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Volume 79 (1976)
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- Issue 5 Pages 853-
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Volume 79, Issue 5
Displaying 1-28 of 28 articles from this issue
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Tatsuo NAKAYAMA, Kikuo OGATA, Kozo NARITA1976 Volume 79 Issue 5 Pages 853-869
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHen's oviduct polysomes are present in the precipitates prepared from the oviduct homogenates by low-speed centrifugation, in contrast to other eukaryotic polysomes. The polysomes possessed synthesizing activity for ovalbumin. The amount of the released form of ovalbumin (soluble ovaibumin) synthesized in cell-free system A or B, consisting of the cell sap and the total ribosomal fraction or the polysomes, respectively, was about a half of that bound to the polysomes (nascent ovalbumin). The amount of soluble ovalbumin synthesized in cell-free system C, consisting of the pH 5 fraction and the polysomes, was only about 5%p of that of nascent ovalbumin. These results indicate that factors required to release nascent ovalbumin from polysomes are present in the pH 5 supernatant fraction.
The soluble and nascent ovalbumins, which were purified by chromatography on a CM-cellulose column and by the use of antiovalbumin antiserum, respectively, seemed to be elongation products, the initiations of which were supposed to occur in the oviducts before preparation of the cell-free system. The initiated chains in vitro were found to exist as nascent peptides bound to polysomes. Thus, the cell-free systems prepared in the present study lacked the ability to complete initiated peptide chains.
The soluble ovalbumin synthesized in the cell-free systems was identical with ovalbumin Al containing two residues of phosphates, which was crystallized from hen's egg-white, and was different from soluble ovalbumin devoid of the prosthetic group (ovalbumin A3) prepared in the oviduct minces. This result suggests that an enzyme necessary for incorporation of the phosphate is present in the cell sap.View full abstractDownload PDF (1354K) -
Tatsuo NAKAYAMA, Kozo NARITA, Kikuo OGATA1976 Volume 79 Issue 5 Pages 871-881
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDithiothreitol or glutathione, essential for amino acid incorporation into polypeptides in cell-free systems, inhibited glucosamine incorporation into ovalbumin, a glycoprotein containing about three residues of N-acetylglucosamine and about five residues of mannose. However, the thiol was necessary for mannose incorporation into ovalbumin in cell-free systems prepared from hen's oviducts. The two monosaccharides were incorporated into soluble ovalbumin (intracellular ovalbumin released from the poly-somes, corresponding to ovalbumin A3 which contains no phosphate and probably no carbohydrate) present in cell sap which was prepared from homogenates of the minced oviducts by ultracentrifugation. It was difficult to study the incorporation of the carbohydrates into ovalbumin in connection with synthesis of the protein using the crude cell-free system.
The incorporation of glucosamine and mannose into various forms of ovalbumin was therefore studied using minced oviducts. It was concluded from the incorporation -of the two radioactive monosaccharides and of amino acids into the extracellular, soluble and nascent protein fractions that all the carbohydrate moieties were not incorporated into the growing polypeptides bound to polysomes, but were incorporated into ovalbumin molecules released from polysomes after synthesis had been com-pleted.View full abstractDownload PDF (904K) -
Kunio TSURUGI, Kikuo OGATA1976 Volume 79 Issue 5 Pages 883-893
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. When the postmitochondrial supernatant (PM-supernatant) from regenerating rat liver was incubated with [3H]methionine, the incorporation of [3H]methionine into the N-terminal residues of nascent peptides on ribosomes was observed. This incorporation was sensitive to a low cocentration (2×10-6M) of pactamycin. The results suggest that PM-supernatant has low but definite activity for the initiation of nascent protein synthesis. Polysomes and cell sap from regenerating rat liver showed negligible pactamycin-sensitive incorporation of [3H]methionine into N-terminal residues of nascent peptides.
2. PM-supernatant from regenerating rat liver was incubated with [35S]methionine in the complete reaction mixture. After addition of ribosomal proteins labelled with [3H]methionine in vivo, ribosomal structural proteins were prepared from the incubation mixture by acetic acid extraction, CM-cellulose column chromatography, Sephadex G-200 gel filtration and finally by two-dimensional acrylamide gel electrophoresis. Incorporation was observed in the greater part of ribosomal proteins on the two-dimensional gel. From the 35S-to-3H ratios of ribosomal protein fractions during the purification procedures, it appeared that the incorporation of labelled methionine into the ribosomal proteins by PM-supernatant was about 3% of that into the total proteins. When [3H]leucine was used, the values were about 4% in the same cell-free system and 5 to 6% in in vivo labelling. The results indicate that ribosomal proteins are synthesized with high efficiency by PM-supernatant from regenerating rat liver.View full abstractDownload PDF (2332K) -
Shonen YOSHIDA, Shigeo MASAKI, Teruo ANDO1976 Volume 79 Issue 5 Pages 895-901
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA synthesizing reactions catalyzed by both large and small species of calf thymus DNA polymerase (DNA polymerase-α and -β) [EC 2.7.7.7] were stimulated to comparable extents by the presence of spermidine or spermine, and it was not possible to differentiate these two species in terms of their sensitivities to polyamines. Optimal concentrations for stimulation were 0.5-1.0mM for spermidine and 2-10 μM for spermine. Excess polyamines strongly inhibited the reactions. The modes of stimulation were as follows:
1) Stimulation was observed with templates bearing long single-stranded sections when either natural DNA or synthetic homopolymer-oligomer duplex was used.
2) The natural DNA-dependent reaction was stimulated by polyamines at suboptimal concentrations of Mg2+; the apparent Km value for Mg2+ was lowered on adding polyamines, while the Vmax value was unchanged. When synthetic homopolymer-oligomer duplex was used as a template, the reaction was stimulated by spermidine even at the optimal concentration of Mn2+.
3) Polyamines markedly influenced the salt requirements of the reactions of DNA polymerase. Spermidine could replace salts such as KCl or NaCl at concentrations less than 1/100 of those of salts.View full abstractDownload PDF (512K) -
Haruaki TOMIOKA, Midori KAGAWA, Shoshiro NAKAMURA1976 Volume 79 Issue 5 Pages 903-915
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe 3β-hydroxysteroid oxidase produced by Streptomyces violascens was purified from the culture broth by procedures including batch-wise treatment on DEAE-cellulose, ammonium sulfate fraction, gel filtration on Sephadex G-75, and column chromatog-raphy on DEAE-cellulose. The highly purified enzyme preparation exhibited no significant absorption maxima in the visible region other than a maximum at 280nm. Optimum pH and temperature for the enzyme activity were approximately pH 7.5 and 50°, respectively. The Michaelis constant (Km) for cholesterol determined under two different experimental conditions were 4.5 and 6.7×10-4M. The enzymatic activity was remarkably inhibited by various metal salts such as FeCl3, FeSO4, AgNO3, etc. On the other hand, neither EDTA nor Fe-chelating agents had any inhibitory effect on the enzymatic activity, while other metal-binding agents, KCN and NaN3, caused significant inhibition. The enzyme activity was inhibited almost completely by N-bromosuccinimide and iodine but not p-chloromercuribenzoate.
The highly purified enzyme did not require any external electron acceptors other than oxygen. In addition, the activity was not influenced by the addition of external electron donors.
The enzyme showed a high substrate specificity for 3β-hydroxysteroids and the relative oxidation rates were 100 for cholesterol, 91 for 5α-cholestan-3β-ol, 83 for pregn-5-en-3β-ol-20-one, 80 for androst-5-en-3β-ol-17-one, 64 for 5a-androstan-3β-of-17-one, etc. The oxidation of cholesterol by the enzyme was remarkably inhibited by the addition of 5α-cholestan-3β-ol, 5α-cholestan-3-one, 5β-cholestan-3β-ol, 5α-cholestane-3β, 5α-diol or 5α-lanosta-8, 24-dien-3β-ol. These findings indicate the present enzyme belongs to the class of 3β-hydroxysteroid oxidase but some of its physical and enzymatic properties obviously differ from those of 3β-hydroxysteroid oxidase of Brevibacterium.View full abstractDownload PDF (891K) -
I. Copurification and SeparationAkira ISHIHAMA, Toshihiko IKEUCHI, Takashi YURA1976 Volume 79 Issue 5 Pages 917-925
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAdenosinetriphosphatase (ATPase) [EC 3.6.1.3] activity has been found to exist in most preparations of DNA-dependent RNA polymerase [EC 2.7.7.6] obtained from Escherichia coli by a number of purification procedures so far established. Electro-phoretic analysis on polyacrylamide gels demonstrated that ATP hydrolysis and RNA synthesis were catalyzed by two distinct enzyme proteins. It appears that the two enzymes are associated or have similar molecular properties. Separation of the two enzymes, the object of the present work, was achieved by three independent methods: ion exchange chromatography on a phosphocellulose column, electrophoresis in glycerol gradients, or high-salt glycerol gradient centrifugation.View full abstractDownload PDF (624K) -
II. Enzymatic Properties and Molecular StructureAkira ISHIHAMA, Toshihiko IKEUCHI, Akira MATSUMOTO, Sadaaki YAMAMOTO1976 Volume 79 Issue 5 Pages 927-936
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn adenosinetriphosphatase (ATPase) [EC 3.6.1.3] copurified with the DNA-dependent RNA polymerase [EC 2.7.7.6] from Escherichia coli was isolated to apparent homo-geneity and some of its functional as well as structural properties were examined. Although the novel ATPase exhibited metal requirements similar to those of Mg2+, Ca2+-ATPase, its response to NaN3 and antisera appeared completely different from that of the Mg2+, Ca2+-ATPase. The purified ATPase was found to be a large protein with a molecular weight of 9.3×105 daltons, composed of identical subunits of 7×104 daltons. When viewed under an electron microscope, the ATPase appeared to be very similar to material previously misidentified as the RNA polymerase. The physiological role of the novel ATPase, however, remains unclear.View full abstractDownload PDF (2875K) -
Takashi MATSUMOTO, Jiro TOBARI, Koji SUZUKI, Tokuji KIMURA, T.T. TCHEN1976 Volume 79 Issue 5 Pages 937-943
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have isolated an iron-sulfur protein from a Pseudomonas species grown on glucose. This protein has different properties from the two known iron-sulfur proteins isolated from other Pseudomonas species: rubredoxin and putidaredoxin.
The iron-sulfur protein was purified to homogeneity by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The absorption spectrum of the oxidized iron-sulfur protein shows a peak at 283nm with shoulders at about 290, 320, and 410nm. The protein contains 4 g atoms of iron and 4 moles of labile sulfur per mole of protein, and has a molecular weight of approximately 14, 000. The amino acid composition of the protein shows a predominance of acidic amino acids. The Pseudomonas protein was found to be active for both photosynthetic nicotinamide nucleotide reduction by chloroplasts and cytochrome c reduction by spinach ferredoxin-NADP+ reductase [EC 1.6.7.1].
On the basis of these results, this protein appears to be unique among all known ferredoxins. From an evolutionary point of view, it appears to be more closely related to Azotobacter ferredoxin than to Desulfovibrio ferredoxin.View full abstractDownload PDF (1215K) -
Hideo KATO, Ikuko OYAMADA, Masumi MIZUTANI-FUNAHASHI, Hachiro NAKAGAWA1976 Volume 79 Issue 5 Pages 945-953
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMethods were developed for the radioisotopic assay of argininosuccinate synthetase [L-citrulline: L-aspartate ligase (AMP-forming), EC 6.3.4.5] and argininosuccinase [L-argininosuccinate arginine-lyase, EC 4.3.2.1]. The assay of argininosuccinate synthetase was based on the separation of [14C]argininosuccinate formed from aspartate and [carbamoyl-14C]citrulline in the presence of ATP from the substrate citrulline. For this, the product was converted to its anhydride form by boiling for 30min at pH 2.0 followed by application on a column of Dowex 50W (pyridine form). Argin-inosuccinic anhydride was eluted with 0.3M pyridine acetate buffer, pH 4.25, while citrulline was eluted with 0.1M pyridine acetate buffer, pH 3.80.
The assay of argininosuccinase was based on the separation of [14C]argininosuc-cinic acid formed from arginine and [U-14C]fumaric acid from the substrate fumarate on a column of Dowex 50W(H+ form). The argininosuccinic acid was adsorbed on the column and eluted with 1M pyridine solution, while fumarate was not adsorbed.
The distributions of these two enzymes in various organs and cell fractions were reinvestigated using these methods.View full abstractDownload PDF (735K) -
Masashi NAKAYAMA, Yoshimi TOMITA, Hiroshi SUZUKI, Kazutosi NISIZAWA1976 Volume 79 Issue 5 Pages 955-966
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn endo-cellulase component [EC 3.2.1.4] of random type; F II, was obtained from “ Cellulase Onozuka, ” a commercial product from Trichoderma viride, and was subjected to partial proteolysis with a protease preparation of the same fungal origin. The resulting modified cellulase was fractionated by two steps of column chromatography, and the resulting patterns, together with the substrate specificity expressed in terms of the randomness of CMC hydrolysis and the immunological properties against anti-F II-rabbit serum, were examined. The chromatographic patterns were very similar to those of cellulase subfractions without proteolytic treatment. Moreover, the immunological response of the modified cellulases from F II was mostly positive and their randomness of CMC hydrolysis was generally lower, compared with subfractions of F II which were not subjected to proteolysis. The subfractions of Peak III, which were obtained from F II by proteolysis, showed mostly negative immunological response and higher randomness of CMC hydrolysis compared with subfactions of Peak III which were not subjected to proteolysis. Thus, some limited proteolysis of cellulase components may, at least in part, be responsible for its multiplicity in vivo.View full abstractDownload PDF (1612K) -
Kyoko OGASAHARA, Kichiko KOIKE, Minoru HAMADA, Tadayasu HIRAOKA1976 Volume 79 Issue 5 Pages 967-975
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of hydrophobic probes, 8-anilinonaphthalene-l-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2, 2'-disulfonate (MBAS), with pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3] was investigated. When ANS or MBAS was mixed with the apoenzyme of lipoamide dehydrogenase, the fluorescence quantum yield of each dye was enhanced markedly and the emission maxima concurrently shifted to the blue. The quantum yield, 0.038, of ANS bound to the apoenzyme, calculated from the corrected emission spectrum, was eight times higher than that in buffer solution, and the value, 0.0090, for bound MBAS was eighteen times higher than that in buffer solution. Moreover, the absorption bands of both ANS and MBAS shifted to the red upon binding with the apoenzyme. A general feature of the absorption spectra of these dyes observed on changing the solvent from polar to apolar was a red shift of the absorption bands. These results indicate that ANS or MBAS bound to the apoenzyme of lipoamide dehydrogenase is situated in a hydrophobic region of the apoenzyme molecule. It was found that 2 moles of each dye was bound per mole of the apoenzyme, which contains two polypeptide chains. The dissociation constants for the ANS- and MBAS-apoenzyme, complexes were estimated to be 1.03×10-5 and 1.54×10-5M, respectively. The enhanced fluorescence of both dyes bound to the apoenzyme decreased linearly upon adding FAD and disappeared at about 2 moles of FAD per mole of the apoenzyme. This suggests that both ANS and MBAS were displaced from their binding sites on the apoenzyme by FAD.
The protein fluorescence spectrum of the apoenzyme had a maximum at 352 nm, which was blue-shifted by 6 nm from that of tryptophan in the buffer. Upon binding ANS or MBAS, the maximum of the protein fluorescence of the apoenzyme returned to 350nm for the holoenzyme, and the fluorescence intensity decreased. Thus, the conformation around some tryptophan residues was affected by the binding of the dyes.
When guanidine hydrochloride (GuHCl) was added to the ANS-apoenzyme complex solution, the enhanced fluorescence due to the bound ANS decreased and the emission maximum concurrently shifted to the red. Further, the maximum of the protein fluorescence of the apoenzyme shifted to the red, indicating the exposure of some tryptophan residues buried in an apolar region of the apoenzyme. Thus, the binding of ANS to the apoenzyme was inhibited by protein denaturation due to GuHCl.
In contrast, the holoenzyme of lipoamide dehydrogenase did not bind ANS or MBAS at all.View full abstractDownload PDF (670K) -
Takahisa KANDA, Kazumasa WAKABAYASHI, Kazutosi NISIZAWA1976 Volume 79 Issue 5 Pages 977-988
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA culture filtrate of Irpex lacteus (Polyporus tulipiferae) was fractionated initially by salting out with ammonium sulfate, and a cellulase [EC 3.2.1.4] fraction with high Avicel-hydrolyzing activity (formerly called Avicelase) was extensively purified by a series of column chromatography procedures. This purified endo-cellulase showed a less random hydrolytic mechanism, and was obtained in a yield of 0.04% with respect to the starting material. Its specific activity was enhanced approximately 30 times over that of the starting material. The cellulase component showed a single peak on both ultracentrifugal and acrylamide disc electrophoretic analyses. Its molecular weight was estimated to be 56, 000.
It contained 12.2% carbohydrate ; the major sugar constituents were glucose and mannose. Regarding the amino acid composition, the contents of aspartic acid and glycine were highest, followed by those of glutamic acid, serine, and threonine.
The cellulase component was not markedly inhibited by most metal ions tested except for Hg2+.
This purified endo-cellulase attacked a series of cellooligosaccharides, β-cellobioside, CM-cellulose, and insoluble cellulosic substrates. In the digests from insoluble substrates, glucose, cellobiose, cellotriose, and cellotetraose were detectable, but the amount of cellobiose was the largest by far. In contrast, cellobiose and glucose were produced in almost equal amounts from β-cellobioside.View full abstractDownload PDF (2467K) -
Takahisa KANDA, Kazumasa WAKABAYASHI, Kazutosi NISIZAWA1976 Volume 79 Issue 5 Pages 989-995
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn endo-cellulase [EC 3.2.1.4] of carboxymethyl-cellulase type (F-1) which was fractionated from culture filtrate of Irpex lacteus and purified to electrophoretic and ultracentrifugal homogeneity, was found to show xylanase [EC 3.2.1.8] activity. The activity was not removed from any of the intermediate fractions during the purification of the initial F-I peak, and the ratio of xylanase to cellulase activity remained almost unchanged through the purification processes. The xylanase activity of F-1 showed not only the same optimal pH, heat stability, and pH stability as its cellulase activity, but also the same mobility as the cellulase activity upon cellulose acetate film and starch zone electrophoreses.
The overall rates of hydrolysis of mixtures of various concentrations of CM-cellulose and xylan by F-1 coincided well with those calculated from the Michaelis-Menten treatment of two substrates competing for the same active site of the enzyme. These results indicate that the xylanase activity of F-1 is intrinsic to the cellulase itself.View full abstractDownload PDF (477K) -
Takahisa KANDA, Kazumasa WAKABAYASHI, Kazutosi NISIZAWA1976 Volume 79 Issue 5 Pages 997-1006
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe substrate specificities of three endo-cellulase [EC 3.2.1.4] components, F-1, F-2, and S-1, obtained from the culture filtrate of Irpex lacteus (Polyporus tulipiferae), were investigated in detail. It was confirmed that the former is of a more random type, belonging to the carboxymethyl-cellulase (CMCase) group, and the latter two are of a less random type, belonging to the Avicelase group. It was found that a mixture of CMCase and Avicelase shows a remarkable synergistic action in the degradation of cotton and Avicel and that CMCase lowers the degree of polymerization of both cotton and CM-cellulose faster than Avicelase, relative to the production of reducing sugar. Thus, it was assumed that cotton and similar cellulosic substrates were degraded mainly by the synergistic action of these cellulase components produced by this cellulolytic fungus.View full abstractDownload PDF (679K) -
III. Inhibition by Gluconolactone of the Hydrolysis of Maltodextrin Catalyzed by Glucoamylase from Rhizopus niveusMasatake OHNISHI, Takashi YAMASHITA, Keitaro HIROMI1976 Volume 79 Issue 5 Pages 1007-1012
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInhibition by gluconic acid-1:5-lactone (gluconolactone) and phenyl a-glucoside of the hydrolysis of maltodextrin catalyzed by glucoamylase [EC 3.2.1.3] from Rhizopus niveus was investigated in relation to the subsite structure of the enzyme. Inhibition by gluconolactone was of the mixed type, whereas that by phenyl α-glucoside was purely competitive. These inhibition types were consistent with a theoretical pre-diction based on the assumption that gluconolactone and phenyl α-glucoside bind mainly to Subsites 1 and 2, respectively.
The inhibitor constant of gluconolactone was determined to be 1.5mM, which is in good agreement with the dissociation constant estimated by difference spectrophotometry (1.5mM) (Ohnishi, M. et at. (1975) J. Biochem. 77, 695-703).View full abstractDownload PDF (424K) -
Itsuo OKUMURA, Kigen KONDO, Yukio MIYAKE, Kimikazu ITAYA, Takehiko YAM ...1976 Volume 79 Issue 5 Pages 1013-1019
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Alaantoinase [EC 3.5.2.5] was isolated from cells of Candida utilis and purified by chromatography on columns of DEAE-cellulose and Sephadex G-200 after treatment with urea to remove urate oxidase [EC 1.7.3.3].
2. The purified allantoinase catalyzed the hydrolysis of allantoin into allantoic acid. However, only a half of the allantoin produced from uric acid by urate oxidase was converted. The rest of the allantoin was unchanged, and showed a negative optical rotation.
3. On the other hand, the combined action of crude urate oxidase and allantoinase resulted in nearly complete conversion of uric acid into allantoic acid. Furthermore, the unpurified allantoinase preparation hydrolyzed racemic allantoin to allantoic acid completely.
4. These results indicate that the urate oxidase produces racemic allantoin from uric acid and that the allantoinase attacks only allantoin of positive optical rotation. The results also suggest that allantoin racemase is present in the yeast cells.View full abstractDownload PDF (482K) -
Masahiko KISUMI, Masaki SUGIURA, Jyoji KATO, Ichiro CHIBATA1976 Volume 79 Issue 5 Pages 1021-1028
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo unnatural amino acids were found as by-products in isoleucine fermentation from threonine by Serratia marcescens. These amino acids were identified as L-norvaline (2-aminopentanoic acid) and L-homoisoleucine (2-amino-4-methylhexanoic acid). Formation of L-norvaline and L-homoisoleucine was not observed when L-leucine was added to the medium. The leucine auxotroph derived from the isoleucine accu-mulator did not produce L-norvaline or L-homoisoleucine even during the accumulation of large amounts of isoleucine. It is suggested that L-norvaline and L-homoisoleucine formation is closely related to leucine biosynthesis.View full abstractDownload PDF (479K) -
VI. Relationship between the Peroxisomal Core and Urate OxidaseHidenori HAYASHI, Kohji TAYA, Tetsuya SUGA, Shinkichi NIINOBE1976 Volume 79 Issue 5 Pages 1029-1034
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1. 7. 3. 3] activity. This preparation had a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3], L-α-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1. 11. 1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC 1.9.3. 1] (mitochondria), acid phosphatase [EC 3.1. 3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation.
The core obtained showed a single protein band in sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight of about 35, 000, When the peroxisomal core was subjected to treatment at various pH's with 0.1M carbonate buffer, urate oxidase was almost completely solubilized at pH 11.0, although approximately 35% of the core protein still remained in the pellet. After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times ; the density of the insoluble protein remaining in the pellet was identical with that of the original core on sucrose density gradient centrifugation.View full abstractDownload PDF (402K) -
Takashi KAWASAKI, Hiroshi SANEMORI, Yoshiko EGI, Shusuke YOSHIDA, Kazu ...1976 Volume 79 Issue 5 Pages 1035-1042
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe biochemical properties of strains (PT-R101 and PT-R108) of Escherichia coli K12 resistant to growth inhibition by pyrithiamine, an antimetabolite of thiamine, have been studied. Intracellular thiamine pyrophosphate concentration in these resistant strains was slightly but definitely higher than that in the parent strain. Thiamine synthesis from the pyrimidine and thiazole moieties of thiamine by cell suspensions was greater in the resistant strains than the parent strain. The activities of enzymes involved in thiamine biosynthesis in these pyrithiamine-resistant strains were 2-3 times higher than the parent strain (3301), except for thiamine-phosphate kinase, which was indetectable in in vitro assay of the activity. However, other evidence indicates that this enzyme is not defective but is functioning in vivo and, furthermore, that the negligible activity of this enzyme did not affect the growth rate of the mutants. The activities of these enzymes were further enhanced when PT-R101 was grown on 5 mM adenine and were reduced almost to zero when the strain was grown on 0.1 μM thiamine in the same way as the parent strain. However, when these resistant strains were grown on a low concentration of thiamine such as 0.05 μM, thiamine synthesis by cell suspensions also decreased, but only to a limited extent compared with the parent strain.
These results suggest that PT-R101 and PT-R108 are altered in the mechanisms of regulation of thiamine biosynthesis. Their altered properties might be due to a reduced binding affinity of the repressor protein, which is involved in the regulation of thiamine synthesis, for the corepressor, thiamine pyrophosphate.View full abstractDownload PDF (706K) -
Toshio ANDO, Hiroshi ASAI1976 Volume 79 Issue 5 Pages 1043-1047
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of HMM to F-actin containing bound 1, N6-ethenoadenosine diphosphate (ε-ADP), a fluorescent analogue of ADP, caused a significant increase in the fluorescence intensity of ε-ADP at 410nm on excitation at 340nm. This increase is regarded as due to a conformational change in the actin molecule induced by HMM binding. The fluorescence intensity increase was not directly proportional to the amount of bound HMM. This phenomenon suggests that a conformational change in neighbouring actin molecules is induced cooperatively by the conformational change of the actin molecule binding HMM.View full abstractDownload PDF (355K) -
Mikiharu YOSHIDA, Fumi MORITA1976 Volume 79 Issue 5 Pages 1049-1052
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubfragment-1 prepared by chymotryptic digestion of myosin was applied to a column of Sepharose-adipic acid hydrazide-ATP in 1mM EDTA, 10mM Tris-HCl (pH 7.6), and 40mM KCl. Ninety-nine per cent of subfragment-1 was adsorbed on the column in this medium. Forty-three per cent of the applied protein was eluted with 6mM ADP in the above buffer and then 52% was eluted with 1mM EDTA, 10mM Tris-HCl (pH 7.6), and 0.7M KCl. The former fraction contained g3 chain and the latter gl chain. These fractions were apparently the same as the two components, p2 and pl, respectively, isolated by ion-exchange chromatography using DEAE-cellulose (Yagi & Otani (1974) J. Biochem. 76, 365-373). No significant difference of ADP binding was found between the two fractions ; both could bind about 0.5 mole per 105g of protein. The preparation of the two subfragment-1 fractions is described.View full abstractDownload PDF (1163K) -
I. Release of Calcium Taken up upon Using ATPMichiki KASAI, Hiroshi MIYAMOTO1976 Volume 79 Issue 5 Pages 1053-1066
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+ taken up by sarcoplasmic reticulum membrane fragments (SRF) upon using ATP could be released rapidly by changing the anion outside the vesicles from methanesulfonate to chloride. It is considered that this anion exchange caused depolarization of the sarcoplasmic reticulum membrane. Similar rapid release of Ca2+ taken up by SRF was also caused by a change from high to low osmotic pressure, probably due to bursting of the membrane. On the basis of experiments in which these two types of Ca2+ release were discriminated, it was concluded that Ca2+ bound inside the membrane was released directly by anion exchange (depolarization). However, Ca2+ release was not caused by cation exchange.
Sucrose inhibited these two types of Ca2+ release. Ca2+ taken up in the presence of oxalate could not be released by any treatment used. Liver microsome fraction also has Ca2+ uptake activity. However, Ca2+ was not released upon anion exchange, but was released upon osmotic change. These results show that Ca2+ release from SRF upon anion exchange is specific to the sarcoplasmic reticulum membrane.
In conclusion, SRF membrane retains the ability to respond to the depolarization caused by ion exchange and can release the accumulated Ca2+.View full abstractDownload PDF (1067K) -
II. Release of Calcium Incorporated without ATPMichiki KASAI, Hiroshi MIYAMOTO1976 Volume 79 Issue 5 Pages 1067-1076
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+ incorporated in vesicles of sarcoplasmic reticulum fragments (SRF) by diffusion could be released rapidly by Changing the ionic environment, by dilution from methanesulfonate (MS) to chloride. This ion exchange is considered to make the membrane potential of SRF inside-negative. Much faster release of Ca2+ was also observed upon osmotic change from high to low. These responses were very similar to the Ca2+release from SRF after take up using ATP, but the release rate was slow in the case of anion exchange.
The behavior of K+, Na+, sucrose, and inulin incorporated in SRF was followed upon similar treatment. These ions and molecules were not released upon ion exchange, but were immediately released by osmotic treatment. Therefore, the Ca+ release upon anion exchange was not due to the bursting of SRF, but to a direct effect such as a membrane potential change of the SRF. The behavior of anions such as Cl- and propionate could not be followed by the same method because of the large permeability of these anions.
It was also shown that Ca+ release upon ion exchange was not a direct effect of pH change.
Liver microsomes did not show Ca+ release upon the same treatment as SRF.View full abstractDownload PDF (751K) -
Yoshihiro MIYAKE, Naoko TAKAYAMA1976 Volume 79 Issue 5 Pages 1077-1087
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe slow reduction of microsomal cytochrome P-450 by dithionite consists of an initial fast and then a slow phase. During reduction of aniline and azide complexes with cytochrome P-450, an intermediate spectrum developed in the fast phase and changed to that of the reduced form in the slow phase. Only the spectra in the slow phase had an isosbestic point. No intermediate spectrum was detectable during reduction of the cyanide complex and native-cytochrome P-450. Carbon monoxide accelerated the reaction, causing complete reduction in the initial phase. The electron spin resonance spectrum of cytochrome P-450 was greatly reduced in the initial phase of reduction with dithionite. These results indicate that reduction of the aniline and azide complexes of cytochrome P-450 involves two steps : first reduction of cyto-chrome P-450 and then some changes in the reduced state, The aniline and cyanide difference spectra of reduced cytochrome P-450 showed peaks at 423nm and 429nm, respectively, while that of azide had a peak at 445 nm and a trough at 404nm.
An easy method to obtain the difference spectrum of reduced minus oxidized cytochrome P-450 using a spectral data processor is reported. The effects of other NADH-nonreducibie pigments on the spectrum is eliminated by this procedure, provided these pigments are rapidly reduced by dithionite. Therefore, the spectrum obtained was slightly differed from that measured by the usual method, especially in the region of 425nm.View full abstractDownload PDF (1952K) -
Takashi MORITA, Sadaaki IWANAGA, Tomoji SUZUKI1976 Volume 79 Issue 5 Pages 1089-1108
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine prothrombin was activated, in both the absence and presence of diisopropyl-phosphofluoridate (DFP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28, 000 and 57, 000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Inter-mediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong α-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35, 000 and 27, 000 daltons. The former was identified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly-, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocatalytically yielded a fragment (inner fragment) of 14, 000 daltons with N-terminal serine and the clotting enzyme α-thrombin [EC 3.4.21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture.
On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin III, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51, 000 and 34, 000 daltons, of which the former Their data, indicating that cleavage of the peptide bond connecting thrombin A and B chains of prothrombin molecule is catalyzed by the venom procoagulant, are in agreement with the results presented here. So far, however, we have no evidence for cleavage of the peptide bond (-Arg-Ser) linking the N-terminal fragment and the inner fragment by the ven-om procoagulant, although they suggested that this occurred. This peptide bond was very susceptible to active intermediate ECV or pro-thrombin ECV, as shown in the present paper. Even in the presence of a high concentration of DFP, cleavage of this bond proceeded rapid-ly during prothrombin activation. Thus, we feel that cleavage of this peptide bond by the venom procoagulant can be ruled out.View full abstractDownload PDF (9767K) -
Satoshi YONEZAWA, Samuel H. HORI1976 Volume 79 Issue 5 Pages 1109-1111
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA hybrid enzyme (LI) of liver phosphorylase [EC 2.4.1.1] (L) and phosphorylase I, which is mainly located in brain, was isolated and its enzymatic and immunological properties were examined. The following results were obtained : (1) AMP stimulated the b forms of the hybrid (LIb), I(Ib), and L(Lb); (2) in the presence of AMP, SO42-stimulated Lb more than LIb and inhibited Ib; (3) in the absence of AMP, SO42-stimulated all three isozymes in the order: Ib<LIb<Lb; (4) on conversion to the a forms, the activities of L, LI, and I increased 35.5-fold, 3-fold, and 1.2-fold, respectively; (5) the relative inhibition potencies of anti-Lb antibody with LIa and LIb were 63% and 4%, respectively of that with La, and those of anti-Ib antibody with LIa and LIb were 42% and 88%, respectively of that with Ia. Since the ratios of the specific activities of purified La and Ia and of Lb and Ib are 70:82 and 2 : 70, respectively (Schliselfeld, 1973), the present findings suggest a 1:1 association of I and L subunits in the hybrid molecule.View full abstractDownload PDF (1243K) -
Masatoshi BEPPU, Tadao TERAO, Toshiaki OSAWA1976 Volume 79 Issue 5 Pages 1113-1117
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMonovalent succinyl-concanavalin A was prepared by photoaffinity labeling of succinyl concanavalin A with p-azidophenyl α-D-mannopyranoside. This concanavalin A derivative showed a weak but definite mitogenic activity against mouse splenic lymphocytes and human peripheral lymphocytes, suggesting that direct receptor crosslinkage on the cell surface is not an obligatory prerequisite for the activation of lymphocytes.View full abstractDownload PDF (333K) -
Yoshiko NARAHASHI, Kaoru YODA1976 Volume 79 Issue 5 Pages 1119-1122
Published: May 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA fifth and new DFP-sensitive alkaline proteinase E, with strong esterase activity toward Ac-(Ala)s-OMe was found in pronase, a protease mixture from St. griseus K-1. Proteinase E was shown to be different from the elastase [EC 3.4, 21.11]-like enzyme or subtilisin [EC 3.4.21.14]-like enzyme, and alkaline proteinases A, B, and C in pronase. Proteinase E was purified to a state appearing homogeneous on polyacryl-amide gel electrophoresis. Its molecular weight was estimated as 26, 600 by gel filtration, It was unstable below pH 5.6. Studies on its actions on acyl-amino acid esters showed that it hydrolyzed the ester bonds of esters of tryptophan, tyrosine, phenylalanine, leucine, and alanine in decreasing order of ease.View full abstractDownload PDF (786K)
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