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Volume 114 (1993)
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Volume 114, Issue 5
Displaying 1-24 of 24 articles from this issue
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Hiroyuki Nakagawa, Mitsuaki Ishihara, Kazuyo Ohashi1993 Volume 114 Issue 5 Pages 623-626
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA group of 33-kDa peptides was prepared from chicken gizzard. Gizzard smooth muscle was extracted with Hasselbach-Schneider's high ionic strength solution. The extract was fractionated by ammonium sulfate precipitation. The soluble fraction obtained at 60% ammonium sulfate saturation was dialyzed against 0.1M KCl. The precipitate formed during the dialysis mainly contained 33-kDa peptides. On SDS-PAGE, two major bands and one minor band were stained reddish purple by Coomassie Brilliant Blue R-250. These peptides, which were insoluble in physiological salt solutions, bundled not only actin filaments but also desmin filaments. Actin was markedly coprecipitated with the 33-kDa peptides on the usual centrifugation at 22, 000×g.View full abstractDownload PDF (2228K) -
Toshihide Okajima, Katsuyuki Tanizawa, Toshio Fukui1993 Volume 114 Issue 5 Pages 627-633
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo highly conservative residues, Va167 and Gln 101, in adenylate kinase are located in the hydrophobic region putatively involved in binding of the adenine ring of AMP. We have performed polymerase chain reaction-based random mutagenesis of the two residues using recombinant chicken muscle adenylate kinase cDNA as a template. The synthetic oligonu-cleotide primers contained A, G, C, T-mixed bases in the codons corresponding to those for Va167 and Gln101. The amplified fragments were ligated with the expression plasmid pKK223-3, and the mutant proteins expressed were identified by immunoblotting. Enzymatically active mutant proteins were selected on the basis of the growth at 45°C of the temperature sensitive Escherichia coli mutant for adenylate kinase. At position 67, various amino acid residues other than Val have been found to restore the growth at 45°C of the ts mutant. In contrast, only Gln, His, and Met could be present at position 101. These results are compatible with the proposal that Va167 contributes to the AMP binding through hydrophobic interactions and Gln101 by forming a hydrogen bond with the adenine ring. Indeed, several purified Va167 and Gln101 mutant enzymes exhibited markedly high Km values for AMP, Whereas the Km values for MgATP were comparable to those of the wild-type enzyme. Substrate specificity for nucleoside monophosphates was changed significantly by the mutagenesis of the two residues.View full abstractDownload PDF (794K) -
Mikiharu Yoshida, Yuji Mizuno, Ikuya Nonaka, Eijiro Ozawa1993 Volume 114 Issue 5 Pages 634-639
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe determined several internal amino acid sequences of dystrophin-associated glyco-protein, A3a (one of the 43DAG doublets), of rabbit skeletal muscle. All the sequences of A3a determined were found in the C-terminal region of dystroglycan, which is the region assumed to be the cytoplasmic domain of 43DAG [Ibraghimov-Beskrovnaya et al. (1992) Nature 355, 696-702]. Therefore, A3a is identical with 43DAG. We raised an antibody (PA3a) against a synthetic polypeptide equivalent to one of the internal amino acid sequences of A3a. The antibody specifically reacted with A3a of rabbit skeletal muscle. PA3a, however, did not react with A3b, the other 43DAG doublet, suggesting that the 43DAG doublets are different proteins from each other. When the human control muscles were examined, PA3a immunohistochemically stained the cell surface membranes and exclu-sively reacted with a single protein similar to A3a in the SDS extracts. The protein was also detected in the SDS extract of the Duchenne muscular dystrophy (DMD) muscle devoid of dystrophin. When the muscle specimens from 30 DMD patients were immunohisto-chemically examined with the antibody, the cell surface membranes were consistently stained. Therefore, we conclude that the dystrophin-associated protein, A3a, is retained in DMD muscles.View full abstractDownload PDF (1939K) -
Kanefusa Kato, Sachiyo Goto, Kaori Hasegawa, Yutaka Inaguma1993 Volume 114 Issue 5 Pages 640-647
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe responses of two low-molecular-weight stress proteins, αB crystallin and HSP28, to various types of stress were determined quantitatively by specific immunoassays in a human glioblastoma cell line (U118MG). Levels of αB crystallin (2-4 ng/mg protein) and HSP28 (1-1.5μg/mg protein) in the soluble fraction from cells that had been cultured at 37°C increased about 100-fold and 3-fold, respectively, within 24h after heat treatment for 15min at 45°C, with a temporary decrease, due to redistribution to the insoluble fraction, during the heat treatment. Exposure of cells to arsenite (NaAsO2, 100μM for 1h) also induced the two proteins with a time course similar to that observed after heat stress, but without a decrease during the stress period. L-Azetidine-2-carboxylate (5mM for 5h) was also effective in inducing the two proteins, but to a lesser extent. Other chemicals, including CdCl2, ZnCl2, AlCl3, ethanol, caffeine, nicotine, NaN3, dibutyryl 3', 5'-cyclic AMP, forskolin, and a phorbol ester, did not induce the two proteins. Expression of αB crystallin and HSP28 mRNAs in cells was enhanced after heat stress and after exposure to arsenite. When cells were challenged with heat stress in the presence of arsenite, the effect on the induction of the two proteins was synergistic. Ethanol (1-2%) enhanced the responses to heat stress or arsenite stress. Glycerol (10% or 1.36M), added to the culture medium during the stress period, completely blocked the expression of the mRNA for αB crystallin and the induction of the two proteins by heat stress, but not that by arsenite stress. These results indicate that the two low-molecular-weight stress proteins, αB crystallin and HSP28, respond analogously to heat and chemical stressors in U118 MG cells, but suggest that the events associated with activation of the heat shock genes by heat stress are different from those associated with the activation by chemical (arsenite) stress.View full abstractDownload PDF (1802K) -
Jerzy Silberring, Hans-Ullrich Demuth, Peter Brostedt, Fred Nyberg1993 Volume 114 Issue 5 Pages 648-651
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo cysteine proteinases, cleaving dynorphins A and B to enkephalins, were isolated from the human spinal cord. These enzymes were found to be competitively inhibited by a new class of synthetic inhibitors: N-peptidyl-O-acyl hydroxylamines. The most potent (Ki<20μM) were the N-terminally protected peptides Z-Phe-Phe-NHO-Ma and Boc-Phe-Gly-NHO-Bz, both containing hydrophobic amino acids at the P2 position. N-Peptidyl-O-acyl hydroxylamines were converted in water solution to the corresponding hydroxamic acids and no cleavage of the peptide bond within the inhibitor sequence was observed after prolonged incubation with the enzymes. It is anticipated that these synthetic compounds may serve as potential pharmacological tools for in vitro studies on dynorphin processing.View full abstractDownload PDF (469K) -
Soh Yamazaki, Ken Sato, Katsuko Suhara, Masao Sakaguchi, Katsuyoshi Mi ...1993 Volume 114 Issue 5 Pages 652-657
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA proline-rich region is present following the signal-anchor sequence in the amino-terminal portion of all known microsomal cytochrome P-450s. To assess the functional significance of the proline residues in this region, we systematically altered these residues of cytochrome P 450 (M1) (P 450 2C11); one, two, and three proline residues out of the five in the region were exchanged for alanine residues. The wild-type and the mutated proteins were expressed in the fission yeast Schizosaccharomyces pombe under the control of nmt1 promoter. The wild-type and the mutated proteins were all highly expressed in the yeast cells (5-9% of the total membrane protein). The expressed wild-type P 450 (M 1) showed a typical carbon monoxide difference spectrum of P-450 and the activity of testosterone hydroxylation, whereas all the mutated proteins constructed in the present study showed no characteristic P-450 spectrum, suggesting that the substitution of the proline residues in this region resulted in a defect of proper heme incorporation. Furthermore, the mutated proteins in which more than one proline residues had been exchanged were more sensitive to trypsin digestion than the wild type. From these results, we propose that the proline residues in the proline-rich region are crucial for the formation of the correct conformation of microsomal P-450 molecules.View full abstractDownload PDF (2332K) -
Terutomo Kohira, Kunio Matsumoto, Akira Ichihara, Toshikazu Nakamura1993 Volume 114 Issue 5 Pages 658-662
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCellular receptors for interleukin-1 (IL-1) bind both IL-lα and IL-1β with the same affinity, and both IL-lα and IL-1β have the same potential in most IL-1-related biological responses, despite their low amino acid sequence homology (26%). Here, we identified and partially characterized a biologically functional novel IL-1 receptor, which specifically binds IL-1β but not IL-lα, present on adult rat hepatocytes. Scatchard analysis of the binding of 125I-IL-1β to isolated adult rat hepatocytes showed that rat hepatocytes express a high affinity receptor for IL-1β with a Kd value of 9.6×10-10M at 1, 200 sites/cell, whereas there was no specific binding of 125I-IL-lα at least up to 10 ng/ml. Specific binding of 125I-IL-1β to hepatocytes was competitively inhibited by unlabeled IL-1β, but not by IL-1α. Cross-linking of 125I-IL-1β to plasma membranes of hepatocytes indicated that the receptor for IL-1β has an apparent molecular mass of 80-100 kDa. Moreover, the formation of a cross-linked complex between 125I-IL-1β and the receptor was specifically inhibited by unlabeled IL-1β, but not by IL-lα. Finally, DNA synthesis of adult rat hepatocytes was specifically inhibited by IL-1β, but not by IL-1α, in a dose-dependent manner. Therefore, (i) adult rat hepatocytes express a novel type of IL-1 receptor, i.e., the IL-1β-specific receptor; and (ii) this IL-1β-specific receptor is biologically functional, being relevant to the specific inhibitory activity of IL-1β on growth of rat hepatocytes.View full abstractDownload PDF (1518K) -
Sukalyan Chatterjee, Weining Jiang, S. Donald Emerson, Masayori Inouye1993 Volume 114 Issue 5 Pages 663-669
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCS 7.4 is the major cold-shock protein specifically expressed to a level as high as 13% of the total cellular protein within the first hour when Escherichia coli cell culture is shifted from 37 to 15°C [Goldstein et al. (1990) Proc. Natl. Acad. Sci. USA 87, 283-287]. It consists of 70 amino acid residues with a very high content of aromatic residues. CS 7.4 was overproduced and purified to homogeneity. Its secondary structure was analyzed by examining circular dichroism at both the far and near-UV regions; the results suggest that the protein is largely β-sheet in conformation. The predominance of β-sheet structure in the protein was confirmed by using Fourier-transform infrared spectroscopy. A folded compact conformation was also verified by fluorescence emission spectroscopy. We evaluated Tm, ΔH, and ΔS from the thermal denaturation profile of the protein. Unusual spectral features observed in the far-UV region are attributed to the high content of aromatic residues. The protein is relatively small and contains no disulfide bonds. However, it is surprisingly stable to heat denaturation.View full abstractDownload PDF (1654K) -
Kuo-Chin Lin, David Shiuan1993 Volume 114 Issue 5 Pages 670-676
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTranscription of Escherichia coli biotin operon is repressed by biotin repressor in the presence of biotinyl-5'-adenylate as a corepressor. To determine precisely the site of action of biotin repressor on the operator sequence, DNaseI footprinting experiments were performed on the PCR-produced biotin operator and its mutants. The results indicate that the repressor binds to the wild-type operator as well as mutated operator sequence at +15 position or -15 position, and protects the 39-base region from nucleotide -19 to +20 of the upper strand, and the 40-base region from nucleotide -22 to +18 of the lower strand, with a few hypersensitive sites. This is consistent with previous speculation that the biotin operator is an approximately 40 by imperfect palindromic DNA sequence capable of binding with two molecules of biotin repressor. However, the protection pattern of the mutant operator which lacks half of the palindromic structure is quite different from the corresponding region of the wild type. Though two repressor monomers bound to the mutant operator, as revealed by parallel binding studies [Lin, Shiuan, and Campbell (1991) Biochim. Biophys. Acta 1090, 317-325], only 12 to 13 by on the DNA sequence was protected, suggesting that one monomer of the repressor dimer is hanging near the DNA backbone of the mutant operator even though the biotin repressor is functioning as a dimer.View full abstractDownload PDF (1966K) -
Yasushi Kawakami, Kazuo Nakamura, Hisako Kojima, Minoru Suzuki, Fuyuhi ...1993 Volume 114 Issue 5 Pages 677-683
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel glycosphingolipid (SEGLx) has been isolated from the plerocercoids of a tapeworm, Spirometra erinacei. From the results of compositional analysis, methylation analysis, exoglycosidase hydrolysis, acid hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance (NMR) analysis, its structure was concluded to be _??_ This is the first report of a glycosphingolipid with a novel carbohydrate structure which is characterized by i) the occurrence of a penultimate glucose molecule attached to the reducing end galactose through a 81-3 linkage and ii) the presence of a fucose attached to a glucose through an α1-3 linkage. The ceramide contained sphinganine or 4-D-hydroxy-sphinganine, and either a nonhydroxy fatty acid with 16, 18, 26, or 28 carbon atoms, or hydroxystearic acid. Proton NMR analysis revealed that the chemical species of both the long chain base and fatty acid moieties affect the chemical shifts of the anomeric proton resonances of not only the reducing terminal galactose but also the penultimate glucose.View full abstractDownload PDF (1617K) -
Shizu Takeda, Yasuhiro Sagara, Kiyoshi Kita, Shunji Natori, Kazuhisa S ...1993 Volume 114 Issue 5 Pages 684-690
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA particulate fraction consisting of heavy organelles such as nuclei and mitochondria was prepared from Ehrlich ascites tumor cells. From this fraction we have purified a GTP-binding protein with a molecular mass of 33 kDa (MTG 33) by guanidine hydrochloride extraction followed by four steps of column chromatography. The Kd value of MTG 33 for GTP was 17 nM. [α-32P] GTP-binding to MTG 33 was inhibited by GTP and GDP, but not appreciably by ATP, CTP, UTP, or GMP. MTG 33 hydrolyzed GTP to GDP at a rate of 4.5 mmol/min/mol protein. Subcellular fractionation analysis of mouse liver revealed that the heavy mitochondrial fraction contained the highest level of MTG 33. Furthermore, dual immunofluorescence examination indicated that the staining of NIH 3T3 cells with anti-MTG 33 antibody is coincident with the distribution of mitochondrial succinate dehy-drogenate. Of the mouse organs examined, the heart contained the highest level of MTG 33. These results strongly suggest that MTG 33 is a GTP-binding protein located in mitochondria.View full abstractDownload PDF (2314K) -
Tomiyoshi Ito, Jiro Hiraiwa, Junji Nishioka, Masaki Iino, Koji Suzuki1993 Volume 114 Issue 5 Pages 691-696
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTissue factor is a cellular membrane glycoprotein that acts as a cofactor for Factor VIIa-catalyzed activation of Factors X and IX in the extrinsic blood coagulation pathway. We prepared fourteen murine monoclonal antibodies to human tissue factor (designated MTF-1 to -14), and purified tissue factor from human placenta by immunoaffinity chromatography using the antibody-coupled Sepharose 4 B. The antibodies were characterized for inhibitory activity against the activation of Factor X by a complex of Factor VIIa and purified placental tissue factor or a recombinant mutant tissue factor whose transmembrane and cytoplasmic domains were deleted. Four MTFs (MTF-6, -7, -8, and -14) markedly inhibited the cofactor activity of tissue factor by directly blocking the interaction with Factor VIIa. Three other MTFs (MTF-1, -4, and -9) moderately inhibited the cofactor activity of the mutant tissue factor, presumably by diminishing the interaction between the extracellular region of the tissue factor and phospholipids. Both groups of MTFs inhibited the cofactor activity of the mutant tissue factor in Factor X activation by Factor VIIa in the absence of phospholipids. These findings suggest that the former four MTFs recognize the binding site for Factor VIIa and the latter three MTFs recognize the site for Factor X in the extracellular region of tissue factor. These MTFs are useful to purify tissue factor by immunoaffinity chromatography and to study the structure-function relationship of tissue factor, and may be useful to establish an immunoassay of tissue factor in body fluids.View full abstractDownload PDF (1689K) -
Yasuna Kobayashi, Yoichi Matsuura, Eiichi Kotani, Teruo Fukuda, Takaak ...1993 Volume 114 Issue 5 Pages 697-701
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe investigated the structural requirements for the induction of hepatic microsomal cytochrome P 450 2B1/2 (P450 2B1/2) and cytochrome P 450 1A1/2 (P 450 1A1/2) by imidazole- and pyridine-containing compounds in rats. Clotrimazole, an azole antifungal drug, and 1-diphenylmethylimidazole preferentially induced P 450 2B1/2 in a dose-depen-dent manner, and slightly induced P 450 1A1/2. 1-Benzylimidazole preferentially induced P 450 1A1/2. 1-Phenylimidazole, which lacks the methylene bridge of 1-benzylimidazole, only induced P 450 1A1/2. In turn, loss of aromaticity of the N-substituted moiety of imidazole, as in 1-cyclohexylmethylimidazole and 1-tert-butylimidazole, resulted in a preferential induction of P450 2B1/2. Likewise, various pyridine-containing compounds showed structure-dependent induction of P 450 species. Namely, 4-diphenylmethyl-pyridine induced P 450 2B1/2.4-Benzylpyridine induced both P 450 2B1/2 and P 450 1A1/2. 4-Cyclohexylmethylpyridine and 4-tent-butylpyridine predominantly induced P 450 2B1/2.4-Phenylpyridine preferentially induced P 450 1A1/2 rather than P 450 2B1/2. Oxygenation products at the methylene bridge, 4-benzoylpyridine and phenyl-4-pyridylmethanol, could not induce P 450 1A1/2. In turn, 2, 4'-dipyridyl induced both P 450 2B1/2 and P 450 1A1/2, but not 2, 2'-dipyridyl. 4, 4'-Trimethylenedipyridine preferentially induced P 450 1A1/2 at very low doses. These findings indicate that imidazole- and pyridine-containing compounds having lipophilic groups are inducers of hepatic P 450, and that such compounds having aromatic groups and taking coplanar conformational structures are potent inducers of P 450 1A1/2.View full abstractDownload PDF (1281K) -
Krishna Singh, Dave Deonarine, Vijayalakshmi Shanmugam, Donald R. Seng ...1993 Volume 114 Issue 5 Pages 702-707
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOsteopontin (OP), purified from rat bone, binds Ca2+ but whether different molecular forms of OPs derived from non-osteogenic sources and non-phosphorylated OP also possess this property remains to be determined. Furthermore, it is not known which specific site or sites of the molecule bind Ca2+. In the present study, following an established procedure, total proteins in the conditioned media from OP-synthesizing cell cultures were separated by SDS-PAGE, transferred to Immobilon-P membranes, and incubated with 45CaCl2, then Ca2+ ions bound to protein bands were analyzed by autoradiography. Purified OPs, and synthetic oligopeptides representing specific domains of the OP molecule were adsorbed on the membrane and processed as described above. Our results show that OPs synthesized by normal rat kidney cells, oncogenically transformed Rat-1 cells, OP purified from human milk, and non-phosphorylated OP secreted by 1α, 25-dihydroxyvitamin D3-treated mouse epidermal JB 6 cells all bind detectable levels of Ca2+ with specificity. We also show that a synthetic peptide representing the domain of OP which contains nine consecutive aspartic acid residues binds Ca2+ with specificity. It is probable, therefore, that a Ca2+-binding site resides in this region of the OP molecule. We conclude that Ca2+-binding is a general property of OP, irrespective of its molecular mass and origin, and the phosphate moieties of OP may not influence the conformation or accessibility of the Ca2+ affinity sites of the molecule.View full abstractDownload PDF (4018K) -
Kunio Fujiwara, Tsunehiro Kitagawa1993 Volume 114 Issue 5 Pages 708-713
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme-linked immunosorbent assay (ELISA) for small molecular haptens needs conjugates of the hapten with larger protein molecules for coating the wells of microtiter plates. The formation of such conjugates is not always reproducible. This makes it difficult to evaluate hapten-protein stoichiometries and to understand the precise orientation of the hapten on the protein. In this paper we describe an assay in which the polyamine spermidine (Spd) is coupled with glutaraldehyde (GA) to carrier poly-L-lysine (PL) or human serum albumin (HSA) coated on polystyrene microtiter wells. Each step of the assay was tested for maximum efficiency. This ELISA detected Spd with excellent reproducibility (coefficient of variation=5.9%), an EC50 of 4.3×10-6M and a detection limit of 0.1×10-6M. The present ELISA was about 100 times more sensitive in detecting Spd than a conventional ELISA for Spd using a Spd-HSA conjugate for coating microtiter wells. The Spd antiserum showed 215% cross-reaction with N1-acetyl-Spd, 30.7% with N8-acetyl-Spd, 10% with spermine, 3.3% with cadaverine, 1.7% with putrescine, and less than 0.43% with 1, 3-diaminopropane in the new ELISA system. A much higher degree of hapten binding to the plate occurred with the present method than with the previously reported method using microtiter wells activated directly with GA. The hapten conjugation is simple, reproducible and should provide a general method for developing ELISAs, not only for polyamines but also for amino acids and small peptides.View full abstractDownload PDF (589K) -
Tomihiko Higuti, Kayo Kuroiwa, Sadamori Miyazaki, Yutaka Yoshihara, Hi ...1993 Volume 114 Issue 5 Pages 714-717
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubunit d of H+-ATP synthase from rat liver mitochondria was isolated from the purified enzyme by reverse-phase high performance liquid chromatography. The partial amino acid sequence of the subunit was determined by automated Edman degradation of the peptide fragments. The nucleotide sequence of subunit d of rat liver H+-ATP synthase was determined from a recombinant cDNA clone isolated by screening a rat hepatoma cell line H4TG cDNA library with a probe DNA. The sequence was composed of 581 nucleotides including a coding region for the import precursor of subunit d and noncoding regions on the 5'- and 3'-sides. The possible precursor of subunit d and its mature polypeptide deduced from the open reading frame consisted of 161 and 160 amino acid residues with molecular weights of 18, 763 and 18, 631, respectively. Subunit d is a hydrophilic protein with an isoelectric point of 6.19. The sequence of the rat subunit d is highly homologous with that of subunit d of bovine heart and slightly similar to that of the subunit d of the yeast mitochondria. However, it had no homology with the sequence of any of the subunits of bacterial or chloroplast H+-ATP synthase.View full abstractDownload PDF (396K) -
Masaaki Ohnuma, Issei Mabuchi1993 Volume 114 Issue 5 Pages 718-722
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn actin filament-severing activity of 45 K protein isolated from sea urchin eggs was abolished when this protein was incubated with phosphatidylinositol-4, 5-bisphosphate (PIP2). This effect was specific to PIP2 since phosphatidylinositol, phosphatidylinositol-4-monophosphate, inositol-1, 4, 5-trisphosphate, and phosphatidylserine did not show such an effect at the same concentration. Digestion of PIP2 with phospholipase C eliminated the effect. On the other hand, PIP2 did not affect either the formation of 45 K protein-actin complex or actin filament-capping activity of the complex. Possible implication of the binding of PIP2 to 45 K protein in cytoskeleton formation after fertilization of sea urchin eggs is discussed.View full abstractDownload PDF (468K) -
Shitsuw Shikata, Kohei Shimada, Yasuo Ohnishi, Sueharu Horinouchi, Ter ...1993 Volume 114 Issue 5 Pages 723-731
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Serratia marcescens serine protease (SSP; 66 kDa) is synthesized as a precursor (preproSSP; 112 kDa) composed of the NH2-terminal signal peptide of 27 amino acids, the mature protease part and a large COOH-terminal domain. When the SSP gene is expressed in Escherichia coli under the control of the tac promoter, the mature enzyme is excreted into the medium through the outer membrane, whereas preproSSP and two proteins, C-1 (40 kDa) and C-2 (38 kDa), processed from the COOH-terminal domain, are accumulated in the membrane fraction. Although treatment of the intact cells with trypsin caused slight truncation of C-1 and C-2, the main parts of C-1 and C-2, both of which are detected in the outer membrane, were resistant to trypsin, even after the cells had been osmotically shocked. Consistent with this, a high content of β-sheet structure in C-2 was suggested by marked heat-modifiability, as determined by their electrophoretic mobilities on SDS-polyacrylamide gel. These findings suggest rigid integration of C-1 and C-2 in the outer membrane. Upon induction of the tac promoter, rapid excretion of SSP into the medium was first accompanied by the accumulation of C-1 in the outer membrane, which was followed by conversion of C-1 to C-2. PreproSSP was not detected during the accumulation of SSP in the medium, but it was gradually accumulated after the accumulation of SSP had reached a plateau. In addition, preproSSP still containing the intact NH2-terminal signal peptide was completely digested with trypsin when added to osmotically shocked cells. It is therefore likely that preproSSP is a dead-end product in the secretory pathway of SSP. This may be explained in terms of a perturbation of the secretory machineries caused by the overexpression of preproSSP, since β-lactamase was also accumulated in the precursor form in the insoluble fraction of the cells accumulating preproSSP. An active-site mutant enzyme containing Thr instead of Ser-341 was still excreted into the medium, but ac-companied by abnormal processing. This abnormal processing was ascribed to both the OmpT protease and an unidentified protease of E. coli.View full abstractDownload PDF (5491K) -
Taketoshi Uzawa, Akihiko Yamagishi, Takuya Ueda, Nobutoshi Chikazumi, ...1993 Volume 114 Issue 5 Pages 732-734
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA continuous cell-free protein synthesis system of an extremely thermophilic eubacterium, Thermus thermophilus HB 27, was constructed. This system produced MS 2 phage RNA translation products at a rate of more than 5μg per hour per 1.9mg of ribosomes at 65°C and the production continued linearly for at least 340min. When no polyamine was added, the system did not produce the proteins. The highest activity was recorded when 0.1mM tetrakis (3-aminopropyl) ammonium and 1.0mM spermine were added simultaneously.View full abstractDownload PDF (306K) -
Sachiko Murase, Noboru Yumoto1993 Volume 114 Issue 5 Pages 735-739
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activity of aspartase (L-aspartate ammonia-lyase, EC 4. 3. 1. 1) from Escherichia coli is enhanced 2- to 3-fold by three types of modification of the enzyme as reported previously; the replacement of Cys-430 with Trp by site-directed mutagenesis, the truncation of the C-terminal region by limited proteolysis, and the acetylation of amino groups with acetic anhydride. To elucidate the molecular basis of such activation, we have compared the kinetic properties of the modified enzymes in this study. Although the modifications caused very similar changes in the kinetic properties, such as increase in kcat, the half-saturating concentration of substrate, and Hill coefficient values, the modified enzymes differed greatly in sensitivity to the activator L-aspartate and the inhibitor Cl- ions. As a result of the mutation, the binding affinity for the activator was greatly decreased without change in the sensitivity to the inhibitor, whereas after acetylation, the sensitivity to the inhibitor was completely lost without decrease in the binding affinity for the activator. After truncation of the C-terminal region, both a large decrease in the binding affinity for the activator and complete loss of sensitivity to the inhibitor occurred, suggesting that this type of activation is equivalent to the former two types combined.View full abstractDownload PDF (507K) -
Naoyuki Yamamoto, Atsuko Akino, Toshiaki Takano1993 Volume 114 Issue 5 Pages 740-745
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cell-wall-associated proteinase from Lactobacillus helveticus CP 790, grown in skim milk, was purified to homogeneity by DEAE ion exchange chromatography in the presence of EDTA. Its molecular weight was estimated to be 45, 000 by SDS-PAGE and also by the recovery of proteinase activity only from this fraction of SDS-PAGE gel slices of crude extract. It had maximum activity at pH 6.5 and 42°C. Since the activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), it was suggested to be a serine-type proteinase. It preferentially hydrolyzed α- and β-casein but did not hydrolyze κ-casein by the purified enzyme. The 10 main peptides liberated from α-casein, and the 15 peptides liberated from β-casein, were isolated by reversed phase HPLC. These peptides were hydrolyzed by HCl and their amino acid compositions were analyzed and identified. Many of peptides were located in the C-terminal parts of α- and β-casein. Peptide bonds cleaved by the proteinase had no clear specificity but Leu-X, Phe-X, Ser-X, Lys-X, Glu-X, and Gln-X sequences frequently appeared.View full abstractDownload PDF (1489K) -
Akiho Yokota, Hiroshi Tokai1993 Volume 114 Issue 5 Pages 746-753
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLysine residues have been suggested to be involved in the hysteretic decrease of the activity of spinach ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) and the binding of ribulose 1, 5-bisphosphate to its regulatory sites [Yokota, A. & Tsujimoto, N. (1992) Eur. J. Biochem. 204, 901-909]. This work identifies the lysine residues and investigates the effects of their chemical modification on the course of RuBisCO reaction. The carbamylated form of RuBisCO reacted with trinitrobenzene sulfonate in three phases; an initial rapid, second slow, and final non-specific reaction. Lys-334 in loop 6, Lys-21, and Lys-128, all from the large subunits, were trinitrophenylated in the first 60-min reaction. Lys-305 of the large subunits was labeled in the next step. The modification of these residues was strongly suppressed in the enzyme form that had undergone hysteretic conformational change after binding 2-carboxyarabinitol 1, 5-bisphosphate at its catalytic sites. Instead, Lys-450 of the large subunits and Lys-71 from the small subunits were newly modifed in the quaternary complex. A higher concentration of 2-carboxyarabinitol 1, 5-bisphosphate reduced the trinitrophenylation of the two residues to half. The modification of the carbamylated form of the enzyme for 30min was expected to arylate Lys-21, Lys-128, and Lys-334 at random, and the course of the reaction of the partially modified enzyme was expected to deviate from that of the unmodified enzyme. Experimental results showed that this was the case. Lys-21 may construct a salt bridge with Glu-53 of the same large subunit, and Lys-128 is close to the peptidic oxygens between Val-331 and Gly-333 of loop 6 of the adjoining subunit in the large-subunit dimer in the quaternary complex. We reason that the decrease in activity in the reaction course may be caused by the interactions of these lysine residues with other important residues.View full abstractDownload PDF (910K) -
Isabel de la Mata, María Pilar Castillón, Juan Manuel Do ...1993 Volume 114 Issue 5 Pages 754-759
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inhibition of β-glucosidase from Trichoderma reesei QM 9414 by several specific reagents was studied. Diethylpyrocarbonate (DEP) nearly abolished the enzyme activity at concentrations above 10mM. The presence of substrate or analogs protected the enzyme against inactivation. The reaction followed pseudo-first order kinetics with a second-order rate constant of 0.02mM-1•min-1. The pH-dependence of the inactivation showed the involvement of a group with a pK of 5.2. Difference spectra at 242 nm and the reversal of the inactivation in the presense of 1M hydroxylamine indicated the modification of histidine residues. Statistical analysis of residual fractional activity versus the number of modified histidine residues indicated that one histidine residue is essential for catalysis. p-Hy-droxymercuribenzoate completely inhibited the enzyme at concentrations of the reagent above 2mM. Substrate or analogs protected the enzyme against inactivation. The reaction followed pseudo-first order kinetics with a second-order rate constant of 0.002mM-1 min-1. Treatment of the modified enzyme with 5, 5'-dithio-bis (2-nitrobenzoic acid) (DTNB) showed that one cysteine residue was essential for activity. At pH 5.0 2-ethoxy-l-ethoxy-carbonyl-1, 2-dihydroquinoline (EEDQ) inactivated the enzyme according to pseudo-first order kinetics with a second-order rate constant of 0.12min-1. The pH-dependence of the inactivation showed the involvement of a group with a pK of 5.64, indicating the modification of a carboxyl group essential for activity.View full abstractDownload PDF (613K) -
1993 Volume 114 Issue 5 Pages 760
Published: 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (21K)
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