111
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPoly(A)-containing RNA has been isolated from the cotyledons of soybean seeds by adsorp-tion on a poly(U)-Sepharose column. Approximately 0.15% of the total soybean RNA applied bound to the column. The bound RNA (poly(A)-containing RNA) was shown to be mRNA by its ability to serve as template in a cell-free system derived from wheat germ. Poly(A)-containing RNA was polydisperse, migrating from approximately 50, 000 to 700, 000 daltons with a mean of 150, 000 daltons in polyacrylamide gel electrophoresis. The size of the poly(A) portion of this RNA was in the range of 55 to 290 nucleotides. The adenylic acid content of the presumed poly(A) fragment was about 95 %.
The radioactive products of translation directed by the poly(A)-containing RNA in the wheat germ cell-free system were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoprecipitation using antisera against beta-conglycinin and glycinin. The results of this investigation show that mRNAs for the subunit proteins of the major components of a soybean storage protein exist in the poly(A)-containing RNA preparation obtained from the cotyledons of dry soybean seeds.View full abstractDownload PDF (673K) -
Kiyohisa UCHIDA, Yasuharu NOMURA, Masumi KADOWAKI, Haruto TAKASE, Nozo ...1978 Volume 84 Issue 5 Pages 1113-1118
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBiliary secretion, fecal excretion, pool size, and turnover frequency of bile acids, as well as serum and liver lipid levels, were determined in spontaneously hypertensive rats (SHR) and compared with those in normotensive control rats (NR) of Wistar Kyoto strain. Serum cholesterol level in SHR was similar to that of NR but markedly increased after the feeding of a 2 % cholesterol diet for one week. Liver cholesterol level in SHR was higher than that in NR for all ages (8 to 77 weeks) and markedly increased after cholesterol feeding. Bile flow and biliary secretion of cholesterol, phospholipid, and bile acids were markedly elevated in SHR. The cholic acid level was greatly increased but levels of other bile acids were not changed or even decreased. The blood pressure of our SHR started to increase at the age of 6 to 7 weeks and attained the maximum at 15 to 16 weeks. Bile flow appeared to increase with the development of hypertension. Fecal excretion of bile acids in SHR, in either total amount or bile acid composition, was not greatly different from that in NR. After cholesterol feeding, fecal excretion of bile acids increased more significantly with SHR. The pool size of bile acids, as well as biliary secretion, increased about 60% in SHR but the turnover frequency and bile acid synthesis were unchanged. It was concluded that the pool size of cholic acid was increased in SHR and that this enhanced the absorption of cholesterol, resulting in a marked accumulation of cholesterol in serum and tissues.View full abstractDownload PDF (415K) -
Kazuo TERAO, Kikuo OGATA1978 Volume 84 Issue 5 Pages 1119-1123
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activity of 60S subunits of rat liver ribosomes in poly(U)-dependent polyphenylalanine synthesis was inhibited by incubation with N-ethylmaleimide. However, when 60S subunits were incubated with iodoacetamide, their activity decreased only slightly. Furthermore, iodoacetamide-pretreated 60S subunits became insensitive to N-ethylmaleimide. Similar results were obtained for the activity of EF-2-dependent GTPase of 60S subunits. As a whole, the labeling patterns of ribosomal proteins on two-dimensional gel electrophoresis were similar for 60S subunits labeled with both 14C-labeled sulfhydryl reagents, although the extent of labeling of some proteins was somewhat different.
These results indicate that the SH groups in the 60S subunits are not directly involved in the activities of the subunits described above.View full abstractDownload PDF (393K) -
Haruo SUZUKI1978 Volume 84 Issue 5 Pages 1125-1132
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mode of inhibition of rabbit globin synthesis by m7G5'p and m7G5'ppp (“cap analogs”) was studied using the rabbit reticulocyte lysate system.
The rate of globin synthesis was measured at various concentrations of both f[35S]Met-tRNAf and the cap analogs. The cap analogs were found to inhibit competitively the incorporation of f[35S]Met into hot trichloroacetic acid-insoluble material.
Nascent chains prelabelled with f[35S]Met were released at various concentrations of m7G5'ppp. The release of nascent chains was not inhibited by m7G5' (Suzuki, H. (1976) FEBS Lett. 72, 309) and m7G5'ppp, and it is therefore concluded that the cap analogs inhibit a step of initiation of globin synthesis.
Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of an 80S/fMet-tRNAf was inhibited by the cap analogs, but not that of a 40S/fMet-tRNAf complex.
These data suggest that a factor which is required for the binding of globin mRNA with 40S/fMet-tRNAf complex forms an inactive complex with the cap analogs, so that the cap analogs inhibit globin synthesis.View full abstractDownload PDF (575K) -
Shintaro KIKUCHI, Makoto ISHIMOTO1978 Volume 84 Issue 5 Pages 1133-1138
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSD-Serine dehydratase [EC 4.2.1.14] was purified from a strain of Klebsiella pneumoniae 140-fold from crude extract with a yield of 5%. This enzyme catalyzed the formation of pyruvate and ammonia not only from D-serine but also from L-serine, and also catalyzed the formation of α-ketobutyrate and ammonia from D-threonine. Km values for D-serine, L-serine, and D-threonine were 2.8mM, 20mM, and 3.6mM, respectively. Km for pyridoxal 5'-phosphate was 2.5μm. The molecular weight was estimated to be 46, 000 by Sephadex G-150 gel filtration and 40, 000 by SDS-polyacrylamide gel electrophoresis. This enzyme was inducible by D-serine. Induction by casamino acids appeared to depend on the presence of D-serine.View full abstractDownload PDF (778K) -
Hiroshi NAKAJIMA, Koichi SUZUKI, Kazutomo IMAHORI1978 Volume 84 Issue 5 Pages 1139-1146
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcetate kinase (AK) of Bacillus stearothermophilus is a tetrameric enzyme composed of 4 identical subunits each having a molecular weight of 40, 000. It gave sigmoidal kinetics with respect to ATP. The native enzyme was hybridized with the succinylated enzyme in order to clarify the subunit structure as well as the relationship between the structure and function of this enzyme. The succinylation of the enzyme was performed with succinic anhydride. Succinylated AK thus obtained had no enzymatic activity. When a mixture of the native and succinylated enzymes was treated with 6m guanidine hydrochloride and dialyzed, only one species of hybrid was obtained. The specific activity and extent of succinylation of the hybrid enzyme were intermediate between those of reconstituted and succinylated enzymes. These results show that hybrid AK is composed of two native and two succinylated subunits.
Hybrid AK had sigmoidal kinetics quite similar to those of reconstituted AK. This suggests that the combination of two subunits is necessary for allosteric behavior. These observations, taken together with the data that 4 mol of alanine was found as the N-terminal amino acid, suggest that acetate kinase of B. stearothermophilus is an (A)2-(A)2 type tetramer consisting of identical subunits.
This conclusion was supported by the results of crosslinking of this enzyme using a bifunctional reagent, hexamethylene diisocyanate.View full abstractDownload PDF (589K) -
Tetsunori TANAKA, Motoharu HIRAI, Teruo NAKAJIMA1978 Volume 84 Issue 5 Pages 1147-1153
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe formation of β-aspartyl-glycine from asparagine and glycine was demonstrated in the supernatant of rat kidney. The enzyme involved in this process was partially purified. Based on the properties of the enzyme reaction and the coincidence of purification rates of this activity and asparaginase, it can be speculated that the enzyme is a kind of asparaginase. Examination of the preference for β-aspartyl donors and acceptors showed that asparagine and glycine were the preferred donor and acceptor, respectively. β-Aspartyl dipeptides also transferred their aspartyl residues to amino acids. Amino acids other than glycine also accepted the aspartyl moiety from the donors.View full abstractDownload PDF (460K) -
Hiroshi HAYAKAWA, Keiko KUMURA, Mutsuo SEKIGUCHI1978 Volume 84 Issue 5 Pages 1155-1164
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUracil-DNA glycosylase, which acts specifically on uracil-containing DNA, was purified 250-fold from an extract of Escherichia coli 1100. The enzyme releases free uracil from DNA, producing alkali-labile apyrimidinic sites in the DNA. The enzyme is active on both native and heat-denatured DNA of phage PBS1, which contains uracil in place of thymine. φX174 DNA which had been treated with bisulfite and then at alkaline pH was susceptible to the action of uracil-DNA glycosylase. Since DNA treated with bisulfite alone was less susceptible to the enzyme, it is likely that the enzyme recognizes deaminated cytosine, namely uracil, but not bisulfite adducts of uracil and cytosine in the treated DNA. DNA treated with nitrite or hydroxylamine was not attacked by the enzyme. Enzyme activity acting on bisulfite-treated DNA was absent from an extract of E. coli mutant BD10 (ung). The mutant exhibited higher sensitivity to bisulfite than did the wild-type strain and was unable to reactivate phage T1 pre-exposed to bisulfite and weak alkali.View full abstractDownload PDF (733K) -
Kenji OEDA, Kenji SHIMIZU, Mutsuo SEKIGUCHI1978 Volume 84 Issue 5 Pages 1165-1169
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme activity specifically active on nitrous acid-treated DNA was found in an extract of Escherichia coli. The enzyme acts on both double- and single-stranded DNAs, treated with nitrous acid, in the presence of EDTA, although the former DNA is a better substrate. Evidence is presented that nitrous acid- and bisulfite-induced types of damage in DNA are recognized by different enzymes: (1) Uracil-DNA glycosylase, purified 250-fold from E. coli 1100, attacks bisulfite-treated DNA but not nitrous acid-treated DNA. (2) Almost equal levels of activity toward nitrous acid-treated DNA were found in wild-type and uracil-DNA glycosylase-deficient strains of E. coli.View full abstractDownload PDF (354K) -
Naoyoshi TANE, Ken HASHIMOTO, Tamotsu KANZAKI, Hideo OHYAMA1978 Volume 84 Issue 5 Pages 1171-1176
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree human malignant melanomas were cultured in pure populations and one tumor was cloned into melanotic and amelanotic cell lines. In the homogenates of these cultured cells, specific collagenase activities were demonstrated by isotope release from 14C-labeled collagen, disc electrophoresis, and specific cleavage of collagen molecules as demonstrated in the segment long spacing form. No significant collagenase activity was observed in the culture media. Interestingly, early cultures had a high collagenase activity in the cells and as they were successively subcultured, the activity diminished. Cysteine completely inhibited the degradation of tropocollagen as determined by disc electrophoresis and EDTA partially inhibited the degradation. It is concluded that human malignant melanoma cells produce a specific collagenase in vitro which can be extracted in early culture directly from the homogenate.View full abstractDownload PDF (2163K) -
Yasuko SHINDO, Takashi HASHIMOTO1978 Volume 84 Issue 5 Pages 1177-1181
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat liver peroxisomes oxidized palmitate in the presence of ATP, CoA and NAD+, and the rate of palmitate oxidation exceeded that of palmitoyl-CoA oxidation. Acyl-CoA synthetase [acid: CoA ligase (AMP-forming); EC 6.2.1.3] was found in peroxisomes. The substrate specificity of the peroxisomal synthetase towards fatty acids with various carbon chain lengths was similar to that of the microsomal enzyme. The peroxisomal synthetase activity toward palmitate (40-100 nmol/min per mg protein) was higher than the rate of palmitate oxidation by the peroxisomal system (0.7-1.7 nmol/min per mg protein). The data show that peroxisomes activate long chain fatty acids and oxidize their acyl-CoA derivatives.View full abstractDownload PDF (321K) -
Tomoko TAKAHASHI, Yukiko TSUCHIDA, Masachika IRIE1978 Volume 84 Issue 5 Pages 1183-1194
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Three forms of glucoamylase [EC 3.2.1.3] were simultaneously purified from a Rhizopus species by (NH4)2SO4 fractionation and successive chromatographies on Sephadex G-75, DEAE-Sephadex, and CM-Sephadex, and were finally separated from each other by means of recycling chromatography on Bio-Gel P-150. The purification achieved was 3-4 fold from crude extract with respect to each glucoamylase; the yields of the three glucoamylases, designated as Gluc1, Gluc2, and Gluc3 in order of content, were 39, 7, and 0.4%, respectively. All the purified enzymes were homogeneous in polyacrylamide gel electrophoresis, isoelectric focusing, and ultracentrifugation.
2. The three glucoamylases were glycoproteins differing in both amino acid composition and carbohydrate content, but showed a common antigenicity in immunodiffusion. The molecular weights of Gluc1, Gluc
2, and Gluc3 were estimated to be 74, 000, 58, 600, and 61, 400, respectively, by sedimentation equilibrium and these values were verified by SDS-polyacrylamide gel electrophoresis. The specific activities of the three enzymes toward starch were in the opposite order to their molecular weights.
3. The three glucoamylases had the same broad pH optima in the range pH 4.5-5.0 and shared a common susceptibility to inactivation by heat, extreme pH, and such divalent cations as Hg2+, Pb2+, and Mn2+, indicating close similarity in enzymatic properties.View full abstractDownload PDF (3086K) -
Yukiko UEHARA, Ben'ichiro TONOMURA, Keitaro HIROMI1978 Volume 84 Issue 5 Pages 1195-1202
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was found that an increase in fluorescence intensity at 340 nm is observed on the binding of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' in the pH range 6-10.
The dissociation constant, K1, of the enzyme-inhibitor complex was determined as a function of pH and temperature by direct fluorometric titration utilizing the single photon counting technique in the protein concentration range of 10-9M. K1 values as low as 10-10M could be obtained with reasonable accuracy by this high-sensitivity detection method.
From the temperature dependence of K1, it was found that the binding is endothermic, and is entirely “entropy-driven” in nature.
The effect of pH on K1 suggested the participation of an ionizable group with pKapp=8.5 in the binding.View full abstractDownload PDF (573K) -
Kentaro HORIUCHI, Daisaburo FUJIMOTO, Masanori FUKUSHIMA1978 Volume 84 Issue 5 Pages 1203-1207
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat liver chromatin prepared from purified nuclei catalyzed the acetylation of histones in nucleosomes at the same level as that of nuclei. The activity of histone acetyltransferase in chromatin was destroyed by heat treatment at 65°C for 5 min. Histories in exogenously added nucleosomes also served as substrate for the enzyme. The sites of acetylation in the nucleosomes appeared to be in the trypsin-digestable N-terminal regions of histones H4, H3, and H2A, as has been reported in an in vivo system.View full abstractDownload PDF (994K) -
I. Characterization of Thiosulfate-FOrming Activity at the Cell Extract LevelKunihiko KOBAYASHI, Eiji KATSURA, Tatsuhito KONDO, Makoto ISHIMOTO1978 Volume 84 Issue 5 Pages 1209-1215
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThiosulfate and sulfide were detected in the sulfite reductase reaction catalyzed by a cellfree extract of photoautotrophically grown Chromatium vinosum.
Hydrogen was consumed upon addition of sulfite to the extract in the presence of hydrogenase and methylviologen. Hydrogen uptake proceeded biphasically. During the first phase, thiosulfate and sulfide were formed concomitant with the decrease in sulfite. After the disappearance of sulfite, hydrogen was consumed with reduced velocity and sulfide accumulated as the final product with the total consumption of three mol of hydrogen per mol of sulfite.
The molecular weight of a major sulfite reductase was estimated to be about 180, 000 by the polyacrylamide disc electrophoresis method using enzyme staining. Arsenite, EDTA, αα'-dipyridyl, cyanide, or azide did not inhibit the activity at the concentration of 1mM. The activity was present in the soluble fraction and was stable at -20°C.View full abstractDownload PDF (1161K) -
Takahisa KANDA, Shingo NAKAKUBO, Kazumasa WAKABAYASHI, Kazutosi NISIZA ...1978 Volume 84 Issue 5 Pages 1217-1226
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cellulase component of Avicelase type was obtained from Driselase, a commercial enzyme preparation from a wood-rotting fungus Irpex lacteus (Polyporus tulipiferae). It showed a single band on SDS-polyacrylamide electrophoresis. The amino acid composition of this cellulase resembled those of cellulase components of endo-type from the same fungus. However, it produced exclusively cellobiose from CMC as well as from water-insoluble celluloses such as Avicel or cotton at earlier stages of hydrolysis. In addition, the hydrolysis of CMC practically stopped after an initial rapid stage. The cellulase showed a strong syner-gistic action with an endo-cellulase of higher randomness (typical CMCase-type) in the hydrolysis of CMC as well as Avicel. In contrast to cellotriose and -tetraose, cellopentaose and -hexaose were attacked very rapidly, and only cellobiose was produced. These results suggest that the cellulase is an exo-type component. However, it mutarotated the products from cellopentaitol in the same direction as endo-cellulases. It represented a relatively large portion of the total cellulase activity, and may play an important role in the degradation of native cellulose in vivo.View full abstractDownload PDF (2441K) -
Kyoden YASUMOTO, Kimikazu IWAMI, Toru FUSHIKI, Hisateru MITSUDA1978 Volume 84 Issue 5 Pages 1227-1236
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSγ-Glutamyltransferase was purified 10, 000-fold from bovine colostrum and was found to resemble the mammalian kidney enzymes with respect to molecular constitution and substrate specificity. The purification process involved separation of membrane material from colostral whey, treatment with papain, and column chromatography on DEAE-Sephadex and Sephadex G-150. The final preparation was apparently homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and double immunodiffusion. It had a molecular weight (as determined by gel filtration) of about 80, 000, a sedimentation coefficient of 5.0 s20, w, and an isoelectric point of pH 3.85, and was composed of two non-identical glycopeptides (with molecular weights of 55, 000 and 25, 000). Treatment with neuraminidase yielded a more negatively charged variant with intact enzymatic activity.
The reaction with γ-glutamyl-p-nitroanilide in the presence of glycylglycine as an acceptor was optimal at about pH 8.5 and at about pH 9.0 in its absence. Hydrolytic reaction, as assessed in terms of glutamate release, was practically absent at high pH. The activation profile by various amino acids and peptides was similar to that observed with the enzymes from other sources; glycylglycine was the best acceptor so far tested. The phosphate-inde-pendent glutaminase activity of the colostral enzyme was much lower than that of the human kidney enzyme either in the presence or absence of maleate; glutamate liberation from glutamine in the presence of maleate proceeded at only about 0.2% of the rate observed for transpeptidation between γ-glutamyl-p-nitroanilide and glycylglycine. Initial velocity measure-ments at various substrate concentrations yielded results which were consistent with a ping-pong mechanism modified by an autotranspeptidation shunt.View full abstractDownload PDF (2055K) -
Immunological Identity and Varying Affinities for Concanavalin AKimikazu IWAMI, Kyoden YASUMOTO, Toru FUSHIKI, Yasuyo TAKIGAWA, Hisate ...1978 Volume 84 Issue 5 Pages 1237-1243
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHighly purified preparations of bovine γ-glutamyltransferase obtained from colostrum, kidney, liver, small intestine, and mammary gland were tested for their immunological prop-erties and binding capacity to concanavalin A (Con A). In double immunodiffusion experiments, the transferases gave rise to a single and completely fused precipitin line against rabbit antiserum obtained by immunization with the colostral enzyme. In addition, the immunological identity was studied by quantitative immunoprecipitation experiments in which each transferase at a fixed level was incubated with various concentrations of rabbit antiserum against the colostral and renal enzymes. It was concluded that the bovine transferases bear common antigenic determinants of identical reactivity and specificity.
The binding capacities of the transferases to Con A-Sepharose were compared in the absence and presence of various concentrations of α-methyl-D-mannoside. The colostral and mammary gland enzymes exhibited the strongest affinity for the lectin, whereas the renal and small intestinal enzymes had little or no affinity. The behavior of the hepatic enzyme was intermediate. These differences in affinity for the lectin persisted after the removal of terminal sialic acid by neuraminidase treatment. In view of the high Con A affinity of the milk enzyme, it was presumed that the transferase molecules present in both fat globule and skim milk membrane surfaces might serve as one of the major Con A-binding sites. This possibility is supported by the fact that the transferase accounted for about one-third of the protein in the “high affinity” fraction under conditions designed to resolve the membrane proteins into “high” and “low affinity” fractions in Con A-Sepharose affinity chromatography.View full abstractDownload PDF (1151K) -
Teizo KITAGAWA, Yutaka ORII1978 Volume 84 Issue 5 Pages 1245-1252
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe resonance Raman spectra of bovine heart cytochrome oxidase were observed with excitation at 488.0 and 514.5nm for various molecular states. The resting enzyme as purified gave two Raman lines in the Band IV region at 1370 and 1357cm-1. On ferricyanide oxidation a single line appeared at 1372cm-1, indicating simultaneous oxidation of the two heme a molecules of cytochrome oxidase by ferricyanide. The resting enzyme was reduced by laser illumination under anaerobic conditions and a single Raman line appeared at 1362cm-1, at the same frequency as Band IV of dithionite-reduced oxidase. This photoreduction was confirmed by the appearance of a 440nm band in the absorption spectrum. In the presence of air, however, a molecular state characterized by the coexistence of two Raman lines in the Band IV region emerged under laser illumination. These two lines were also produced by oxygen bubbling of the dithionite-reduced oxidase. The heme a species yielding the lower-frequency counterpart must have ensued from some form of dimeric cytochrome oxidase, because this line was lost when the oxidase was dissociated into monomers by treatment with sodium dodecyl sulfate. Since the absorption spectra of the resting, aerobically laser-illuminated, and oxygen-bubbled oxidases apparently resemble the spectrum of the ferricyanide-oxidized oxidase, the oxidase having the two Raman lines in the Band IV region is suggested to be in the same oxidation state as, but in a different conformational state from, the ferricyanide-oxidized oxidase. The Raman spectra indicate the presence of a low-spin heme iron for both ferricyanide-oxidized and dithionite-reduced cytochrome oxidase. Addition of cyanide to reduced cytochrome oxidase resulted in a Raman spectrum quite similar to that of ferroheme a bis-imidazole cyanhydrin, suggesting the formation of cyanhydrin at the peripheral formyl group but not the replacement of axial ligands with cyanide in the oxidase thus treated.View full abstractDownload PDF (648K) -
Purification and CharacterizationMichio YAZAWA, Hideto KUWAYAMA, Koichi YAGI1978 Volume 84 Issue 5 Pages 1253-1258
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Ca2+-dependent activator protein of myosin light-chain kinase, which was identified as the bovine brain modulator protein of cyclic nucleotide phosphodiesterase, was isolated from rabbit skeletal muscle. The purified protein binds about 2Ca2+ per mol in a medium containing 5mM MgCl2, 10μm CaCl2, and 0.1M KCl at pH 6.8. The Ca2+ binding caused a conformational change of the activator protein which was measured by difference ultraviolet absorption spectroscopy. In the same Ca2+ concentration range as that causing the conformational change, activation of myosin light-chain kinase was observed.View full abstractDownload PDF (1129K) -
Michio YAZAWA, Koichi YAGI1978 Volume 84 Issue 5 Pages 1259-1265
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSModulator-deficient myosin light-chain kinase from rabbit skeletal muscle was purified by modulator protein-Sepharose 4B affinity chromatography. The purified protein showed a single band (MW 80, 000) on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and it exists as a monomer in the native state as determined by gel filtration. The modulatordeficient myosin light-chain kinase (MW 80, 000), modulator protein (MW 16, 500) and Ca2+ were essential for the kinase activity. The half-maximal activity of the kinase in the presence of excess modulator protein with 10mM MgCl2 was at pCa 5.1, where full activity of actomyosin-ATPase is observed in the presence of the troponin-tropomyosin system. Assuming a rapid equilibrium between myosin light-chain kinase and the two substrates, ATP and g2 light-chain, Km values for ATP and g2 light chain were evaluated as 0.28mM and 0.024mM, respectively. Vm/e was 5.7s-1.View full abstractDownload PDF (1623K) -
Purification and CharacterizationTsuneo MIYAHARA, Atsushi TAKEDA, Akira HACHIMORI, Tatsuya SAMEJIMA1978 Volume 84 Issue 5 Pages 1267-1276
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCatalase [EC 1.11.1.6] from goat liver was purified by acetone and ammonium sulfate fractionations followed by DEAE-cellulose and Bio-Gel A-1.5m column chromatography. The purified enzyme was found to be homogeneous ultracentrifugally and the molecular weight of the native goat liver catalase (GLC) was estimated by gel filtration to be approximately 220, 000. SDS-gel electrophoresis results indicated that catalase from goat liver consists of four apparently identical subunits, with a molecular weight of about 60, 000; this is similar to the subunit structure of bovine liver catalase (BLC). The enzyme activity of GLC was slightly less than that of BLC, which apparently correlated with the lower R.Z. value (A405nm/A280nm) of GLC than of BLC. The optimal pH of the enzyme activity of GLC was found to be around 7, the same as that of BLC. The CD spectrum of native GLC generally resembled that of BLC, although the band intensities and band positions differed slightly. However, the α-helix content of GLC was found to be about 30% by CD measurement, slightly less than that of BLC. The effect of pH on the stability of GLC was studied in terms of α-helical conformation and the fluorescence of tryptophanyl residues and 8-anilinonaphthalene-1-sulfonate (ANS) bound to the enzyme. These results indicated that GLC may dissociate into subunits at extreme pH's. On the other hand, precise densitometric tracing of disc electrophoresis gels indicated heterogeneity of the purified GLC; there were four components, with isoelectric points of 5.80, 5.97, 6.01, and 6.10. It was found that three out of the four components varied electrophoretically on treatment with thiol reagents such as 2-mercaptoethanol (ME) and dithiothreitol (DTT).View full abstractDownload PDF (1261K) -
Tatsushi OKA, Kazuyuki MORIHARA1978 Volume 84 Issue 5 Pages 1277-1283
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSα-Chymotrypsin [EC 3.4.21.1] catalyzed the syntheses of peptide bonds with various N-acylated amino acids or peptides having aromatic or hydrophobic amino acid residues at the C-terminal position as carboxyl components, and amino acid derivatives, peptides or their derivatives as amine components. A neutral pH was most efficient and quite high concentrations of α-chymotrypsin and starting materials were required for synthesis. For amine components, hydrophobic or bulky amino acid residues were useful at the N-terminal posi-tion. Stereospecificity was also observed at the N-terminal position of amine components. Peptide synthesis was not usually seen when the products were soluble in the reaction mixture. This could be partly overcome by increasing the concentration of either the carboxyl or the amine component to more than ten times that of the other.View full abstractDownload PDF (494K) -
Masa'aki TSUDA, Tasuku HONJO, Akira SHIMIZU, Den'ichi MIZUNO, Shunji N ...1978 Volume 84 Issue 5 Pages 1285-1290
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSImmunoglobulin k type light chain mRNA (L, mRNA) accumulated in parallel with secretion of immunoglobulin M in cultured mouse spleen cells activated by lipopolysaccharide. Actinomycin D suppressed the accumulation of k chain mRNA completely without affecting the degradation rate of k chain mRNA. The half life of k chain mRNA was about 9h. Available evidence indicates that lipopolysaccharide stimulates de novo synthesis of k chain mRNA. The accumulation of k chain mRNA was markedly suppressed by inhibitors of DNA or protein synthesis such as hydroxyurea, cytosine arabinoside and cycloheximide.View full abstractDownload PDF (416K) -
I. Its Enzymatic Properties and Modification with p-Bromophenacyl BromideKiyoshi KONDO, Hiroko TODA, Kozo NARITA1978 Volume 84 Issue 5 Pages 1291-1300
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ1-Bungarotoxin purified from the venom of Bungarus multicinctus has phospholipase A activity, which was assayed by titrating fatty acids released from phosphatidyl choline with alkali (the pH-stat titration method) and by scanning carbonized spots of the fatty acids on TLC plates (the TLC analysis method). The toxin as the phospholipase was very stable in acidic media and retained full activity even after incubation at 37°C for 20h, but the toxin was labile above pH 9. The loss of activity in alkaline media might be caused by precipitation near the isoelectric point (pI9.5) of the toxin followed by irreversible denaturation. The toxin as the enzyme was active over a broad pH range (pH 6-9), but the optimum pH for hydrolysis of dispersed phosphatidyl choline was 8.0 as determined by both methods, pH-stat titration and TLC analysis.
The phospholipase activity of the toxin decreased on modification with p-bromophenacyl bromide (BPB). The inactivation followed pseudo-first-order kinetics. The complete loss of enzymatic activity of the toxin was accompanied by the loss of one histidine residue in the toxin. The pH dependence of the inactivation rate suggested that the modified residue has pKa 6.9. This pKa value also indicated that the modified residue might be a histidine participating in the phospholipase activity of the toxin.View full abstractDownload PDF (1348K) -
II. Identification of the Histidine Residue of β1-Bungarotoxin Modified by p-Bromophenacyl BromideKiyoshi KONDO, Hiroko TODA, Kozo NARITA1978 Volume 84 Issue 5 Pages 1301-1308
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ1-Bungarotoxin modified with p-bromophenacyl bromide (BPB) was reduced and carboxymethylated, and the resulting two constituent RCM-polypeptide chains (the RCM-A and B chains) were separated. The RCM-A chain was found to be modified by BPB by measuring its UV absorption spectrum and was shown to have lost one histidine residue by analyzing its amino acid composition. To determine the location of the modified histidine residue in the A chain of the toxin, the RCM-A chain was digested with TPCK-trypsin, and the resulting peptides were fractionated by gel filtration followed by DEAE-cellulose chromatography. The modified residue was finally identified as histidine-48 in the A chain by Edman degradation and from the amino acid composition of the BPB-modified peptide. The amino acid sequence around the modified histidine residue in the A chain is highly homologous with those of porcine pancreas phospholipase A2 and presynaptic toxin, notexin. We conclude that histidine-48 in the A chain participates in the phospholipase A activity of β1-bungarotoxin.View full abstractDownload PDF (501K) -
Yousuke SEYAMA, Akihiko KAWAGUCHI, Shigenobu OKUDA, Tamio YAMAKAWA1978 Volume 84 Issue 5 Pages 1309-1314
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new assay method for fatty acid synthetase using mass fragmentography is described. The amounts of fatty acids synthesized in deuterated water are determined by mass fragmentography by monitoring the intensities of m/e 77 and m/e 74 fragment ions using hepta-decanoic acid as an internal standard. This method is suitable for any fatty acid synthetase which produces several species of fatty acids and is also applicable to crude enzyme preparations. This procedure is more sensitive and specific than the conventional spectrophotometric method. Fatty acid synthetases from rat liver and Brevibacterium ammoniagenes are analyzed by this method.View full abstractDownload PDF (419K) -
Swaraj K. PATNAIK1978 Volume 84 Issue 5 Pages 1315-1317
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (182K) -
Showbu SATO, Takahisa SHINOMIYA1978 Volume 84 Issue 5 Pages 1319-1321
Published: November 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA site-specific endonuclease has been isolated from Therinus thermophilus HB8 and named TthHB8I. It recognizes the same sequences as TaqI from Thermus aquaticus YT-1 does. The amount of TthHB8I in the cells was comparable to that of TaqI. T. thermophilus HB8 has an advantage over T. aquaticus YT-1 for preparation of a TaqI-like enzyme since it is easier to obtain T. thermophilus HB8 cells in quantity.View full abstractDownload PDF (799K)
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