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Volume 125, Issue 1
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Sakihito Kitajima, Fumihiko Sato1999 Volume 125 Issue 1 Pages 1-8
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHigher plants accumulate several kinds of “pathogenesis-related (PR)” proteins in response to infection by pathogens such as fungi or viruses. Gene expression of one group of PR proteins is known to be mediated by phytohormone ethylene. Here we describe the signal transduction system from the ethylene receptor ETR to transcription factors, ERFs. Ethylene-inducible PR genes are expressed constitutively in roots and cultured cells even when are not infected. We discuss the mechanisms of this pathogen-independent expression of PR genes and describe recent findings in the study of molecular mechanisms of antifungal activities of the PR proteins. Genes of PR-1 and -5 proteins have now been identified in the genomes of various species of organisms, including humans and nematodes. PR proteins may contribute to the innate immunity of plants as well as to that of other organisms.View full abstractDownload PDF (3057K) -
Masahiro Kawabata, Kohei Miyazono1999 Volume 125 Issue 1 Pages 9-16
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembers of the TGF-β superfamily regulate the growth and differentiation of various types of cells. Smads are recently identified proteins that mediate intracellular signaling of the TGF-β superfamily. Smads are grouped into three classes depending on their structure and functions. R-Smads are phosphorylated by type I serine-threonine kinase receptors for TGF-β superfamily members. R-Smads then associate with Co-Smads. Smad4 is the only vertebrate Co-Smad identified thus far, and is required for the signaling pathways of different ligands. The heteromeric Smad complex translocates into the nucleus, where it activates target genes. Anti-Smads inhibit signaling by R-Smads and Co-Smads. Smads bind to DNA directly or indirectly via other DNA binding proteins. R-Smads interact with transcriptional coactivators, and have intrinsic transactivation activity. Elucidation of the functions of Smads will provide the framework for research on TGF-β superfamily signaling.View full abstractDownload PDF (1802K) -
Sachiko Hashimoto, Man Xu, Yutaka Masuda, Toshihiro Aiuchi, Shigeo Nak ...1999 Volume 125 Issue 1 Pages 17-23
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-Hydroxyisovalerylshikonin (β-HIVS), which was isolated from the plant, Lithospermium radix, inhibited the growth of various lines of cancer cells derived from human solid, tumors at low concentrations between 10-8 and 10-6M. When HL-60 cells were treated with 10-6M β-HIVS for 3 h, characteristic features of apoptosis, such as DNA fragmentation, nuclear fragmentation, and activation of caspase-3-like activity, were observed. The most characteristic features of the effect of β-HIVS were the remarkable morphological changes induced upon treatment of HL-60 cells with β-HIVS, as visualized on the staining of actin filaments with phalloidin labeled with tetramethylrhodamine B isothiocyanate. Moreover, activation of MAP kinases, such as ERK2, JNK and p38, was detected after treatment with 10-6M β-HIVS preceding the appearance of the characteristics of apoptosis, and the features of the activation of these MAP kinases were quite different from those of Fas and anticancer drug-induced apoptosis. The activation of JNK by β-HIVS was not inhibited by inhibitors of caspases, suggesting that JNK is located either upstream or independent of the caspase signaling pathway. β-HIVS did not inhibit the activity of topoisomerase II. These results indicate that β-HIVS induces apoptosis in HL-60 cells through a mechanism unlike those reported for anti-Fas antibodies and etoposide.View full abstractDownload PDF (3025K) -
Hirofumi Komori, Noriaki Sasai, Fujihiko Matsunaga, Chieko Wada, Kunio ...1999 Volume 125 Issue 1 Pages 24-26
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA replication initiator protein (RepE54) complexed with iteron DNA at its binding site was crystallized by the hanging drop vapor diffusion method. The crystals belong to monoclinic space group C2 with unit cell dimensions of a=108.4 Å, b=81.9 Å, c=73.9 Å, and β=121.5°, where one molecule of the protein-DNA complex exists per asymmetric unit. They diffract X-rays up to 2.6 Å resolution with synchrotron radiation.View full abstractDownload PDF (2277K) -
Teruaki Akao, Shuichi Hashimoto, Kyoichi Kobashi, Yuji Hidakat1999 Volume 125 Issue 1 Pages 27-30
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA peptide has been isolated from pronase digest of bovine serum albumin as the stimulatory factor of streptolysin S (SLS) production by Streptococcus pyogenes, and its primary structure has been deduced [Akao et al. (1992) Infect. Immun. 60, 4777-4780]. To determine the essential structure for the stimulation, a peptide (P-1) having the deduced structure, in which three peptide fragments are linked by two disulfide bonds, and shorter analogs (P-2 to P-4) of peptide P-1 were chemically synthesized. Another peptide (P-5), in which Ala is, inserted between the two Cys residues in the middle peptide chain of P-1, was also synthesized. These synthetic peptides were identified by mass spectrometry and analysis of amino acid compositions. The synthetic P-i stimulated SLS production in a dose-dependent manner. Other peptide analogs also showed remarkable stimulation of SLS production. Treatment of P-1 with performic acid resulted in loss of its stimulatory activity, indicating that disulfide bridges of the peptides are necessary for their activity on SLS production. These results suggest that the unique primary structure of three peptide chains linked by two disulfide bridges is requisite for the stimulatory effect on SLS production.View full abstractDownload PDF (486K) -
Kiyotaka Oshikawa, Shigeyuki Terada1999 Volume 125 Issue 1 Pages 31-35
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo platelet aggregation inhibitors, ussuristatin 1(US-1) and 2 (US-2), were newly isolated from the venom of Chinese viper (Agkistrodon ussuriensis) by SP-Toyopearl 650M column chromatography and reverse-phase HPLC. The M1-s of these polypeptides were estimated to be about 8, 000 by SDS-PAGE. Analytical gel filtration revealed that US-2 exists as a dimer. Both polypeptides comprised 71 amino acids, whose sequences showed high similarities to those of other disintegrins. US-1 had a typical Arg-Gly-Asp (RGD) sequence, which is responsible for blocking the binding of fibrinogen to the receptor. In US-2, the corresponding sequence was Lys-Gly-Asp (KGD). US-1 strongly suppressed platelet aggregation induced by ADP, collagen, thrombin, and epinephrine with IC50=17-33 nM. US-2 also inhibited the platelet aggregation, but the IC50s were about ten times higher. US-1 also dose-dependently inhibited the adhesion of human melanoma cells to fibrinogen and fibronectin, while US-2 did not inhibit the cell adhesion to fibronectin. This indicates that the KGD-bearing disintegrin is a specific inhibitor for the fibrinogen receptor.View full abstractDownload PDF (1405K) -
Ken Yanagisawa, Akiko Kosaka, Hiroyuki Iwahana, Makoto Nakanishi, Shin ...1999 Volume 125 Issue 1 Pages 36-40
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSContact inhibition is a well-known phenomenon, but the details of its mechanism are poorly understood. Recently, cyclin-dependent kinase inhibitors have been studied intensively with respect to their regulatory role in the cell cycle, and of them, p27Klp1 is particularly involved in contact inhibition. p27Klp1 is believed to be regulated primarily through posttranscriptional mechanisms. We now report that cyclin-dependent kinase inhibitors, including p27, are regulated differently at the mRNA level during contact inhibition in murine BALB/c-3T3 fibroblasts. The mRNA expression of p15, p16, and p27 was up-regulated as the cell density increased, but, on the contrary, the mRNA level of p21clp1/WAF1/Sdl1 markedly decreased when the cells became confluent. The protein levels of these genes were regulated in the same way as their mRNA levels, and cyclin-dependent kinase-2 activity was markedly inhibited on density-mediated growth arrest of the cells. These results indicate that the regulation of mRNA expression of cyclin-dependent kinase inhibitors appears to contribute to their protein levels and to the arrest of cell growth through contact inhibition.View full abstractDownload PDF (3861K) -
Yorishige Imamura, Toyohiko Migita, Masaki Otagiri, Tominari Choshi, S ...1999 Volume 125 Issue 1 Pages 41-47
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme responsible for the ketone-reduction of 4-benzoylpyridine (4BP) was purified 350-fold to homogeneity from the cytosolic fraction of rabbit heart. The purified enzyme exhibited a molecular mass of 110 kDa on gel filtration, and 27 kDa on SDS-PAGE, indicating that it is a tetrameric protein composed of four identical-size subunits. Aromatic aldehydes, ketones, and menadione were effective substrates for the enzyme. Flavonoids were potent inhibitors of the enzyme, but barbiturates or pyrazole was not. Based on this substrate specificity and inhibitor sensitivity, the enzyme was taken to be a carbonyl reductase. Kinetic studies led us to conclude that the reduction of 4BP by the enzyme follows an ordered Bi Bi mechanism. The enzyme also appeared to catalyze the redox (oxidation-reduction) cycling of menadione to produce the superoxide radical. Further-more, we provide evidence that a hydrophobic pocket, which corresponds to a straight-chain alkyl group of five carbon atoms in length, is located in the substrate-binding site of the enzyme.View full abstractDownload PDF (814K) -
Takanori Satoh, Hiroshi Shinoda, Keisuke Ishii, Masayuki Koyama, Nobuh ...1999 Volume 125 Issue 1 Pages 48-57
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete primary structure of inorganic pyrophosphatase [EC 3. 6. 1. 1] from Bacillus stearothermophilus (ATCC 12016) was determined at the amino acid level by automated Edman degradation. The subunit of the enzyme consists of 164 amino acid residues with a calculated molecular mass of 18, 796. The amino acid sequence of the enzyme is almost identical to that of thermophilic bacterium PS-3. Based on the determined primary structure, a PCR-amplified semi-synthetic gene was constructed and expressed in Escherichia coli JM109. The recombinant Bst. PPase showed the same characteristics and activity as the authentic enzyme, and exhibits higher thermostability than the E. coli enzyme. Furthermore, we prepared tyrosine-substituted variants by site-directed mutagenesis to elucidate the role of two highly conserved tyrosines (Y46 and Y130). As a result, two variants, Y46F and Y130F, lost most of their enzyme activity, whereas their conformations were unaffected. However, the wild-type and two variants exhibited different thermo-stability behaviors in the presence or absence of Mg2+. Therefore, these tyrosines may contribute to the structural integrity of the active site of the enzyme.View full abstractDownload PDF (3248K) -
Hiroshi Shinoda, Manabu Hattori, Atsushi Shimizu, Tatsuya Samejima, Ta ...1999 Volume 125 Issue 1 Pages 58-63
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the role of Va175 in the oligomeric structure of trimeric inorganic pyrophosphatase (PPase) [EC 3. 6. 1. 1] from Bacillus stearothermophilus (Bst.), we used site-directed mutagenesis to prepare variants in which Va175 was replaced by Ala, Phe, Leu, Ile, Lys, Gln, and Asp. As a result, the variants in which valine is replaced by hydrophobic residues such as Ala, Phe, Leu, and Ile (V75A, F, L, and I) show almost the same level of enzyme activity and thermostability as the wild type enzyme, whereas variants with hydrophilic residue replacements such as Lys, Gln, and Asp (V75K, Q, and D) showed gross reductions in enzyme activity and thermostability. The dissociation of V75K and V75D from trimer to monomers occurred rapidly as the temperature rose, while V75F, V75L, and V751 dissociated more slowly than the wild type. There was no particular effect of heat treatment on the dissociation of V75A or V75Q, but these variants were slightly dissociated even in the native state. Thus, we conclude that Va175 may locate at the interface between the monomers and its hydrophobic interactions with its surroundings may play a key role in the thermostability and oligomeric subunit interactions of the enzyme.View full abstractDownload PDF (1669K) -
Shigeyuki Terada, Junko Hori, Satoshi Fujimura, Eiji Kimoto1999 Volume 125 Issue 1 Pages 64-69
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA non-hemorrhagic proteinase, brevilysin L6 (L6), has been purified to homogeneity from Agkistrodon halys brevicaudus venom by gel filtration and DEAE-Toyopearl 650M chromatography. It is an acidic protein with an isoelectric point of 4.8, and its molecular mass was estimated to be 21.5 kDa by SDS-PAGE. The optimum pH of L6 was about 9. EDTA and o-phenanthroline inhibited the proteolytic activity, suggesting that L6 is a metalloprotease. Cysteine also inhibited the activity of L6, but glutathione did not. The protein was stable in the pH range of 5-8.5 and below 40°C. Calcium ions had no effect on the proteolytic activity of L6 or on its thermal stability. The enzyme preferentially cleaved X-Leu, X-Phe, X-Val, and X-His bonds. L6 showed weak α-fibrinogenase activity. The complete amino acid sequence of L6 was also determined by manual Edman degradation. L6 is a nonglycosylated single-chain polypeptide consisting of 203 residues with an N-terminal pyroglutamic acid and a calculated molecular weight of 22, 713 Da. Its entire sequence is highly homologous to those of other metalloproteases from various snake venoms. A zinc-binding motif, HEXXHXXGXXH, is located at residues 143-153 in the sequence of L6.View full abstractDownload PDF (2340K) -
Kazuma Iwai, Tomohiro Mega, Sumihiro Hase1999 Volume 125 Issue 1 Pages 70-74
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe presence of free oligomannosides in cytosol has been demonstrated by metabolic radiolabeling, and Manα1-2Manα1-2Manα1-3(Manα1-6)Manβ1-4GlcNAc was detected as the main free oligomannoside in Chinese hamster ovary cells [Kmiécik, D., Herman, V., Stroop, C. J. M., Michalski, J. C., Mir, A. M., Labiau, O., Verbert, A., and Cacan, R. (1995) Glycobiology, 5, 483-494]. In the present paper, the isomeric structures and amounts of oligomannosides in the cytosol fraction of hen oviduct were analyzed by pyridylamination and exoglycosidase digestion. Hen oviduct was used since our group has already characterized the cytosolic neutral α-mannosidase and endo-β-N-acetylglucosaminidase obtained from the same source. The amounts of Man2GlcNAc, Man3GlcNAc, Man4GlcNAc, and Man5GlcNAc were 0.6, 0.6, 0.5, and 0.8 nmol/g tissue, respectively, but Man6GlcNAc-Man9GlcNAc were not detected. The isomeric structures of the Man3GlcNAc-Man5GlcNAc found were (Manα1-2)0-2Manα1-3(Manα1-6)Manβ1-4GlcNAc, which were compatible with the substrate specificities of cytosolic endo-β-N-acetylglucosaminidase and neutral α-mannosidase, indicating that these enzymes participate in the formation of the oligomannosides present in the cytosol.View full abstractDownload PDF (1792K) -
Shoji Narutaki, Ben M. Dunn, Kohei Oda1999 Volume 125 Issue 1 Pages 75-81
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPseudomonas sp. 101 carboxyl proteinase (PCP) and Xanthomonas sp. T-22 carboxyl proteinase (XCP), the first and second unique carboxyl proteinases from prokaryotes to be isolated and characterized, are not inhibited by the classical carboxyl proteinase inhibitor pepstatin. In this study, we elucidated their subsite preferences by using a series of synthetic chromogenic substrates, Lys-Pro-Ile(P3)-Glu(P2)-Phe*Nph-Arg(P2')-Leu(P3') (Nph is p-nitrophenylalanine, Phe*Nph is the cleavage site) with systematic substitutions at the P3, P2, P2', and P3' positions. Among 45 substrates tested, the best substrate for PCP had a Leu replacement at the P2 position (kcat=27.2 s-1, Km=4.22 μM, kcat/Km=6.43 μM-1•s-1), and that for XCP had an Ala replacement at the P3 position (kcat=79.4 s-1, Km=6.05 μM, kcat/Km=13.1 μM-1•s-1). PCP and XCP preferred such charged amino acid residues as Glu, Asp, Arg, or Lys at the P2' position. This suggested that the S2' subsites of PCP and XCP are occupied by hydrophilic residues, similar to that of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [Shibata et al. (1998) J. Biochem. 124, 642-647]. In contrast, the S2' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydophilic nature of the S2' subsite appears to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases.View full abstractDownload PDF (861K) -
Kazutomo Toyofuku, Ikuo Wada, Kuninori Hirosaki, Jong Sung Park, Yoshi ...1999 Volume 125 Issue 1 Pages 82-89
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced _??_2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When α-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immuno-precipitation of calnexin from extracts of [35S] methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.View full abstractDownload PDF (4364K) -
Hikaru Sakamoto, Jun Kitahara, Yasuhito Nakagawa1999 Volume 125 Issue 1 Pages 90-95
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe liberation of arachidonic acid and the production of prostaglandin D2 (PGD2) were significantly influenced by peroxide and the level of intracellular glutathione (GSH). The productions of free arachidonic acid and PGD2 in RBL-2H3 cells were enhanced considerably by exposure to tert-butyl hydroperoxide (t-BHP). The liberation of arachidonic acid induced by t-BHP was not inhibited by EGTA. The productions of PGD2 and arachidonic acid induced by t-BHP were significantly facilitated by the depletion of intracellular GSH using buthionine sulfoximine (BSO) or diethyl maleate (DEM), although the depletion of GSH had no effect on the production of PGD2 induced by A23187. t-BHP failed to activate the conversion of free arachidonic acid to PGD2, since the formation of PGD2 from exogenously added arachidonic acid was not enhanced by treatment with t-BHP. The level of lipid hydroperoxides in t-BHP-treated cells was significantly elevated by treatment with DEM. These results suggest that hydroperoxides increase the free arachidonic acid available for the synthesis of PGD2 by activating phospholipase A2 (PLA2) and that the depletion of GSH by DEM accelerates the activation of PLA2 by raising peroxide levels in cells. Thus, the observed alterations in GSH levels are large enough to cause increased PGD2 synthesis in RBL-2H3 cells exposed to oxidative stress.View full abstractDownload PDF (2131K) -
Takashi Sato, Tadashi Kageura, Tsutomu Hashizume, Misako Hayama, Kazuy ...1999 Volume 125 Issue 1 Pages 96-102
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo study the involvement of sphingolipids in glycerophospholipid metabolism, the contribution of ceramide to the activation of group IV cytosolic phospholipase A2 (cPLA2) was investigated in platelets using cell-permeable C6-ceramide (N-hexanoylsphingosine). The addition of ceramide led to potentiation of thrombin-induced activation of cPLA2 and mitogen-activated protein kinase (MAPK) as well as arachidonic acid release and lysophos-phatidyicholine formation. However, ceramide by itself did not induce any response. The arachidonic acid release due to the synergistic action of ceramide and thrombin was inhibited by PD98059, a MAPK kinase inhibitor. Ceramide also stimulated thrombin-induced protein kinase C (PKC) activation, but ceramide by itself failed to do so. Further-more, ceramide synergistically enhanced diacylglycerol (DAG) formation and Ca2+ mobilization with thrombin, and also DAG formation with Ca2+-ionophore A23187. The DAG formation in response to ceramide with thrombin or A23187, as well as arachidonic acid release with thrombin were completely inhibited by U73122, a phospholipase C (PLC) inhibitor. These results suggest that ceramide triggers PLC activation through its synergistic action with thrombin, and subsequently potentiates the sequential PKC-MAPK cascade-cPLA2 pathway, thus resulting in enhancement of arachidonic acid release.View full abstractDownload PDF (953K) -
Atsuto Inoue, Tomoko Hayakawa, Eri Otsuka, Asako Kamiya, Yoshirou Suzu ...1999 Volume 125 Issue 1 Pages 103-108
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe reported previously that C-type natriuretic peptide (CNP) promotes the differentiation and mineralization of osteoblastic cells [Am. J. Physiol. 270 (Cell Physiol. 39): C1311-C1318, 1996]. However, little information is available about the mechanism of action of CNP in differentiating osteoblastic cells. Using the technique known as differential display-polymerase chain reaction, we attempted to identify the mRNAs whose levels are regulated by CNP in mouse clonal preosteoblastic MC3T3-E1 cells. One species of mRNA whose level was increased by CNP was 99% homologous to the 3'-untranslated region of a mouse gene for biglycan (BGN), a small proteoglycan. BGN is known to be involved in bone formation by osteoblastic cells. Therefore, we investigated the relationship, during the formation of mineralized nodules, between CNP and BGN using calvarial osteoblast-like cells (ROB cells) from newborn rats, that are a good model for studies on bone formation in vitro. Northern blot analysis revealed that transcription of the mRNA for BGN was up-regulated by CNP in ROB cells on days 6 and 8, whereas no effect of CNP was observed on days 3 and 12. Brief treatment with 10-7M CNP on days 3 through 9 exclusively enhanced the deposition of calcium, a result that suggests that CNP might regulate the expression of mineralization-related genes and, probably, the gene for BGN during a specific time period.View full abstractDownload PDF (2621K) -
Hiroshi Hamana, Takao Shinozawa1999 Volume 125 Issue 1 Pages 109-114
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the role of the C-terminal region on the activity and thermostability of orotate phosphoribosyltransferase (OPRTase, EC 2. 4. 2. 10) from Thermus thermophilus, four C-terminal amino acid-deleted OPRTases (1, 2, 3, and 5 residues deleted) were constructed. The activities of all the mutant OPRTases were lower than that of wild-type OPRTase at all temperatures investigated (50-80°C). V- and EV-OPRTase, mutants with Val and Glu-Val deletions, respectively, showed 63 to 75% of the activity of wild-type OPRTase at the temperatures investigated. EEV- and PLEEV-OPRTase, with Glu-Glu-Val and Pro-Leu-Glu-Glu-Val deletions, respectively, had activities of 22 to 35% of the wild-type. The Km values for orotate of all mutant OPRTases were more than 4-fold higher than that of the wild-type (25 μM). On the other hand, the Km for PRPP of the wild-type was 34 μM, and there were no significant differences between the wild-type and mutant OPRTases. The kcat values of the V- and EV-OPRTases were similar to that of the wild-type, but those of the EEV- and PLEEV-OPRTases were less than 50% that of the wild-type. The optimum temperature of all mutant OPRTases, 70°C, was 10°C lower than that of the wild-type. The remaining activities of wild-type and V-OPRTase after incubation at 90βC for 20min were 70 and 60% of the non-treated OPRTase activity, respectively. Although the remaining activity of EV-OPRTase was only 14% of the non-treated OPRTase activity, the addition of 200mM KCl during heat treatment increased it to 70%. Circular dichroism spectroscopy revealed that V- and EV-OPRTase denature more easily than the wild-type OPRTase. The results suggest that the C-terminal valine and glutamic acid residues are important for the activity and thermostability of T. thermophilus OPRTase.View full abstractDownload PDF (1681K) -
Katsuya Baba, Takako K. Abe, Susumu Tsunasawa, Shoji Odani1999 Volume 125 Issue 1 Pages 115-122
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFatty acid-binding protein (FABP) was purified from the liver of the Amphibia, Rana catesbeiana, by gel filtration and ion-exchange chromatography. The complete primary structure of the frog liver FABP was determined by protein analysis. Two isoforms, I and II, were separated by reversed phase HPLC, and found to differ by 10 atomic mass units as measured by ion-spray ionization mass spectrometry. A detailed analysis of enzymatic peptides revealed a single Pro (isoform I)/Ser (isoform II) replacement at position 16. It seems remarkable that a rather neutral point mutation results in the nearly complete separation of the two isoforms by reversed phase chromatography. Homology modeling suggests the location of this site on the first helix of the helix-turn-helix domain and the presence of a single thiol group of cysteine-91 at the inside of the ligand-binding cavity. Binding studies using a natural fluorescent fatty acid, cis-parinaric acid, showed a lower Kd value for the serine form and large enhancement of fluorescence intensity upon glutathione-thiolation at cysteine-91. Examination of phylogenetic relationships identified the frog liver protein as a mammalian liver type FABP, and suggested a change in the vertebrate liver FABP gene expression at the bony fish/cartilagenous fish boundary.View full abstractDownload PDF (1045K) -
Kunihiko Itoh, Osamu Nakagomi, Kaori Suzuki, Kazuyuki Inoue, Hitoshi T ...1999 Volume 125 Issue 1 Pages 123-129
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe prepared and characterized human monoclonal Fab fragments to rotavirus from IgG1 κ combinatorial libraries (designated as N and O) constructed from peripheral blood lymphocytes (PBLs) from two healthy individuals. Approximately 30-fold enrichment in eluted phage was obtained in these libraries after five rounds of panning against rabbit polyclonal antibody-captured human rotavirus (HRV) Wa strain. Forty-eight clones from each library. were tested for reactivity to HRV Wa in an enzyme-linked immunosorbent assay (ELISA), and the identities of positive clones were determined by BstNI fingerprinting. As a result, eight individual clones (five from N library and three from O library) were isolated. In testing the cross-reactivity of Fabs against a panel of self- or non-self antigens, all Fab clones were found to be specific for HRV Wa. Fab clones from the two libraries showed distinct characteristics with respect to their reaction patterns with Wa and crossreactivities with rotavirus strains, and displayed variable heavy (VH) chain gene usage, although they recognized the VP6 protein as determined by immunoblotting. The distinct epitope recognition by Fabs from two libraries suggests different courses of humoral immune response to rotavirus during infection in the two individuals.View full abstractDownload PDF (1821K) -
Yumiko Saito, Tomoko Yamanushi, Toshihiko Oka, Akihiko Nakano1999 Volume 125 Issue 1 Pages 130-137
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe yeast SAR1 gene encodes a low-molecular-weight GTPase which is essential for the formation of transport vesicles from the endoplasmic reticulum (ER). To understand how the Sar1p function is regulated in its GTPase cycle, we searched for multicopy suppressors of sar1 temperature-sensitive mutants and identified SEC12, SED4, truncated SEC16, and EKS1. EKS1 turns out to be identical to HRD3, which was independently isolated as a gene implicated in the degradation of an HMG-CoA reductase isozyme, Hmg2p. In this paper, we show that the product of EKS1/HRD3 is a type-I transmembrane glycoprotein and resides in the ER. The eks1/hrd3 disrupted cells are normal in growth and transport of cargo proteins, but missecrete BiP (Kar2p). The overexpression of EKS1/HRD3, which stabilizes Hmg2p, did not affect the stability of wild-type or mutant Sarlp or any early Sec proteins we examined. These results suggest that the role of Ekslp/Hrd3p is not involved in the ER protein degradation in general but rather required for the maintenance of the ER membrane functions. The novel genetic interactions unveiled between SAR1, SEC12, SEC16, and SED4 will provide useful information as to how the complex machinery of vesicle budding operates.View full abstractDownload PDF (3771K) -
Hideto Miyoshi, Kazuhiro Takegami, Kimitoshi Sakamoto, Tatsushi Mogi, ...1999 Volume 125 Issue 1 Pages 138-142
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNatural aurachin C is the most potent inhibitor of oxidation of ubiquinols by cytochromes bo and bd from Escherichia coli. To probe the structural properties of the substrate oxidation site in the ubiquinol oxidases, we synthesized a systematic set of aurachin C analogues (N-hydroxy-4-quinolone derivatives) and examined how their structure affects their activity towards cytochromes bo and bd, which are structurally unrelated. We found that the presence of the 3-methyl group of the 2-n-decyl and 2-n-undecyl derivatives increased the inhibitory potency towards both enzymes, probably due to a local steric congestion that allows favorable interaction of the alkyl tail with the enzyme. Increase in the chain length of the 3-alkyl tail of the 2-n-undecyl derivatives decreased the inhibitory potency only in cytochrome bo, indicating that the binding site for the alkyl tails of cytochrome bo is smaller than that of cytochrome bd. Based on these findings, we discuss the differences in the molecular mechanism of substrate oxidation by these two terminal ubiquinol oxidases.View full abstractDownload PDF (616K) -
Takashi Wada, Toshio Yamazaki, Seiki Kuramitsu, Yoshimasa Kyogoku1999 Volume 125 Issue 1 Pages 143-150
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe region containing the RNA polymerase α subunit (RNAPα) gene (rpoA) and the ribosomal protein genes of a thermophilic eubacterial strain, Thermus thermophilus (Tt) HB8, was cloned from a genomic DNA library by Southern hybridization. The gene order in this region is rpl36-rps13-rps11-rps4-rpoA-rpl17, which is identical to that in some other eubacteria. The rpoA gene encodes a 315 amino acid residue protein with a molecular weight of 35, 013, the amino acid sequence showing 42% identity to that of Escherichia coli.(Ec). From the results of comparison of the amino acid sequence and the predicted secondary structure of the C-terminal domain of Tt RNAPα (Tt αCTD) with those of Ec, the overall folding is expected to be similar. However, amino acid residues Asn268 and Cys269 in Ec αCTD, which are essential for its interaction with DNA or regulatory proteins, were replaced by His and Ser, respectively, in Tt αCTD. By means of a T7-based expression system in Ec cells, Tt RNAPa was overexpressed and purified. The high thermostability of Tt RNAPα was demonstrated by the CD spectra.View full abstractDownload PDF (1563K) -
Keigo Bunai, Kouhei Yamada, Kenji Hayashi, Kouji Nakamura, Kunio Yaman ...1999 Volume 125 Issue 1 Pages 151-159
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe precursors of β-laetamase fusion proteins having the signal peptide of Bacillus subtilis alkaline protease (pAprE-B1aH6) or penicillin binding protein 5* (pPBP5*-BlaH6) accumulated in B. subtilis cells in the absence of SecA or Ffh. Using the five purified precursors of secretory proteins including the two fusion proteins, B. subtilis Ffh and SecA, we analyzed the protein targeting mechanism of B. subtilis in vitro. B. subtilis SecA recognized the completely translated precursors of secretory proteins to which Ffh also bound. Moreover, B. subtilis SecA-precursor complex formation was enhanced 15- to 30-fold when the precursor and Ffh were incubated first and then SecA was added, but not vice versa. We also found that B. subtilis SecA directly interacted with Ffh in vitro. These results indicate that B. subtilis SecA and Ffh interact to function cooperatively in a protein translocation pathway including other protein factors, and that Ffh, as well as SecB in Escherichia coli, enhances the binding of SecA to presecretory proteins in B. subtilis cells.View full abstractDownload PDF (4052K) -
Yoichiro Hama, Hiroki Nakagawa, Kiyonori Mochizuki, Toshihisa Sumi, Hi ...1999 Volume 125 Issue 1 Pages 160-165
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis paper describes a new HPLC method for the sugar analysis of algal galactans containing 3, 6-anhydrogalactose, which is readily destroyed during usual acid hydrolysis and methanolysis. By anhydrous mercaptolysis of galactans at 60°C for 6 h with the newly developed solvent system, 0.5 N HCl/[ethanethiol:methanol (2:1, v/v)], component sugars, including 3, 6-anhydrogalactose, were liberated quantitatively as their diethyl dithioacetals. The resultant sugar diethyl dithioacetals were found to have strong UV-absorption (absorption maximum of 3, 6-anhydrogalactose diethyl dithioacetal in water, 191-192 nm; molar extinction coefficient, 4, 400). The sugar diethyl dithioacetals were then resolved simultaneously within 15min by reversed phase HPLC with 30% acetonitrile as the eluent and detected by UV-absorption at 215 nm. Amounts of sugar diethyl dithioacetals less than 50 pmol can be determined without further derivatization.View full abstractDownload PDF (685K) -
Yoko Shiga, Shunichi Kametani, Takeshi Kadokura, Hiroshi Akanuma1999 Volume 125 Issue 1 Pages 166-172
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycogen is a storage compound that provides both carbon and energy, but the mechanism for the regulation of its metabolism has not been fully clarified. Recently, we found a new glycogenolytic pathway in rat liver in which glycogen is first metabolized to 1, 5-anhydrofructose (AnFru) and then to 1, 5-anhydroglucitol (AnGlc-ol). Because the amounts of glycogen and AnFru are closely related in various rat organs and the second reaction, AnFru to AnGlc-ol, is strongly inhibited in the presence of glucose, we expected that this pathway might play a regulatory role in glycogen metabolism. Here we evaluate the expected involvement of AnGle-ol and AnFru in the regulatory mechanism in Escherichia coli C600. Having established the presence of this new glycogenolytic pathway in E. coli C600, we further show that the conversion of AnFru to AnGlc-ol is activated only after the exhaustion of glucose in the medium, and that as little as 5 μM AnGlc-ol in the medium acutely accelerates the degradation of glycogen by 40%. We consider the role of AnGle-ol in the mechanism that controls glycogen metabolism in E. coli to be as follows. When glucose is abundant, E. coli accumulate glycogen, a fraction of which is steadily degraded so that the amount of AnFru is about 1/1, 000 of glycogen on a weight basis. When glucose is depleted and the demand for glycogen utilization is elevated, AnFru, which has accumulated mostly in the medium, is promptly taken up and converted to AnGle-ol, which accelerates glycogen degradation. We also suggest the possibility that AnGlc-ol is one of the extracellular signaling molecules for bacteria.View full abstractDownload PDF (863K) -
Kyoko Tanaka, Hiroshi Iguchi, Shigeru Taketani, Rieko Nakata, Sadako T ...1999 Volume 125 Issue 1 Pages 173-176
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA facile cleavage of peptide bonds of apolipoprotein B (apoB) by radical reaction is reported. When human LDL was subjected to oxidative damage using Cu2+, extensive degradation of apoB was observed based on immunoblotting. The degradation of apoB was inhibited by radical scavengers (β-mercaptoethanol, butylated hydroxytoluene, and probucol) and promoted by a radical initiator [2, 2'-azobis(2-amidinopropane)dihydrochloride]. When human serum was treated with Cu2+, a similar cleavage pattern of apoB was observed. The cleaved apoB proteins were also detected in normal serum on the basis of immunoblots. These results suggest that apoB is highly reactive toward radicals in vitro and in vivo, with reaction resulting in the cleavage of peptide bonds.View full abstractDownload PDF (1780K) -
Shinsaku Maruta, Yasuo Uyehara, Kazuaki Homma, Yasunobu Sugimoto, Kats ...1999 Volume 125 Issue 1 Pages 177-185
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the presence of MgADP, a novel phosphate analogue of gallium fluoride (GaFn) forms a ternary complex with the myosin subfragment-1 (S-1), in the same way that has been previously reported with aluminum fluoride (AIF4-), beryllium fluoride (BeFn), scandium fluoride (ScFn), and vanadate (Vi), and this complex formation may mimic different states along the ATPase kinetic pathway. This novel complex has been characterized and compared with other complexes to ascertain whether it forms a transition-state analogue of myosin ATPase. The complex formed quickly, although several times slower than the BeFn complex. The half-life of the myosin•ADP•GaFn complex was about 50h at 4°C. The formation of the myosin•ADP•GaFn complex was accompanied by an increase in trypto-phane fluorescence, similar to that observed upon the addition of ATP, but slightly lower than that of the M**•ADP•P_??_ complex. Upon addition of GaFn to acto-myosin•ADP, actomyosin did not dissociate, and the S-1•ADP•GaFn complex was scarcely decomposed by actin, like the AIF4- and ScFn complexes but unlike the BeFn and Vi complexes. The conformations at the localized region of SHl, SH2, and RLR, which are very accessible to the binding of ATP, were studied by fluorescent labeling and chemical modification, and the results suggested that these conformations are very similar to that of the M**•ADP•P_??_ state. Small-angle X-ray solution scattering showed that the radius of gyration value decreases by about 3 Å when S-1 forms an S-1•ADP•GaFn complex, suggesting that the shape of the complex becomes compact or rounded in shape, similar to that in the presence of ATP or complexes with other phosphate analogues, and thus mimics the myosin**•ADP•P_??_ state closely. The overall results may indicate that the complex mimics a somewhat different transient state from that of other complexes but has a similar global conformation along the ATPase kinetic pathway.View full abstractDownload PDF (1123K) -
An Azide-Insensitive Superoxide Dismutase from a Hyperthermophilic Archaeon, Sulfolobus solfataricusShigeyuki Yamano, Tadashi Maruyamat1999 Volume 125 Issue 1 Pages 186-193
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe superoxide dismutase (SOD) gene of Sulfolobus solfataricus, a hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized. When the protein was expressed in E. coli, it formed a homodimer that contained both Mn and Fe. Metal reconstitution experiments of the SOD with Fe or Mn showed that only the Fe-reconstituted SOD was active. Substitution of Tyr88 to Phe did not affect the metal specificity of the enzyme. The Fe-reconstituted SOD was extremely resistant to thermal denaturation; e.g. 96% of the initial activity was retained after heating at 95°C for 2 h. Fe-reconstituted SOD was not inhibited by azide, but fluoride inhibition was observed. This suggests that some steric hindrance in the substrate funnel of the enzyme prevents the access of N3- but allows O2- and F- access to the active site.View full abstractDownload PDF (2068K) -
Sakura Tsukita, Shuuji Koyanagi, Kumiko Nagata, Hiromasa Koizuka, Hiro ...1999 Volume 125 Issue 1 Pages 194-201
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHelicobacter pylori is a microaerophilic Gram-negative spiral bacterium residing in human stomach. A cb-type cytochrome c oxidase that terminates the respiratory chain was purified to near homogeneity by solubilizing the membranes with Triton X-100 and applying anion exchange, Cu-chelating, and gel filtration chromatographies. Redox- and CO-difference spectra and pyridine ferrohaemochrome analysis showed the enzyme to contain three haems C, one low-spin protohaem, and one high-spin protohaem that probably forms a dioxygen-reducing bimetalic center with a copper atom. The enzyme actively oxidizes soluble cytochrome c from this bacterium (TNmax of about 250 s-1) with a Km of 0.9 μM. Yeast cytochrome c and N, N, N', N'-tetramethyl p-phenylenediamine (TMPD) are also oxidized at similar maximal velocities with larger Km's. Oxygen pulse experiments on resting cells in the presence of ascorbate plus TMPD or L-lactate indicated that this sole terminal oxidase pumps H+, although the H+ pumping activity by proteoliposomes recon-stituted from the enzyme and P-lipids was low. Two main bands with haem C at 58 and 26 kDa were observed upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and succeeding protein and haem staining. Sequencing of the operon encoding the subunits of the enzyme revealed the presence of ccoNOQP. N-terminal analysis of the 58 kDa band showed 15 or 13 amino acids coinciding with the amino acid sequences deduced from the DNA of ccoN and ccoO. CcoN, the largest subunit bearing two protohaems and copper, and ccoO, a mono-haem cytochrome subunit form a protein complex with an apparent molecular mass of 58 kDa, even in the presence of sodium dodecyl sulfate. The 26 kDa band is tentatively assumed to be ccoP with two haems C.View full abstractDownload PDF (2151K) -
Kazumi Hiraga, Sailaja S. Seeram, Shin-ichi Tate, Naoki Tanaka, Masats ...1999 Volume 125 Issue 1 Pages 202-209
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStreptomyces metalloproteinase inhibitor (SMPI) is the only inhibitor to show “standard mechanism inhibition” against metalloproteinases. SMPI is a globular protein with an exposed loop containing the reactive site, C64-V65. To analyze the importance of basic residues in the reactive site loop of SMPI, mutants were constructed for R60, K61, and R66 (R60A, K61A, R66A, R60/K61A, 60/61/66A, and 60/61/66E). The mutants involving only R60, K61, and R60/K61 residues, respectively, showed strong inhibitory activity and were stable against enzyme activity. Both the triple mutants showed very weak inhibitory activity and underwent rapid degradation. The addition of basic residues to the loop (V62R and T63R) did not cause any further increase in inhibitory activity. These results suggest that basic residues in the reactive site loop play some role in maintaining a stable enzyme-inhibitor complex. The R66 mutant showed reduced activity and was rapidly degraded by enzymes. It was concluded that R66 is essential for maintaining a strong hydrophobic interaction with the Sl' hydrophobic pocket of the enzyme. To investigate the roles of the disulfide bridge and the P68 residue near the reactive site, C64/69S and P68T mutants were constructed. These mutants showed very weak inhibitory activity and were rapidly degraded by enzymes. These results suggest that the disulfide bridge and P68 residue are very essential for SMPI to function as an inhibitor.View full abstractDownload PDF (3557K) -
Masaaki Ito, Shoji Narutaki, Ken-ichi Uchida, Kohei Oda1999 Volume 125 Issue 1 Pages 210-216
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, is the first example from a prokaryote of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1, 2-epoxy-3(p-nitrophenoxy)propane. To identify the catalytic residue(s) of PCP, chemical modification was carried out using carboxyl residue-specific reagents, carbodiimides. PCP was inactivated effectively by N, N'-dicyclohexylcarbodiimide (DCCD) with pseudo-first-order kinetics. For the inactivation, 0.7 mol DCCD was involved per 1 mol PCP. The effects of pH and methanol on the inactivation showed that two carboxyl residues (Asp and/or Glu) were involved in the reaction. The inactivation by DCCD was prevented by a competitive inhibitor, tyrostatin, or a synthetic substrate in a concentration-dependent manner. Based on these data, differential labeling of PCP with DCCD was carried out: Firstly, PCP was treated with cold DCCD in the presence of tyrostatin. After removal of the tyrostatin, which covered the substrate binding site, by dialysis, the PCP was treated with [14C]DCCD to label carboxyl residue(s) essential for its function. Two labeled peptides were isolated by HPLC from a trypsin digest of cold- and [14C]DCCD modified PCP. On analysis of their amino acid sequences, it was revealed that the [14C]-DCCD was bound to Asp140 and Glu222 of PCP, respectively. Based on these data, it was strongly suggested that Asp140 and Glu222 of PCP were involved in its catalytic function or substrate binding.View full abstractDownload PDF (879K)
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