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Volume 124 (1998)
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- Issue 4 Pages 687-
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Volume 124, Issue 4
Displaying 1-26 of 26 articles from this issue
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Hideto Watanabe, Yoshihiko Yamada, Koji Kimata1998 Volume 124 Issue 4 Pages 687-693
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAggrecan, a large aggregating proteoglycan, is one of the major structural components of cartilage. Its core protein contains three glubular domains and two glycosaminoglycan-attachment domains. These domains play various roles to maintain cartilage structure and function. An N-terminal globular domain binds hyaluronan and link protein to form huge aggregates. The chondroitin sulfate (CS) chains attach to the CS domain and provide a hydrated, viscous gel that absorbs compressive load. Two autosomal recessive chondrodys-plasias, cartilage matrix deficiency (cmd) in mice and nanomelia in chicken are both caused by aggrecan gene mutations. Cmd homozygotes die shortly after birth, while the heterozygotes are born normal. However, cmd heterozygotes develop late onset of spinal disorder, which suggests aggrecan as a candidate gene predisposing individuals to spinal problems. Nanomelia is a useful model to elucidate intracellular trafficking of proteoglycans. Further studies on aggrecan will lead to prophylaxis and treatment of joint destructive diseases such as osteoarthrosis and to elucidation of cartilage development, which is essential for skeletal formation.View full abstractDownload PDF (3285K) -
Yuri Aoyama, Tadao Horiuchi, Osamu Gotoh, Mitsuhide Noshiro, Yuzo Yosh ...1998 Volume 124 Issue 4 Pages 694-696
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA CYP51-like gene (Z80226) of Mycobacterium tuberculosis was expressed in Escherichia coli. The product exhibited absorption spectra characteristic of P450. The expressed P450 formed a stoichiometric complex with ketoconazole, one of the specific ligands of CYP51. These findings indicate that the CYP51-like gene of M. tuberculosis actually encodes a P450 having active-site environments similar to those of CYP51, confirming the predicted orthologous nature of this gene to eukaryotic CYP51. Although eukaryotic CYP51s are membrane-binding proteins, the expressed product was accumulated only in the soluble fraction of the host cells.View full abstractDownload PDF (423K) -
Sachiko Okamoto, Yoshihiko Watanabe, Yoshinobu Takakura, Mitsuru Hashi ...1998 Volume 124 Issue 4 Pages 697-701
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDouble-stranded polyriboinosinic acid:polyribocytidylic acid (poly I:poly C) is a powerful inducer of type I interferons (IFNs). However, the dose of poly I:poly C required for efficient IFN induction is so high as occasionally to be cytotoxic. In this work, we examined the IFN-inducibility of poly I:poly C complexed with several cationic reagents in mouse fibroblast L cells and found that Lipofectin and LipofectACE can induce the production of a substantial amount of type I IFNs (mostly β-type) even at a two-order lower dose compared with poly I:poly C alone. Such effects of poly I:poly C were optimal at 0.1 μg/ml for 2-10 μg/ml of Lipofectin and LipofectACE. These conditions caused no significant cytotoxicity in the recipient cells. Furthermore, a short treatment (less than 10 min) with the complexes was sufficient for the maximum induction. This IFN induction method was applicable to other cell types and other species including human. Hence, our observations may pave the way for clinical application of the IFN inducer.View full abstractDownload PDF (1750K) -
Wei Luo, Wen-Zhang Xie, Ji-Hong Bai, Hai-Meng Zhou1998 Volume 124 Issue 4 Pages 702-706
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe toxicity of NO3- and NO2- to mammals has been widely publicized. However, the kinetic mechanism of inhibition of human muscle creatine kinase by NO3- and NO2- has not been explored. The kinetic theory of the substrate reaction during the modification of enzyme activity previously described by Tsou (Adv. Enzymol. Related Areas Mol. Biol. 1988, 61, 381-436) has been applied to a study of the kinetics of slow reversible inhibition of human muscle creatine kinase by planar anions (NO3- and NO2-). The kinetic equation of the substrate reaction was derived from theoretical analysis and experimental data, then simplified. The microscopic rate constants for the reaction of the inhibitors with the enzyme were obtained from the simplified equation for the substrate reaction in the presence of the inhibitors. The results show that the apparent forward rate constant A is dependent on ATP concentration, indicating competition between the inhibitor (NO3- or NO2-) and ATP. The results also suggest that binding of creatine-MgADP and the anion with the enzyme is very tight, since their binding constants are much higher than those for normal substrates.View full abstractDownload PDF (566K) -
Masahito Suiko, Yoichi Sakakibara, Rabia Awan-Khan, Hiroshi Sakaida, H ...1998 Volume 124 Issue 4 Pages 707-711
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUpon two-dimensional thin-layer separation, the sulfated L-3, 4-dihydroxyphenylalanine (L-DopaS) generated enzymatically was found to co-migrate with only one of the two ninhydrin-stained spots corresponding to the two sulfated forms (3-O-sulfate and 4-O-sulfate) of synthetic L-DopaS. To clarify precisely the identity of the enzymatically generated L-DopaS, the two sulfated forms of synthetic L-DopaS were separated and purified using high performance liquid chromatography. Purified L-Dopa 3-O-sulfate and L-Dopa 4-O-sulfate were identified by 1H-nuclear magnetic resonance (NMR) spectrometry and used as standards in the analysis of the L-DopaS generated during metabolic labeling of HepG2 human hepatoma cells or enzymatic assay using recombinant human monoamine (M)-form phenol sulfotransferase. The results obtained demonstrated unequivocally the generation of L-Dopa 3-O-sulfate, indicating the specificity of the M-form phenol sulfotransferase being for the meta-hydroxyl group of L-Dopa.View full abstractDownload PDF (1973K) -
Tadashi Ueda, Miyako Tsurumaru, Taiji Imoto1998 Volume 124 Issue 4 Pages 712-716
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method for evaluating the association and dissociation rate constants of interaction between a lysozyme and its substrate analogue, an immobilized p-aminophenyl-tri-N-ace-tyl-β-chitotrioside, by means of surface plasmon resonance has been developed. Site-speci-fic immobilization of p-aminophenyl-tri-N-acetyl-β-chitotrioside, which is a product of p-nitrophenyl-tri-N-acetyl-β-chitotrioside, on carboxymethyldextran linked to the sur-face of the cuvette of the instrument, IAsys, was carried out by catalysis with EDC/NHS. The kinetic parameters of the interaction between hen or human lysozyme and the immobilized substrate analogue indicated that a larger dissociation constant of the human lysozyme-immobilized substrate analogue complex depended on a smaller association rate constant. The kinetic parameters of the interaction between the immobilized substrate analogue and a mutant hen lysozyme, in which Argl4 and Hisl5 are deleted, with higher activity than the wild type hen lysozyme were measured. It was suggested that the higher activity of the mutant lysozyme was due to faster removal of the substrate from the active site cleft and/or the formation of a stabler and better complex as to hydrolysis.View full abstractDownload PDF (1777K) -
Shou Takashima, Hiroshi Iikura, Akira Nakamura, Makoto Hidaka, Haruhik ...1998 Volume 124 Issue 4 Pages 717-725
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn exoglucanase gene was cloned from a cellulolytic fungus, Humicola grisea. DNA sequencing of this gene, designated as exo1, revealed that it contained four introns in the coding region. The deduced amino acid sequence of EXO1 was 451 amino acids in length and showed 57.7% identity with that of H. grisea cellobiohydrolase 1 (CBH1), but lacked the typical domain structures of a cellulose-binding domain and a hinge region. Transcrip-tional analysis of the exo1 and cbh1 genes showed that the expression of these genes was induced by Avicel, and repressed in the presence of glucose. The exo1 gene was expressed in Aspergillus oryzae, and the recombinant EXO1 protein was purified. EXO1 and CBH1 produced by A. oryzae showed relatively higher activity toward Avicel, but showed much lower activity toward carboxymethyl cellulose (CMC) and p-nitrophenyl-β-D-cellobioside (PNPC), than H. grisea endoglucanase 1 (EGL1). The addition of a cellulose-binding domain and a hinge region to EXO1 caused decreases in its enzymatic activities as well as the deletion of the cellulose-binding domain from CBH1. EXO1 showed relatively weak or no synergistic activity toward Avicel with H. grisea endoglucanases, but showed a significant level of apparent synergism with H grisea CBH1 and Trichoderma reesei EGLI. CBH1 showed a significant level of apparent endo-exo synergism with H. grisea and T. reesei endoglucanases. H. grisea has at least two different types of major exoglucanase components and shows strong cellulolytic activity through synergism with cellulase components including EXO1 and CBH1.View full abstractDownload PDF (3428K) -
Naoya Kojima, Yuriko Tachida, Shuichi Tsuji1998 Volume 124 Issue 4 Pages 726-737
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the effect of α1, 6-linked fucose modification of N-glycans on the expression of polysialic acids (PSAs), the expression of PSAs in a fucose-lacking mutant of Chinese hamster ovary (CHO) cells, Lec13, was compared with that in CHO K1 cells. PSA synthase activity in these cells and the antennary structures of N-glycans associated with the neural adhesion molecule (NCAM), which is a major PSA-carrying glycoprotein, did not differ between the two types of cells. Metabolic labeling of cells with [3H] glucosamine for 48 h followed by immunoprecipitation with anti-PSA monoclonal antibodies revealed that the amount of labeled PSA-carrying glycoproteins obtained from Lec13 cells was 10-times less than that from Kl cells, although the incorporation of [3H] glucosamine into total extracts and NCAM was almost the same. In contrast, when cells were pulse labeled with [35S]-methionine followed by a 1 h chase, there was not such a great difference in PSA-carrying protein synthesis between K1 and Lec13 cells. However, during a prolonged chase period, PSA-carrying proteins rapidly decreased in Lec13 cells, whereas those in Kl cells did not change. The degradation of PSA-carrying glycoproteins in Lec13 cells was partly prevented when the cells were grown in fucose-containing medium. Therefore, fucose modification of core N-glycans may affect the efficient expression of PSAs through the intracellular stability of PSA-carrying glycoproteins.View full abstractDownload PDF (4924K) -
Yasutaka Wada, Koichi Ishiguro, Tomohiko J. Itoh, Tsuneko Uchida, Hiro ...1998 Volume 124 Issue 4 Pages 738-746
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphorylation of tau, a heat-stable neuron-specific microtubule-associated protein, by cdk5 was stimulated in the presence of microtubules (MTs). This stimulation was due to an increased phosphorylation rate and there was no increase in total amount of phosphorylation. Two-dimensional phosphopeptide map analysis showed that MTs stimulated phos-phorylation of a specific peptide. Using Western blotting with antibodies that the recognized phosphorylation-dependent epitopes within tau, the phosphorylation sites stimulated by the presence of MTs were found to be Ser202 and Thr205 (numbered according to the human tau isoform containing 441 residues). MT-dependent phosphorylation at Thr205 was observed in situ in rat cerebrum primary cultured neurons. Stimulated phosphorylation at Ser202 and Thr205 decreased the MT-nucleation activity of tau, which is in contrast to MT-independent phosphorylation at Ser235 and Ser404.View full abstractDownload PDF (3233K) -
Hiroaki Iijima, Masaaki Miyazawa, Juro Sakai, Kenta Magoori, Mitsuko R ...1998 Volume 124 Issue 4 Pages 747-755
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe very low density lipoprotein receptor (VLDLR) gene contains an exon encoding a region of clustered serine and threonine residues immediately outside the membrane-spanning sequence, and this region has been proposed to be the site of clustered O-linked carbo-hydrate chains. Two forms of VLDLR transcripts, with and without the O-linked sugar region, are generated through alternative splicing. Reverse transcription polymerase chain reaction with RNAs from various rabbit tissues revealed that the VLDLR transcript with the O-linked sugar region (type-1 VLDLR) is the major transcript in heart and muscle, while the VLDLR transcript without the O-linked sugar region (type-2 VLDLR) predominates in non-muscle tissues, including cerebrum, cerebellum, kidney, spleen, adrenal gland, testis, ovary, and uterus. Hamster fibroblasts expressing type-2 VLDLR bound with relatively low affinity to β-migrating very low density lipoprotein compared with type-1 VLDLR-transfected cells. In contrast, the internalization, dissociation, and degradation of the ligand were not significantly impaired in either type of VLDLR-transfected cell. The receptor proteins in type-2 VLDLR-transfected cells underwent rapid degradation and accumulated in the culture medium, while those in type-1 VLDLR-transfected cells were stable and resistant to proteolytic cleavage. Analysis of the O-linked sugars of both types of transfected cells suggested that the O-linked sugar region is the major site for O-glyco-sylation.View full abstractDownload PDF (5395K) -
Susan A. Rotenberg, Jianwei Zhu, Hans Hansen, Xiao-dong Li, Xiao-guang ...1998 Volume 124 Issue 4 Pages 756-763
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing a combined pharmacological and genetic approach, we have identified as 260-280 in the C2 region as a critical factor in the catalytic function of protein kinase Cα (PKCα). Progressive truncations from the N-terminus as well as selected internal deletion mutants were expressed in Saccharomyces cerevisiae and tested for altered sensitivity to dequalinium, a PKC inhibitor whose target site was previously mapped to the catalytic domain. PKC mutants representing truncations of up to 158 amino acid residues (aa) from the N-terminus (ND84 and ND158) displayed 60-63% inhibition of kinase activity by 50 μM dequalinium, somewhat more sensitive than the wild-type PKCα enzyme (45% inhibition). Mutant ND262, lacking N-terminal as 1-262, was inhibited by almost 72% with 50 μM dequalinium, but mutant ND278, which lacked an additional 16 aa, was inhibited by only 9% of total activity. This result suggests that a C-terminal segment of the C2 region (aa 263-278) influences inhibition by dequalinium at low micromolar concentrations. An internal deletion mutant (D260-280) which retains the entire primary structure of PKCa except for as 260-280, was similarly inhibited by only 4% with 50 μM dequalinium. In the absence of dequalinium and despite the presence of a nearly complete regulatory domain, this mutant exhibited constitutive activity (both in vitro and in a phenotypic assay with S. cerevisiae) that could not be further stimulated even by the potent activator TPA. Taken together, our findings suggest that, in the native structure of PKCα, the segment described by as 260-280 regulates PKCα activity and influences the sensitivity of PKCα to dequalinium.View full abstractDownload PDF (2079K) -
Long-sen Chang, Shinne-ren Lin, Chun-chang Chang1998 Volume 124 Issue 4 Pages 764-768
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTaiwan cobra (Naja naja atra) phospholipase A2 (PLA2) was inactivated by arginine-specific reagents, phenylglyoxal and 1, 2-cyclohexanedione. Kinetic analyses of the modification reaction revealed that the inactivation of PLA2 followed pseudo-first-order kinetics and the loss of activity was correlated with the incorporation of one molecule of modification reagent per PLA2 molecule. This was confirmed by the results of amino acid composition determination, that showed that a marked decrease in enzymatic activity was associated with the modification of one arginine residue. Tryptic cleavage of the modified protein and microsequencing revealed that Arg-30 was the functionally essential residue. The incorporation of a modifier into the PLA2 did not significantly affect the secondary structure of the enzyme, as revealed by the CD spectrum, and Ca2+-binding of the modified PLA2 was unaffected. Nevertheless, the nonpolarity of the active site of PLA2 markedly decreased with the arginine modification, as evidenced by the decreases in the enhancement of Trp and 8-anilinonaphthalene sulfonate fluorescence. These results, together with those of X-ray crystallographic analysis of N. naja atra PLA2 [Scott et al. (1990) Science 250, 1541-1546], demonstrate that Arg-30 is one of the residues involved in the interfacial binding of a PLA2 molecule with its substrate.View full abstractDownload PDF (1760K) -
Masahiro Iwakura, Tatsuyuki Takenawa, Tsutomu Nakamura1998 Volume 124 Issue 4 Pages 769-777
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteins appear to contain structural elements which determine the folded structure. If such elements are present, the order of structural elements in the primary structure, i.e. the chain topology, can be disregarded for building of the folded tertiary structure, when they are properly connected to each other by proper linkers. To experimentally examine this, “topological” mutants (designated as GHF33 and GHF34) of Escherichia coli dihydrofolate reductase (DHFR) were designed and constructed by switching two amino acid sequence parts containing the βF strand and βG-βH strands in the primary sequence. In this way, the chain topology of wild-type DHFR, βA→αB→βB→αC→βC→βD→αE→βE→αF→βF-→βG-→βH-, was changed to βA→αB→βB→αC→βC→βD→αE→βE→αF→βG-→βH-→βF-. Such topological mutant proteins were stably expressed and accumulated in E. coli cells, and highly purified. Molecular mass measurements of the purified proteins and their proteolytic fragments confirmed that GHF33 and GHF34 had the designed sequence. In terms of kcat, the GHF33 and GHF34 proteins showed about 10 and 20% of the DHFR activity of the wild-type with Km values of 3.3 μM (GHF33) and 2.1 μM (GHF34), respectively. The topological mutants showed a cooperative two-state transition in urea-induced unfolding experiments with ΔGH2O values of 4.0 and 4.8 kcal/mol. Whereas, the Km and ΔGH2O values for wild-type DHFR were 0.9 μM and 6.1 kcal/mol, respectively. The significance of the topological mutations was discussed with respect to protein folding and protein evolution.View full abstractDownload PDF (1043K) -
Susumu Tsunasawa, Satomi Nakura, Tetsuo Tanigawa, Ikunoshin Kato1998 Volume 124 Issue 4 Pages 778-783
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3. 4. 19. 3, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus VS protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P. f. Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys 142, His 166, and Glu 79. On the other hand, a unique short stretch sequence (positions around 175-185) that is absent in bacterial Pcps was found in P. f. Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the “International Congress on Exthermophiles '98” p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required.View full abstractDownload PDF (824K) -
Yasuhiro Tomita, Dong-Ho Kim, Kenta Magoori, Takahiro Fujino, Tokuo T. ...1998 Volume 124 Issue 4 Pages 784-789
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe report herein the identification of a novel member of the low-density lipoprotein receptor (LDLR) family termed LDLR-related protein 4 (LRP 4). Murine LRP 4 cDNA encodes a 1113-amino-acid type II membrane-like protein with eight ligand-binding repeats in two clusters. Southern blot analysis of genomic DNA from several different organisms suggests the presence of LRP 4 homologues in chicken lacking the gene encoding apolipoprotein E, which is recognized by the ligand-binding repeats of LDLR. LRP 4 transcripts were detected almost exclusively in heart in mouse and humans. Despite the presence of the ligand-binding repeats, COS cells transfected with LRP 4 did not show surface-binding of β-migrating very-low-density lipoprotein, suggesting that LRP 4 plays a role in a pathway other than lipoprotein metabolism.View full abstractDownload PDF (3273K) -
Ayumi Koike-Takeshita, Tanetoshi Koyama, Kyozo Ogura1998 Volume 124 Issue 4 Pages 790-797
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmong prenyltransferases that catalyze the sequential condensation of isopentenyl diphos phate with allylic diphosphate to produce prenyl diphosphates with various chain lengths and stereochemistries, medium-chain prenyl diphosphate synthases are exceptional in that they comprise two dissociable heteromeric protein components. These components exist without binding with each other under physiological conditions, and neither of them has any prenyltransferase activity by itself. In order to elucidate the precise molecular mechanism underlying expression of the catalytic function by such a unique two-component system, we examined the possibility of forming a hybrid between two of the components of three different medium-chain prenyl diphosphate synthases, components I and II of heptaprenyl diphosphate synthase from Bacillus subtilis, components I' and II' of heptaprenyl diphosphate synthase from Bacillus stearothermophilus, and components A and B of hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26. As a result, only the hybrid-type combination of component I and component II' gave distinct prenyltransferase activity. The hybrid-type enzyme catalyzed the synthesis of heptaprenyl diphosphate and showed moderate heat stability, which lay between those of the natural enzymes from B. subtilis and B. stearothermophilus. There is no possibility of forming a hybrid between the heptaprenyl and hexaprenyl diphosphate syntheses.View full abstractDownload PDF (5125K) -
Dong-Seok Lee, Kazunori Anzai, Naohide Hirashima, Yutaka Kirino1998 Volume 124 Issue 4 Pages 798-803
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe phospholipid translocation from the outer to the inner leaflet of synaptic vesicles isolated from the electric organ of the Japanese electric ray, Narke japonica, was measured using fluorescent phospholipid probes. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS) with a fluorescent NBD-labeled short acyl chain at the sn-2 position was mixed with purified synaptic vesicles and the probe in the outer leaflet of the membranes was reduced with dithionite to quench the fluorescence from time to time. The percentage of fluorescence remaining after the dithionite treatment served as an index for the phospholipid translocation. The results obtained indicated that about 30, 13, and 9% of NBD-PE, NBD-PS, and NBD-PC, respectively, were translocated from the outer to the inner leaflet in 3h. Thus, the translocation activity in synaptic vesicle membranes was much higher for PE than for PS, in contrast to the previous results obtained with plasma membranes, including synaptosomal membranes. The percentages of the phospholipid in the inner leaflet at equilibrium were estimated to be 41, 31, and 14% for PE, PS, and PC, respectively. The translocation was inhibited by pretreatment with an SH reagent, iodoacetamide, indicating the involvement of a proteinaceous translocator. These data may provide a biochemical basis for elucidating the mechanisms of membrane fusion and exocytosis at nerve endings.View full abstractDownload PDF (1926K) -
Seiichi Taguchi, Shigeru Yamada, Shuichi Kojima, Haruo Momose1998 Volume 124 Issue 4 Pages 804-810
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have been focusing on the potent involvement of the molecular interaction between a protease and a protease inhibitor in the physiological or morphological regulation of Streptomyces cells producing them [Taguchi et al. (1995) J. Bacteriol. 177, 6638-6643; Suzuki et al. (1997) J. Bacteriol. 179, 430-438]. In this study, an extracellular protease, termed SAM-P 26, was isolated as a target of endogenous protease inhibitor (SSI) from the culture medium of an SSI non-producing mutant strain derived from Streptomyces albogriseolus S-3253. Complete amino acid sequence determination revealed that SAM-P 26 is identical to a protein encoded by the SAM-P20D gene, which was previously found to be located downstream of the gene for SAM-P20, another target protease of SSI. Based on the sequence homology, SAM-P 26 was categorized as a member of the chymotrypsin family like SAM-P 20. Sequence similarity between SAM-P26 and SAM-P 20 was immunologically demonstrated by Western blot analysis using anti-SAM-P 20 antiserum. The molecular mass (26 kDa) of SAM-P 26 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was much higher than that calculated from the amino acid sequence of SAM-P 26 (18, 376.8 Da) and that of the S-pyridylethylated form (18, 808.4 Da) of SAM-P 26 determined by Matrix-assisted Laser Desorption/Ionization-Time of Flight/Mass Spectrometry. Analytical gel-filtration analysis revealed that SAM-P 26 exists as a monomer (18.8 kDa) in the native state. The results as to substrate specificity and inhibitor sensitivity indicated SAM-P 26 exhibits chymotrypsin-like activity. For the proteolytic activity, the optimal pH was 10.5 and the optimal temperature was 60°C. The complex formation of SAM-P 26 with SSI was confirmed by native-PAGE analysis.View full abstractDownload PDF (3553K) -
Ralf Wilhelm, Aharon Abeliovich, Ali Nejidat1998 Volume 124 Issue 4 Pages 811-815
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAxenic cultures of the ammonia-oxidizing bacterium Nitrosomonas europaea were starved of ammonia (energy source) for up to 342 d. During this time the bacteria retained the ability to respond instantly to ammonia (1mM) or hydroxylamine (0.1mM) amendment by oxidizing it to nitrite without initial protein synthesis. In vivo, the ability to oxidize amended ammonia stayed almost constant during the starvation period, but a drop in the hydroxylamine oxidation rate (to 33%) was observed after 4 wk of starvation when exogenous hydroxylamine was supplied as sole energy source. In contrast, it has been shown that the level and in vitro activity of hydroxylamine oxidoreductase were not significantly affected during the starvation period. Only minor changes were detected between the protein patterns on one-dimensional SDS-PAGE of growing and starved cells. Thus, it is concluded that the activities of the energy-generating enzymes in N. europaea were not affected during long-term ammonia starvation.View full abstractDownload PDF (1975K) -
Yumiko Saito, Keitarou Kimura, Toshihiko Oka, Akihiko Nakano1998 Volume 124 Issue 4 Pages 816-823
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSarlp belongs to a unique subfamily of the small GTPase superfamily and is essential for the formation of vesicles that transport proteins from the endoplasmic reticulum to the Golgi apparatus. We have obtained mutants of the yeast SAR1 gene, which show several different phenotypes in cell growth and protein transport [Nakano, A., Otsuka, H., Yamagishi, M., Yamamoto, E., Kimura, K., Nishikawa, S., and Oka, T. (1994) J. Biochem. 116, 243-247; Yamanushi, T., Hirata, A., Oka, T., and Nakano, A. (1996) J. Biochem. 120, 452-458]. In this study, we have purified five mutant Sarl proteins using an Escherichia coli expression system and characterized their biochemical properties in detail. Three of them prefer GDP binding to GTP binding and are thus regarded as GDP-form mutants, and one is insensitive to the GTPase-activating protein and is almost fixed in the GTP-bound state. The GDP mutants are defective in vesicle formation in vitro, whereas the GTP mutant can drive vesicle formation but not the overall transport to the Golgi. These mutants will be useful for further understanding of the regulation of the GTPase cycle of Sarlp.View full abstractDownload PDF (968K) -
Naotada Ishihara, Tohru Komiya, Masao Sakaguchi, Akio Ito, Katsuyoshi ...1998 Volume 124 Issue 4 Pages 824-834
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondria have two independent protein-import machineries, one in the outer membrane (the Tom system) and the other in the inner membrane (the Tim system). Here, we have characterized the initial steps of precursor import into rat liver mitoplasts. The import reaction was separated into two stages, consisting of precursor binding to the mitoplasts at 0-10°C, and a subsequent chase reaction at 30°C. This assay revealed four distinct precursor-import steps: ΔΨ-dependent initial binding of the precursor, precursor transfer to the Tim23-Tim17 stage, -dependent translocation of the presequence across the inner membrane, and the complete translocation of the mature portion of the precursor. Antibodies against the intermembrane space domain of Tim23 inhibited neither the precursor binding nor the subsequent translocation of the presequence across the inner membrane. In contrast, the antibodies inhibited the complete translocation of the mature domain of the precursor across the inner membrane. Immunoprecipitation with anti-Tim23 IgGs revealed that the precursor-Tim23 complex increased with time and temperature after the initial targeting of the precursor to the mitoplasts. These results suggest that the precursor is first targeted to the inner membrane component ΔΨ-dependently, then transferred to the Tim system consisting of Tim23-Tim17, and finally imported into the matrix.View full abstractDownload PDF (9364K) -
Takanori Kumagai, Masanori Sugiyama1998 Volume 124 Issue 4 Pages 835-841
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA gene, blmA, encodes a bleomycin (Bm)-binding protein, designated BLMA, from Bmproducing Streptomyces verticillus and confers resistance to Bm in Streptomyces and Escherichia coli cells. In the present study, by transfection of the gene into COS-1 cells with a plasmid designated pEF-BOS/blmA, which contains a strong promoter from the human polypeptide chain elongation factor 1α, we transiently overproduced BLMA at a high level of approximately 4% of the whole cell protein. Although NIH/3T3 cells transfected with pEF-BOS/blmA, designated NIH/3T3-BR cells, stably expressed BLMA, its expression level was about 0.1% of the total protein. Using an anti-BLMA monoclonal antibody reported previously [Sugiyama et at. (1995) FEBS Lett. 362, 80-84], we revealed that BLMA is localized in the nucleus of pEF-BOS/blmA-transfected COS-1 and NIH/3T3-BR cells. Semi-permeabilized nuclear transport experiments showed that BLMA penetrates the nuclear envelope by energy- and transporter-independent passive diffusion, suggesting that the karyophilic nature of BLMA may be due to the acidic nature of the protein. NIH/3T3-BR cells were 130-fold more resistant to Bm than the host cells. NIH/3T3 cells exhibited a swollen nuclear envelope and a malformed spindle body and overexpressed at least 4 kinds of stress proteins including calreticulin and mitochondrial matrix protein P1 when exposed to 25 μg/ml of Bm, whereas NIH/3T3-BR cells grew without morphological alteration and expressed no stress proteins under the same conditions. Furthermore, reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression of interleukin-6, an inflammatory cytokine, is activated by addition of Bm in NIH/3T3 cells, but not in the NIH/3T3-BR cells. These results suggest that BLMA contributes to protection of mammalian cells from the inflammatory effect of Bm.View full abstractDownload PDF (5328K) -
Yoshiyuki Ishii, Syuji Sonezaki, Yasushi Iwasaki, Eiichi Tauchi, Yoshi ...1998 Volume 124 Issue 4 Pages 842-847
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDnaJ is a molecular chaperone, which contains a zinc finger-like motif and cooperates with DnaK to mediate the folding of newly synthesized and denatured proteins. DnaJ was overproduced and purified using the maltose binding protein (MBP) fusion vector. The fusion protein (MBP-DnaJ) was expressed in a soluble form in Escherichia coli and purified to homogeneity using amylose resin in a single step. The UV-visible absorption spectrum of MBP-DnaJ showed peaks at 355 and 475 nm. Moreover, these absorption peaks disappeared upon treatment with ethylenediaminetetraacetic acid (EDTA) or p-hydroxymercuriphenylsulfonic acid (PMPS). Inductively coupled plasma (ICP) spectrometry demonstrated that MBP-DnaJ contains Fe ions as well as Zn ions. MBP-DnaJ mediated the replication of the λ phage in vivo, stimulated the ATPase activity of DnaK and prevented the aggregation of denatured rhodanase, indicating that fusion of MBP to the N-terminal of DnaJ does not affect the functions of DnaJ. To study the roles of bound metal ions, metal-free MBP-DnaJ, and MBP-DnaJ containing 2 Zn ions were prepared. MBP-DnaJ containing Fe and Zn ions, and MBP-DnaJ containing 2 Zn ions stimulated the ATPase activity of DnaK, prevented the aggregation of denatured rhodanase and bound to DNA to similar extents. On the other hand, metal-free MBP-DnaJ showed much lower DNA-binding ability and lower ability to prevent rhodanese aggregation. Therefore, the bound metal species do not affect the function of the zinc finger-like motif of DnaJ, whereas removal of the metal ions from DnaJ diminishes its binding ability as to DNA and denatured proteins.View full abstractDownload PDF (3267K) -
Norio Inokuchi, Shigeru Saitoh, Hiroko Kobayashi, Tadashi Itagaki, Tak ...1998 Volume 124 Issue 4 Pages 848-856
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA base non-specific and acid RNase was isolated from cellular slime mold (Dictyostelium discoideum) cells in a homogeneous state (about 2.4 kDa) by SDS-polyacrylamide gel electrophoresis. The RNase (RNase Ddl) has a pH optimum of 5.0. The amino acid sequence of RNase DdI was determined by a combination of protein chemistry, a search of Data base, Dicty cDB and further sequence analysis of cDNA from the same bank. RNase DdI consists of 198 amino acid residues, and about 13.3, 0.9, 1.2, 3.3, and 1.0 residues of mannose, xylose, glucose, GlcNAc, and GalNAc, respectively. RNase DdI has two characteristic conserved segments of the RNase T2 family, and thus belongs to the RNase T2 family. Considering the fact that most of the RNase activity of D. discoideum is present in the Iysosomal fraction [Wiener and Ashworth (1970) Biochem. J. 118, 505-512], it was concluded that the lysosomal RNase in D. discoideum is a member of the RNase T2 family. The amino acid sequence of RNase DdI is highly homologous with that of Physarum polycephalum RNase (RNase Phyb), and its amino acid sequence seems to be similar to those of plant/animal type RNases, rather than fungal RNases. The location of RNase DdI in the phylogenetic tree of the RNase T 2 family was estimated.View full abstractDownload PDF (3873K) -
Naoya Kimura, Makoto Uchida, Shigenori Nishimura, Haruki Yamaguchi1998 Volume 124 Issue 4 Pages 857-862
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have recently revealed that the intramolecular Asn-glycans promote the refolding of reductively denatured bovine pancreatic RNase B, and that extramolecular Asn-glycans of both high-mannose and complex types also markedly stimulate the oxidative refolding of RNase B and its nonglycosylated form, RNase A [Yamaguchi, H. and Uchida, M. (1996) J. Biochem. 120, 474-477; Nishimura et al. (1998) J. Biochem. 123, 516-520]. The present investigation was undertaken to see whether this function of Asn-glycans is specific to the refolding of pancreatic RNases; i.e., extramolecular Asn-glycans were examined for their effects on the oxidative refolding of hen egg white lysozyme and bovine a-lactalbumin by monitoring changes in activity, dynamic volume, intrinsic fluorescence, and affinity for a fluorescent probe, 1-anilino-8-naphthalenesulfonate. Asn-glycans of both high-mannose and complex types markedly stimulated the oxidative refolding of these proteins, giving similar results to those previously obtained with RNases, though differences attributable to the characteristics of individual proteins were observed in the promotive effects. Thus it seems probable that Asn-glycans generally promote the proper folding of denatured polypeptides.View full abstractDownload PDF (720K) -
Hirofumi Tsuga, Kimihiko Kameyama, Tatsuya Haga1998 Volume 124 Issue 4 Pages 863-868
Published: October 01, 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDesensitization of human muscarinic acetylcholine receptor m 2 subtypes (hm 2 receptors) stably expressed in chinese hamster ovary cells was measured as decreases in the carbamylcholine-stimulated [35S]GTPγS binding activity in membrane preparations after pretreatment of cells with carbamylcholine. The extent of carbamylcholine-stimulated [35S] GTPγS binding activity was found to decrease to 64% following pretreatment of cells with 10 μM carbamylcholine for 30min, and under the same conditions 51-59% of hm 2 receptors were sequestered/internalized as assessed by decreases in the [3H] N-methylscopolamine binding activity on the cell surface. A similar reduction in the carbamyleholine- stimulated [35S]GTPγS binding activity was observed by pretreatment of cells with 5 nM propylbenzylylcholine mustard, which irreversibly bound to and inactivated 58% of the hm 2 receptors. When the cells were pretreated with 10 μM carbamylcholine in the presence of 0.32M sucrose, which is known to inhibit clathrin-mediated endocytosis, no sequestration/ internalization of hm 2 receptors was observed, and the extent of carbamylcholine-stimulated [35S] GTPγS binding activity did not change. These results indicate that desensitization of hm 2 receptors may be caused by reduction of receptor number on the cell surface through sequestration/internalization rather than by loss of the function of receptors.View full abstractDownload PDF (768K)
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