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Volume 81, Issue 1
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Properties of Acetylated Spinach FerredoxinRyuichi MASAKI, Keishiro WADA, Hiroshi MATSUBARA1977 Volume 81 Issue 1 Pages 1-9
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe acetylatien of spinach ferredoxin by acetic anhydride modified about four moles of amino groups. The absorption spectra, CD spectra, the fluorescence of sole tryptophan residue and the biological activity of acetylated ferredoxin were investigated.
An equilibrium existed between two different states, D- and N-form, of the acetylated ferredoxin and was dependent on the cation concentration. D-form completely reverted to N-form upon the binding of one mole of cation, Na+ or Mg2+. Although the N-form was in-distinguishable from native ferredoxin in every property tested, the D-form was significantly different from the N-form or native ferrcdoxin and was very unstable, especially at low salt concentrations. It is suggested that the amino groups might be important in maintaining the protein conformation by forming salt linkages, but may not be essential for the activity.
Furthermore, since the D-form, unlike the N-form and native ferredoxin, was inactive in the ferredoxin-NADP+ reductase [EC 1. 6. 7. 1] assay system and had no inhibitory effect in this system, it was considered to be incapable of forming a complex with ferredoxin-NADP+ reductase. On the other hand, the N-form of the mcdified ferredoxin was as active as native ferredoxin. It is suggested that amino groups of spinach ferredoxin are not essential for the redox reaction of ferredoxin or for complex formation with the reductase.View full abstractDownload PDF (637K) -
I. Fractionation and PropertiesKiso AKAHANE, Soichi MUROZONO, Kunio MURAYAMA1977 Volume 81 Issue 1 Pages 11-18
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method is described for the fractionation of reduced and alkylated proteins of fowl feather. Fowl feather extracts were chromatographed on a Sephadex G-75 column in 4M urea con-taining 1 M NaC1 and separated into four fractions, GF-1, 2, 3, and 4. The elution patterns were used to compare the components of different feather parts, barbs, rachis+medulla, and calamus. In all cases, GF-3 was the main fraction and the percentages with respect to the total peak area found for barbs, rachis+medulla, and calamus were about 65%, 74%, and 93%, respectively. Each of the fractions was examined by polyacrylamide disc gel electro-phoresis and all were heterogeneous. The slowly moving bands mainly corresponded to fraction GF-1, intermediate bands to GF-2 and 3, and faster bands to GF-4. Many other polypeptide chains, which have not been found previously, were newly separated from three minor fractions of fowl body feather. The molecular weights of fractions GF-2 and 3 were estimated by calibrated gel filtration to be 33, 000 and 10, 500, respectively. Marked differ-ences were found in the amino acid compositions of various fractions from fowl feather. The GF-1 fraction and insoluble residue had very similar compositions; in both cases the contents of serine, glycine, and proline were lower and those of helix-favoring amino acids, namely, lysine, tyrosine, and methionine, were higher than those found in other fractions.View full abstractDownload PDF (603K) -
II. Isolation of Some Proteins from BarbsKunio MURAYAMA, Kiso AKAHANE, Soichi MUROZONO1977 Volume 81 Issue 1 Pages 19-24
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour fractions, GF-1, 2, 3, and 4, which had been separated from S-carboxymethylated (SCM-) proteins of fowl feathers by gel filtration, were each chromatographed on a DEAE-cellulose column in 0.05 M Tris-HC1 buffer (pH 8.5) containing 8 M urea. The major fraction, GF-3, was further separated into seven peaks; the first four were shown to be single com-ponents by polyacrylamide disc gel electrophoresis. Chromatograms of GF-1 and 2 showed broad peaks which appeared at nearly the same volume as in GF-3. The components from GF-3 had very similar amino acid compositions except that the SCM-cysteine content showed a tendency to increase in the order of elution from the column. SCM-extract prepared from barbs of the wing feathers of a fowl was more heterogeneous than that taken from the body feathers.
A combination of gel filtration on Sephadex G-75 and chromatography on DEAF-cellu-lose was found to be more effective for the isolation of soluble SCM-proteins.View full abstractDownload PDF (1576K) -
Conversion to a Homologous Inactive Preneocarzinostatin Due to Change of Asparagine 83 to Aspartic Acid 83 Accompanied by Conformational and Biological AlterationsHiroshi MAEDA, Kenji KUROMIZU1977 Volume 81 Issue 1 Pages 25-35
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amide content of neocarzinostatin (NCS), an antitumor protein, has been determined by analysing asparagine and glutamine in the Pronase-aminopeptidase M digests of tetra-S-carboxymethyl-NCS and carboxyl-modified NCS (modified with a water-soluble carbodiimide and [14C] glycine methyl ester). Preneocarzinostatin (PRE) was separated and purified from a crude NCS preparation by CM-cellulose column chromatography. PRE was found to contain one mole less asparagine than NCS, and asparagine was deamidated to aspartic acid in PRE. A time-dependent conversion of NCS to PRE at pH 3.2 at 4° or in 0.1M acetic acid at 26° was studied in two ways; first, by quantitative determination of NCS and PRE by CM-cellulose column chromatography and second, by following the release of free NH3, during dialysis in an air-tight container. Within experimental error, PRE was indistinguishable from NCS in amino acid content after acid hydrolysis, as well as in apparent molecular weight as determined by SDS-disc gel electrophoresis (10%. acrylamide), and N- and C-terminal amino acid residues. Both NCS and PRE shared a common antigenicity as determined by Ouchterlony's agar diffusion method. Only a slight difference between the two in electro-phoresis on a cellulose acetate membrane and on a peptide map of the tryptic digest was demonstrated. PRE, however, was completely devoid of biological activity. In addition to the chromatographic difference, a conformational difference was observed by CD spectro-scopy, namely, an apparently looser structure of PRE was indicated by the shallowness of the trough in the 240-265 nm region. This interpretation was supported by the finding that digestions by Pronase were more extensive with PRE than with NCS. These results indicate an important role of the single asparagine residue (Asn 83) of NCS in the biological activity, which is evidently governed by the conformation.View full abstractDownload PDF (1225K) -
Yoshio HOJIMA, Mari ISOBE, Hiroshi MORIYA1977 Volume 81 Issue 1 Pages 37-46
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kallikrein inhibitor contents of human and animal plasma were determined with glandular kallikreins [EC 3.4.21.8]. One ml of plasma could inactivate 20-700 kallikrein units (KU). Rat plasma was the most potent and inactivated 230-700 KU. However, no enzyme capable of inactivating kallikrein could be found in this plasma. Two fractions which inhibited hog pancreatic kallikrein, a fraction corresponding to az macroglobulin and a fraction which was eluted prior to albumin, were separated from rat plasma by Sephadex G-200 gel filtration. The former inhibitor could inhibit hog pancreatic kallikrein action on Nα-benzoyl-L-arginine ethyl ester (BAEE) as well as in the dog vasodilator assay.
The other inhibitor was partially purified from rat plasma. One mg of the preparation inhibited 67 KU and the hydrolysis of 5.8 μmoles/min of BAEE by hog pancreatic kallikrein [EC 3. 4. 21. 8]. The inhibitor also inhibited other glandular and plasma kallikreins, trypsin [EC 3. 4. 21. 4], α-chymotrypsin [EC 3. 4. 21. 1], etc. The optimal pH of the inhibitor was 7.5-8. The inhibitor was unstable below pH 5, and was destroyed by heating at temperature above 60°. The isoelectric point of the inhibitor was determined by Ampholine fccusing to be 4.4, and its molecular weight was estimated to be 73, 000 by Sephadex G-100 and G-150 filtrations. Several experimental results suggested that this inhibitor differed from α1-anti-trypsin.View full abstractDownload PDF (696K) -
VII. Stereospecificity in the Enzyme-like Hydrolysis of Benzoyl-L (D)-arginine-p-nitroanilides by Copoly (Cys, Glu)Junzo NOGUCHI, Norio NISHI, Seiichi TOKURA, Umeji MURAKAMI1977 Volume 81 Issue 1 Pages 47-55
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe substrate specificity in the hydrolysis of L-, DL-, and D-BAPA (benzoylarginine-p-nitro-anilide) by copoly (L-Cys, L-Glu) and copoly (D-Cys, D-Glu) was studied, and enzyme-like stereosp-, cific hydrolyses by poly-α-amino acids were identified for the first time. The L-type copolymer hydrolyzed L-BAPA faster than D-BAPA and the rates (v) of BAPA hydrolyses by. L-type copolymer were found to be in the order vL>vDL>vD. On the other hand, the D-type copolymer hydrolysed D-BAPA faster than L-BAPA and the rates of BAPA hydrolyses by D-type copolymer were in the order vD>vDL vL. In all cases, the reaction followed Michaelis-Menten kinetics when the substrate concentration was corrected, and the optimum conditions of the reaction were pH 6.0 and 40°. The activity appeared after a certain amount of BAPA had combined with the polymer. D- and L-substrates combine competitively with the polymer and the different rates of hydrolysis are presumably due to the different substrate configura-tions in relation to the conformation of the active site in the polymer. The polymer shows activity near the range of random coil conformation, where some α-helical conformation is still present. Only some of the cysteine residues in the copolymer are involved in the hydrolytic activity.View full abstractDownload PDF (644K) -
Genichiro OSHIMA, Kinzo NAGASAWA1977 Volume 81 Issue 1 Pages 57-63
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPeptidyldipeptide hydrolase [angiotensin I-converting enzyme, EC 3. 4. 15. 1] was inhibited by inorganic and organic phosphorus compounds tested, except for β-glycerophosphate, 5'-AMP, and 5'-ADP, at the reagent concentrations used. Orthophosphate and pyrophosphate nonspecifically inhibited the enzyme activity.
The enzyme was also inhibited specifically by carboxylates. The degree of inhibition by aliphatic monocarboxylates increased in proportion to their chain length up to C14. Aromatic and ω-phenylalkylcarboxylates also inhibited the enzyme activity. The enzyme was noncom-petitively inhibited by acetate, 3-phenylpropionate and laurate. The K1's for acetate, 3-phenylpropionate, and laurate were 60, 3.3, and 2.5mM, respectively.View full abstractDownload PDF (491K) -
II. Effects of Chemical Modifications on the Activity of ThermolysinHaruyuki OSHIMA, Teruo ABE, Kenji TAKAHASHI1977 Volume 81 Issue 1 Pages 65-70
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. When thermolysin was treated with a 100-fold molar excess of 2, 4, 6-trinitrobenzene-l-sulfo-nate at pH 8.0 and 37° for 7 h, all 12 amino groups in the enzyme were almost completely trinitrophenylated. The fully trinitrophenylated enzyme still retained more than 80% of its original activity. The amino groups are therefore not essential for activity.
2. When treated with a 100- to 1, 000-fold molar excess of N-acetylimidazole at pH 6.5 and 23° for 2h, thermolysin lost about 54% of its activity with concomitant acetylation of 21 tyrosine residues out of the total of 28 residues. The reaction did not easily proceed any further. This partially inactivated enzyme regained almost full activity upon treatment with hydroxylamine. These modified tyrosine residues are therefore not directly involved in the active site.
3. Thermolysin was not inactivated by treatment with about 100- to 150-fold molar excess of 2-hydroxy-5-nitrobenzyl bromide or dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide at pH 6.0 and room temperature for 1h both in the presence and absence of 8M urea. Thus the three tryptophan residues in the enzyme are not accessible to these reagents. When treated with a 4.3- to 43-fold molar excess of N-bromosuccinimide over tryptophan, the enzyme was inacti-vated to varying extents, depending on the reaction conditions used. In this case, the tyrosine residues appeared to be the most rapidly modified, but tryptophan and histidine residues were also modified with extensive inactivation at higher concentrations of the reagent. The presence of 8M urea retarded the inactivation.View full abstractDownload PDF (486K) -
Tsuneyuki NAGAOKA, Akira HACHIMORI, Atsushi TAKEDA, Tatsuya SAMEJIMA1977 Volume 81 Issue 1 Pages 71-78
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new method of affinity chromatography using blue dextran-Sepharose 4B resin was estab-lished to purify NADP+-dependent isocitrate dehydrogenase [EC 1. 1. 1. 42] from Bacillus stearotherinophilus in high yield. The purified preparation was found to be homogeneous on disc gel electrophoresis. The SH groups of the enzyme were modified with 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to determine the number of SH groups per molecule and their contribution to the enzyme activity. One SH group was titrated with DTNB per subunit (the native enzyme consisted of two subunits) and after complete denaturation with 4 M guanidine-HCl the number of titratable SH groups remained unchanged. ORD and CD measurements showed that the α-helical conformation of the polypeptide backbone was unaffected by DTNB modification, though the near ultraviolet CD spectrum was evidently altered. The fluores-cence derived from tryptophanyl residue(s) was quenched by the modification to 30% of the native level, which may indicate the presence of SH in the vicinity of tryptophanyl residue(s). A remarkable decrease of the enzyme activity was detected upon modification with DTNB, but there was some discrepancy between the rate of inactivation and that of modification of SH groups. The presence of substrate and Mg2+ gave partial protection against modification of the SH groups by DTNB. Complete protection of the native enzyme activity against heat-ing at 65° was observed in the presence of substrate and Mg2+, but the thermostability of the enzyme was markedly reduced by modification of the SH groups.View full abstractDownload PDF (664K) -
Eiji SHIMADA, Go MATSUMURA1977 Volume 81 Issue 1 Pages 79-91
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHyaluronic acid was prepared from adult rabbit skin. Defatted skin powder suspended in 0.5M NaCl was homogenized, and total glycosaminoglycans were precipitated from this 0, 5 M NaC1 extract with cetylpyridinium chloride, then redissolved successively with increasing concentrations of NaCl and finally 0.5N NaOH. Hyaluronic acid, the major acid glycosami-noglycan in the 0.5M NaCl extract, was purified and fractionated by DEAE-Sephadex chro-matography. The molecular weights ranged from 1×104 to 7.2×104.
Alternatively, hyaluronic acid was obtained from adult rabbit skin without mechanical powdering and homogenizing. Defatted skin pieces were suspended in water and heated at 100°, then the extract was digested with pronase followed by DNase [EC 3. 1. 4. 5]. Glyco-saminoglycans were excluded in gel filtration with Sephadex G-75. Hyaluronic acid and dermatan sulfate, the two major glycosaminoglycans of this tissue, were separated by gel chromatography on Sepharose 4B. The molecular weight of this hyaluronic acid ranged from 1.6×105to 1.3×106.
Yields of hyaluronic acid by these two methods were similar. Hyaluronic acid was prob-ably degraded by the mechanical treatments in the first method. Other factors affecting the viscosity of the tissue extract were examined.View full abstractDownload PDF (850K) -
Separation of Four Forms of the Rabbit Skeletal Muscle EnzymeMasanao KOBAYASHI, Kanefusa KATO1977 Volume 81 Issue 1 Pages 93-97
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphoprotein phosphatase [phosphoprotein phosphohydrolase EC 3. 1. 3. 16] in the soluble fraction of rabbit skeletal muscle, when assayed with phosphorylase a [EC 2. 4. 1. 1] from rabbit skeletal muscle and phosphohistone as substrates, was resolved into three active fractions (Fractions I, II, and III in order of elution) by DEAE-cellulose column chromatography.
Sucrose density gradient centrifugation showed that these fractions were composed of subfractions of different molecular size (I: 7.3S and 4S; II: 8S and 4S; III; 6.7S). Components with larger molecular size in the major fractions, II and III, were dissociated to a molecular size similar to that of the smallest component on freezing in the presence of mercaptoethanol.
These results indicate that phosphoprotein phosphatase from skeletal muscle occurs in multiple forms very similar to those of the liver enzyme reported previously (Kobayashi, Kato and Sato (1975) Biochirn. Biophys. Acta. 373, 343-355).View full abstractDownload PDF (378K) -
Masatake OHNISHI, Takashi YAMASHITA, Keitaro HIROMI1977 Volume 81 Issue 1 Pages 99-105
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA transition-state analog, gluconolactone, was found to partially quench the protein fluores-cence of glucoamylase [EC 3. 2. 1. 3] from Rhizopus niveus. The interaction between glucono-lactone and the enzyme was studied statically and kinetically at pH 4.5 in terms of fluorescence change. The dissociation constant Kd of the enzyme-analog complex determined by fluoro-metric titration at 25° (Kd=1.6 mM) was in good agreement with that obtained by difference spectrophotometric titration (Ohnishi, M. et al. (1975) J. Biochem. 77, 695-703) and with the inhibitor constant determined for the hydrolysis of maltodextrin (Ohnishi, M. et al. (1976) J. Biochem. 79, 1007-1012).
The kinetics of the interaction were studied by the fluorescence stopped-flow method. The dependence of the apparent first-order rate constant, kapp, on gluconolactone concentration showed a saturation curve, consistent with a two-step mechanism involving a rapid bimolec-ular association followed by a slow unimolecular isomerization process. The dissociation constant, KI, for the rapid bimolecular process and the forward and backward rate constants for the isomerization were obtained at 25° and 5°, and the activation parameters were evaluated.
It was found that the isomerization process, but not the bimolecular association, is accom-panied by fluorescence intensity change, indicating that the former process involves a micro-environmental change of a tryptophan residue(s) of the enzyme.
Maltose was found to decrease the rate of interaction of gluconolactone with the enzyme by competing with the analog for the active site.View full abstractDownload PDF (971K) -
III. Isolation and Characterization of a Novel Globoside Containing Mannose from Spermatozoa of the Fresh-Water Bivalve, Hyriopsis schlegeliiTaro HORI, Mutsumi SUGITA, Junko KANBAYASHI, Osamu ITASAKA1977 Volume 81 Issue 1 Pages 107-114
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree globosides were isolated from spermatozoa of the fresh-water bivalve, Hyriopsis schle-gelii by mild alkaline hydrolysis, acetone precipitation, Unisil column chromatography and preparative thin-layer chromatography. These globosides are unique in their sugar chains, since they contain mannose instead of galactose found so far in all globosides of mammals and other animals examined. The main globoside in the spermatozoa was characterized as Glc-NAcβ (1→2) Manβ (1→3) Manβ (1→4) Glcβ (1→1)-ceramide by partial acid hydrolysis, analysis of its anomeric configuration with chromium trioxide, methylation analysis and enzymatic hydrolysis. The globoside contained normal saturated fatty acids ranging in length from C16 to C21, palmitic and stearic acids being predominant. Its main long-chain was octadeca-4-sphingenine.View full abstractDownload PDF (2391K) -
Yoshio YAMAKAWA, Akira OHSAKA1977 Volume 81 Issue 1 Pages 115-126
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Phospholipase C [EC 3. 1. 4. 3] was purified from the culture filtrate of Clostridinrn perfringens by successive chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-100. During the purification it was noted that, beside the monomer form of the enzyme which was purified, a part of the enzyme existed in active polymerized forms.
2. The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line in immunodiffusion with the National Standard gas gangrene (C. per-fringens) antitoxin, indicating the homogeneity of the preparation.
3. The specific lecithin-hydrolyzing activity of the purified preparation was comparable to that of a preparation obtained by affinity chromatography, which had the highest specific activity previously reported.
4. The molecular weight of the purified enzyme was estimated to be 43, 000 by SDS-polyacryl-amide gel electrophoresis, although the same preparation gave a molecular weight of 31, 000 as determined by gel filtration on Sephadex G-150. From this and the above finding that apart of the enzyme exists in active polymerized forms, the discrepancy among reported values for the molecular weight of C. perfringens phospholipase C can be accounted for.
5. For maximum hydrolytic activity toward lecithin, the enzyme required sodium deoxy-cholate (SDC) and Ca2+ ions. In the presence of 6 mm Ca2+, the optimal molar ratio of SDC to lecithin for maximal hydrolytic activity was about 0.5 for dipalmitoyl lecithin and about 1.0 for egg lecithin. The effects of various divalent cations on the enzymatic hydrolysis were also investigated.
6. The effects of sodium deoxycholate and Ca2+ ions on the enzymatic hydrolysis are discussed, based on their possible roles in mixed micelle formation.View full abstractDownload PDF (2083K) -
Akemi SUZUKI, Shizuo HANDA, Ineo ISHIZUKA, Tamio YAMAKAWA1977 Volume 81 Issue 1 Pages 127-134
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeminolipid (1-O-alkyl-2-O-acy1-β-3'sulfogalactosyl glycerol) was detected on a micro-scale by high performance liquid chromatography (HPLC) using a UV detector. The benzoylation method of McCluer and Evans ((1973) J. Lipid Res. 14, 611-617) was used to convert semino-lipid to a derivative with UV absorption. Total lipid from germinal tissue was benzoylated and the product was separated by Florisil and Sephadex LH-20 column chromatographies. The seminolipid obtained in the effluent from the Sephadex LH-20 column was determined by HPLC. Eighty percent recovery of 35S-labelled seminolipid was obtained by this method. The method was used to determine the seminolipid content of boar spermatozoa:980 nmoles of seminolipid was found per gram of packed cells.View full abstractDownload PDF (2520K) -
Shin-ichi AKIYAMA, Michihiko KUWANO, Mikio YAMAMOTO, Hideya ENDO1977 Volume 81 Issue 1 Pages 135-141
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of the dye aurintricarboxylic acid (ATA) on RNA polymerases [EC 2. 7. 7. 6] solu-bilized from rat liver was studied. Complete inhibition of RNA synthesis in vitro was observed when 3-5μg/ml of ATA was added to the reaction mixture at time 0, while 40-70μg/ml of a rifampicin derivative, AF/013, was required to produce the same extent of inhibition. RNA formation, however, continued at a rate of one-half that of the control when ATA was given after the onset of RNA synthesis in a dose capable of completely blocking RNA formation if administered at time 0. ATA was found to interact with RNA polymerizing enzyme itself and competed specifically with the binding of RNA polymerase to template DNA. Preincuba-tion of the enzyme with DNA at 37° before adding dye made the DNA-enzyme complex partly resistant to the drug. RNA-synthesizing activity resistant to ATA increased when nucleoside triphosphates, especially GTP, were added to the preincubation mixture in the presence of Mn2+. However, ATA only slightly affected RNA synthesis in nuclei isolated from rat liver.View full abstractDownload PDF (477K) -
Takaharu KAWADA, Takao FUJISAWA, Kazuo IMAI, Kikuo OGATA1977 Volume 81 Issue 1 Pages 143-152
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEmploying livers from rats fed on a protein-free diet for two weeks, the effects of protein deficiency on both biosynthesis and degradation of rRNA were investigated and the following results were obtained.
1. Protein deficiency led to a decrease of total liver RNA content per DNA to about 80% of that in normal rat liver.
2. From the kinetics of rRNA labelling with [14C] orotic acid in vivo, the half-lives of cyto-plasmic rRNA's of normal and protein-deficient rat livers were determined to be 6.2 and 5.1 days, respectively. Furthermore, considering the pool size of rRNA in rat liver, the turnover rate of cytoplasmic rRNA was calculated to be 0.212 pmole/min/mg of nuclear DNA in normal rats and 0.240 pmole/min/mg of nuclear DNA in protein-deficient rats.
3. From the electrophoretic patterns of nucleolar RNA's of both groups of rat livers labeled with [S4C]orotic acid, the time courses of the specific activities of nucleolar 45S, 32S, and 28S rRNA's were analysed and the half-life of each nucleolar RNA in both groups of rat livers was determined. Nucleolar 45S, 32S, and 28S RNA's had half-lives of 6.0, 15.9, and 26.5min in normal rats, respectively, and 5.5, 19.4, and 22.9min in protein-deficient rats, respec-tively.
Considering the pool size of each nucleolar RNA obtained from the electrophoretic pattern, the turnover rates of 45S, 32S, and 28S RNA's were calculated to be the same, i.e., 0.189 pmoles/min/mg of nuclear DNA, in normal rat liver and 0.372, 0.372, and 0.358 pmoles/ min/mg of nuclear DNA in protein-deficient rat liver, respectively.
4. These results indicate that protein deficiency increased both the rate of degradation of cytoplasmic rRNA and that of nucleolar rRNA synthesis in rat liver. While in normal rat liver the rates of rRNA synthesis and degradation were rather similar, the rate of rRNA synthesis in protein-deficient rats was about 1.5 times higher than that of its degradation. Therefore, the decrease of total liver RNA content in protein deficiency might be accounted for by stimulated degradation of rRNA in the nucleus.
5. The activities of RNase in nuclear fractions of both groups of rat livers were compared. Both activities of nuclear acid RNase and especially that of the free form of alkaline RNase in protein-deficient rat liver were higher than those in normal rat liver.View full abstractDownload PDF (765K) -
I. Removal of Extracellular and Intracellular Proteins and Their Effects on Protein SynthesisTatsuo NAKAYAMA, Kozo NARITA1977 Volume 81 Issue 1 Pages 153-162
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe minced oviducts prepared from laying hens contained considerable amounts of extracel-lular proteins (about 60mg per g of the wet oviducts), which could largely be removed (about 80%) by washing four times with chilled Krebs-Ringer-Tris buffer, pH 7.6.
When the washed, minced oviducts were incubated at 37° with the buffer, intracellular proteins were increasingly secreted to the extracellular fluids as the incubation time was in-creased and the amounts of intracellular proteins decreased concomitatnly, suggesting that the rate of secretion of intracellular proteins was faster than that of protein synthesis under the conditions used (without addition of amino acids to the medium). Repeated 5-min incuba-tions (five to seven times) resulted in the secretion of intracellular proteins in amounts of 100mg or more per g of the wet oviducts.
The secretion rate of intracellular proteins was almost constant at 37° regardless of the volume of buffer used for the incubation, and no secretion occurred at 4° even after 5 h. It ap-peared that a 30 min period was required for the secretion of intracellular proteins from the oviducts at. 37° and that the rate of secretion was not affected appreciably by the amount of secreted proteins present in the extracellular fluids.
The protein synthesis activity in the washed, preincubated minced oviducts was independ-ent of the amount of intracellular proteins involved but was dependent upon that of secreted proteins present in the extracellular fluids.View full abstractDownload PDF (759K) -
Yutaka ORII, Masayuki MORITA1977 Volume 81 Issue 1 Pages 163-168
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method was established to estimate the pH change of several buffer solutions on freezing by using a combination of pH indicators. Among more than 30 buffer solutions examined, almost half exhibited a pH change in the temperature range between freezing point and 220°K; the results were tabulated. Glycerol was found to suppress the pH changes because of its “salt buffer” effect.View full abstractDownload PDF (426K) -
Purification, Crystallization, and Isolation of a Binary Complex with NADHHiroyuki HASEGAWA1977 Volume 81 Issue 1 Pages 169-177
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDihydropteridine reductase [EC 1. 6. 99. 7] was purified from bovine liver in 50% yield and crystallized. The physicochemical properties of the purified enzyme were quite similar to those of sheep liver dihydropteridine reductase. During the course of purification, however, the enzyme was found to be separated into 2 major peaks together with minor peaks by column chromatography on CM-Sephadex, and one of the major peaks was identified as a binary complex of the enzyme with NADH. The reductase-NADH complex was also prepared in vitro and crystallized. Upon addition of quinonoid-dihydropterin to the complex, NADH was oxidized and released from the enzyme. The amount of bound NADH was calculated to be 2 moles per mole of the reductase. The occurrence of the reductase-NADH complex in bovine liver extract as a predominant form was in accord with the pyridine nucleotide specificity for NADH as a coenzyme. The results further support the view that NADH is the natural coenzyme of this reductase.View full abstractDownload PDF (1264K) -
Application to D-Amino Acid OxidaseKihachiro HORIIKE, Kiyoshi SHIGA, Akio ISOMOTO, Toshio YAMANO1977 Volume 81 Issue 1 Pages 179-186
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have mathematically analyzed ligand-induced monomerization and dimerization in a protein monomer-dimer equilibrium system, in which the monomer has one and the dimer two binding sites. These dimer sites have the same binding constants for the first ligand but may cooperatively interact when one of them is occupied by a ligand molecule. In this system, the apparent dimerization constant and the apparent molecular weight are functions of free ligand concentration, and depend on the intrinsic binding constants of the ligand molecule to the monomer and the dimer. The behavior of these functions is classified into 17 cases accord-ing to the values of the three intrinsic binding constants, and some calculated examples are shown graphically for selected parameters. The theory was also applied to D-amino acid oxidase [EC 1.4.3.3], a flavoprotein, and the pH dependence of the apparent dimerization constant and the apparent molecular weight in the presence of ligand, p-aminobenzoate, were studied theoretically using parameters obtained in our previous experiments (5).View full abstractDownload PDF (470K) -
IV. Purification and Characterization of NADH-cytochrome b5 ReductaseSachiko KUBOTA, Yuzo YOSHIDA, Hiroshi KUMAOKA1977 Volume 81 Issue 1 Pages 187-195
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe presence of NADH-cytochrome b5 reductase [EC 1. 6. 2. 2] in microsomes from anaerobi-cally grown yeast was confirmed by its isolation and purification. The purified preparation of the reductase showed an apparent molecular weight of 27, 000 daltons. The reductase appeared to contain loosely-bound FAD as a prosthetic group. The reductase required NADH as a specific electron donor, and could reduce some redox dyes as well as cytochrom b5. The reductase, however, could not reduce cytochrome c. Michaelis constants of the reductase for NADH and calf liver cytochrome b5 were 6.3 and 1.5 μM, respectively, and optimal pH for cytochrome b5 reduction was 5.6.
Although some differences exist between the properties of NADH-cytochrome b5 reductase from yeast and from mammalia, the results indicate a functional similarity of the present enzyme to mammalian NADH-cytochrome b5 reductase in the microsomal electron-transport system.View full abstractDownload PDF (638K) -
V. Purification and Characterization of NADPH-cytochrome c ReductaseSachiko KUBOTA, Yuzo YOSHIDA, Hiroshi KUMAOKA, Atsuko FURUMICHI1977 Volume 81 Issue 1 Pages 197-205
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA flavoprotein catalyzing the reduction of cytochrome c by NADPH was solubilized and purified from microsomes of yeast grown anaerobically. The cytochrome c reductase had an apparent molecular weight of 70, 000 daltons and contained one mole each of FAD and FMN per mole of enzyme. The reductase could reduce some redox dyes as well as cytochrome c, but could not catalyze the reduction of cytochrome b5. The reductase preparation also catalyzed the oxidation of NADPH with molecular oxygen in the presence of a catalytic amount of 2-methyl-l, 4-naphthoquinone (menadione). The Michaelis constants of the reductase for NADPH and cytochrome c were determined to be 32.4 and 3.4 μM, respectively, and the optimal pH for cytochrome c reduction was 7.8 to 8.0. It was concluded that yeast NADPH-cytochrome c reductase is in many respects similar to the liver microsomal reductase which acts as an NADPH-cytochrome P-450 reductase [EC 1. 6. 2. 4].View full abstractDownload PDF (693K) -
Toyoki KATO, Yuji TONOMURA1977 Volume 81 Issue 1 Pages 207-213
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembranous vesicles (microsomes) were isolated from plasmodia of the acellular slime mold, Physarurn polycephalum. The microsomes were about 0.2 μm in diameter, and about 10 nm thick. The main protein component of the vesicles had a molecular weight of 100, 000 daltons.
Calcium ions were taken up by the microsomes only in the presence of Mg2+-ATP. The maximum amount of Call ions accumulated in the microsomes was 0.24 μmole/mg protein. The Call uptake was not accelerated by oxalate.
The ATPase [EC 3. 6. 1. 3] activity required Ca2+ ions for full activation. The concentra-tion of Ca2+ions required for half-maximum activation was about 1 μM. The Km and Vm values were 53 μM and 1.6 pmole/(mg.min), respectively. About 0.2 mole of Ca2+ ions was taken up by the microsomes, coupled with the hydrolysis of 1 mole of ATP.
The ATPase activity and Ca2+ uptake of the microsomes were not inhibited by sodium azide. Furthermore, electron microscopic examination showed that mitochondrial contamina-tion was slight.
These results suggest that a vesicular calcium transport system, analogous to the sarco-plasmic reticulum in skeletal muscle, is involved in regulation of the Ca2+ concentration in plasmodia of Physarum.View full abstractDownload PDF (1262K) -
Purification, Characterization, and FunctionKoscak MARUYAMA, Sumiko KIMURA, Tetsuro ISHII, Masaaki KURODA, Kazuyo ...1977 Volume 81 Issue 1 Pages 215-232
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-Actinin, a minor regulatory protein of muscle, was purified from skeletal muscles of rabbit and chicken by DEAE-Sephadex chromatography. β-Actinin consisted of two subunits, βI and βII, with chain weights of 37, 000 and 34, 000 daltons, respectively. The amino acid compositions were similar, though not identical. It appears that each of the two subunits is associated in solution. β-Actinin had the following effects on actin: (1) inhibition of reas-sociation of F-actin fragments; (2) inhibition of network formation of F-actin; (3) inhibition of growth of F-actin fragments; (4) retardation of depolymerization of F-actin and (5) accelera-tion of polymerization of G-actin. All these actions of β-actinin can be explained in terms of action as an “ending factor.” Experimental evidence favored the view that, β-actinin is bound to one end of the F-actin filament, namely to the end opposite to the direction of poly-merization. Fluorescence-labeled anti-β-actinin stained the middle portion of the A band of myofibrils. Based on the finding that the stain was unchanged on removal of myosin, it is suggested that β-actinin is located at the free ends of the I filaments of myofibrils. Thus is seems likely that β-actinin functions as an ending factor for actin filaments.View full abstractDownload PDF (3410K) -
I. Purification and CharacterizationKazuyo OHASHI, Sumiko KIMURA, Kazuo DEGUCHI, Koscak MARUYAMA1977 Volume 81 Issue 1 Pages 233-236
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new minor regulatory protein designated as I-protein has been isolated from skeletal muscles of the chicken and the rabbit. The yield of purified I-protein was small: 5-10mg per kg of muscle. I-protein was found to have a molecular weight of 50, 000 and high contents of Asp and Glu, accounting for more than one third of the total amino acids.View full abstractDownload PDF (783K) -
II. LocalizationKazuyo OHASHI, Tomoh MASAKI, Koscak MARUYAMA1977 Volume 81 Issue 1 Pages 237-242
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit antiserum raised against I-protein was used for immunofluorescent staining of chicken myofibrils. The FITC-conjugated anti-I-protein antibody stained A-band regions except at their middle regions. According to the conditions used, the myofibrils stained by their fluores-cent antibody showed slightly different patterns, i.e., the nonstained regions in the center of the A-bands were wider. On fixing with glutaraldehyde, myofibrils were stained in the A-band regions except at their middle regions. Therefore, I-protein may be localized at A-bands except for the center.View full abstractDownload PDF (2353K) -
III. FunctionKoscak MARUYAMA, Sachio KUNITOMO, Sumiko KIMURA, Kazuyo OHASHI1977 Volume 81 Issue 1 Pages 243-247
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSI-protein inhibited the Mg-activated ATPase [EC 3. 6. 1. 3] activity of actomyosin by approxi-mately 50% at low ionic strength. Concomitantly, the onset of superprecipitation was re-tarded. I-protein was found to bind to myosin, but not to F-actin.
The inhibitory action of I-protein occurred only in the absence of Ca ions in the troponin-tropomyosin-actin-myosin system. Addition of Ca ions abolished the effect. Thus, it is very likely that I-protein prevents unnecessary hydrolysis of ATP in the relaxed state of muscle.View full abstractDownload PDF (960K) -
Evidence for the K. S. T. Mechanism with Two Enzyme-ATP Complexes and Two Phosphorylated Intermediates of High-Energy TypeMotonori YAMAGUCHI, Yuji TONOMURA1977 Volume 81 Issue 1 Pages 249-260
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Kinetic properties of the [3H] ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase [EC 3. 6. 1. 3] were investigated, using Nal-treated microsomes from bovine brain, and the following results were obtained.
1. The rates of the Na+-dependent exchange reaction in the steady state were measured in a solution containing 45μM free Mg2+, 100mM NaCl, 80 μM ATP, and 160 μM ADP at pH 6.5 and 4-5°. The rate and amount of decrease in phosphorylated intermediate on adding ADP, i.e., the amount of ADP-sensitive EP, were measured while varying one of the reaction param-eters and fixing the others mentioned above. Plots of the exchange rate and the amount of ADP-sensitive EP against the logarithm of free Mg2+ concentration gave bell-shaped curves with maximum values at 50-60 μM free Mg2+. Plots of the exchange rate and the amount of ADP-sensitive EP against pH also gave bell-shaped curves with maximum values at pH 6.9-7. They both increased with increase in the concentration of NaCl to maximum values at 150-200 mM NaCl, and then decreased rapidly with increase in the NaCl concentration above 200 mM The dependences of the exchange rate and the amount of ADP-sensitive EP on the concentration of ADP followed the Michaelis-Menten equation, and the Michaelis constants, Km, for both were 43 μM. The dependence of the exchange rate on the ATP concentration also followed the Michaelis-Menten equation, and the Km value was 30 μM. The amount of ADP-sensitive EP increased with increase in the ATP concentration, and reached a maximum value at about 5 μM ATP.
2. The Na+-dependent [3H] ADP-ATP exchange reaction was started by adding [3H]ADP to EP at low Mg2+ concentration. The reaction consisted of a rapid initial phase and a slow steady phase. The amount of [3H] ATP formed during the rapid initial phase, i.e. the size of the ATP burst, was equal to that of ADP-sensitive EP, and was proportional to the rate in the steady state. At high Mg2+ concentration, the rate of Na+-dependent exchange in the steady state was almost zero, and EP did not show any ADP sensitivity. However, rapid formation of [3H]ATP was observed in the pre-steady state, and the size of the ATP burst increased with increase in the KCl concentration.
From these findings, we concluded that an enzyme-ATP complex (E2ATP) formed at low Mg2+ concentration is in equilibrium with EP+ADP, that the rate-limiting step for the exchange reaction is the release of ATP from the enzyme-ATP complex, that the ADP-insensitive EP_??_produced at high Mg2+ concentration is in equilibrium with the enzyme-ATP complex, and that the equilibrium shifts towards the enzyme-ATP complex on adding KCl. Actually, the ratio of the size of the ATP burst to the amount of EP was equal to the reciprocal of the equilibrium constant of step_??_determined by a method previously reported by us.View full abstractDownload PDF (844K) -
Hiroshi SHINKAI, Tomoyuki KAWAMOTO, Hisae HORI, Yutaka NAGAI1977 Volume 81 Issue 1 Pages 261-263
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCollagenase inhibitors with molecular weights of about 6, 000 and 12, 000 were isolated from latent chick skin collagenase treated with 3M NaI and from the culture medium of embryonic skin explants. It is suggested that these inhibitors, which are possibly derived from connective tissue macromolecule metabolites, are candidates for regulating factors of collagenase activity in vivo.View full abstractDownload PDF (211K) -
Tadashi YOSHIDA, Goro KIKUCHI1977 Volume 81 Issue 1 Pages 265-268
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHeme oxygenase was purified to apparent homogeneity from pig spleen microsomes. The purified heme oxygenase showed an apparent molecular weight of 157, 000±7, 000 daltons when estimated by gel filtration. On SDS-polyacrylamide gel electrophoresis, the heme oxygenase preparation gave a single protein band showing a minimum molecular weight of about 26, 000 daltons. Heme oxygenase could readily bind with heme and the resulting heme complex gave an absorption maximum at 406 nm. The heme bound to the enzyme protein was found to be a good substrate for the heme oxygenase reaction.View full abstractDownload PDF (1000K) -
A Possible Initiation Complex for Gramicidin A BiosynthesisKatsuhiko AKASHI, Kou KUBOTA, Kiyoshi KURAHASHI1977 Volume 81 Issue 1 Pages 269-272
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA partially purified fraction (Component I) which catalyzed the ATP-PPi exchange reaction dependent only on valine and glycine among the five constituent amino acids of gramicidin A was obtained from crude extracts of Bacillus brevis ATCC 8185, which produces both gramicidin A and tyrocidines. This fraction was found to bind valine and glycine as thioesters. The addition of a formylTHFA synthesizing system to Component I brought about the formation of Component I-bound formylvaline and formylvalylglycine.View full abstractDownload PDF (852K) -
A New Type of Regulatory ProteinTakashi MIKAWA, Teruhiko TOYO-OKA, Yoshiaki NONOMURA, Setsuro EBASHI1977 Volume 81 Issue 1 Pages 273-275
Published: January 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe physiological activity of gizzard ‘troponin’ fraction ((1975) J. Biochem. 78, 859) was shown to be due to the 80, 000 dalton component.View full abstractDownload PDF (219K)
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