All issues
Volume 104 (1988)
- Issue 6 Pages 867-
- Issue 5 Pages 679-
- Issue 4 Pages 489-
- Issue 3 Pages 317-
- Issue 2 Pages 159-
- Issue 1 Pages 1-
Volume 104, Issue 5
Displaying 1-35 of 35 articles from this issue
- |<
- <
- 1
- >
- >|
-
Yukiteru Katsube, Nobuo Tanaka, Akio Takenaka, Tohru Yamada, Tairo Osh ...1988 Volume 104 Issue 5 Pages 679-680
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene coding for 3-isopropylmalate dehydrogenase of Thermus thermophilus was cloned and expressed in Escherichia colt. The extracted enzyme was crystallized in a suitable size for X-ray crystallographic studies. The crystals have a space group of P3121 or P3221 with a=b=78.6 Å and c=157.4 Å.View full abstractDownload PDF (1175K) -
Toshiyuki Nishio, Minoru Kamimura, Masakazu Murata, Yoshiyasu Terao, K ...1988 Volume 104 Issue 5 Pages 681-682
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lipase from Pseudomonas fragi 22.39 B catalyzed the transesterification in ester and alcohol mixtures without any other solvent. Activated esters, such as vinyl and phenyl esters, were excellent acyl donors for the reaction, and the activity was enhanced by increasing the carbon number of the fatty acid fraction of the esters. Primary alcohols were esterified faster than secondary ones in this reaction system, while tertiary alcohols such as α-terpineol did not react at all. The lipase exhibited stereoselectivity in the esterification of alcohols such as 2-octanol.View full abstractDownload PDF (217K) -
Shirou Kirita, Ken-ichirou Morohashi, Toshihide Hashimoto, Hidefumi Yo ...1988 Volume 104 Issue 5 Pages 683-686
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing pcP-450 (11β)-2 cDNA (Morohashi et al. (1987) J. Biochem. 102, 559-568) as the probe, a different cDNA clone, pcP-450 (11β)-3, was isolated from a cDNA library of bovine adrenal cortex. The restriction enzyme map of pcP-450 (11β)-3 was highly homologous but not identical with that of pcP-450 (11β)-2. Nucleotide sequence determination revealed the substitutions of 14 nucleotides and 3 amino acids between pcP-450 (11β)-2 and-3. Blotting analysis involving two different oligonucleotide probes specific to these two cDNAs indicated that at least two kinds of P-450 (11β) mRNA were expressed in individual animals and that a least two kinds of P-450 (11β) genes exist in the bovine genome.View full abstractDownload PDF (450K) -
Michihiro Sumida, Minoru Hamada, Akihiro Shimowake, Chie Morimoto, Hir ...1988 Volume 104 Issue 5 Pages 687-692
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation=1.0 μM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accerelated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [γ-32P] ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation=3.0 μM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0°C in the presence of 1 μM ATP and 10 μM Ca2+ (half time=0.2s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time=0.1s). The rate of EP decomposition at 0°C after adding EGTA was about 6.7 times slower (rate constant: kd=4.3×10-3s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.View full abstractDownload PDF (1765K) -
Kenta Nakai, Minoru Kanehisa1988 Volume 104 Issue 5 Pages 693-699
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to make better use of the information contained in rapidly expanding amino acid sequence data, a new method to predict various modification sites of proteins from their primary structures is presented. It is also applicable to the prediction of other functional sites in proteins. Here we show the examples of N-glycosylation and serine/threonine phosphorylation sites. The method is essentially an elaboration of consensus sequence pattern matching based on stepwise discriminant analysis. The occurring amino acids near a potential modification site are represented by six numerical values which reflect various properties of amino acids. Longer-range effects around these sites are also considered. The stepwise procedure enabled us to automatically select effective features for discrimination. A computer program with our method first identifies potential modification sites by a sequence pattern, NX(S/T) for N-glycosylation or (S/T) for phosphorylation, and then decides by discriminant analysis whether a potential site is likely to be a true modification site. The prediction accuracy in the second step of discrimination was about 60% for glycosylation sites and about 80% for phosphorylation sites.View full abstractDownload PDF (901K) -
Aritsune Uchida, Shinya Ebata, Keishirou Wada, Hiroshi Matsubara, Yuza ...1988 Volume 104 Issue 5 Pages 700-705
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of the major ferredoxin component isolated from a dinoflagellate, Peridinium bipes, was completely determined. Staphylococcus aureus V8 proteolytic, tryptic and chymotryptic peptides of Cm-ferredoxin were prepared and sequenced. The sequence was Phe-Lys-Val-Thr-Leu-Asp-Thr-Pro-Asp-Gly-Lys-Lys-Ser-Phe-Glu-Cys-Pro-Gly-Asp-Ser-Tyr-Ile-Leu-Asp-Lys-Ala-Glu-Glu-Glu-Gly-Leu-Glu-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ser-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Val-Leu-Thr-Gly-Ser-Ile-Asp-Gln-Ser-Asp-Gln-Ala-Phe-Leu-Asp-Asp-Asp-Gln-Gly-Gly-Asp-Gly-Tyr-Cys-Leu-Thr-Cys-Val-Thr-Tyr-Pro-Thr-Ser-Asp-Val-Thr-Ile-Lys-Thr-His-Cys-Glu-Ser-Glu-Leu. It was composed of 93 amino acid residues with 7 cysteine residues, the highest number found among the chloroplast-type ferredoxins so far sequenced. A cysteine residue was found for the first time at the 89th Position in a chloroplast-type ferredoxin. Calculation of the numbers of amino acid differences among chloroplast-type ferredoxins indicates that the Peridinium ferredoxin is far divergent not only from higher plant ferredoxins but also from blue-green algal ferredoxins.View full abstractDownload PDF (569K) -
Yoshinori Inaoka, Shunji Naruto1988 Volume 104 Issue 5 Pages 706-711
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPropioxatins A and B are inhibitors of enkephalinase B, which hydrolyzes enkephalin at the Gly-Gly bond. In order to clarify the structure-activity relationships of propioxatin, several compounds were synthesized and their inhibitory activity for not only enkephalinase B but also enkephalinae A was examined. The hydroxamic acid group in propioxatin was primarily essential for coordinating the metal ion in the active site of the enzyme. Among devalyl propioxatin A derivatives, the proline-containing compounds inhibited enkephalinase B and others inhibited both enzymes. An alteration of the character of the P3', amino acid valine in propioxatin A, e.g. amidation of carboxylic acid or replacement of the side chain, caused a 2 to 400-fold decrease of the inhibitory activity for enkephalinase B or an appearance of enkephalinase A inhibition with K1 values in the micromolar range. Substitution of the proline by alanine also resulted in a 1, 000-fold loss of inhibitory activity for enkephalinase B. Propioxatin A was the most potent and specific inhibitor of enkephalinase B among the synthesized compounds. These potent and specific inhibitory effects were caused by the P2' proline residue, the P3' valine side chain and its free carboxylic acid. Each of the S1', S2', and S3' subsites in an enkephalinase B active site has a large and hydrophobic pocket, but the arrangement might be unique. The results could explain why enkephalinase B does not hydrolyze longer peptides.View full abstractDownload PDF (635K) -
Hitoshi Shimano, Hiroyuki Aburatani, Natsuko Mori, Shun Ishibashi, Tak ...1988 Volume 104 Issue 5 Pages 712-716
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbits fasted for 9 and 14 d exhibited 6- and 8-fold increase in plasma cholesterol level, respectively. As one of the mechanisms causing hypercholesterolemia in fasted rabbits, the decreased activity of hepatic low-density lipoproteins (LDL) receptor has been reported (Stoudemire, J. B., Renaud, G., Shames, D. M., & Havel, R. J. (1984) J. Lipid Res. 25, 33-39). In order to demonstrate the down-regulation of hepatic LDL receptor on a molecular basis, we carried out immunoblotting of the liver membranes with a specific antibody against LDL receptors and blot hybridization of hepatic RNAs with cDNA of LDL receptor. Immunoblotting showed that LDL receptors in fasted rabbits were markedly decreased, and blot hybridization of RNAs showed a significant decrease in mRNA level of hepatic LDL receptor in fasted rabbits. Further, significant decreases both in LDL binding to liver membrane and in hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in fasted rabbits were demonstrated. From these results, we concluded that hypercholesterolemia in fasted rabbits is caused by the impaired catabolism of LDL due to down-regulated biosynthesis of hepatic LDL receptor.View full abstractDownload PDF (1737K) -
Akira Shirahata, Toshisuke Takeshima, Keijiro Samejima1988 Volume 104 Issue 5 Pages 717-721
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMonospecific antiserum to rat spermidine synthase was prepared by immunization of rabbits with purified enzyme protein from rat prostate, and its usefulness for analysis of spermidine synthase protein in not only rat tissues but also several other mammals was demonstrated by Western blotting and immunotitration of the enzyme activity. Application of the antiserum for elucidating the relationship between the enzyme activity and protein in normal rat tissues strongly suggested that marked difference in spermidine synthase activity among rat tissues depends solely on the difference in the amount of enzyme protein. Also, application of the antiserum for analyzing spermidine synthase from liver of mouse, rat, guinea pig, pig, and human, showed that the enzymes had a similar subunit molecular weight of 35, 000 and a cross-reactivity with the antiserum, exhibiting almost the same immunoreactivity to mouse enzyme as to rat enzyme. Thus, it was suggested that the antiserum would be useful for further studies of mammalian spermidine synthase from the viewpoints of enzymology and molecular biology.View full abstractDownload PDF (1635K) -
Akiyoshi Tamura, Takashi Kawate, Mari Ogata, Tatsuhiko Yagi1988 Volume 104 Issue 5 Pages 722-726
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnti-sera for hydrogenase, cytochrome c3, and desulfoviridin (abbreviated as anti-hyd, anti-c3, and anti-dvn, respectively) were raised in mice, and used to locate these antigens in cells of Desulfovibrio vulgaris Miyazaki. The activity of the intact cells to absorb H2 with methyl viologen or sulfite as an electron acceptor was cumulatively inhibited by treating the cells with anti-hyd and anti-c3, but unaffected by anti-dvn treatment. The activity of the intact cells to produce H2 from formate was also inhibited by anti-c3 treatment, but the inhibition by anti-hyd treatment was not significant. The fluorescent antibody technique applied to intact cells of D. vulgaris Miyazaki indicated that both hydrogenase and cytochrome c3, are localized on the surface of the cell. These results are not exactly in conformity with the hydrogen-cycling hypothesis for proton gradient formation in the energy metabolism in Desulfovibrio. The procedure described in the present paper provides a new technique to elucidate the roles of proteins by applying anti-sera to intact cells without destroying the cellular structure.View full abstractDownload PDF (2764K) -
Yasuzo Nishina, Hiromasa Tojo, Retsu Miura, Yoshihiro Miyake, Kiyoshi ...1988 Volume 104 Issue 5 Pages 727-733
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPicolinate binds to the anionic semiquinoid form of D-amino acid oxidase (IMO), and the complex formed has a broad absorption band in the long-wavelength region extending beyond 800 nm, which is reminiscent of a charge transfer interaction. The binding has a stoichiometry of 1:1 with respect to the enzyme. The dissociation constant at 25°C was 30μM at pH 7.0. The pH dependence (pH 7.0-8.3) of the dissociation constant indicates that one proton is associated with the complex formation, and suggests that picolinate able to bind to the anionic semiquinoid enzyme is in the cationic form protonated at the nitrogen atom. By adding dithionite to the oxidized DAO solution containing pyruvate and various amines, a similar anionic semiquinoid DAO complex having a broad long-wavelength absorption band, appeared. Resonance Raman spectra with excitation at 623.8nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methylamine indicate that the complex consists of the anionic semiquinoid DAC, and N-methyl-α-iminopropionate produced from pyruvate and methylamine, and that the imino group must be protonated. This supports the proposal that the presence of a positively charged group in the vicinity of flavin is required for the stabilization of the anionic semiquinoid flavin. The results also suggest that the broad absorption band is derived from the charge transfer interaction between the anionic semiquinoid flavin and the imino acid, in which the flavin C (4a)-N (5) locus and the locus containing H-N+-=C-COO- of the amino acid are important for the interaction. The charge-transfer complexes of anionic semiquinoid DAO hardly react with a reducing agent (dithionite or photoreduction) or an oxidizing agent (oxygen), different from free anionic semiquinoid DAO. These phenomena can be explained by the complexation (charge-transfer complex) with ligands over the flavin C (4a)-N(5) locus.View full abstractDownload PDF (787K) -
Toshihiro Fujii, Jun Ozawa, Yoshiro Ogoma, Yoshiyuki Kondo1988 Volume 104 Issue 5 Pages 734-737
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChicken gizzard muscle caldesmon has been examined for ability to interact with tropomyosin from chicken gizzard muscle by using fluorescence enhancement of tropomyosin labeled with dansyl chloride (DNS) and affinity chromatography. The binding of caldesmon to tropomyosin was regulated by Ca2+ and calmodulin, i. e., at low ionic strength most of the caldesmon bound to tropomyosin-Sepharose 4B was co-eluted by adding calmodulin only in the presence of Ca2+, but not in its absence. This regulation by Ca2+ and calmodulin was also suggested by fluorescence measurements. Actin-and calmodulin-binding sites on the caldesmon molecule were located in the 38K fragment (Fujii, T., Imai, M., Rosenfeld, G. C., & Bryan, J. (1987) J. Biol. Chem. 262, 2757-2763). When 38K-enriched fraction was applied to the tropomyosin-Sepharose, the 38K fragment was retained by the column and could be eluted by adding Ca2+ and calmodulin.View full abstractDownload PDF (463K) -
Yuko Kamada, Hisako Muramatsu, Makoto Kawata, Hiroyoshi Takamizawa, Ta ...1988 Volume 104 Issue 5 Pages 738-741
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPoly-N-acetyllactosamines were prepared from Ehrlich carcinoma cells cultured in the presence of [14C] galactose. Methylation analysis indicated that 31% of the galactose was in the non-reducing end. Of it, 77% was cleaved by a-galactosidase, and 56% was released as a disaccharide by endo-β-galactosidase C. Methylation analysis confirmed that the released disaccharide was mostly Galα1→3Gal. Therefore, Galα1→3Gal structure, not Galα1→3 (Galα1→6) Gal structure, was the major α-galactosyl structure in the poly-N-acetyllactosamines synthesized. Furthermore, α-galactosidase digestion did not change the content of disubstituted galactosyl residues. Thus, Galα1→3 (Galαl→6) Gal structure, which was suggested to be the sole non-reducing terminal structure of poly-N-acetyllactosamines of Ehrlich carcinoma cells, was not detected in significant amounts under the present experimental conditions.View full abstractDownload PDF (458K) -
Maija-Liisa Rasilo, Tatsuya Yamagata1988 Volume 104 Issue 5 Pages 742-754
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA large-sized glucose polymer was isolated by pronase digestion from line PC12 pheo-chromocytoma cells metabolically labeled with [1-3H] galactose. The polymerwas included on a column of concanavalin A-Sepharose and could be eluted with 10mM methyl-α-mannoside. Its slight retention in a column of Bio-Gel A-5m suggested that its molecular weight was in the several millions. Glucose was the component monosaccharide and there were two minor lipophilic components present. The polymer was digested with α-amylase into a series of oligosaccharides and was cleaved by glucoamylase into glucose residues. The disaccharide obtained by digestion with α-amylase was identified as maltose in several HPLC systems and by NMR spectroscopy. NMR measurement revealed the trisaccharide to be maltotriose. Susceptibility of the polymer molecule to α-amylase, and the digestion products obtained, indicated a resemblance to glycogen. An analysis for saccharide compositions before and after reduction of the polymer suggested the presence of an aglycon part.Contrary to expectations based on the presence of this moiety, the polymer displayed good solubility in neutral organic solvents. Two-thirds of the glucose polymer was also soluble in 10% TCA. A similar glucose polymer was isolated from neuronal cells of rat embryos metabolically labeled with [1-3H] galactose. Mouse neuroblastoma cells did not synthesize the polymer.View full abstractDownload PDF (1490K) -
Hideo Kubo, Atsushi Irie, Fuyuhiko Inagaki, Motonori Hoshi1988 Volume 104 Issue 5 Pages 755-760
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMelibiosylceramide (Galαl-6G1cβ1-1Cer) was found as the sole ceramide dihexoside from the eggs of the sea urchin, Anthocidaris crassispina. Ceramide monohexoside of the eggs consisted only of glucosylceramide (Glc1β1-1Cer). These lipids were purified by successive column chromatographies on DEAE-Sephadex A-25, silicic acid and Florisil, and identified by gas-liquid chromatography, negative-ion fast atom bombardment mass spectrometry and proton nuclear magnetic resonance spectroscopy as well as methylation analysis. Long-chain base compositions of both lipids were almost identical and comprised n-C18-phytosphingosine and small amounts of its homologs (C17-C19). Fatty acid compositions were qualitatively very similar, but the glucosylceramide contained more 2-hydroxy fatty acid than the melibiosylceramide. Although the chain length of fatty acids was distributed over a wide range, six major fatty acids, namely 22:1, 23:1, 24:1, 22h:1, 23h:1 and 24h:1, constituted more than 92% of the fatty acid content in these lipids.View full abstractDownload PDF (1333K) -
Hiroyuki Shimasaki, Noriyuki Hirai, Nobuo Ueta1988 Volume 104 Issue 5 Pages 761-766
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe fluorescence characteristics of product (I), formed during the lipid peroxidation of rat liver phosphatidylcholine liposomes containing glycine, and fluorescent product (II), derived from the reaction of malonaldehyde with glycine, were examined to elucidate the mechanism of fluorescent chromophore formation. Fluorescent product (I) had a fluorescence emission maximum at 430 nm when excited at 360 nm; its fluorescence intensity decreases in alkaline medium, but is restored by readjustment of pH to neutrality. In contrast, fluorescent product (II) exhibited an emission maximum at 458 nm, and the fluorescence was quenched at acidic pH. The fluorescent substances formed during the lipid peroxidation of hemoglobin-free human erythrocyte ghost membranes had similar fluorescence characteristics to produt (I). Gel filtration experiments showed that molecular size of fluorescent product (I) was larger than that of fluorescent product (II). The thiobarbituric acid-reactive substances released from peroxidizing liposomal phospholipids had a larger molecular size than malonaldehyde, and produced little or no fluorescence with glycine. It is concluded that the precursor of the fluorescent product formed during the lipid peroxidation of membrane phospholipids differs from malonaldehyde. The mechanism of the formation of blue emitting fluorscent material, believed to be a component of lipofuscin, seems to involve peroxidized phospholipids of the membrane.View full abstractDownload PDF (659K) -
Makio Hayakawa, Ichiro Kudo, Motowo Tomita, Shoshichi Nojima, Keizo In ...1988 Volume 104 Issue 5 Pages 767-772
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn our previous report (Hayakawa, M., Kudo, I., Tomita, M., & Inoue, K. (1988) J. Biochem. 103, 263-266), we have shown that phospholipases A2, purified from rat platelet membrane fractions and an extracellular medium of thrombin-stimulated rat platelets were essen-tially identical to each other. Both purified enzymes were digested with proteases, and the resulting peptides were subjected to primary sequence determination. The sequence analysis of the HPLC-separated peptides and the alignment of the sequences showed a tentative primary structure of rat platelet phospholipase A2, which was composed of 125 amino acid residues. It showed 47% homology with snake venom Agkistrodon halys blomhoffli phospholipase A2.View full abstractDownload PDF (581K) -
Masaru Himeno, Hiroshi Koutoku, Hiroshi Tsuji, Keitaro Kato1988 Volume 104 Issue 5 Pages 773-776
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcid phosphatase in rat liver lysosomal contents, C-APase I, was purified about 5, 700-fold over the homogenate with 8.0% recovery, to apparent homogeneity as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included; preparation of crude lysosomal contents, DEAE Sephacel ion exchange chromatography, hydroxylapatite chromatography, and gel filtr ation with Sephacryl S-300. The enzyme is composed of three identical subunits with an apparent molecular weight of 48K. The enzyme contains about 11% carbohydrate and the carbohydrate moiety was composed of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine in a molar ratio of 20 : 3 : 11 : 1. Sialic acid was not detected in the enzyme. Antisera against the purified C-APase I were raised in goat and the C-APase I was rapidly purified with high yield (10%) by using the specific antibodies coupled to Sepharose 6B.View full abstractDownload PDF (1756K) -
Katsura Inoue, Seiki Kuramitsu, Kenji Aki, Yasushi Watanabe, Toshio Ta ...1988 Volume 104 Issue 5 Pages 777-784
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSilvE gene of Escherichia coli was inserted into the region downstream of the tac promotor. As a result, the branched-chain amino acid aminotransferase was overproduced by about a hundred-fold in E. coli W3110. The overproduced aminotransferase was purified from cell extracts about 40-fold to homogeneity. Chemical and physicochemical analyses confirmed that it was a product of the ilvE gene. The enzyme existed in a hexamer with a subunit molecular weight of 34, 000; the double trimer model of the enzyme presumed by the previous chemical cross-linking experiments (Lee-Peng, F.-C. et al. (1979) J. Bacteriol. 139, 339-345) was supported by electron micrographs. The circular dichroic (CD) spectrum of branch-chain amino acid aminotransferase had double negative maxima at 210 and 220 nm. The α-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. The absorption spectrum of the enzyme showed two peaks at 330 and 410 nm. There was no pH-dependent spectral shift. The CD spectrum of the coenzyme, pyridoxal 5'-phosphate, had negative peaks at 330 and 410 nm. These spectral properties of branched-chain amino acid aminotransferase were quite different from those of E. coli aspartate aminotransferase. Each subunit bound approximately 1 mol of pyridoxal 5'-phosphate. A lysyl residue, which forms a Schiff base with the aldehyde group of the pyridoxal 5'-phosphate, was identified in the primary structure of the enzyme.View full abstractDownload PDF (2126K) -
Yasushi Yamazoe, Norie Murayama, Miki Shimada, Kiyomi Yamauchi, Kiyosh ...1988 Volume 104 Issue 5 Pages 785-790
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCharacteristics of a typical male-dominant reaction, dealkylation of n-propoxycoumarin, in rat livers were studied in relation to microsomal testosterone 6β-hydroxylase. The depropylation was more than 10-fold higher in the liver of male than female adult rats, but the sex-related difference was eliminated by neonatal castration. Hypophysectomy of adult male rats, which decreased the rates of male-specific P-450-male-dependent reactions, increased the depropylation of propoxycoumarin, while the rate was decreased by either intermittent injection or continuous infusion of human growth hormone to hypophysectom-ized rats. With regard to age-related difference, microsomal depropylation was detectable at neonate and reached a maximal level at 14 to 20 d of age, but was abruptly diminished only in female rats at puberty. These changes are in good agreement with those of testosterone 6β-hydroxylation and the content of a male-specific P-4506β-1/PB-1. In reconstituted systems using extracted microsomal lipids, P-4506β-1/PB-1 and P-450-male catalyzed the depropylation of propoxycoumarin. However, the microsomal depropylation was inhibited by antibodies which recognize P-4506β-1/PB-1, but not P-450-male. These results indicate that microsomal depropylation of propoxycoumarin is catalyzed mainly by a male-specific P-4506β-1/PB-1 in livers of untreated rats.View full abstractDownload PDF (795K) -
Keith R. Jarvie, Hyman B. Niznik, Natalie H. Bzowej, Philip Seeman1988 Volume 104 Issue 5 Pages 791-794
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ligand binding subunit of the D2 dopamine receptor (Mr_??_94, 000) can be visualized by autoradiography following photoaffinity labeling with [125I] N-azidophenethylspiperone and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following removal of sialic acids with the exoglycosidase, neuraminidase, [125I]N-azidophenethylspiperone photoincorporated into a protein of Mr=54, 000 with the appropriate pharmacological profile for D2 receptors. The desialylated D2 receptor bound dopaminergic agonists with high affinity and was capable of coupling to a functional G-protein as indexed by: 1) pertussis-toxin mediated [32P] ADP ribosylation of proteins of Mr=42, 000 and 39, 000, and 2) the conversion of the agonist high affinity form of D2 receptors to one displaying low affinity for agonists in the presence of guanine nucleotides. These data suggest that sialic acid residues do not contribute significantly to the ligand binding characteristics of D2 receptors despite the large change produced in the estimated molecular mass of the binding subunit.View full abstractDownload PDF (3013K) -
Yuko Tawada, Ken-Ichi Furukawa, Munekazu Shigekawa1988 Volume 104 Issue 5 Pages 795-800
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of cAMP on ATP-induced intracellular Ca+ mobilization in cultured rat aortic smooth muscle cells was investigated. Treatment of cells for 3 min at 37°C with dibutyryl cAMP, a membrane-permeable analogue of cAMP, at concentration up to 500 μM resulted in 1.5- to 1.7-fold increase in the peak cytosolic Ca2+ concentration when cells were stimulated with 3 to 200 μM ATP either in the presence or absence of extracellular Ca2+. Similar results were obtained when 0.5 mM 8-Br-cAMP or 10 AM forskolin was used instead of dibutyryl cAMP. In contrast to the Ca2+ response, dibutyryl cAMP did not affect ATP-induced formation of inositol trisphosphate (IP3). Furthermore, the dibutyryl cAMP treatment did not affect the size of the Ca2+ response elicited by 10 μM ionomycin. These results suggest that intracellular cAMP potentiates the ATP-induced Ca2+ response by enhancing Ca2+ release from the intracellular Ca2+ store(s), rather than by increasing the ATP-induced production of IP3 or by increasing the size of the intracellular Ca2+ store. Using saponin-permeabilized cells, we have shown directly that cAMP enhances Ca2+ mobilization by potentiating the Ca2+ -releasing effect of IP, from the intracellular Ca2+ store.View full abstractDownload PDF (752K) -
Yasumitsu Takagi, Ken-ichirou Morohashi, Shun-ichiro Kawahata, Mitiko ...1988 Volume 104 Issue 5 Pages 801-806
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESScDNA clones of the mRNA for rat liver carboxyesterase El, one of thecarboxyesterases exclusively located on the luminal side of microsomal vesicles, were isolated. Sequence analysis of 2 kbp long cDNA revealed the primary structure of carboxyesterase El, which consisted of 549 amino acids (Mr 60, 171.71) and contained an extra peptide of 18 amino acids at the NH2, -terminus of the mature enzyme. Comparison of the deduced primary structure and sequences of some proteolytic fragments of the purified enzyme indicated the multiplicity of the enzyme. The extra peptide at the NH2-terminal had features in common with the signal peptides of most secretory proteins. However, no polar amino acid residues existed before the hydrophobic core of the signal peptide. A new interpretation is proposed to explain how the signal peptide without the NH2-terminal polar residues works. A tetrapeptide (KDEL) which was shown to keep a few microsomal proteins in the lumen of the endoplasmic reticulum was not found in the primary structure of carboxyesterase El, which suggested the existence of another mechanism for retention of proteins in thelumen of endoplasmic reticulum. Carboxyesterase El showed significant homology with the COOH-terminal portion of thyroglobulin.View full abstractDownload PDF (759K) -
Haruhisa Tsukamoto, Kazuyoshi Azuma, Takahiro Miyauchi, Hirofumi Usui, ...1988 Volume 104 Issue 5 Pages 807-816
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpecific activities of tyrosine tubulin kinase in the particulate fractions from ratcerebel-lum, medulla oblongata, hypothalamus, striatum, midbrain, and cerebralcortex ranged within 30% of each other and more than 3 times higher than those in the solublefractions. In the cerebral cortex, tyrosine protein kinase activity toward tubulin and tyrosine-glutamate (1 : 4) copolymers was mainly distributed in the plasma membrane and the microsome fractions. The kinase activity in cerebral cortex particulate fractions was quantitatively solubilized and separated into two peaks, kinase I and kinase II, by Sephacryl S-300 gel filtration in the presence of 0.2% Nonidet P-40 and 0.2 M NaCl. Kinases I and II were each resolved into 5 active peaks (I-1→5 and II-1→5) by casein-Sepharose column chromatography. The molecular weights of these kinases were estimated from the s20.w values to be 59, 000-65, 000. The Km values of II-1→5. for tubulin were nearly 10 times higher than those of 1-1 However, the Km values of the two groups of kinases for tyrosine-glutamate copolymers were not so significantly different. About 60% of the copolymers kinase activity in 1-3, 1-4, 11-3, and 11-4 was immunoprecipitable with a saturating amount of monoclonal antibody against pp60c-src. Incubation of the immuno-precipitates with ATP resulted in the autophosphorylation of a 60kDa protein in 1-3 and 1-4, and a 52 kDa protein in 11-3 and 11-4. Immunoblotting also indicated 1-3 and 1-4 as 60 kDa bands and 11-3 and 11-4 as 52 kDa bands on SDS-polyacrylamide gels. The relative specific activities of 1-3, 1-4, 11-3, and 11-4 were 1 : 1 : 6 : 3 with tubulin and 1 : 1 : 9 : 7 with tyrosine-glutamate copolymers. V8 peptide maps of 32P-labeled 1-3, 1-4, 11-3, and 11-4 revealed that 1-3 and 1-4 were pp60c-src and the neuronal variant, pp60s-src, respectively, and suggested that 11-3 and 11-4 could be derived from pp60s-src and pp60s-src, respectively, hv removing the 8 kDa amino termini.View full abstractDownload PDF (3149K) -
Hiroshi Kitagawa, Hiroshi Nakada, Yoshito Numata, Akira Kurosaka, Shig ...1988 Volume 104 Issue 5 Pages 817-821
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA murine monoclonal antibody, designated as MSW 113, was generated using a human colonic cancer cell line, SW 1116, as the immunogen. MSW 113 was shown to be directed mainly to mucin-type oligosaccharide with sialyl-Lea antigens. The reactivity of MSW 113 to sialyl-Lea was stronger than that of NS 19-9, which is believed to be raised against the same determinant group. MSW 113 binds to sialyl-Lea-ol, LS-tetrasaccharide a, and disialyllacto-N-tetraose with higher affinities, compared to NS 19-9. These two antibodies could clearly be distinguished in that MSW 113 bound to sialic acid but not to fucose, whereas NS 19-9 bound to fucose but not to sialic acid. Thus, MSW 113 is directed more toward sialic acid-containing terminal structures while NS 19-9 is directed toward fucose-containing internal structures. MSW 113 was found to be useful for detecting antigens in the bloodstream of patients, especially those with pancreas cancer. Even NS 19-9 negative patient sera were positive for MSW 113.View full abstractDownload PDF (1189K) -
Akiko Takasuga, Hiroyuki Adachi, Fumitoshi Ishino, Michio Matsuhashi, ...1988 Volume 104 Issue 5 Pages 822-826
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2. This amino acid sequence was verified by sequencing the labeled tryptic peptide containing the labeled chymotryptic peptide region. A mutant PBP 2 (thiol-PBP 2), constructed by site-directed mutagenesis to replace Ser330 with Cys, lacked the penicillin-binding activity. These findings provided evidence that Ser330 near the middle of the primary structure of PBP 2 is the penicillin-binding active-site residue, as predicted previously on the basis of the sequence homology. Around this active site, the sequence Ser-Xaa-Xaa-Lys was observed, which is conserved in the active-site regions of all E. coli PBPs so far studied, class A and class C β-lactamases, and D-Ala carboxypeptidases. The COOH-terminal amino acidof PBP 2 was identified as His633.View full abstractDownload PDF (2729K) -
Kenji Kinoshita, Naoyuki Taniguchi, Akira Makita, Masaki Narita, Kiyos ...1988 Volume 104 Issue 5 Pages 827-831
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-N-Acetylhexosaminidase (hexosaminidase) I, which has an intermediate charge character between those of hexosaminidases A (αβ2) and B((ββ)2), was purified 1, 500-fold from human placenta by procedures including chromatographies on concanavalin A (Con A)-Sepharose and an immunoadsorbent column. The isolated hexosaminidase I was heat-stable, and antigenically cross-reactive to anti-β chain-IgG but not to anti-α chain-IgG. The results of substrate specificity experiments using 3H-labeled natural substrates indicated that the hexosaminidase I hydrolyzed Gb4 Cer to Gb3 Cer but not GM2 to GM3. The tryptic peptide map of the hexosaminidase I was similar to that of hexosaminidase B, though some differences were observed. The hexosaminidase I after treatment with neuraminidase or endo-β-N-acetylglucosaminidase H was partly converted to less acidic forms. Treatment of the hexosaminidase I with acid phosphatase did not change the charge character. Therefore hexosaminidase I is an acidic variant form of hexosaminidase B, possibly resulting from sialylation and the presence of phosphodiester bonds at the carbohydrate moiety.View full abstractDownload PDF (2495K) -
Kaeko Kamei, Saburo Hara, Tokuji Ikenaka, Sawao Murao1988 Volume 104 Issue 5 Pages 832-836
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of a lysozyme, (B-enzyme), from Bacillus subtilis YT-25 wasdetermined by conventional methods. B-Enzyme comprised 117 amino acid residues and had a heterogeneous sequence in the amino-terminal region. The amino acid sequenceof B-enzyme was different from those of all other lysozymes the sequences of which are known. However, the partial amino acid sequence of Ser (74) to Ser (97) of B-enzyme was homologous with that of the active-site region of hen egg-white lysozyme (Ser (36) to Ser (60)), which includes one of the catalytic amino acids, Asp (52). It is interesting that B-enzyme has an amino acid sequence homologous with that of the gagprotein p25 of the AIDS virus ARV-2.View full abstractDownload PDF (504K) -
Takashi Kamogashira, Maki Sakaguchi, Yasukazu Ohmoto, Keiko Mizuno, Ry ...1988 Volume 104 Issue 5 Pages 837-840
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing the expression system for site-specific mutagenesis in Escherichia coli, we have made deletion mutants at the N-terminal or C-terminal region of human interleukin-1β (IL-1β) consisting of 153 amino acids. The truncated mutants showed that at least 147 amino acids (numbers 4-150) in IL-1β are necessary for the exertion of biological activity. When we changed the arginine at the 4th position (Arg4) in IL-1β to other specific amino acids, there was a marked difference in the relative extent of biological and receptor binding activities among the mutants. The order of the mutants was Arg4=Lys4>Gln4>Gly4=des-Arg4>Asp4. Our results demonstrate that the arginine residue at the 4th position in IL-1β is important, but not essential, for IL-1β to exhibit its biological and receptor binding activities, and the positive charge at this site plays a key role for IL-1β to exert the activities.View full abstractDownload PDF (1614K) -
Chikara Masuta, Shigeru Kuwata, Yoichi Takanami1988 Volume 104 Issue 5 Pages 841-846
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFull-length cDNA of a satellite RNA (Strain Y) which induces bright yellow symptoms on tobacco plants infected with cucumber mosaic virus (CMV) was cloned and sequenced. The published sequence of the satellite RNA was revised with three possible differences (residues 161, 167, and 173) and a nucleotide insertion at residue 234. The satellite cDNA was then inserted into a commercially available transcription vector. In vitro transcription products from the recombinant plasmid harbored 24 non-viral bases at their 5' ends and had very low infectivity when coinoculated with CMV. After removal of the extra 5' sequence of the transcripts with RNase H, the infectivity of the transcripts increased markedly. Analysis of the effects of extra 5' sequences of several lengths confirmed the importance of natural 5' ends for biological activity of the satellite. Trimming down to 6-9 extra bases at the 5' end enhanced the infectivity of the transcripts by 10-fold, although the specific activity of the natural satellite is still 100-fold higher. Dideoxynucleotide sequence analysis proved that the progeny satellite RNA did not retain the 24 non-viral bases atthe 5' end of the transcript from pIBI 31-MC.View full abstractDownload PDF (3131K) -
Toru Yamada, Toru Inoue, Toshirou Nishida, Eisuke Furuya, Kunio Tagawa1988 Volume 104 Issue 5 Pages 847-850
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAccumulation of pyrophosphate induced by acetate administration was investigated in rat liver in situ and in perfused rat liver. Intraperitoneal injection of acetate into rats increased the pyrophosphate concentration in the liver to about 2 μmol/g liver, which was 200 times that in control liver. Perfusion of liver with acetate alone did not result in accumulation of pyrophosphate. However, the further addition of a Ca2+ -mobilizing hormone, such as noradrenaline or angiotensin II, together with glucagon to the perfusion medium containing 1 mM acetate caused accumulation of pyrophosphate to a similar level to that observed in vivo. Acetate, glucagon and a Ca2+ -mobilizing hormone were all required for accumulation of pyrophosphate in perfused liver. Omission of Ca2+ from the perfusion medium or addition of a Ca2+-antagonist reduced the accumulation significantly. The two kinds of hormones, glucagon and an α -agonist, either singly or in combination, did not affect the rate of acetate utilization. These results show that liver cells accumulate a large amount of pyrophosphate during acetate metabolism at high intracellular levels of Ca2+ that can be realized by the synergistic actions of the two kinds of hormones.View full abstractDownload PDF (544K) -
Jun-ichi Kajihara, Mitsuo Enomoto, Keiko Nishijima, Masahiko Yabuuchi, ...1988 Volume 104 Issue 5 Pages 851-854
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe physicochemical properties of purified recombinant human copper-zinc superoxide dismutase (r-hSOD) were compared with those of human placental copper-zinc superoxide dismutase (h-SOD). No differences were found in specific activity, metal contents, amino acid composition, and tryptic peptide map. The spectrophotometric properties including UV, ESR, and CD spectra were also similar. The result of isoelectric gel electrophoresis showed that the difference in isoelectric point (pI) was derived from acetylation of the N-terminal amino acid (alanine) in h-SOD. In SDS-polyacrylamide gel electrophoresis, both SODs showed the same behavior and enzymic activity was retained only under non-reducing conditions. ESR analysis of the denatured enzyme suggested that the high stability was derived from the structure of the active site around copper. Experiments using other metal-substituted SODs (Cu, Co in place of zinc) suggested that zinc contributed to the stability and the unique electrophoretic behavior of the enzyme.View full abstractDownload PDF (1053K) -
Jun-ichi Kajihara, Mitsuo Enomoto, Kazuo Katoh, Keiichi Mitsuta, Masah ...1988 Volume 104 Issue 5 Pages 855-857
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relation between ESR-detectable Cu(II) and Cu, Zn-superoxide dismutase activity was examined. The Cu(II) spin numbers per one unit of SOD were 6.26×1012 (±0.51 ×1012) spins in several preparations of recombinant human Cu, Zn-SOD, native placental, and erythrocyte SOD. Measurement could be performed over a wide range of pH (4.0-10.0), preferably at temperatures below -40 °C. The data obtained by this method correlated well to the results obtained by the method of Fridovich et al. using the xanthine-xanthine oxidase system (correlation coefficient 0.995). The specific activity of SOD was proportional to the Cu(II) content measured by ESR, but not to the total Cu content measured by atomic absorption. This indicates that it is important to measure the Cu(II) content for determining Cu, Zn-SOD activity.View full abstractDownload PDF (291K) -
Hideto Kuwayama, Setsuro Ebashi1988 Volume 104 Issue 5 Pages 858-861
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1) Taking myosin light chain kinase (MLCK) activity as the index, bovine brain extract was fractionated by the use of anion-exchange chromatography, cation-exchange chromatogra-phy, and calmodulin affinity chromatography. The kinase activity of the fraction thus obtained was elevated up to about 12, 400 times over that of the original crude extract. 2) The fraction mentioned above was subjected again to anion exchange chromatography. The kinase activities were divided into two parts, i.e., part I which contained the 155 kDa component and part II which was virtually free of 155 kDa component. The MLCK activity of part I was considerably lower than that of part II. 3) Part I was subjected to gel filtration using AcA 34 gel and the 155 kDa component was isolated. The fraction contained the 155 kDa component in a homogeneous state and showed myosin specific kinase activity, which was about 2×105 times that of the original crude extract. 4) The high kinase activity of part II seemed to be ascribable to the 130 kDa component, in accord with the report of Hathaway, Adelstein, and Klee (J. Biol.Chem. 256, 8183-8189, 1981).View full abstractDownload PDF (1729K) -
Hideto Kuwayama, Masashi Suzuki, Ritsuko Koga, Setsuro Ebashi1988 Volume 104 Issue 5 Pages 862-866
Published: 1988
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1) Two protein components, 155 and 130 kDa in their electrophoretic molecular weights, respectively, were isolated in a homogeneous state from bovine aorta; they showed both the superprecipitation-inducing effect on desensitized natural actomyosin and the myosin light chain kinase (MLCK) action on gizzard myosin. 2) The superprecipitating activity of the 155 kDa component was 5 time higher than that of the 130 kDa component on the basis of equivalent MLCK activity. 3) The same procedure was applied to bovine stomach, giving rise to a 155 kDa component in a homogeneous state as in the case of aorta, but the 130 kDa component thus prepared was contaminated by higher molecular weight components. 4) If compared on the basis of equivalent MLCK activity, bovine stomach 155 kDa component showed more than 10 times higher superprecipitating activity than the fraction that contained the 130 kDa component as the main constituent. 5) The discrepancy between the superprecipitating activity and MLCK activity mentioned above was discussed in relation to the Ca2+ regulation mechanism in smooth muscle contraction. The possibility that the 130 kDa component might be a proteolytic product of the 155 kDa component was also discussed.View full abstractDownload PDF (1532K)
- |<
- <
- 1
- >
- >|