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Stereochemistry of Hydrogen Incorporation from Reduced Pyridine Nucleotide
Akihiko KAWAGUCHI, Tsutomu YOSHIMURA, Kazuki SAITO, Yousuke SEYAMA, Ta ...
1980 Volume 88 Issue 1 Pages
1-7
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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The stereochemical course of the enoyl reduction catalyzed by fatty acid synthetase was investigated using the enzymes from rat liver and
Brevibacterium ammoniagenes. Deuterium-labeled fatty acids were synthesized by incubating the synthetases with either 4
R-[4-
2H
1]- or 4
S-[4-
2H
1] NADPH. The deuterium-labeled fatty acids thus produced were subjected to the action of a stereospecific enzyme, acyl-CoA oxidase. The deuterium-labeled fatty acids and 2, 3-dehydroacyl thioesters, the products of acyl-CoA oxidase, were methylated and analyzed for deuterium content by gas chromatography-mass spectrometry. These experiments provided information to determine the configuration at the C-3 position of fatty acids formed by fatty acid synthetases. The results suggested that the stereochemistry of hydrogen (as hydride) incorporation from reduced pyridine nucleotides during enoyl reduction was different between rat liver and
B. ammoniagenes synthetases: the enoyl reduction of rat liver enzyme involved the
re-attack of hydride and that of
B. ammoniagenes enzyme involved the
si-attack of hydride.
2
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IV. The Amino Acid Sequence of the Activation Peptide Segment of Japanese Monkey Pepsinogen
Takashi KAGEYAMA, Kenji TAKAHASHI
1980 Volume 88 Issue 1 Pages
9-16
Published: July 01, 1980
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The complete amino acid sequence of the activation peptide segment of the major component of Japanese monkey pepsinogens was determined. The pepsinogen was converted to pepsin by incubation at pH 2.0 and 14°C for 17min. The activation peptides were separated from the resulting pepsin and purified by chromatography on columns of sulfopropyl (SP)-Sephadex. One 25-residue peptide and two 22-residue peptides were isolated and their amino acid sequences were determined. The results showed that the former peptide was derived from the aminoterminal half (residues no. 1 to 25) and the latter peptides from the carboxyl-terminal half (residues no. 26 to 47) of the activation segment. The latter two peptides were identical except for a single amino acid replacement. The complete amino acid sequence of the whole activation peptide segment was thus deduced as follows:
1Ile-Ile-Tyr-Lys-Val-Pro-Leu-Val-Arg-
10Lys-Lys-Ser-Leu-Arg-Arg-Asn-Leu-Ser-Glu-
20His-Gly-Leu-Leu-Lys-Asp-Phe-Leu-Lys-Lys-
30His-Asn-Leu-Asn-Pro-Ala-Ser-Lys-Tyr-Phe-
40Pro-
GlnLys-Ala-Glu-Ala-Pro-Thr-
47Leu.
The whole chain was composed of 47 residues, and cleaved into two polypeptides by the cleavage of
25Asp-
26Phe bond during activation. Two amino acids were detected at residue-41,
i.e., glutamine and lysine, indicating the presence of two closely related pepsinogens in the sample used. The total number of residues (47) of the whole activation peptide segment is 3 and 2 residues larger than those of porcine pepsinogen and bovine pepsinogen, respectively. The differences are 15 and 22 residues as compared with porcine pepsinogen and bovine pepsignogen, respectively.
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Masao HYODO, Kenshi SUZUKI
1980 Volume 88 Issue 1 Pages
17-25
Published: July 01, 1980
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When the sequence of DNA chain elongation in intact mouse cells was analyzed by pulselabeling the cells with [
3H]thymidine followed by sedimentation in an alkaline sucrose gradient, four classes of DNA, different in size, were observed. They were the Okazaki-type initial fragment, two high molecular weight DNA classes which we designated as DNA intermediates I and II, and the bulk of chromosomal DNA. When the size of DNA synthesized in permeabilized cells (cells treated with detergent to make them permeable to nucleoside triphosphates) was analyzed by the same method, we found that one of the intermediate DNAs, DNA intermediate I, was the major product of the
in vitro DNA replication and further elongation of the DNA chain from DNA intermediate I to II was lacking. But when a soluble fraction released from the cells after treatment with Triton X-100 was added to the permeabilized cells, the activity of joining chains of DNA intermediate I to form DNA intermediate II was partially restored. From the size of the two DNA intermediates and the way they were elongated as revealed by the sedimentation analysis, DNA intermediate I and II seem to correspond to replicon-size DNA and clustered replicon-size DNA, respectively. And our results suggest that there exists some unknown factor or process which is required for the joining of completed replicon-size DNA at the terminals.
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Isolation and Four Variant Sequences
Tomoko HAYASHI, Yoshihide OHE, Hiroaki HAYASHI, Koichi IWAI
1980 Volume 88 Issue 1 Pages
27-34
Published: July 01, 1980
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The amino acid sequence of human spleen histone H2A was investigated as a study in histone evolution, following previous investigation of human spleen histone H2B [Ohe, Y., Hayashi, H., & Iwai, K. (1979)
J. Biochem. 85, 615-624]. A mixture of H2A, H3, and H4, obtained from the whole histone by CM-cellulose chromatography, was fractionated into individual histones by Bio-Gel P-10 chromatography. The H2A fraction was further purified by Bio-Gel P-60 chromatography, and two subfractions, H2A (1) and H2A (2), were obtained in a ratio of about 2:1. Subfraction H2A (1) gave 21 tryptic peptides with reasonable recoveries, all of which, except for one, showed the compositions expected from the sequence of calf thymus H2A, including the partially phosphorylated N-terminal peptide. The one exception was due to a partial substitution of arginine for lysine at residue 99. Thus it was deduced that H2A (1) contains two variants; variant I accounts for 40% of the total and has the sequence of 129 amino acids identical with that of calf thymus H2A, and variant 2 accounts for 30% of the total and contains one substitution (Arg for Lys-99). Subfraction H2A (2) also gave the expected tryptic peptides, together with three unexpected peptides. From the sequence determination of such peptides and/or the position assignment of substituted residues, H2A (2) was deduced to contain two variants; each accounts for 15% of the total, variant 3 contains two substitutions (Ser for Thr-16 and Met for Leu-51), and variant 4 contains the same two substitutions and one deletion (His-123 or 124). The three substitutions and one deletion found in human H2A are compared with those in the known H2A sequences of other mammals and lower eukaryotes, and the implications are discussed.
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Kazushi TANABE, Yukari N. TAGUCHI, Akio MATSUKAGE, Taijo TAKAHASHI
1980 Volume 88 Issue 1 Pages
35-38
Published: July 01, 1980
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Steady-state kinetic studies of DNA polymerase α purified from mouse myeloma MOPC104E cells have been carried out. The results of initial velocity analysis with or without sodium pyrophosphate, a product inhibitor, indicated that the reaction mechanism of this enzyme can be categorized as an ordered Bi Bi type where the concentration of the ternary complex is very low.
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Masashi KOBAYASHI, Makoto IWASAKI, Yukio SHIGETA
1980 Volume 88 Issue 1 Pages
39-44
Published: July 01, 1980
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The time-course of insulin binding to isolated adipocytes pretreated with chloroquine (0.1mM) showed that no steady state condition was reached and that the binding kept increasing with the time of incubation up to three hours. Sixty-one percent of the bound labeled insulin could not be dissociated from these cells by 100 μg/ml of unlabeled insulin, whereas 86% dissociated from the control cells. Chromatography revealed that the bound intact insulin, which had the ability to bind to insulin receptors of adipocytes, was increased and that degraded insulin decreased in the chloroquine treated cells. However, these cells showed normal insulin stimulation of 2-deoxy-glucose uptake. The data suggest that insulin is internalized in adipocytes after binding to insulin receptors and that insulin is degraded at the site, probably lysosomes, where chloroquine inhibits this degradation process.
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Shigeki SHIBAHARA, Tadashi YOSHIDA, Goro KIKUCHI
1980 Volume 88 Issue 1 Pages
45-50
Published: July 01, 1980
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In the pig spleen the specific activity of heme oxygenase was two to three times higher in smooth microsomes than in rough microsomes, whereas the total heme oxygenase activities recovered in the two microsomal fractions were similar. Free and bound polysomes were isolated from pig spleen and nascent peptides on these polysomes were analyzed by employing [
3H]puromycin and a heme oxygenase-specific rabbit antibody (IgG). It was shown that free polysomes are the major site of heme oxygenase synthesis. In addition, cell-free synthesis of heme oxygenase was performed in a reticulocyte lysate system with free and bound polysomes isolated from pig spleen, and the results obtained again indicated that heme oxygenase is synthesized predominantly on free polysomes. The heme oxygenase newly synthesized on free polysomes may be incorporated first into the rough portion of endoplasmic reticulum either before or after its release from polysomes, although the specific activity of this enzyme at the steady state is considerably higher in the smooth region.
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Chieh-Ju LIANG, Katsuko YAMASHITA, Akira KOBATA
1980 Volume 88 Issue 1 Pages
51-58
Published: July 01, 1980
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The sugar chain of bovine pancreatic ribonuclease B was released from the polypeptide moiety by hydrazinolysis and by endo-β-
N-acetylglucosaminidase H digestion, and reduced with NaB[
3H]
4 after
N-acetylation. The radioactive oligosaccharide mixtures thus obtained were fractionated by Bio-Gel P-4 column chromatography, and their structures were studied by sequential exoglycosidase digestion, methylation study, periodate oxidation and acetolysis. The results show that bovine pancreatic ribonuclease B has one of the following sugar chains as asparagine-linked carbohydrate.
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Miho TAKAHASHI
1980 Volume 88 Issue 1 Pages
59-68
Published: July 01, 1980
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The substrate specificity and the action mechanism of a nuclease named nuclease TT1, from the culture broth of an extreme thermophile,
Thermus thermophilus HB8, were investigated. The enzyme is nonspecific for the sugar moiety and cleaves both single- and double-stranded DNAs, rRNA, tRNA and oligonucleotides irrespective of chain length to produce 5'-mononucleotides exonucleolitically. The action mechanism is processive and the enzyme shows no porality of degradation. The minimal unit as a substrate is a 5'-dinucleotide. The rate of hydrolysis is independent of a terminal phosphate group. The substrate lacking a 5'-phosphoryl group is degraded to leave the 5'-terminus and the penultimate nucleotide (NpN) as a core. The substrate possessing a 3'-phosphoryl group is degraded to leave the mononucleoside 5', 3'-diphosphates (pNp). However, NpN and pNp are gradually degraded by a large dose of the enzyme to produce a 5'-mononucleotide. The enzyme is free from nonspecific phosphatase and phosphodiesterase activities. Application of this enzyme to determine the sequence of oligonucleotides is shown.
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Hiroko KOSAKA, Mamoru ISEMURA, Teruo ONO, Yukio NISHIMURA, Keitaro KAT ...
1980 Volume 88 Issue 1 Pages
69-75
Published: July 01, 1980
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A commercial preparation of bovine liver β-glucuronidase contained two distinct enzyme species, both of which catalyze the hydrolysis of 4-methylumbelliferyl α-L-iduronide. The species with a molecular weight of about 290, 000 was devoid of phenyl α-L-iduronidase activity and exhibited 4-methylumbelliferyl β-D-glucuronidase activity. The species with a molecular weight of about 78, 000 was active towards phenyl α-L-iduronide but lacked the latter activity. Studies of the kinetics, of inhibition and heat inactivation suggested that the hydrolysis of 4-methylumbelliferyl α-L-iduronide is due to the β-glucuronidase in the case of the 290, 000-dalton species. The highly purified β-glucuronidase preparations derived from rat preputial gland and liver lysosomes also exhibited 4-methylumbelliferyl α-L-iduronidase activity. Thesefindings support the view that β-glucuronidase can hydrolyze certain α-L-iduronide bonds and raise the possibility that β-glucuronidase may play a role in the catabolism of iduronic acid-containing glycosaminoglycans.
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Shuzo OTANI, Isao MATSUI, Satoshi NAKAJIMA, Megumi MASUTANI, Yasuhiro ...
1980 Volume 88 Issue 1 Pages
77-85
Published: July 01, 1980
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Ornithine decarboxylase activity increased rapidly in guinea pig lymphocytes and reached a peak 5h after the addition of A 23187 to the cell culture. The optimum dose of A 23187 for the induction of ornithine decarboxylase was similar to that for the stimulation of DNA synthesis. Induction of ornithine decarboxylase by A 23187 or phytohemagglutinin was inhibited by the addition of ethylene glycol bis(β-aminoethylether)-
N, N'-tetraacetic acid to the cell cultures and the inhibition was overcome by the addition of CaCl
2, but not MgCl
2. The effects of phytohemagglutinin on Ca
2+ influx and efflux were less than those of A 23187. In contrast, phytohemagglutinin was more potent than A 23187 in stimulating ornithine decarboxylase activity. These results suggest that Ca
2+ is necessary to induce ornithine decarboxylase, but the extent of increase of cytosolic Ca
2+ concentration does not necessarily coincide with that of ornithine decarboxylase activity.
Treatment with low concentrations of cytochalasin B inhibited A 23187-induced ornithine decarboxylase activity, but not phytohemagglutinin-induced activity. The addition of dibutyryl cAMP or cholera toxin to cell cultures inhibited phytohemagglutinin-induced ornithine decarboxylase activity, while it rather enhanced A 23187-induced activity. Phytohemagglutinin and A 23187 in combination failed to induce ornithine decarboxylase activity more effectively than phytohemagglutinin alone, but in combination with lipopolysaccharide, A 23187 had an additive effect. These results suggest that there are substantial differences between A 23187 and phytohemagglutinin as regards the mechanism of induction of ornithine decarboxylase, although both mitogens act on the same lymphocyte population.
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I. Synthesis of 1 α, 24 (R) and 1 α, 24 (S)-Dihydroxy-[24-3H] Vitamin D3 and Their Metabolism in the Rat
Seiichi ISHIZUKA, Kiyoshi BANNAI, Tatsuyuki NARUCHI, Yoshinobu HASHIMO ...
1980 Volume 88 Issue 1 Pages
87-95
Published: July 01, 1980
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Tritium labeled 1 α, 24 (R)-dihydroxyvitamin D
3 and 1 α, 24 (S)-dihydroxyvitamin D
3 were synthesized and the metabolism and distribution of these compounds in the rat were studied. The 1 α, 24 (R)-dihydroxyvitamin D
3 was assumed to be gradually metabolized to 1 α, 24 (R) 25-trihydroxyvitamin D
3 in the liver and released into the blood, since relatively large amounts of the administered compound appeared in the serum as 1 α, 24 (R) 25-trihydroxyvitamin D
3. In contrast, large amounts of the unchanged compound and only very small amounts of 1 α, 24 (R) 25-trihydroxyvitamin D
3 were found in other tissues. The distribution pattern of 1 α, 24 (S)-dihydroxyvitamin D
3 was similar to that seen with 1 α, 24 (R)-dihydroxyvitamin D
3; the major portion was recovered as the 25-hydroxylated metabolite in the serum whereas in the small intestine and bone large amounts of the unchanged compound and a trace amount of the metabolite were found. It was also clarified by liver perfusion experiments that 25-hydroxylations of these compounds take place in the liver and the resulting products are released into the blood. The 25-hydroxylations of 1 α, 24 (R)-dihydroxyvitamin D
3 and 1 α, 24 (S)-dihydroxyvitamin D
3 in the liver were not inhibited by the addition of each metabolite, 1 α, 24 (R) 25-trihydroxyvitamin D
3 or 1 α, 24 (S) 25-trihydroxyvitamin D
3, suggesting that the hydroxylation reaction is catalyzed by the enzyme similar to that for 1 α-hydroxyvitamin D
3 25-hydroxylation. From these results, it was suggested that 1 α, 24 (R)-dihydroxyvitamin D
3 and 1 α, 24 (S)-dihydroxyvitamin D
3 perform their activities for intestinal calcium transport and bone calcium mobilization without undergoing the 25-hydroxylation reaction.
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Nakao ARIGA, Hirohiko KATSUKI
1980 Volume 88 Issue 1 Pages
97-102
Published: July 01, 1980
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o-Chlorobenzylchloride, a simple aromatic halogen compound, was found to inhibit the growth of
Saccharomyces cerevisiae and to lower the contents of sterols and fatty acids. The growth inhibition was considerably alleviated by the presence of sterols such as ergosterol and cholesterol and of unsaturated fatty acids such as oleate and linolenate. Inspection of effect of the inhibitor on the electron transport system related to the biosyntheses of these compounds revealed that the cytochrome contents and some enzyme activities in the system of the inhibited cells were much lower than those of the control cells. The features of the inhibition were similar to those of inhibition for other organisms by the hypocholesterolemic compounds such as triparanol and benzmalecene.
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Bunzo MIKAMI, Shigeo AIBARA, Yuhei MORITA
1980 Volume 88 Issue 1 Pages
103-111
Published: July 01, 1980
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The behavior of SH groups of soybean β-amylase was investigated by chemical modification. Two SH groups out of a total of five in the native enzyme reacted with 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB), iodoacetamide or monoiodoacetate at high ionic strength, accompanied by inactivation of the enzyme. At low ionic strength, only one SH group was accessible to DTNB without inactivation. By using the reaction with iodoacetamide, the dissociation constants of these two SH groups and rate constants for the reaction were determined. The most reactive SH group, which was independent of the inactivation, reacted with monoiodoacetate 185 times faster than the essential SH group. Next, selective modification of one SH group with monoiodoacetate was carried out. The modified enzyme, having activity equal to that of the native enzyme, was purified by ion exchange column chromatography. It was successively treated with DTNB or iodoacetamide in order to achieve selective modification of the essential SH group. The enzyme modified further with DTNB was fully reactivated by 2-mercaptoethanol treatment. It also recovered 65% of the original enzymatic activity on treatment with cyanide but only 7% with sulfite.
Maltose and cyclohexadextrin protected the essential SH group from modification, the former being more effective. The ultraviolet absorption spectrum of soybean β-amylase was changed by modification of the essential SH group, and the spectrum was also changed by the binding of maltose. The change induced by maltose was influenced by modification of the essential SH group. It was concluded that the essential SH group of soybean β-amylase does not participate in the catalysis, but is situated near the binding site of maltose.
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Yoshio TSUJITA, Akira ENDO
1980 Volume 88 Issue 1 Pages
113-120
Published: July 01, 1980
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The intracellular localization of two forms of membrane-bound acid protease (M
1 and M
2) [EC 3. 4. 23. 6] of
Aspergillus oryzae (Tsujita, Y. & Endo, A. (1978)
Eur. J. Biochem. 84, 347-353) was investigated. When the mycelia were treated with wall-lytic enzymes, M
1 remained in the cells but most of M
2 was solubilized and released. The cell wall fraction obtained by mechanical disruption of the mycelia contained less than 5% of the total acid protease activity in the cells.
Subcellular fractionation of the membranes obtained from burst spheroplasts showed that the acid protease was present in both rough and smooth microsomes. Acid protease M
1 was predominant in the former and M
2 in the latter, possibly on the surface of the cytoplasmic membranes.
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Shoji MATSUSHITA, Retsu MIURA, Toshio YAMANO, Yoshihiro MIYAKE
1980 Volume 88 Issue 1 Pages
121-129
Published: July 01, 1980
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The reverse reaction of D-amino acid oxidase with chloropyruvate and ammonia was studied. An intermediate in the reverse reaction was observed spectrophotometrically on mixing D-amino acid oxidase with chloropyruvate and ammonia aerobically. This intermediate was visibly green and was stable for more than an hour at room temperature. It had an absorption spectrum characterized by peaks at 382 and 450 nm and a broad band which extended beyond 600 nm. These features were identical with those observed in the aerobic forward reaction of β-chloro-D-alanine with the enzyme. Reduction of the intermediate with NaB
3H
4 produced tritiated β-chloroalanine. When a limited amount of D-alanine was added to the intermediate, it was partially reduced, forming another state with spectral features similar to those obtained by partial reoxidation of the D-alanine-reduced enzyme with chloropyruvate and ammonia. When the enzyme in the intermediate obtained by mixing the enzyme with chloropyruvate and ammonia was reduced by treatment with sodium dithionite or by photoreduction in the presence of EDTA and 3-methyllumiflavin, β-chloroalanine was produced, but there was no detectable production of either alanine or pyruvate, which would have been obtained if elimination of chloride had occurred. These results suggest that the green intermediate is a complex of the oxidized enzyme with ketimino acid and that this is a common intermediate in the reverse reaction with chloropyruvate and ammonia and in the forward reaction with β-chloro-D-alanine. To explain the results in the reverse reaction consistently with the forward reaction with β-chloro-D-alanine, a reaction mechanism is proposed in which oxidation or elimination occurs concertedly with abstraction of the α-proton.
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Toshihiko SUGANUMA, Tamio MIZUKAMI, Ken-ichi MOORI, Masatake OHNISHI, ...
1980 Volume 88 Issue 1 Pages
131-138
Published: July 01, 1980
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An α-amylase [EC 3. 2. 1. 1] from
Streptomyces praecox was purified and its characteristic action, the conversion of maltotriose (G
3) to maltose (G
2) without appreciable formation of glucose (G
1), was investigated. Isoelectric focusing or the glycogen adsorption procedure was employed after chromatography. Isoelectric focusing showed that the enzyme preparation after chromatographic separation comprises three isozymes. The preparation from the glycogen adsorption procedure showed the highest specific activity of any preparation of this enzyme ever obtained. Product analysis with uniformly labeled G
3 revealed that at a high concentration (18mM) of G
3, much more G
2 is produced than G
1 (the product ratio G
2/G
1 is over 20), while at a lower concentration (10 μM) the reaction mixture was composed of nearly equimolar amounts of glucose and maltose. Based on the product analysis of reducing endlabeled G
3 in addition to the above findings, the following conversion mechanism is proposed:
Streptomyces α-amylase catalyzes transglycosylation to produce maltotetraose (G
4) as a transient product which is immediately degraded into two molecules of G
2 by a subsequent hydrolytic reaction,
i.e., two molecules of G
3 are converted into three molecules of maltose without appreciable formation of glucose.
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Naoki OKAMURA, Keiko HANAKURA, Masako KODAKARI, Sadahiko ISHIBASHI
1980 Volume 88 Issue 1 Pages
139-144
Published: July 01, 1980
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Superoxide anion production in polymorphonuclear leukocytes obtained from guinea pig peritoneal cavities was stimulated by treatment with 5 μg/ml of cytochalasin D
in vitro. The stimulation was further enhanced by the addition of either 10
-5-10
-7M vinblastine or 10
-3-10
-6M colchicine to cytochalasin D. Higher concentrations of these anti-microtubular agents were inhibitory to the stimulating effect of cytochalasin D on the superoxide anion production, while lower concentrations had almost no effect. In the resting leukocytes,
i.e. without cytochalasin D, the anti-microtubular agents by themselves had no effect on the superoxide anion production. The cooperation of cytochalasin D and the anti-microtubular agents was also observed in the stimulation of NADPH oxidase activity and bactericidal function of the leukocytes.
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Michio MUGURUMA, Yoshiko MUGURUMA, Toshiyuki FUKAZAWA
1980 Volume 88 Issue 1 Pages
145-149
Published: July 01, 1980
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1. Z-line constituents contributing to the formation of the contraction bands in chicken myofibrils were investigated by using the rabbit calcium-activated factor (CAF).
2. CAF digestion hampered the formation of the contraction bands and increased the amount of soluble proteins which are apparently derived from the Z-line. Among several proteins released by CAF digestion, a 95, 000 dalton component, which corresponds to α-actinin, was predominant, followed by a 220, 000 dalton component and several other proteins.
3. An apparent inverse relationship between the formation of the contraction bands and the release of Z-line proteins was observed. It is concluded that the presence of Z-line, including at least the two components mentioned above, is essential for the formation of the contraction bands.
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Satoshi MARUYAMA, Shintaro SUGAI
1980 Volume 88 Issue 1 Pages
151-158
Published: July 01, 1980
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The magnesium-induced conformational changes of yeast 5 S ribosomal RNA have been studied by means of difference absorption spectroscopy and circular dichroism. A hypochromic difference absorption spectrum with a trough at _??_260nm was observed together with a slight increase and blue shift of the peak at 265nm and a significant increase of the absolute magnitude of the trough at 208nm in the circular dichroism spectrum. The magnesium concentration dependence of the difference absorption gave a Hill coefficient of 2.0±0.2. All of the equilibrium data indicate that magnesium ions produce a more ordered form of the molecule. Transient kinetic studies using a magnesium concentration jump were carried out. The kinetic curves were biphasic, each phase being associated with a relatively slow unimolecular conformational change induced by magnesium binding. A simple scheme was able to accommodate all of the equilibrium and kinetic data, and the kinetic parameters were calculated. Activation parameters for the unimolecular steps suggest that the conformational changes of the 5 S RNA involved the breaking of several base pairs and tertiary folding.
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Yoshihiro SOKAWA
1980 Volume 88 Issue 1 Pages
159-166
Published: July 01, 1980
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A new protein retained by poly (I): poly (C)-Sepharose was induced together with dsRNA-dependent enzymatic activities, a protein kinase and 2', 5'-oligoadenylate synthetase (2, 5 A synthetase), in interferon-treated mouse L 929 cells; it had an apparent molecular weight of 50, 000 (50 K) and was not phosphorylated by the protein kinase. The kinetics of the induction of the poly (I): poly (C)-binding 50 K protein were similar to those of dsRNA-dependent protein kinase and 2', 5'-oligoadenylate synthetase, and their inductions were all dependent on the interferon dose added, though a relatively higher dose was required for the 50 K protein.
When the interferon preparation was heated to 100°C in the presence of sodium dodecyl sulfate, its effect on cells of inducing the activity of 2', 5'-oligoadenylate synthetase was preserved completely, indicating that the interferon molecule itself is responsible for the induction of the synthetase. Since the induction of the enzymatic activity was inhibited by addition of either actinomycin D or cycloheximide, it may not be an activation of a latent enzyme but a
de novo synthesis of the enzyme.
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Shin-ichi SUGIMOTO, Isamu SHIIO
1980 Volume 88 Issue 1 Pages
167-176
Published: July 01, 1980
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Component B of chorismate mutase of
Brevibacterium flavum, the first enzyme specific for phenylalanine and tyrosine biosynthesis, was purified to near homogeneity. The molecular weights of component B and its subunit were estimated to be 25, 000 and 13, 500, respectively. Component A (previously purified) or B alone did not show any chorismate mutase activity but together they showed activity. The enzyme activity was not proportional to the amount of the enzyme. The optimum pH of the reaction was 8.0. Double-reciprocal plots of the reaction rate against chorismate concentration curved upwards. S
0.5 and Hill coefficient values were estimated to be 5.5mM and 3.1, respectively. The chorismate mutase component A and 3-deoxy-D-
arabino-heptulosonate 7-phosphate synthetase activities of component A were labile and were stabilized by tryptophan, dithiothreitol or cobalt ions. Phenylalanine and tyrosine inhibited the enzyme activity partially and competitively. The simultaneous presence of phenylalanine and tyrosine caused cumulative inhibition at saturated concentrations. The concentrations of phenylalanine, tyrosine, and phenylalanine plus tyrosine (each) giving 50% inhibition under the standard conditions were 0.0041, 0.095, and 0.0023mM, respectively. Tryptophan activated the enzyme about 6-fold. The concentration giving the half-maximum activation was 0.0023mM. Furthermore, tryptophan overcame the inhibition caused by phenylalanine and tyrosine. The tryptophan activation affected only S
0.5, not the maximum velocity or the Hill coefficient. β-2-Thienylalanine,
m-fluorophenylalanine, α-methylphenylalanine, and phenylalanine hydroxamate inhibited the enzyme in the same way as phenylalanine, while tyrosine hydroxamate and α-methyltyrosine inhibited it in the same way as tyrosine. 4-Methyltryptophan, 5-fluorotryptophan, 6-fluorotryptophan, tryptophan hydroxamate, and α-methyltryptophan activated the enzyme in the same way as tryptophan.
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Yoichi NAKAMURA
1980 Volume 88 Issue 1 Pages
177-181
Published: July 01, 1980
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Solubilized Ca
2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C
12E
8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with P
1.
The time course of decay of E
32P formed with 4 μM AT
32P in the presence of 10mM CaCl
2 and 10mM MgCl
2 (forward reaction) was measured by adding 0.4mM unlabeled ATP and 10mM P
1 at pH 6.0 and 30°C. The rate of E
32P decay was accelerated by 0.4mM ADP. On the other hand, when the time course of decay of E
32P formed with 10mM
32P
1 in the presence of 5mM EGTA and 10mM MgCl
2 (backward reaction) was measured by adding 0.4mM unlabeled ATP and 15mM CaCl
2, the rate of E
32P decay was unaffected by 0.4mM ADP.
AT
32P was produced on adding ADP to E
32P formed with AT
32P in the presence of 10mM CaCl
2 and 10mM MgCl
2, while no AT
32P was produced on adding ADP to E
32P formed with
32P
1 in the presence of 5mM EGTA and 10mM MgCl
2, even when 15mM CaCl
2 was added simultaneously with ADP.
View full abstract
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Hisao KATO, Noriaki ADACHI, Yasuo OHNO, Sadaaki IWANAGA, Katsumi TAKAD ...
1980 Volume 88 Issue 1 Pages
183-190
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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Fluorogenic peptides, peptidyl-4-methylcoumaryl-7-amides (MCA), containing COOH-terminal lysine residues, were newly synthesized and tested as substrates for plasmin. Among six peptidyl-MCA's, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA were found to be useful for the specific and sensitive assay of plasmin. The
Km values estimated from Lineweaver-Burk plots for these substrates using human and bovine plasmins were in the region of 10
-4M. Boc-Glu-Lys-Lys-MCA was slightly hydrolyzed by bovine plasma kallikrein, and Boc-Val-Leu-Lys-MCA was slightly hydrolyzed by human and hog urinary kallikreins and hog pancreatic kallikrein. However, both of the fluorogenic peptides were essentially unaffected by urokinase, α-thrombin, Factor Xa, Factor IXa, Factor XIa, and Factor XIIa. It was confirmed that plasmin hydrolyzed Boc-Glu-Lys-Lys-MCA, cleaving the lysyl-MCA bond, but not the lysyl-lysyl bond.
These fluorogenic peptides were resistant to human plasmin activated by streptokinase. Boc-Glu-Lys-Lys-MCA was not hydrolyzed by human plasmin or plasminogen in the presence of more than a 5-fold molar excess of streptokinase. The sensitivity of Boc-Val-Leu-Lys-MCA to human plasmin was also reduced, but plasmin retained 35% of the maximum activity even in the presence of a 20-fold molar excess of streptokinase. These results suggest that streptokinase-plasmin complex has essentially no activity towards Boc-Glu-Lys-Lys-MCA.
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Fumiyuki YAMAKURA, Koji SUZUKI
1980 Volume 88 Issue 1 Pages
191-196
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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The Cd-, Cr-, and Mn-substituted enzymes of iron-superoxide dismutase from
Pseudonionas ovalis were prepared from the apoenzyme by using the alkaline treatment method described before (
1) with a slight modification. The Cd-substituted enzyme had 1.16g atoms of Cd per mol of enzyme and no visible absorption. The Cr-substituted enzyme had 1.27g atoms of Cr per mol of enzyme and had absorption maxima at 530 nm and 670 nm with a shoulder around 370 nm. The Mn-substituted enzyme had 1.27g atoms of Mn per mol of enzyme and had absorption shoulders around 470 nm and 600 nm. The Cd-, Cr-, and Mn-substituted enzymes had no enzymatic activity. The reactivity of the four sulfhydryl groups and circular dichroism spectra of the Cd-, Cr-, and Mn-substituted enzymes were similar to those of the Fe-reconstituted enzyme. These results indicate that these metals may bind to the same site as Fe in the enzyme. The fluorescence emission intensities of the Cd-, Cr-, and Mn-substituted enzymes were 0.75, 0.46, and 0.69 times that of the apoenzyme, respectively.
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Miho OHTA-FUKUYAMA, Yoshihiro MIYAKE, Shigenori EMI, Toshio YAMANO
1980 Volume 88 Issue 1 Pages
197-203
Published: July 01, 1980
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Choline oxidase from
Alcaligenes sp. catalyzed the oxidation of choline and betaine aldehyde to betaine with concomitant consumption of oxygen and production of hydrogen peroxide. The values of
Km for choline and betaine aldehyde were 0.87 and 6.2mM, respectively. The molecular weight of the enzyme was estimated to be 66, 000 by SDS-gel electrophoresis and 72, 000 by gel-filtration using a high performance liquid chromatograph.
The prosthetic group of the enzyme was identified as 8α-[N (3)-histidyl]-FAD from the electrophoretic mobility at pH 6.25 of the hydrolysate of the methylated histidylflavin. The visible absorption spectrum of the enzyme showed peaks at 358 and 453 nm and a shoulder at about 480 nm. The covalently bound FAD was reduced on addition of either choline or betaine aldehyde under anaerobic conditions and was reoxidized by aeration. The enzyme was found to contain 1 mol of FAD per mol enzyme.
Amino acid analysis of a purified flavin peptide gave the following molar ratios of amino acids to flavin: Pro (1), Asp+Asn (3), Ser (1), His (1), and Arg (1). Aspartic acid was the N-terminal amino acid. The partial sequence of amino acids in the flavin peptide was as follows:
Asp-Asn-Pro-Asn (His FAD, Ser, Arg)
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Miho OHTA-FUKUYAMA, Yoshihiro MIYAKE, Kiyoshi SHIGA, Yasuzo NISHINA, H ...
1980 Volume 88 Issue 1 Pages
205-209
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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Absorption and circular dichroism spectra of cholesterol oxidase from
Schizophylluni commune and choline oxidase from
Alcaligenes sp. were measured and compared. The prosthetic group of cholesterol oxidase is 8α-[N (1)-histidyl]-FAD (
1, 2), while that of choline oxidase is 8α-[N (3)-histidyl]-FAD (
3).
In the CD spectra of the two enzymes in either the oxidized or reduced state, the corresponding bands in the visible region are of approximately the same intensity and shape but of opposite sign. A notable feature in the CD spectra of the two enzymes after light irradiation is the appearance of a CD band in the longer wavelength region (550-650 nm) and the opposite signs of the CD band in this region in the two enzymes. The similarity of the shape and intensity of the CD spectra of the two enzymes suggests that the environments surrounding the flavin moieties are very similar, and the sign reversal of the CD bands suggests that the mutual orientations between the transition moment of flavin and that of its environment differ in the two enzymes.
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I. Temperature-Induced Changes in Mycolic Acid Molecular Species and Related Compounds in Mycobacterium phlei
Seiko TORIYAMA, Ikuya YANO, Masamiki MASUI, Emi KUSUNOSE, Masamichi KU ...
1980 Volume 88 Issue 1 Pages
211-221
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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Molecular species of two major subclasses of mycolic acids from
Mycobacterium phlei, α-mycolic acids (M
1) and dicarboxy mycolic acids (M
3), were separated gas-chromatographically and identified mass-spectrometrically. The mycolic acid compositions of extractable and cell wall bound lipids were markedly influenced by growth temperature. Increasing growth temperature from 20°C to 50°C resulted in an increase in longer chain species of both mycolic acid subclasses with a concomitant decrease in shorter chain homologues. The most abundant molecular species were C
76 and C
58 of M
1 and M
3 in the 20°C grown cells, while the 50°C grown cells contained C
80 in M
1 and C
62 in M
3, most abundantly. Changes in mycolic acid composition occurred rapidly after growth temperature was raised from 20°C to 50°C with an increase in C
62 and a concomitant decrease in C
58. Mass fragmentographic analysis revealed that an increase in total carbon numbers of mycolic acids was caused by the elongation of straight chain alkyl unit, without any changes in α-branch. Changes in the molecular species composition of secondary alcohols presumably derived from the ester mycolic acids were also observed and an increase in longer species (C
20-ol-2) with a concomitant decrease in shorter ones (C
18-ol-2) was noted as the temperature rose. An increase in the growth temperature also resulted in a decrease in unsaturated fatty acids in extractable lipids.
These observations suggest that mycobacteria alter the molecular species composition of mycolic acid subclasses and phospholipids, in response to growth temperature, to maintain a suitable membrane function.
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Hiroshi SANEMORI, Takashi KAWASAKI
1980 Volume 88 Issue 1 Pages
223-230
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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The existence of thiamine pyrophosphokinase [EC 2. 7. 6. 2] in procaryotic cells was first demonstrated in
Paracoccus denitrificans (
J. Bacteriol. (1976)
126, 1030-1036). The enzyme was therefore purified from this organism to determine its molecular structure and properties.
Thiamine pyrophosphokinase which was purified 620-fold from
P. denitrificans showed a single band on both polyacrylamide and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular weight in the latter case was calculated to be 23, 000. Gel filtration analysis using Sephadex G-150 gave a molecular weight of 44, 000, indicating that this enzyme contains at least two identical subunits. Although sedimentation equilibrium analysis gave a molecular weight of 96, 000, indirect evidence suggests that the form having this molecular weight is an aggregate of the functional dimer. The activity of the purified enzyme required thiamine, ATP, and Mg
2+, and the enzyme catalyzed the pyrophosphorylation of thiamine by ATP.
Km values for thiamine and ATP were 10 μM and 0.38mM, respectively. The activity was competitively inhibited by pyrithiamine, giving a
K1 value of 19 μM. Oxythiamine and chloroethylthiamine were very weak inhibitors of the enzyme. The activity was also inhibited by the product, TPP.
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Purification and Physicochemical Characterization
Quang Khai HUYNH, Ryuzo SAKAKIBARA, Takehiko WATANABE, Hiroshi WADA
1980 Volume 88 Issue 1 Pages
231-239
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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Glutamic oxaloacetic transaminase isozymes were purified simultaneously to homogeneity from rat liver with high yields. Three subforms of mitochondria) isozyme and three subforms of cytosolic isozyme were separated by chromatography on CM-Sephadex and electrophoresis on polyacrylamide gel.
The general enzymatic properties of the purified isozymes such as their kinetic parameters, isoelectric points, molecular weights, amino acid compositions, NH
2-terminal amino acid sequences and COOH-terminal amino acids were determined. Most of these properties of the isozymes are similar to those of the corresponding isozymes from other sources, such as rat brain and pig and human heart. In amino acid compositions, cytosolic isozyme from rat liver has more proline and glycine and less arginine, threonine and leucine than pig heart cytosolic isozyme; the mitochondrial isozyme has more glutamic acid and glycine and less serine than the corresponding pig heart isozyme. The NH
2-terminal amino acid sequences of GOT isozymes from rat liver were identical with those of the GOT isozymes from pig heart up to the 10 th residues except for the 5 th residues. The subforms of mitochondrial isozyme from rat liver were generated on storage at 4°C for 4-8 weeks.
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Isolation and Fractionation of Oligosaccharides Liberated by Hydrazinolysis
Hideo YOSHIMA, Seiichi TAKASAKI, Akira KOBATA
1980 Volume 88 Issue 1 Pages
241-246
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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The plasma membrane glycoproteins of calf thymocytes were converted to glycopeptides by exhaustive pronase digestion. Glycopeptides with asparagine-linked sugar chains were separated from those with mucine-type sugar chains by Bio-Gel P-10 column chromatography. The asparagine-linked sugar chains were released as oligosaccharides from the peptide moiety by hydrazinolysis and labeled by reduction with NaB [
3H]
4. The radioactive oligosaccharides were fractionated into fifteen acidic components and ten neutral components by combination of paper electrophoresis and Bio-Gel P-4 column chromatography. The acidic nature of all fifteen acidic components can be ascribed to their
N-acetylneuraminic acid residues. The Bio-Gel P-4 column chromatographic patterns of the neutral oligosaccharide fraction and of the neutral fraction obtained on sialidase treatment of the pooled acidic oligosaccharide fraction were totally different, indicating that the acidic oligosaccharides are not simple sialyl derivatives of the neutral oligosaccharides.
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Tokuzo NISHINO, Shingo HATA, Takashi OSUMI, Hirohiko KATSUKI
1980 Volume 88 Issue 1 Pages
247-254
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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We previously proposed the occurrence of multiple pathways in the ergosterol biosynthesis of yeast, based on the results of examination of
14C-incorporation into sterols from L-[methyl-
14C]methionine which was given to the intact cells of yeast. This led us to investigate the validity of the pathways by experiments with the cell-free system. Highly active cell-free extracts could be prepared by disruption of yeast cells with a Vibrogen Cell Mill in the presence of 0.1mM dithiothreitol. This preparation catalyzed
14C-incorporation from [
14C]methionine into ergosterol with a high yield. This preparation was found to be favorable for elucidation of ergosterol synthesis, since only a small amount of radioactivity was incorporated into fatty acid ester form of sterols which were reported to be inactive as a substrate for sterol synthesis reaction. Time course experiment of
14C-incorporation from [
14C]methionine into various sterols under aerobic conditions showed that ergosta-8, 24 (28)-dien-3β-ol was a precursor for ergosta-7, 24 (28)-dien-3β-ol and that radioactivities were converted through ergosta-5, 7, 24 (28)-trien-3β-ol and ergosta-5, 7, 22, 24 (28)-tetraen-3β-ol into ergosterol with time. In contrast, similar experiments under anaerobic conditions showed that ergosta-7, 24 (28)-dien-3β-ol accumulated and very little conversion of radioactivity into ergosterol occurred. In addition, the results indicated that oxygen was required for the introduction of double bond into 22 position as well as into 5 position. The results obtained with the cell-free system supported the validity of the proposal of multiple pathways of ergosterol synthesis in the intact cells.
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Toshio ANDO, Hiroshi ASAI
1980 Volume 88 Issue 1 Pages
255-264
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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Systematic studies were carried out to determine how the dynamic quenching of fluorescence was affected by electrostatic interaction between charges carried by the fluorophore and the quencher. 1,
N6-Ethenoadenosine oligophosphates (ε-ATP, ε-ADP, ε-AMP, and ε-Ad) were used as fluorophores; these compounds have the same luminous group (ε-adenine ring) with variously charged phosphate groups. Acrylamide and the iodide (I
-), well known to be effective quenchers carrying zero and one negative charge, respectively, were used as quenchers. The thallium (I) (Tl
+) was also found to be an effective quencher as a result of studies of the quenching activity of various positively charged ions. Results which agreed qualitatively with those expected were obtained for the charge effects on the dynamic quenching and the effects of ionic strength on it. That is, the repulsive force exerted between I
- and phosphate groups decreased the quenching rate, while the attractive force between TI
+ and phosphate groups enhanced the quenching rate, and increasing ionic strength of solutions mitigated these charge effects. The results obtained, however, were not quantitatively consistent with theoretical expectations for ionic reaction rates in solutions. One reason for the quantitative disagree-ment is that the negatively charged phosphate groups are located at a distance from the luminous group. By theoretical analysis of the quenching data, the distance was calculated to be 9.6Å+
Δr, where
Δr changes according to the number of phosphate groups. Furthermore, through a theoretical analysis of the disagreement, we found a new factor, the “size effect” of a luminous group, which modifies the charge effects on dynamic quenching. The size effect provides information on the gradient of the electric potential generated by the phosphate groups. These studies provide a basis for the study of electric potential at local regions of proteins.
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I. Effect of ATP
Toshio ANDO, Hisao FUJISAKI, Hiroshi ASAI
1980 Volume 88 Issue 1 Pages
265-276
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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Electric potentials at regions near the two specific thiol groups, SH
1 and SH
2, of the heavy meromyosin (HMM) molecule were studied by the fluorescence quenching technique. The effects of binding of ATP to HMM upon the electric potentials were also studied.
N-(
p(2-Benzimidazolyl)phenyl)maleimide (BIPM) was used as a thiol-directed fluorescent reagent. Prior to the labeling of SH
2 with BIPM, the SH
1 group was blocked with
N-ethylmaleimide (NEM). Iodide ions (I
-), thallium ions (Tl
+), and acrylamide were used as quenchers of fluorescence. The sign of the electric potential was collectively determined from the dependence of the Stern-Volmer constants upon the ionic strength of solutions.
1. The region near SH
1 was at a negative electric potential, whereas the electric potential at the region near SH
2 was almost zero.
2. On the addition of ATP, the fluorescence intensity of BIPM bound to SH
1 was unchanged, whereas that of BIPM bound to SH
2 was greatly decreased to about 50% of the original level. The fluorescence intensity recovered as the added ATP was split into ADP and orthophosphate, and became saturated. The saturated level of the fluorescence intensity was, however, smaller than the original one, due to binding of the produced ADP to HMM.
3. On the addition of ATP, the negative electric potential at the region near SH
1 was unchanged, whereas a negative electric potential with large gradient was newly introduced at the region near SH
2. The value of the newly introduced electric potential was calculated on the basis of various assumptions. These results are discussed in connection with the functions of myosin.
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Kaoru SEGAWA, Sadaaki KAWAI
1980 Volume 88 Issue 1 Pages
277-280
Published: July 01, 1980
Released on J-STAGE: November 18, 2008
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The localization of the
src-encoded protein kinase was examined by fractionating cellular extracts from rat cells transformed by a wild type and a temperature-sensitive mutant of Rous sarcoma virus (SR-A 3 Y 1 and ts 68 3 Y 1 cells). It was found to be specifically localized in the post-microsomal supernatant (PMS) fraction. Furthermore, it was noticed that a protein with a molecular weight of 16, 000 (16 K-protein) in the PMS fraction was phosphorylated
in vitro when the PMS fraction from ts 68 3 Y 1 cells was preincubated at 33°C, but not at 42°C. This protein was phosphorylated when the fraction from SR-A 3 Y 1 cells was preincubated at 33°C and at 42°C. Similar temperature-sensitive phosphorylation of 16 K-protein was also observed in the PMS fraction from ts 68 3 Y 1 cells labeled
in vivo with [
32P]-orthophosphate at 33°C. These results suggest that this 16 K-protein might be a candidate for the endogenous acceptor for the src-encoded protein kinase.
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Yoshimasa KAWABATA, Nobuhiko KATUNUMA, Yukihiro SANADA
1980 Volume 88 Issue 1 Pages
281-283
Published: July 01, 1980
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In adult rats the activity of proline oxidase is high in the liver and kidney and moderate in the brain and heart, but it is not detectable in the lung, skeletal muscle, spleen, or small intestine. The activity in the liver is 1.5 times higher in females than males. Administration of estrogen to male rats increased the activity in liver and decreased that in kidney. Administration of a high protein diet increased the activity in the liver only. Administration of hydrocortisone or dexamethasone did not affect the activity in any tissue.
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II. Synthesis of Enzyme-Bound Octanoyldiaminobutyric Acid
Sadaaki KOMURA, Kiyoshi KURAHASHI
1980 Volume 88 Issue 1 Pages
285-288
Published: July 01, 1980
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A partially purified enzyme of
Aerobacillus polyaerogenes, which produces polymyxin E, activates L-2, 4-diaminobutyric acid and binds it as a thioester. The incubation of the enzyme preparation with octanoyl coenzyme A and L-2, 4-diaminobutyric acid in the presence of ATP and an ammonium sulfate fraction yields octanoyldiaminobutyric acid thioesterified to the enzyme.
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1980 Volume 88 Issue 1 Pages
289
Published: 1980
Released on J-STAGE: November 18, 2008
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