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Volume 115 (1994)
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Volume 115, Issue 4
Displaying 1-28 of 28 articles from this issue
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Haruo Suzuki1994 Volume 115 Issue 4 Pages 623-628
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe first catalytic antibodies (abzymes) were reported in 1986. Since then abzymes catalyzing various types of reactions have been reported. There have been a number of reviews on the abzymes, so in this review I intended not to overlap them, but briefly described (i) the background of abzyme production, (ii) the catalytic mechanism of the enzymic reaction, and (iii) some examples of monoclonal and polyclonal abzymes. Recent advancements as to abzyme studies are the invention of an assay system for catalytic activity during cloning, the estimation of the solvent effect on abzyme-catalyzed decarbox-ylation, the production of abzymes catalyzing disfavored chemical reactions and cocaine hydrolysis, and the feasibility of the usage of polyclonal abzymes. Several advantages of polyclonal abzyme studies were also pointed out.View full abstractDownload PDF (739K) -
Ujwal Shinde, Masayori Inouye1994 Volume 115 Issue 4 Pages 629-636
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA large number of prokaryotic as well as eukaryotic proteins are produced with amino terminal propeptides. These amino-terminal extensions are essential for mediating proper folding of their corresponding proteins and are also termed as intramolecular chaperones. Though these propeptides are highly specific and unique in their function, several common features have been identified and indicate that the overall mechanism by which they function may be very similar.View full abstractDownload PDF (874K) -
Takako Kitani, Sachiko Okuno, Hitoshi Fujisawa1994 Volume 115 Issue 4 Pages 637-640
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalmodulin-dependent protein kinase IV (CaM-kinase IV) is a Ca2+-responsive multifunctional protein kinase which occurs abundantly in the brain and thymus. A human eDNA clone encoding CaM-kinase IV was isolated from a Jurkat cell cDNA library and its nucleotide sequence was determined. The cDNA sequence encoded a protein consisting of 473 amino acids with a molecular weight of 51, 925. The nucleotide sequence for the coding region and the deduced amino acid sequence showed 81 and 80% identities with those of the rat enzyme, respectively. Western blot analysis, using a polyclonal antibody raised against the recombinant human CaM-kinase IV, which was expressed in Escherichia coli, revealed two bands corresponding in mobility to molecular weights of 60, 000 and 61, 000, respectively, in a Jurkat cell extract. The antibody also cross-reacted with both isoforms of CaM-kinase IV from rat cerebellum, the apparent molecular weights being 62, 000 and 64, 000, respectively.View full abstractDownload PDF (1128K) -
Shinji Kawai, Sunao Takeshita, Makoto Okazaki, Reiko Kikuno, Akira Kud ...1994 Volume 115 Issue 4 Pages 641-643
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA library prepared from the mouse osteoblastic cell line, MC3T3-El, was screened for the presence of specifically expressed genes by employing subtraction hybridization/differential screening methods. A cDNA clone was identified and sequenced, encoding a protein designated as osteoblast specific factor 3 (OSF-3) comprising 199 amino acids. RNA dot blot analysis indicated weak OSF-3 expression in thymus, spleen, brain, lung, testis, and heart, and high expression in kidney and liver. A homology search of an amino acid sequence database revealed a strong relationship of OSF-3 to the MER5 (gene preferentially expressed in murine erythroleukemia cells) protein and human pag (proliferation associated gene) product. This indicates that OSF-3 plays an intrinsic role in the proliferation and/or differentiation of bone cells.View full abstractDownload PDF (386K) -
Hidetake Miyata, Hiroyuki Hakozaki, Hiroshi Yoshikawa, Naoya Suzuki, K ...1994 Volume 115 Issue 4 Pages 644-647
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to determine the relative motions of an actin filament and a myosin molecule upon hydrolysis of one ATP, an in vitro motility assay, in which individual actin filaments slide over heavy meromyosin molecules bound to a substrate, was combined with an optical trapping technique. An actin filament, attached to a gelsolin-coated bead, was captured with an optical trap. The surface-bound heavy meromyosin molecules pulled the filament against the trapping force, which resulted in back and forth motions of the actin-bound bead. The number of heavy meromyosin molecules interacting with an actin filament (at most 1/μm filament) and the ATP concentration (≤0.5 μM) were chosen so as to facilitate detection of each “pull.” Calculation of the centroid of the bead image revealed abrupt displacements of the actin filament. The frequency of such displacements was between 0.05 and 0.1 per 1 s per 1 μm actin filament, being consistent with calculated values based on the reported bimolecular binding constants of ATP and the actomyosin rigor complex. The distribution of the displacements peaked around 7 nm at a trapping force of 0.016 pN/nm, but it became broader, and some displacements were as large as 30 nm, when the trapping force was reduced to 0.0063 pN/nm, suggesting that the force generation due to the structural change of a myosin head may be insufficient to explain such displacements.View full abstractDownload PDF (431K) -
Shoji Watabe, Hiroyuki Kohno, Hiroshi Kouyama, Tomoko Hiroi, Nagasumi ...1994 Volume 115 Issue 4 Pages 648-654
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have purified SP-22, a substrate protein for mitochondria) ATP-dependent protease in bovine adrenal cortex. Native SP-22 showed an Mγ of 350, 000±20, 000, and was composed of more than 10 molecules of an Mγ 21, 600 subunit. Subcellular and submitochondrial fractionation of adrenocortical tissues revealed that SP-22 was localized in the mitochondrial matrix, suggesting that SP-22 is a natural substrate for ATP-dependent protease, a matrix enzyme. The concentration of SP-22 in adrenocortical mitochondria) fractions was 16±3 μg/mg proteins (mean±SD, n=6) as determined by radioimmunoassay using specific anti-SP-22 antibody. Adrenal cortex showed the highest concentration among the 15 bovine tissues tested, followed by liver, renal cortex, adrenal medulla, heart, and renal medulla. We determined the amino acid sequence of SP-22, which is composed of 195 amino acids. Amino acid 47 was not identified by the sequencer. FAB-mass spectrometry of AA47-AA55 fragment revealed that AA47 was cysteine-sulfinic acid (Cys-SO2H). By a homology search in the NBRF-PIR data base, SP-22 was found to be 91% homologous to murine erythroleukemia cell MER-5 protein, which may have an important role in the induction of differentiation. SP-22 was also homologous to the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium, thiol-specific antioxidant in Saccharomyces cerevisiae, and some other proteins. Since a segment around AA47 was highly conserved, this residue may be important for the biochemical functions of SP-22.View full abstractDownload PDF (1845K) -
Taiju Kuramoto, Katsutoshi Fukuda, Akira Ohshima, Kenji Kihira, Takahi ...1994 Volume 115 Issue 4 Pages 655-658
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis paper describes the determination of the glucurono-conjugated position in two bile alcohol glucuronides excreted in urine of a patient with cerebrotendinous xanthomatosis by a nuclear magnetic resonance study. The urine sample was extracted with reversed-phase resin, and chromatographed on a reversed-phase partition column and a silica gel column to isolate glucurono-conjugates of 5β-cholestane-3α, 7α, 12α, 25-tetrol and 5β-chole-stane-3α, 7α, 12α, 23, 25-pentol. Proton and carbon-13 nuclear magnetic resonance spectra of the natural tetrol glucuronide were identical with those of the chemically synthesized tetrol glucuronide, 7α, 12α, 25-trihydroxy-5β-cholestane-3α-O-β-D-glucopyranosyluronic acid. Hence, the glucurono-conjugated position of the natural tetrol glucuronide was determined to be the C-3 position. By comparison of the 13C chemical shift data with that of the unconjugated pentol, 5β-cholestane-3α, 7α, 12α, 23, 25-pentol, the glucurono-conjugated position of the natural pentol glucuronide was determined to be C-23. Thus the natural pentol glucuronide can be formulated as 3α, 7α, 12α, 25-tetrahydroxy-5β-cholestane-23-O-β-D-glucopyranosyluronic acid. The difference in the glucurono-conjugated position between the 25-tetrol glucuronide and the 23, 25-pentol glucuronide indicates that the former is not the biosynthetic precursor of the latter.View full abstractDownload PDF (433K) -
Takeo Yamaguchi, Satoko Yamada, Eiji Kimoto1994 Volume 115 Issue 4 Pages 659-663
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo study the effect of cross-linking of membrane proteins on vesiculation of human erythrocytes by dimyristoylphosphatidylcholine (DMPC), red cells were treated with diamide at atmospheric pressure or 100 MPa and then incubated with DMPC in buffers of pH 6.5-8.5. Irrespective of buffer pH, the amount of released vesicles increased upon cross-linking of membrane proteins but approached the control level upon reduction of the cross-linking by dithiothreitol. Similar enhancement of vesicle release was also observed in N-ethylmaleimide-treated red cells. Hemolysis during vesiculation was observed only in red cells treated with diamide at 100 MPa. Furthermore, the composition of membrane proteins in released vesicles was analyzed by SDS-PAGE. Membrane vesicles released from intact red cells or the cells treated with diamide at atmospheric pressure contained band 3 as a major membrane protein. On the other hand, membrane vesicles from red cells treated with diamide at 100 MPa contained protein 4.1 in addition to band 3 and the orientation of these proteins was similar to that in intact cells. These results indicate that the amount and membrane protein composition of DMPC-induced vesicles are much affected by chemical modification of SH-groups in red cell membrane proteins.View full abstractDownload PDF (1726K) -
Subramanian Dhandayuthapani, Mohammed Jameela Banu, Yoshiko Kashiwabar ...1994 Volume 115 Issue 4 Pages 664-669
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSElongation factor Tu (EF-Tu) plays an important role in protein biosynthesis and is susceptible to antibiotics in prokaryotes like Escherichia coli. In order to understand the primary structure of EF-Tu in the intracellular pathogenic bacterium Mycobacterium leprae, the gene (tuf gene) coding for this protein was cloned and sequenced. The gene contains a coding region of 1, 188 by with GUG as start codon. The deduced amino acid sequence has 396 amino acids with a molecular weight of 43.6 kDa. Putative GTP-binding sites are located at amino acid positions 19-24, 83-87, and 138-141. Comparison of M. leprae EF-Tu amino acid sequence with those of M. tuberculosis, Micrococcus luteus, E. coli, and Salmonella typhimurium reveals 74-95% homology. Mitochondrial EF-Tu of Saccharomyces cerevisiae (62%) and ehioroplast EF-Tu of Arabidopsis thalina (65.6%) also show strong homology with that of M. leprae. In contrast, the EF-Tu of the archaebacterium Halobacterium marismoruti exhibits relatively less homology (36.7%). Southern hybridization of M. leprae tuf gene with genomic DNA of slow growing and fast growing mycobacteria and related species like Corynebacterium fascians and Nocardia asteroides suggests that the gene is highly conserved in these organisms.View full abstractDownload PDF (3171K) -
Sabu Kasai1994 Volume 115 Issue 4 Pages 670-674
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSP-flavin-bound luciferase, P-flavin-free luciferase, and P-flavin-bound β-subunit of luciferase were prepared from Photobacterium phosphoreum using hydrophobic interaction chromatography after conventional purification using DEAE-cellulose chromatography and gel-filtration. The P-flavin-bound luciferase preparation contained about 20% P-flavin-free luciferase not removable by the present procedure. Since the specific activity of the P-flavin-bound luciferase preparation was about 20% of that of the P-flavin-free luciferase, it was concluded that the P-flavin-bound luciferase is an enzyme-product complex and has no more luciferase activity. Unlike the absorption spectrum of FP390 or other flavoproteins, that of P-flavin-bound luciferase preparation has a high absorption peak around 370 nm and resembles the spectrum synthesized by superposing the P-flavin-free luciferase spectrum on the P-flavin-bound β-subunit spectrum: the P-flavin-bound β-subunit spectrum is similar to that of FP390, while that of P-flavin-free luciferase has an absorption peak around 370 nm but practically no peak around 450 nm. In addition, P-flavin-free luciferase exhibits a weak but distinct NADH-FMN oxidoreductase activity. These results suggest that a prosthetic group, which absorbs around 370 nm, binds to the luciferase and that this compound is required to yield P-flavin; and they support the hypothesis that the physiological function of bacterial luciferase is to produce P-flavin. Furthermore, the presence of P-flavin-bound β-subunit of the luciferase in the cell extract supports the hypothesis that physiological function of the lux operon is the biosynthesis of FP390 including its prosthetic group.View full abstractDownload PDF (1435K) -
Simone Beninati, Donald R. Senger, Eleonora Cordella-Miele, Anil B. Mu ...1994 Volume 115 Issue 4 Pages 675-682
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOsteopontin (OP) is a component of extracellular, bone, and urinary stone matrices, but the mechanism by which it is stably incorporated into such matrices remains unknown. By SDS-PAGE analysis of [125I] OP, treated with a catalytic amount of TG, we first demonstrate both intra- and intermolecular covalent cross-linking of OP. Most importantly, the analysis of the products generated from reactions containing OP, Fn, and TG by SDS-PAGE, autoradiography, and Western blotting using either OP or Fn antibody, and quantitation of. TG-catalyzed ε-(γ-glutamyl) lysine isopeptide formation between OP and Fn demonstrate, for the first time, covalent cross-linking between these two proteins. Similar reactions in the presence of polyamine substrates of TG show OP-Fn intermolecular cross-linking via N, N-bis- (γ-glutamyl) polyamine formation. Finally, immunoprecipitation of 125I-labeled NRK cell surface proteins with anti-OP and anti-Fn antibodies, SDS-PAGE analysis, and autoradiography provides critical evidence for nonreducible OP-Fn cross-linking in vivo. These results clearly suggest that TG-mediated cross-linking between OP and Fn repre-sents one of the most likely mechanisms by which OP becomes covalently linked to bone matrix, urinary stone matrix, and to ECM.View full abstractDownload PDF (4597K) -
Akio Tonouchi, Atsushi Fujita, Satoru Kuhara1994 Volume 115 Issue 4 Pages 683-688
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe identified a yeast gene encoding Flocculent Specific Protein (FSP) produced excessively in the SFL1 gene-disrupted flocculent strain. The sequenced gene encodes a 430 amino acid protein and is mainly composed of multiple repeats of Ser-Asn-Asn-X-Asp-Ser-Tyr-Gly. The FSP gene disruption of the flocculent strain decreased the degree of flocculation, so FSP may be one factor concerned with yeast flocculation. A gene database search indicated that the FSP gene is identical with the DDR48 gene.View full abstractDownload PDF (2747K) -
Tomoharu Himeshima, Tomomitsu Hatakeyama, Nobuyuki Yamasaki1994 Volume 115 Issue 4 Pages 689-692
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of SJL-I, a lectin from the sea cucumber, Stichopus japonicus, was determined by sequence analysis of peptides derived on enzymatic and chemical fragmentation of the protein. SJL-I consists of 143 amino acid residues and its molecular mass was calculated to be 15, 837 Da. Comparison of the sequence of SJL-I with a database revealed that SJL-I exhibits apparent homology with C-type lectins, especially with those of marine invertebrates. The highest homology (identity 28.6%) was found with echinoidin, a lectin from the sea urchin, Anthocidaris crassispina. Comparison of the sequence of SJL-I with those of other C-type lectins indicated that the conserved amino acids are relatively abundant in the C-terminal half of their carbohydrate-recognition domains (CRDs), that can be considered to be involved in binding with Ca2+ as well as carbohydrates.View full abstractDownload PDF (462K) -
Carmichael J. A. Wallace, Yoshikazu Tanaka1994 Volume 115 Issue 4 Pages 693-700
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have expressed the gene for human cytochrome c, and six mutants of the native sequence, in yeast defective in its own iso-1-cytochrome c gene. All constructs support strong growth in strict aerobic metabolism, and substantial amounts of protein could be extracted from permeabilized cells. The purified analogs, Cysl4Ala, Gly37Arg, Arg38Lys, Arg38Gly, Gly84Ser, and Thr28Ile, Gly84Ser, were examined for changes in functional properties, since the majority of these residues are strongly or absolutely conserved. Indeed, although growth rates of the host yeast strains were very similar, there was great divergence in both physicochemical and biological properties, which have been rationalized in terms of changes to the stability of the cytochrome fold, and to the dipole moment of the protein. Interestingly, although modification of electrostatic properties in some mutants can apparently produce a twofold increase in electron transfer efficiency, such changes are not evolutionarily acceptable. The “improvement” is illusory. We suggest that an associated decrease in the stability of the heme crevice offsets any advantage of increased transfer rates.View full abstractDownload PDF (1160K) -
Masumi Eto, Katsuyoshi Isonishi, Yasuhiro Fukui, Fumi Morita, Norio Ni ...1994 Volume 115 Issue 4 Pages 701-707
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs reported previously, the synthetic heptapeptide having the amino acid sequence around the reactive Cys (SH1) of myosin heavy chain, IRICRKG-NH2, inhibited acto-myosin subfragment-1 (S-1) ATPase activity and half inhibition (K1/2) was observed at a peptide concentration of 0.06mM. The inhibitory ability of the peptide was found to be decreased to one-fifth by acetylation of its N-terminal α-amino group. A similar effect of N-acetylation was observed with a nonapeptide, EGIRICRKG-NH2, and an undecapeptide, VLEG-IRICRKG-NH2. These results indicate that N-terminal-free synthetic peptides do not act as proper analogs of the corresponding segment of S-1 heavy chain against F-actin. We isolated a longer peptide extending from Thr682 to Lys709 in S-1 heavy chain, with two Cys residues corresponding to SH1 and SH2. This peptide, having 28 residues (28peptide), inhibited acto-S-1 ATPase activity with a K1/2 of 0.23mM. A cosedimentation binding assay indicated that the 28peptide completely dissociated acto-S-1 in the presence of ATP. This behavior is different from that observed with the N-terminal-free synthetic heptapeptide, and thus the 28peptide might be an analog of the corresponding segment. There is a possibility that the region corresponding to the 28peptide in S-1 heavy chain may bind directly with F-actin and may be involved in determining the acto-S-1 link during the steady state of the acto-S-1 ATPase reaction.View full abstractDownload PDF (879K) -
Shintaro Kamei, Yu-ichi Kamikubo, Tsutomu Hamuro, Hidehiro Fujimoto, M ...1994 Volume 115 Issue 4 Pages 708-714
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRhesus monkey cDNA for tissue factor pathway inhibitor (TFPI) was cloned by means of the reverse transcriptase-polymerase chain reaction, using liver mRNA, and its nucleotide sequence was determined by sequencing five independent clones. Monkey TFPI was found to have a signal peptide of 28 amino acid residues and to be a mature protein of 276 amino acid residues, in which three and seventeen amino acid residue substitutions compared to human TFPI were found, respectively. All the cysteine residues, three putative carbohydrate-linked asparagine residues, and the P1 amino acid residues of each of the three Kunitz inhibitor domains were conserved in the two species. Recombinant monkey TFPI (rTFPI) was isolated from the culture medium of transformed Chinese hamster ovary cells. Amino acid sequence analysis and immunoblotting analysis, using polyclonal and monoclonal antibodies, showed that the carboxyl-terminal basic part of Rhesus monkey rTFPI had been truncated. The inhibitory activity of monkey rTFPl was compared with that of human rTFPI without the carboxyl-terminal basic part. The prothrombin time of human plasma was slightly more prolonged by the addition of monkey rTFPI than by that of human rTFPI. However, no significant differences were found between the potencies of human and monkey rTFPI as to the inhibition of factor Xa and tissue factor-factor VIIa complex.View full abstractDownload PDF (2913K) -
Tetsuro Yoshimura, Shigenori Maezawa, Keiichi Kameyama, Toshio Takagi1994 Volume 115 Issue 4 Pages 715-723
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of lipid composition, temperature, and ionic strength on critical events in the membrane fusion reaction induced by clathrin, such as membrane binding and selfassociation of clathrin, membrane aggregation, and actual fusion, and the size of fused liposomes, were examined using large unilamellar vesicles (LUV) containing acidic phospholipids. When membrane fusion and aggregation of LUV with different lipid compositions were initiated at 25°C in 0.1 M NaCl by lowering the pH of the medium from 7.40 to 4.75 in the presence of clathrin, two types of reaction processes were observed: fusion and aggregation of LUV containing phosphatidylserine and phosphatidic acid occurred slowly with a long fusion lag-phase and reached a high level, whereas the two reactions of LUV containing phosphatidylglycerol and phosphatidylinositol were induced faster with a much shorter fusion lag-time and leveled off in a shorter time. Similar differences in the fusion reactions were observed in media at different temperatures and ionic strengths with any type of LUV: slow and extensive aggregation and fusion occurred at low temperature and/or high ionic strength, but faster, less extensive reactions occurred at high temperature and/or low ionic strength, indicating that the two types of reaction pattern are due to the dependencies of the aggregation and fusion reactions on the temperature and ionic strength, but not on the lipid composition. The sizes of fused liposomes were large under the former conditions but smaller under the latter, and the ratio of the rates of self-association to membrane binding of clathrin was found to be correlated with the rates and levels of these critical events and the sizes of fused liposomes. These results suggest that the fusion reaction and the size of fused liposomes are modulated by the balance between the self-association and membrane binding reactions of the protein. A novel fusion mechanism is proposed to explain the modulation of the size of fused liposomes.View full abstractDownload PDF (1027K) -
Yukio Shirasawa, Toshio Osawa, Akikazu Hirashima1994 Volume 115 Issue 4 Pages 724-729
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe prolyl endopeptidase (PEP) gene of human T cells was amplified by the PCR method and cloned in Escherichia coli. The complete gene consisted of 2, 130 nucleotides corresponding to 710 amino acid residues with a calculated molecular mass of 80, 750. The nucleotide sequence of this clone revealed that T cell PEP DNA is 48, 50, and 91% homologous to those of Flavobacterium meningosepticum, Aeromonas hydrophila, and porcine brain PEP, respectively. This gene was fused to the lacZ sequence from E. coli and expressed as a fused protein in E. coli. This fused protein exhibited PEP activity, which was inhibited by Z-Pro-prolinal, a specific inhibitor of PEP. The fused protein was purified on a β-galactosidase specific affinity column. A polyclonal antibody was raised against the purified protein. Immunological characterization suggested that this protein is different from cytosol-soluble PEP.View full abstractDownload PDF (1651K) -
Sumio Ishijima, Masamiti Tatibana1994 Volume 115 Issue 4 Pages 730-737
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the effects of growth factors on intracellular free Mg2+ concentrations ([Mg2+]1) in single Swiss 3T3 fibroblasts, using microfiuorometry of a Mg2+ -sensitive dye mag-fura-2. We had already noted an increase in [Mg2+]1 after exposure to bombesin for 30-60 min [Ishijima, S., Sonoda, T., & Tatibana, M. (1991) Am. J. Physiol. 261 (Cell Physiol. 30), C1074-C1080] . In the present work, we found that bombesin also induced early changes in [Mg2+]1. The [Mg2+]1 reached peak values within 15 s in most cells, and the significant rise lasted only for 1-2min. The extent of the increase varied from cell to cell (0-600 μM above basal). On the average, the [Mg2+], was increased from basal 0.33 to 0.54mM. Since the time course was similar to that of [Ca2+]1 changes, and the dye mag-fura-2 also binds Ca2+, we evaluated Ca2+ interference with measurement of [Mg2+]1. The contribution of Ca2+ binding would be below 20% of the mag-fura-2 signal. The bombesin-induced [Mg2+], increase was not dependent on external Mg2+, but the omission of external Ca2+ decreased by 60% the [Mg2+]1 increase, and the Ca2+ channel blocker, nicardipine inhibited by 90% the [Mg2+]1 response. This inhibition was partially reversed by raising the concentration of external Ca2+. Two structurally distinct tyrosine kinase inhibitors, genistein and lavendustin A, almost completely inhibited the [Mg2+]1 response. These results suggest that bombesin rapidly induces Mg2+ mobilization from the intracellular pool, through external Ca2+- and tyrosine kinase-dependent mechanisms.View full abstractDownload PDF (1024K) -
Hideyuki Shibata, Masato Nagaoka, Masashi Sakai, Haruji Sawada, Tsunek ...1994 Volume 115 Issue 4 Pages 738-742
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA pure complex of staphylokinase and plasmin was prepared by affinity chromatography with lysine-Sepharose, which enabled the simple analysis of the mechanism of plasminogen activation by staphylokinase. We used a truncated staphylokinase (SAK), which lacks the 10 amino acid residues at the NH2 terminal of native staphylokinase. The purity of this complex was confirmed by the native PAGE profile. Image analysis of the SDS-PAGE profile revealed that the molar ratio of plasmin and SAK in the complex was about 1:1. Using this SAK-plasmin complex, the kinetic parameters for the activation of Glu- or Lys-plasminogen were determined. The kinetic constant, kcat/Km, obtained when Lysplasminogen was used as a substrate was approximately 10 times higher than that obtained when Glu-plasminogen was used. This plasminogen activation property of the SAK-plasmin complex was comparable to that of other plasminogen activators, such as streptokinase, urokinase, and tissue-type plasminogen activator (t-PA). This SAK-plasmin complex will simplify the elucidation of plasminogen activation by SAK. Through kinetic studies, the fibrin specificity and participation of plasminogen activator inhibitor will be clarified.View full abstractDownload PDF (1833K) -
Masataka Tanabe, Tsuneo Asano, Norihiko Moriya, Hiromu Sugino, Atsushi ...1994 Volume 115 Issue 4 Pages 743-751
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree Streptoverticillium anticoagulants, SAC I, II, and III, which strongly inhibit human intrinsic blood coagulation, were each isolated in a homogeneous form from a culture fluid of Streptoverticillium cinnamoneum subsp. cinnamoneum IFO 12852. SAC I, II, and III are simple proteins with molecular weights of around 12, 000, and with isoelectric points of 9.7, 9.7, and 9.9, respectively. Their amino acid compositions are similar and each SAC possesses two disulfide bonds. The COOH-terminal residue of each of these proteins is phenylalanine. Together with the similarity of their protein chemical properties, the results of NH2-terminal amino acid sequence analysis of these SAC proteins strongly suggested that the deletion of Ser-Leu and Ser-Leu-Tyr from the NH2-terminus of SAC I (Ser-Leu-Tyr-Ala-Pro-...) results in the generation of SAC II and III, respectively. The amount of each SAC necessary to double the partial thromboplastin time was around 5 μg/ml. SAC I inhibited activated human factor XII and human plasma kallikrein. It also inhibited, but to a lesser extent, activated factor X. The inhibition constants (K1) of SAC I toward activated factor XII and plasma kallikrein were 5.3×10-8 and 7.2×10-9 M, respectively. The SACs also inhibited some microbial serine proteases such as subtilisin Carlsberg and, to a lesser extent, mammalian serine proteases including bovine trypsin and α-chymotrypsin. Of these three inhibitors, only SAC I inhibited metalloproteases such as thermolysin in addition to these serine proteases.View full abstractDownload PDF (1186K) -
Masataka Tanabe, Kenji Kawahara, Tsuneo Asano, Koichi Kato, Atsushi Ka ...1994 Volume 115 Issue 4 Pages 752-761
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of SAC I, a novel protein inhibitor of blood coagulation produced by Streptoverticillium cinnamoneum subsp. cinnamoneum IFO 12852, was determined. Automated Edman degradation was employed for its fragment peptides, which were obtained by specific cleavage procedures including tryptic, chymotryptic, and peptic digestions, and cyanogen bromide treatment. SACI is composed of 110 amino acid residues with a molecular weight of 11, 642. It has two intramolecular disulfide bonds, Cys31-Cys46 and Cys68-Cys98, but no cysteine residues. The overall sequence homology of SACI is 58% to plasminostreptin, a protein protease inhibitor produced by Streptomyces antifibrinolyticus, and 52% to S-SI, Streptomyces subtilisin inhibitor, produced by Streptomyces albogriseolus. Subtilisin [EC 3. 4. 21. 14]-modified SACI, which was prepared by incubating SACI with subtilisin, had a newly-generated amino terminal sequence, Glu71-Trp72-Asn73-, in addition to the original amino terminal sequence, and a newly-generated carboxyl terminal arginine in addition to the original phenylalanine. These results clearly show that the Arg70-Glu71 bond was specifically cleaved on the limited proteolysis with subtilisin and that the bond is the reactive site for subtilisin.View full abstractDownload PDF (1050K) -
Takashi Murakami, Takayasu Kobayashi, Takayuki Terasawa, Motoko Ohnish ...1994 Volume 115 Issue 4 Pages 762-766
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree molecular species of Mg2+-dependent protein phosphatase (MPPs-1, -2, and -3) were isolated by DEAE cellulose column chromatography and gel filtration from an extract of Saccharomyces cerevisiae. MPP-1 was further purified 150-fold by chromatography using thio-phosphorylated myosin light chain-agarose. MPPs-1, -2, and -3 were distinct from the major acid and alkaline phosphatases, and their activities were not affected by okadaic acid, microcystin-LR or Ca2+, and calmodulin, resembling the enzymatic properties of type 2C protein phosphatase of mammalian cells. The apparent molecular masses of MPPs-1, -2, and -3 on gel filtration were 53, 112, and 128 kDa, respectively. It was demonstrated that MPP-1 is a globular protein of 53-55 kDa and that MPPs-2 and -3 are oligomeric proteins that dissociate upon sucrose density gradient centrifugation, generating catalytic proteins of about 50 kDa. Since the substrate specificities of MPPs-1, -2, and -3 differed from each other both before and after sucrose density gradient centrifugation, it was suggested that the catalytic proteins of these three enzymes are distinct molecular species.View full abstractDownload PDF (631K) -
Yukiko Konami, Chizu Ishida, Kazuo Yamamoto, Toshiaki Osawa, Tatsuro I ...1994 Volume 115 Issue 4 Pages 767-777
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe primary sequence of 247 amino acids of Maackia amurensis hemagglutinin (MAH) was determined using a protein sequencer. After digestion with endoproteinase Lys-C, Asp-N, Arg-C, or Glu-C of MAH, the resulting peptides were purified by reversed phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. The primary sequence of MAR was compared with those of several legume lectins, and it was found that the amino acid sequence of the putative carbohydrate-binding domain of MAH exhibited a high degree of homology with those of di-N-acetylchitobiose-binding Cytisus sessilifolius lectin I (CSA-I), Laburnum alpinum lectin I (LAA-I), and Ulex europaeus lectin II (UEA-II). In the legume lectins whose primary sequences have already been determined several amino acid residues involved in carbohydrate-binding were found to be conserved. Very interestingly, in the primary sequence of MAH, one amino acid residue corresponding to the conserved amino acid, asparagine, in the primary sequences of all other legume lectins was shown to be substituted by aspartic acid. This is the first report of the occurrence of an exceptional amino acid residue among the conserved amino acid residues in the carbohydrate-binding domain of the legume lectins.View full abstractDownload PDF (899K) -
Tomomi Gotoh, Yougo Haraguchi, Masaki Takiguchi, Masataka Mori1994 Volume 115 Issue 4 Pages 778-788
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLiver-selective transcription of the gene for rat arginase, an ornithine cycle (urea cycle) enzyme, is induced by glucocorticoids in a delayed secondary manner; the mRNA induction by the hormones requires de novo protein synthesis, and is preceded by a time lag of several hours. We searched for a DNA element mediating the glucocorticoid induction of the arginase gene with a transient transfection system using hepatoma cell lines. Within the 233-base pair region that is located 11 kilobases downstream from the transcription start site and that spans the junction of intron 7 and exon 8, we detected an enhancer element that is glucocorticoid-responsive and hepatoma cell-selective. The time course of the glucocor-ticoid induction through this enhancer element was delayed compared to that through the primary glucocorticoid-responsive mouse mammary tumor virus promoter. Footprint analysis revealed four protein-binding sites in this enhancer region. In gel retardation analysis, each site exhibited a complicated profile characterized by a number of shifted bands, some of which were tissue-selective and others ubiquitous. Gel shift competition and antibody supershift/inhibition analysis demonstrated that two of the four sites are recognized by members of the CCAAT/enhancer binding protein (C/EBP) family, some of which are liver-enriched.View full abstractDownload PDF (6481K) -
Masafumi Odaka, Chitose Kaibara, Toyoki Amano, Tadashi Matsui, Eiro Mu ...1994 Volume 115 Issue 4 Pages 789-796
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResidue Tyr-341 of the F1-ATPase β subunit from a thermophilic Bacillus strain, PS3, was mutagenized to leucine, cysteine or alanine. Each of the mutated β subunits was isolated and its affinity for ATP-Mg was examined by means of difference circular dichroism and differential titration calorimetry. The Kd values for ATP-Mg obtained were: βY341 (wild type), 0.015mM; βY341L, 0.7mM; βY341C and βY341A, >3mM. All the mutant β subunits could be reconstituted into the α3β3γ complex with α and γ subunits. The α3β(mutant)3γ complexes hydrolyzed ATP with apparent Vmax values larger than that of the α3β(WILD)3γ complex. The apparent Km values of the α3β(mutan)3γ complexes increased in parallel with the Kd values for ATP-Mg of the isolated mutant β subunits. These results indicate that residue βY341 is directly involved in the catalytic ATP-Mg binding and is a major Km-determining residue of F1-ATPase.View full abstractDownload PDF (1004K) -
Michiko Mandai, Masayoshi Iwaki, Yoshihito Honda1994 Volume 115 Issue 4 Pages 797-802
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe performed a comparative study on tyrosine hydroxylase (TH), a rate-limiting enzyme in catecholamine biosynthesis, in two ocular tissues, the retina and the iris-ciliary body. Immunoblotting analysis and gel filtration study suggested no significant difference in their subunit structure and their oligomeric form. The optimal pH of TH was 6.0 in retina and 6.3 in iris-ciliary body. However, at the physiological pH (7.25), the retinal TH had only 15% of the maximum activity while TH in the iris-ciliary body had 70% of the maximum activity. In hydroxylapatite chromatography, both extracts showed different elution profiles; the major TH activity in retina appeared earlier (200mM, phosphate concentration) than that in iris-ciliary body (300mM). When these enzymes were phosphorylated by catalytic subunit of cAMP-dependent protein kinase, most of the activity shifted to the later peak in both enzymes. Also, the activity of dephosphorylated TH in iris-ciliary body shifted to the earlier peaks. These results indicate that native TH in retina is basically less phosphorylated and thus exists in a less activated form than that in iris-ciliary body.View full abstractDownload PDF (1488K) -
Itaru Nitta, Takuya Ueda, Kimitsuna Watanabe1994 Volume 115 Issue 4 Pages 803-807
Published: April 01, 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe found that poly (U) -dependent polypbenylalanine synthesis on Escherichia coli ribosomes was extremely accelerated in the presence of a high concentration of pyridine. In this reaction, chemical energy sources, such as ATP and GTP, and soluble protein factors are not required, but the template and ribosomes are essential for the progress of the reaction. The reaction was inhibited by the antibiotics which inhibit the usual bacterial translation process on the ribosomes. These observations clearly demonstrate that the pyridine-catalyzed amino acid condensation reaction proceeds on the ribosome.View full abstractDownload PDF (4437K)
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