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Volume 126 (1999)
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Volume 126, Issue 4
Displaying 1-23 of 23 articles from this issue
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Saori Takahashi, Keitaro Takahashi, Takahiro Kaneko, Hironobu Ogasawar ...1999 Volume 126 Issue 4 Pages 639-642
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResearch Institute of Food and Brewing (ARIF), 4-26 Sanuki, Arayamachi, Renin binding protein (RnBP) is a proteinous renin inhibitor firstly isolated from porcine kidney. Recently, the protein was identified as the enzyme, N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. The GlcNAc 2-epimerase activity of recombinant human RnBP was specifically inhibited by SH-reagents such as N-ethylmaleimide, 5, 5'-dithiobis-2-nitro-benzoate, and iodoacetic acid, indicating that the most probable reactive site is a cysteine residue. To identify the active site residue (s), we have constructed ten cysteine residue mutants (C41S, C66S, C104S, C125S, C210S, C239S, C302S, C380S, C386S, and C390S) for human GlcNAe 2-epimerase and expressed them in Escherichia coli cells. The relative specific activities of C41S, C66S, C125S, C210S, C239S, C302S, C386S, and C390S are nearly the same to that of the wild-type enzyme. The specific activity of the C104S mutant is 26% of that of the wild-type enzyme. The expression of the C380S mutant in E. coli cells was detected on Western blotting, whereas GIcNAc 2-epimerase activity was not detected in the extract. These results indicate that Cys380 is essential for the enzymatic activity of human GlcNAc 2-epimerase.View full abstractDownload PDF (1822K) -
Yuanyuan Li, Guozhong Tao, Hiroyuki Nagasawa, Hiroshi Tazawa, Akira Ko ...1999 Volume 126 Issue 4 Pages 643-649
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the function of pICln, its localization in subcellular organellae was investigated. A specific polyclonal anti-pICln antibody detected the soluble 38-kDa pICln exclusively in the cytosols of rat heart, lung, liver, spleen, skeletal muscle, testis, and brain, but not rat kidney. pICln-associated proteins in skeletal muscle were also analyzed. Native-gradient PAGE showed a single 340-kDa protein band reactive to anti-pICln antibody. This band also stained with anti-actin antibody. Two-dimensional PAGE and immunoprecipitation analysis indicated that all of the pICln was present in association with actin of a constant length: the molecular ratio of pICln to actin was roughly 1:7. In addition, all actin in the cytosol fractions was found in association with pICln. These results suggest the possibility that skeletal muscle pICln controls the length of cytosolic F-actin.View full abstractDownload PDF (2904K) -
Tetsuo Toraya, Kazunari Yoshizawa, Masataka Eda, Tokio Yamabetl1999 Volume 126 Issue 4 Pages 650-654
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe direct ion-dipolar interactions between potassium ion (K+) and the two hydroxyl groups of the substrate are the most striking feature of the crystal structure of coenzyme B12-dependent diol dehydratase. We carried out density-functional-theory computations to determine whether K+ can assist the 1, 2-shift of the hydroxyl group in the substratederived radical. Between a stepwise abstraction/recombination reaction proceeding via a direct hydroxide abstraction by K+ and a concerted hydroxyl group migration assisted by K+, only a transition state for the latter concerted mechanism was found from our computations. The barrier height for the transition state from the complexed radical decreases by only 2.3 kcal/mol upon coordination of the migrating hydroxyl group to K+, which corresponds to a 42-fold rate acceleration at 37°C. The net binding energy upon replacement of the K+-bound water for substrate was calculated to be 10.7 kcal/mol. It can be considered that such a large binding energy is at least partly used for the substrateinduced conformational changes in the enzyme that trigger the homolytic cleavage of the Co-C bond of the coenzyme and the subsequent catalysis by a radical mechanism. We propose here a new mechanism for diol dehydratase in which K+ plays a direct role in the catalysis.View full abstractDownload PDF (360K) -
Yasuo Yamazaki, Yasuo Shikamoto, Kenji Fukudome, Masao Kimoto, Takashi ...1999 Volume 126 Issue 4 Pages 655-661
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA membrane-associated prothrombin activator (MAPA) was found on various cultured cells derived from non-hematopoietic cells [Sekiya, F. et al. (1994) J. Biol. Chem. 269, 32441-32445]. In this study, we investigated the enzymatic properties of this enzyme using protease inhibitors. While the metalloproteinase inhibitor, o-phenanthroline, had no effect, some Kunitz type serine protease inhibitors attenuated MAPA activity. Recombinant tissue factor pathway inhibitor (rTFPI) also markedly reduced the activity (IC50, 1.3±0.6×10-10M). MAPA activity is, therefore, most likely to be due to factor Xa. We evaluated the effect of exogenous factor Xa on MAPA activity. Factor Xa-dependent prothrombin activation was observed on fibroblast cells (apparent Kd, 1.47±0.72nM). Activation was also observed on glial and neuronal cells, which expressed MAPA activity. These results imply that membrane-bound factor Xa results in MAPA activity on these cells. Therefore, we consid-ered the involvement of factor Va, a component of prothrombinase, in this activity. We examined whether or not the prothrombinase complex is assembled on these cells. Prothrombin was activated in a manner dependent on both exogenous factor Xa and factor Va (apparent Kd of 0.51-1.81 nM for factor Va). These results indicate that the prothrom-binase complex forms specifically on various extravascular cells. Although the prothrom-binase complex can be assembled on monocytes and lymphocytes, it is not known why these cells can activate prothrombin specifically. These cells which have the capacity for prothrombin activator activity could also activate factor X; i.e. cells with factor X activation activity were able to convert prothrombin. These observations suggest that thrombin was generated via two procoagulant activities; factor X activation and subse-quent prothrombinase complex formation on the surface of these cells. This mechanism may explain the various pathological states involving or resulting from extravascular thrombin and fibrin formation.View full abstractDownload PDF (668K) -
Chifumi Fujii, Shingo Morii, Michiaki Kadode, Shizue Sawamoto, Masafum ...1999 Volume 126 Issue 4 Pages 662-667
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTetranitromethane treatment of 3-ketosteroid-Δ1-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitro-methane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, See, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The Km values for 4-androstene-3, 17-dione of the Y- 104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The I, values for 1, 4-andro-stadiene-3, 17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0.014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.View full abstractDownload PDF (2014K) -
Kyosuke Sato, Yasuzo Nishina, Chiaki Setoyama, Retsu Miura, Kiyoshi Sh ...1999 Volume 126 Issue 4 Pages 668-675
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe standard redox potential of acrylyl-CoA/propionyl-CoA couple (C3) was determined to be 69 mV (vs. standard hydrogen electrode) at pH 7 and 25°C. This value implies that the 2, 3-dehydrogenation of propionyl-CoA is thermodynamically much more unfavorable than that of longer acyl-CoAs because the standard redox potentials of crotonyl-CoA/butyryl-CoA (C4), octenoyl-CoA/octanoyl-CoA (Cs), and hexadecenoyl-CoA/palmitoyl-CoA (C16) are all about -10 mV. The unusually high standard redox potential of the acrylyl-CoA/ propionyl-CoA couple is thought to be one of the reasons that in mammals propionyl-CoA is not metabolized by β-oxidation as in the case of longer acyl-CoAs, but by a methyl-malonyl-CoA pathway. The obvious structural difference between C3 and C4 (and longer) is whether an H or the C(4) atom is connected to -C(3)H=C(2)H-C(1)O-S-CoA. The molecular orbital calculations (MOPAC) for the enoyl and acyl forms of C3 and C4 revealed that this structural feature is the main cause for the higher standard redox potential of the C3 couple. That is, the C(4)-C(3) bond is stabilized by the dehydrogenation to a greater degree than the H-C(3) bond.View full abstractDownload PDF (2227K) -
Jens Gebken, Barbara Ldders, Holger Notbohm, Harald H. Klein, Jiirgen ...1999 Volume 126 Issue 4 Pages 676-682
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe formation and organization of skeletal tissue is strongly influenced by mechanical stimulation. There is increasing evidence that gravitational stress has an impact on the expression of early response genes in mammalian cells and may play a role in the formation of extracellular matrix. In particular, osteoblasts may be unique in their response to gravitational stimuli since in these cells microgravity has been reported to reduce collagen synthesis, while in fibroblasts the opposite effect was observed. Here, we have investigated the influence of hypergravity induced by centrifugation on the collagen synthesis of human osteoblast-like cells (hOB) and studied the possible involvement of the mitogen-activated protein (MAP) kinase signaling cascade. Collagen synthesis was significantly increased by 42±16% under hypergravity at 13×g, an effect paralleled by the enhanced expression of the collagen I alpha 2 (COLIA2) mRNA. No difference was seen in the proportion of collagen types I, III, and V synthesized by hOB. Hypergravity induced a markedly elevated phosphorylation of the p44/42 MAP kinases (ERR 1/2). The inhibition of this pathway suppressed the hypergravity-induced stimulation of both collagen synthesis as well as COL1A2 mRNA expression by about 50%. Our results show that the collagen synthesis of non-transformed hOB is stimulated under hypergravitational conditions. This response appears to be partially mediated by the MAP kinase pathway.The formation and organization of skeletal tissue is strongly influenced by mechanical stimulation. There is increasing evidence that gravitational stress has an impact on the expression of early response genes in mammalian cells and may play a role in the formation of extracellular matrix. In particular, osteoblasts may be unique in their response to gravitational stimuli since in these cells microgravity has been reported to reduce collagen synthesis, while in fibroblasts the opposite effect was observed. Here, we have investigated the influence of hypergravity induced by centrifugation on the collagen synthesis of human osteoblast-like cells (hOB) and studied the possible involvement of the mitogen-activated protein (MAP) kinase signaling cascade. Collagen synthesis was significantly increased by 42±16% under hypergravity at 13×g, an effect paralleled by the enhanced expression of the collagen I alpha 2 (COLIA2) mRNA. No difference was seen in the proportion of collagen types I, III, and V synthesized by hOB. Hypergravity induced a markedly elevated phosphorylation of the p44/42 MAP kinases (ERR 1/2). The inhibition of this pathway suppressed the hypergravity-induced stimulation of both collagen synthesis as well as COL1A2 mRNA expression by about 50%. Our results show that the collagen synthesis of non-transformed hOB is stimulated under hypergravitational conditions. This response appears to be partially mediated by the MAP kinase pathway.The formation and organization of skeletal tissue is strongly influenced by mechanical stimulation. There is increasing evidence that gravitational stress has an impact on the expression of early response genes in mammalian cells and may play a role in the formation of extracellular matrix. In particular, osteoblasts may be unique in their response to gravitational stimuli since in these cells microgravity has been reported to reduce collagen synthesis, while in fibroblasts the opposite effect was observed. Here, we have investigated the influence of hypergravity induced by centrifugation on the collagen synthesis of human osteoblast-like cells (hOB) and studied the possible involvement of the mitogen-activated protein (MAP) kinase signaling cascade. Collagen synthesis was significantly increased by 42±16% under hypergravity at 13×g, an effect paralleled by the enhanced expression of the collagen I alpha 2 (COLIA2) mRNAView full abstractDownload PDF (2987K) -
Chao-Tan Guo, Osanmu Nakagomi, Masami Mochizuki, Hideharu Ishida, Mako ...1999 Volume 126 Issue 4 Pages 683-688
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRotavirus is the most common cause of severe gastroenteritis in infants and children worldwide. The cell attachment of most animal rotaviruses, which belong to the neuraminidase-sensitive strains, requires sialic acid residues on the host cell menbranes. On the other hand, most human rotaviruses are classified as neuraminidase-insensitive strains. The involvement of gangliosider on the host cell surface in human rotavirus infection was investigated by rotavirus and the blocking of target cellular receptors. In host cells (MA104 cells) pretreated with Arthrobacter ureafaciens neuraminidase, which were still infected by human rotaviruses (KUN and MO strains), GM3 was hydrolyzed markedly by the neuraminidase, while GM1a was not hydrolyzed at all. Infection by the rotaviruses was strongly inhibited by exogenous gandlioside GM1a, but not GA1. Infection was also inhibited by pretreatment of the MA104 cells with cholera toxin B-subunit, witch specifically blocked ganglioside GM1a on the plasma membrane. The treatment of MA104 cells with the endoglycoceramidase attenuated human rotavirus intection. From these findings, we concluded that GM1a on the plasma menebrane of the host cells was involved in the infection by human rotavirus KUS and MO strains.View full abstractDownload PDF (1882K) -
Seiichi Taguchi, Akiyoshi Ozaki, Takamasa Nonaka, Yukio Mitsui, Haruo ...1999 Volume 126 Issue 4 Pages 689-693
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new cold-adapted protease subtilisin BPN' mutant, termed m-51, was successfully isolated by use of an evolutionary program consisting of two-step in vitro random mutagenesis, which we developed for the screening of mutant subtilisins with increased activity at low temperature. The m-51 mutant showed 70% higher catalytic efficiency, expressed by the kcat/Km value, than the wild-type at 10°C against N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as a synthetic substrate. This cold-adaptation was achieved mainly by the increase in the kcat value in a temperature-dependent manner. Genetic analysis revealed that m-51 had three mutations, Ala'Thr at position -31 (A-31T) in the prodomain, Ala→Val at position 88 (A88V), and Ala→Thr at position 98 (A98T). From kinetic parameters of the purified mutant enzymes, it was found that the A98T mutation led to 30% activity increase, which was enhanced up to 70% by the accompanying neutral mutation A88V. The A-31T mutation severely constrained the autoprocessing-mediated maturation of thee pro-subtilisin in the Escherichia coli expression system, thus probably causing an activity-non-detectable mutation in the first step of mutagenesis. No distinct change was observed in the thermal stability of any mutant or in the substrate specificity for m-51. In the molecular models of the two single mutants (A88V and A98T), relatively large displacements of alpha carbon atoms were found around the mutation points. In the model of the double mutant (A88V/A98T), on the other hand, the structural changes around the mutation point counterbalanced each other, and thus no crucial displacements occurred. This mutual effect may be related to the enhanced activity of the double mutant.View full abstractDownload PDF (1436K) -
Kimimitsu Oda, Yoshihiro Amaya, Mariko Fukushi-Irié, Yasuko Kin ...1999 Volume 126 Issue 4 Pages 694-699
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA soluble form of tissue-nonspecific alkaline phosphatase was purified to apparent homogeneity from the culture media of Sf9 cells which had been infected with recombinant baculoviruses encoding human tissue-nonspecific alkaline phosphatase (TNSALP). To facilitate purification, an oligonucleotide consisting of 6 tandem codons for histidine and a stop codon was engineered into the TNSALP cDNA. The molecular mass of the enzyme purified through a nickel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. That of the native enzyme was 90 kDa as estimated by gel filtration, indicating that the purified soluble TNSALP is dimeric. The enzyme was used for production of antibodies specific for human TNSALP. Immunoblotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells. However, upon digestion with peptide: N-glycosidase F, the 80-kDa TNSALP of human origin and the soluble enzyme of insect origin migrated to the same position on SDS-polyacrylamide gel, indicating that the size difference between the two enzymes is ascribed to N-linked oligosaccharides. The antibodies prepared against the purified TNSALP were found to be useful also for immunoprecipitation and immunofluore-scence studies.View full abstractDownload PDF (1945K) -
Keita Sutoh, Hideki Kato, Takao Minamikawa1999 Volume 126 Issue 4 Pages 700-707
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLittle or no endopeptidase activity was detected in extracts of dry mature wheat seeds, but when they were allowed to imbibe water in darkness, the activity expressed per seedling increased notably after d 1, reached a maximum on d 3 and then decreased. Two major endopeptidases, named WEP-1 and WEP-2, were present in the 50-70% saturated ammonium sulfate fraction of d-3 seedlings, and could be separated by hydrophobic column chromatography. WEP-1 was further purified and identified as a 31-kDa polypeptide that was immunoreactive to antiserum raised against REP-1, a major rice cysteine endopeptidase. Experiments with proteinase inhibitors revealed that WEP-1 and WEP-2 are cysteine and serine endopeptidases, respectively. The two enzymes differed in substrate specificity, pH dependence, and the ability to digest major wheat seed proteins. Determination of its amino-terminal amino acid sequence indicated the similarity of WEP-1 to other cereal cysteine endopeptidases which are involved in the digestion of seed storage proteins. The expression of WEP-1 in de-embryonated seeds was induced in the presence of gibberellic acid and its effect was eliminated by abscisic acid. In addition to WEP-1 and WEP-2, a legumain-like asparaginyl endopeptidase was identified in the extract of seedlings on hydrophobic chromatography. The asparaginyl endopeptidase may function in the early step of mobilization of wheat storage proteins in germinated seeds.View full abstractDownload PDF (3763K) -
Hirotada Fujii, Michael G. Finnegan, Michael K. Johnson1999 Volume 126 Issue 4 Pages 708-714
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe spin state of the heme in superoxide (O2•-)-producing cytochrome b558, purified from pig neutrophils was examined by means of room-temperature magnetic circular dichroism (MCD) under physiological conditions. Cytochrome b558 with varying amounts of low-spin and high-spin heme was prepared by either pH adjustment or heat treatment, and theO2•--forming activity in a cell-free system was found to correlate with the low-spin heme content. The possibility that the O2•--forming activity results from a transient high-spin ferric heme form that is induced during activation by anionic amphophils has also been investigated. EPR spectra of cytochrome b558 activated by either arachidonic acid or myristic acid, showed that a transient high-spin ferric species accounting for approximate-ly 50% of the heme appeared in the presence of arachidonic acid, but not in the presence of myristic acid. Hence the appearance of a transient high-spin ferric heme species on activation with an amphophil does not afford a common activation mechanism in the NADPH oxidase system. The EPR results for cytochrome b558 activated with arachidonic acid showed that the transient high-spin ferric heme can bind cyanide. However, the high-spin ferric heme does not contribute to the O2•- production of cytochrome b558 in cellfree assays in the presence of cyanide.View full abstractDownload PDF (2788K) -
Tatsuya Oda, Noriko Sadakata, Nobukazu Komatsu, Tsuyoshi Muramatsu1999 Volume 126 Issue 4 Pages 715-721
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlthough the depletion of reduced glutathione (GSH) has been observed in a variety of apoptotic systems, little is known about the mechanism of GSH depletion. In this study we used polarized MDCK cells to study the GSH flux during ricin-induced apoptosis. Here we report that the specific accumulation of GSH occurred in the basolateral medium during ricin treatment with similar kinetics to in apoptotic changes such as an increase in caspase-3 like activity and DNA fragmentation, while there was no significant increase in the GSH level in apical medium. These results suggest that GSH effiux occurred through a GSH-specific channel or transporter located in the basolateral membrane domain of polarized MDCK cells undergoing apoptosis. Treatment with other protein toxins such as modeccin, Pseudomonas toxin, and diphtheria toxin, which can induce apoptotic cell death, also resulted in selective GSH efflux from the basolateral side. Thus, GSH effiux through a specific transporter may be a common step of apoptosis induced by these toxins, while these toxins have different intoxication mechanisms leading to protein synthesis inhibition. Pretreatment of cells with Z-Asp-CH2-DCB, a caspase family inhibitor, inhibited ricininduced basolateral GSH efflux as well as DNA fragmentation, suggesting that the activation of caspases, i.e. those that are inhibited by Z-Asp-CH, -DCB, is implicated in the opening of the GSH transporter.View full abstractDownload PDF (1313K) -
Hiroshi Hori, Tomoko Nembai, Yasuhisa Miyata, Tetsuya Hayashi, Kentaro ...1999 Volume 126 Issue 4 Pages 722-730
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo new forms of proteasomes, designated as the endoplasmic reticulum (ER) membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome (ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE analysis revealed that the purified ERa and ERb proteasomes were composed of multiple subunits similar to the cytosolic 20S proteasome. However, electrophoretic, structural and immunochemical differences between the ERa, ERb and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D) PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate extract of the trypsin-treated microsomal fraction yielded a trypsin-modified form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no ERa proteasome was obtained from the 0.0125% sodium cholate extract of the trypsin-treated microsomes, suggesting that ERa and ERb are ER membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and cy tosolic 20S proteasomes exhibited similar specificities as to peptide hydrolyzing activity, although differences in their activities were noted in the presence of SDS and phospholipid. With respect to the proteolysis of protein substrates, only the ERb proteasome cleaved β-casein, and it also degraded reduced and carboxymethylated lysozyme considerably faster than the cytosolic 20S and ERa protea-somes. Collectively our results suggest that the ERa and ERb proteasomes may play roles in intracellular proteolysis distinct from that of the cytosolic 20S proteasome.View full abstractDownload PDF (3128K) -
Satoru Mizuguchi, Kei Amada, Mitsuru Haruki, Tadayuki Imanaka, Masaaki ...1999 Volume 126 Issue 4 Pages 731-737
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33, 633. The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH2-ter-minus. The good agreement of the molecular weights estimated by SDS-PAGE (35, 000) and gel filtration (38, 000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C18), tricaprylin and triolein. Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C2 to C14 indicated that HDE shows a relatively broad substrate specificity. However, comparison of the kcat/Km, values indicated that the C10-C14 substrates are the most preferred ones. Such a preference for substrates with long acyl chains may be a characteristic of HDE.View full abstractDownload PDF (1334K) -
Shigeru Minoguchi, Toshio Ikeda, Shigeyoshi Itohara, Takeshi Kaneko, H ...1999 Volume 126 Issue 4 Pages 738-747
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe RBP-L gene encodes a DNA binding protein that is structurally related to RBP-J, the mammalian homolog of Drosophila Suppressor of Hairless. Although the RBP-L protein binds the same DNA sequence as RBP-J, the in vivo function of this protein remains largely unknown. In order to investigate the role of this protein, we generated RBP-L mutant mice by targeted disruption involving replacement of the protein-coding sequence in the first exon with an in-frame fusion of the nlacZ eDNA. The homozygous mutant mice appeared morphologically normal and fertile. Unexpectedly, we found the possible existence of additional promoter (s) downstream of the first exon whose activity was not fully disrupted in the mutant mice. The promoter upstream of the first exon is regulated in a cell typespecific manner so that transcription is active in neurons but almost inactive in lung where the downstream promoter is active. The specific expression of the β-galactosidase fusion protein was detected in layer VI of the cerebral cortex, in the pyramidal cell layer of the hippocampus, and in the granule cell layer of the dentate gyros. Furthermore, we found that the upstream promoter activity in neurons might be regulated by some neuronal activity.View full abstractDownload PDF (4668K) -
Takashi Shimokawa, Mitsuko Masutani, Shingo Nagasawa, Tadashige Nozaki ...1999 Volume 126 Issue 4 Pages 748-755
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPoly (ADP-ribose) glycohydrolase (Parg) is the main enzyme of poly (ADP-ribose) degradation. To understand its structure-and-function relationship, we purified Parg from rat testis 9, 740-fold using an improved affinity column; the purified product was a 60 kDa protein. Based on the determined sequences of three peptide fragments, degenerated primers were synthesized and a Parg cDNA comprising 3, 974 nucleotides, encoding a 109 kDa protein, was isolated. The 60 kDa Parg purified from rat testes corresponded to the C-terminal half of the 109 kDa deduced peptide. When recombinant rat Parg was expressed as a glutathione S-transferase fusion protein in Escherichia coli, Parg activity was observed for the full-length and C-terminal half proteins but not in for the N-terminal half protein. Taken together, these data indicate that the catalytic domain of Parg is located in the C-terminal half. Further, we newly identified the presence of a potential nuclear export signal in the N-terminal half in addition to the previously reported nuclear localization signals in rat and other mammalian Pargs. Northern blot analysis showed the ubiquitous expression of a single 4.0 kb Parg mRNA in various rat tissues. The findings suggest that the 60 kDa Parg is produced by post-transcriptional processing.View full abstractDownload PDF (3270K) -
Shiro Kominami, Takeshi Yamazaki, Toshihisa Koga, Hiroshi Hori1999 Volume 126 Issue 4 Pages 756-761
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (581K) -
Hiroshi Mizuguchi, Miki Nakatsuji, Shinsuke Fujiwara, Masahiro Takagi, ...1999 Volume 126 Issue 4 Pages 762-768
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA polymerase from Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504 4510]. However, even when KOD DNA polymerase was used for PCR, troubles with nonspecific DNA amplification and primer dimer formation still remain because of undesirable DNA polymerase activity during the first denaturing step of PCR. In order to inhibit this undesirable DNA polymerase activity (hot start PCR), two neutralizing monoclonal antibodies (mAbs), 3GS and βG1, to KOD DNA polymerase were obtained. Both of these antibodies belong to subclass IgG1, κ. Kd values were 7.3×10-8for 3G8 and 1.1×10-6 for βG1. Nucleotide sequencing of eDNAs of these monoclonal antibodies revealed their sequences to differ in their CDRs (complementarity determining region). Exonuclease activity measurement and epitope mapping revealed that the epitope for 3G8 is located in conserved regions among a-like (family B) DNA polymerases (Region II), and the epitope for βG1 is located in the 3'-5' exonuclease domain. When hot start PCR with each of these mAbs was performed, the specificity of target gene amplification became much higher than in reactions without monoclonal antibody. Furthermore, this method can easily be applied to long distance PCR (>17.5 kbp).View full abstractDownload PDF (2865K) -
Hiroyuki Kaji, Rumi Yada-Wakatabe, Takashi Uehira, Mayumi Terai, Atsus ...1999 Volume 126 Issue 4 Pages 769-775
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA rat cystatin A cDNA clone was isolated from a λ ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor. The obtained clone contained a partial coding region of the inhibitor, lacking the 5'-untranslated region and coding sequence for the NH, -terminal 13 residues. The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level. To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2. In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0.5 mg of the purified recombinant protein per 1 liter culture being produced. Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA. The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group. Using the expression system, two kinds of point mutation, the conservative Va154 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain. This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A.View full abstractDownload PDF (2256K) -
Nikhat Manzoor, Mohd. Amin, Luqman Ahmad Khan1999 Volume 126 Issue 4 Pages 776-780
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of ATP hydrolysis by plasma membrane H+-ATPase from Candida albicans has been investigated by following the kinetics of H+ liberation/absorption and the UV difference spectrum in a stopped flow spectrophotometer. A distinct pre-steady state phase of ATP hydrolysis could be defined. While the rapid mixing of P1 and ATPase produced no transient pH changes, the mixing of ADP leads to the release of 1 H+ per molecule of ATPase. Rapid mixing of ATP with ATPase releases about 2 H+ per molecule of ATPase, of which around 1.3 H+ are reabsorbed. The magnitudes of both H+ release and absorption were found to be independent of ATP concentration. The rate of H+ release (k1) shows ATP dependence while the rate of H+ absorption is independent of ATP concentration. The rate of H+ liberation with ADP, on a concentration basis, was far less as compared with ATP, indicating a low affinity of the ATPase for ADP. No change in the difference spectrum was observed with ADP. The stoichiometry of ATP binding to PM-ATPase was found to be unity from UV-difference spectrum studies. The kf values for H+ release and for the appearance of a difference spectrum following the addition of ATP were found to be similar beyond a 1:1 ratio of ATP:ATPase. The results obtained lead us to propose a 4-step kinetic scheme for the mechanism of ATP hydrolysis.View full abstractDownload PDF (397K) -
Akira Watanabe, Tohru Yoshimura, Bunzo Mikami, Nobuyoshi Esaki1999 Volume 126 Issue 4 Pages 781-786
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlanine racemase of Bacillus stearothermophilus has been proposed to catalyze alanine racemization by means of two catalytic bases: lysine 39 (K39) abstracting specifically the a-hydrogen of D-alanine and tyrosine 265 (Y265) playing the corresponding role for the antipode L-alanine. The role of K39 as indicated has already been verified [Watanabe, A., Kurokawa, Y., Yoshimura, T., Kurihara, T., Soda, K., and Esaki, N. (1999) J. Biol. Chem. 274, 4189-4194]. We here present evidence for the functioning of Y265 as the base catalyst specific to L-alanine. The Y265→Ala mutant enzyme (Y265A), like Y265S and Y265F, was a poor catalyst for alanine racemization. However, Y265A and Y265S catalyzed transamination with D-alanine much more rapidly than the wild-type enzyme, and the bound coenzyme, pyridoxal 5'-phosphate (PLP), was converted to pyridoxamine 5'-phosphate (PMP). The rate of transamination catalyzed by Y265F was about 9% of that by the wild-type enzyme. However, Y265A, Y265S, and Y265F were similar in that L-alanine was inert as a substrate in transamination. The apo-form of the wild-type enzyme catalyzes the abstraction of tritium non-specifically from both (4'S)- and (4'R)-[4'-3H] PMP in the presence of pyruvate. In contrast, apo-Y265A abstracts tritium virtually from only the R-isomer. This indicates that the side-chain of Y265 abstracts the α-hydrogen of L-alanine and transfers it supra-facially to the pro-S position at C-4' of PMP. Y265 is the counterpart residue to K39 that transfers the a-hydrogen of D-alanine to the pro-R position of PMP.View full abstractDownload PDF (519K) -
Osamu Sato, Yasuo Ogawa1999 Volume 126 Issue 4 Pages 787-797
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRegulation of the enzymatic activity of heterotrimeric smooth muscle myosin phosphatase (SMMP) by MgATP was examined using phosphorylated myosin (P-myosin), heavy meromyosin (P-HMM), subfragment-1 (P-Si), and 20 kDa myosin light chain (P-MLC20) as substrates. The activity toward P-myosin and P-HMM was dose-dependently reduced by MgATP, whereas that toward P-Sl or P-MLC20 was unchanged. The reduction was mainly due to a decrease in the affinity of SMMP for the substrate with the unchanged maximum activity. This regulation is entirely new in the respect that the responsible molecule is the substrate, not SMMP. Because P-myosin derived from myosin stored in 50% glycerol at -20°C was insensitive to MgATP, the proper integrity of P-myosin is required. Coexisting myosin did not affect this regulation, but it inhibited the SMMP activity in the absence of MgATP. With P-myosin, the enzyme activity was biphasically steeply dependent on the ionic strength. This requires that determinations are conducted with a fixed ionic strength. The Q10 value was about 2, which was quite similar to that for myosin light chain kinase. These results suggest that the rate of dephosphorylation of P-myosin is lowered at rest, but that it may reach a value comparable to the rate of phosphorylation of myosin in the sarcoplasm with the increased level of P-myosin during muscle activation. This regulation by MgATP may underlie the “latch mechanism” in some respects.View full abstractDownload PDF (1028K)
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