巻号一覧
109 巻, 6 号
選択された号の論文の23件中1～23を表示しています
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Masao Sakaguchi, Chiharu Ueguchi, Koreaki Ito, Tsuneo Omura1991 年 109 巻 6 号 p. 799-802
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーTo find factors participating in protein translocation in yeast, we screened a yeast genomic library for genes which, when introduced into Escherichia coli, suppressed secY24, a temperature sensitive mutation of an essential integral membrane protein (SecY) required for protein export. We isolated and characterized a gene (YSY6) which improved the translocation of the OmpA protein in mutant strain IQ85(secY24). It could also suppress another mutant [rpl0215(Am)], in which the level of expression of the SecY protein is decreased at high-temperature. The YSY6 gene encodes a small amphiphilic peptide consisting of 65 amino acids, which can be expressed in E. coli cells.抄録全体を表示PDF形式でダウンロード (2809K) -
Kazuhisa Nakayama, Masahiro Hosaka, Kiyotaka Hatsuzawa, Kazuo Murakami1991 年 109 巻 6 号 p. 803-806
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーWe cloned and sequenced a cDNA from a library of mouse pituitary AtT-20 cells which are known to cleave an endogenous and various foreign prohormones at dibasic sites. This cDNA encodes a novel 753-residue protein, named PC3, which is structurally related to the yeast Kex2 protease involved in precursor cleavage at dibasic sites and to recently identified mammalian Kex2-like proteins, furin and PC2. Among examined cell lines and tissues, PC3 mRNA was only detected in AtT-20 cells. The substrate specificity of PC3 expressed in mammalian cells was similar to that observed in AtT-20 cells. We conclude that PC3 is a resident prohormone processing endoprotease in AtT-20 cells.抄録全体を表示PDF形式でダウンロード (1395K) -
Hiroyuki Takashima, Shigetoshi Amiya, Yuji Kobayashi1991 年 109 巻 6 号 p. 807-810
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーConformational analysis of neocarzinostatin, an antibiotic protein with antitumor activity, in its holo-state in solution was carried out by NMR-spectroscopy. The results showed a β-barrel structure for the carrier protein, in which the chromophore is tightly enwrapped and thus shielded from exposure to the solvent. The three-dimensional structure of this complex led to the proposal of a possible mechanism for the exposure or release of the active principle due to subtle environmental changes, e. g., in pH.抄録全体を表示PDF形式でダウンロード (328K) -
Tetsuya Taura, Koreaki Ito1991 年 109 巻 6 号 p. 811-815
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーIt is not known whether the activity of the Escherichia coli protein export system changes during the division cycle. To address and answer this question, we took two approaches. First, we pulse-labeled a random culture and size-fractionated the labeled cells in the presence of inhibitors of secretion. Second, we pulse-labeled synchronously growing cells at different phases in the cell cycle. In the latter experiment, we used a new method of synchronization in which a random culture was simply filtered through glass fiber filters. In both cases, the proportions of unprocessed precursor molecules were measured by immunoprecipitation and gel electrophoresis for some representative periplasmic and outer membrane proteins. Within the sensitivity limits of these methods, we could not detect any significant variation in the precursor labeling for cells of different ages. Thus, E. coli cells appear to secrete proteins continuously throughout the division cycle.抄録全体を表示PDF形式でダウンロード (2426K) -
Kunihiko Konno1991 年 109 巻 6 号 p. 816-821
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーThermal treatment of squid myosin subfragment-1 (S-1) in the presence of EDTA results in a rapid inactivation of ATPase, a marked turbidity increase, and a dissociation of light chains. These effects were suppressed by addition of calcium ion. Different light chain binding in EDTA-medium from that in Ca-medium was demonstrated by the tryptic digestion of native squid S-1; the two types of light chain are both resistant to trypsinolysis in Ca-medium, whereas they are readily degraded in EDTA-medium. S-1 heavy chain was converted into three fragments with sizes of 27, 47, and 22 kDa in both media. However, trypsinolysis of S-1 inactivated in Ca-medium generated no such heavy chain fragments that survived, while the two types of light chain survived. These light chains were isolated as a complex lacking any heavy chain fragments, and the complex formation was Ca-sensitive. It is concluded that regulatory and essential light chains are present on S-1 as a complex whose formation is mediated by calcium ion, and this binding might alter the S-1 conformation so as to confer resistance to thermal treatment.抄録全体を表示PDF形式でダウンロード (3967K) -
Kazuyuki Honda, Kazuo Ono, Tsutomu Mori, Hideo Kochi1991 年 109 巻 6 号 p. 822-827
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーClofibrate administration to rats caused both the activation and induction of the branched-chain 2-oxo acid dehydrogenase complex in the liver; the former phenomenon occurred within the first 6 h after clofibrate administration whereas the latter occurred after 12 h. Essentially the same results were obtained with primary cultures of rat hepatocytes in the presence of 0.5 mM clofibrate, though about three-fourths of the enzyme complex in control cells (without clofibrate addition) was inactivated during a culture for 44 h, with little reduction of the enzyme amount. This was also confirmed by immunotitration analysis with antibodies raised against the purified decarboxylase and transacylase components of the enzyme complex. On the other hand, the activity of dihydrolipoamide dehydrogenase (a constituent of the complex) was little affected by clofibrate administration. The half lives of the decarboxylase and transacylase components in the primary cultures were estimated to be in the range of 22-26 h, and were unchanged in the presence of clofibrate, when determined with the use of cycloheximide and by a pulse-chase experiment. On the contrary, the rates of synthesis of these two enzyme components had increased to about 1.9-fold after 32 h cultivation in the presence of clofibrate. Thus, the increase in the synthesis of both the components resulted in induction of the complex.抄録全体を表示PDF形式でダウンロード (639K) -
Ken Watanabe, Tadashi Baba, Shin-ichi Kashiwabara, Akiko Okamoto, Yuji ...1991 年 109 巻 6 号 p. 828-833
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーThe genomic region carrying the mouse acrosin gene, including the 5'-flanking sequence, has been isolated and characterized. The acrosin gene consists of five exons separated by four introns. Organization of this gene is very similar to those of the genes for other typical serine proteases, except for the phase class of the first intron. Riboprobe mapping and primer extension analyses showed that the start site of transcription initiation in the acrosin gene is heterogeneous, including three major sites. Thus, the structure and organization of the mouse acrosin gene are different from those of the human gene [Keime, S., Adham, I. M., & Engel, W. (1990) Eur. J. Biochem. 190, 195-200] in two respects: the number of transcription initiation sites and the phase class of the third intron. The putative promoter regions of the mouse and human acrosin genes lack typical sequences of TATA, CAAT, and GC boxes, but contain a consensus sequence, GGGTGGG, known to be specific for the phosphoglycerate kinase-2 gene, and the protamine-1 and 2 genes that are uniquely expressed during spermatogenesis.抄録全体を表示PDF形式でダウンロード (624K) -
Osamu Ohara, Tohru Nakano, Hiroshi Teraoka, Hitoshi Arita1991 年 109 巻 6 号 p. 834-839
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーThe isoform expression patterns of actin and actin-binding proteins have been reported to be good markers for phenotypic modulation of rat vascular smooth muscle cells. In order to elucidate the regulatory mechanism of actin and tropomyosin isoform expression on a molecular basis, we examined the effects of various agents on the isoform expression patterns of actin and tropomyosin at the mRNA level in smooth muscle cells. We found that cAMP-elevating agents induced drastic decreases in the amounts of α-smooth muscle type actin and α-tropomyosin transcripts, the expression of other actin and tropomyosin isoforms being also repressed, but to a lesser extent. The results of the experiment involving RNA synthesis inhibitors strongly suggest that activation of some mRNA-specific degradation machinery by cAMP might be responsible at least for the rapid disappearance of α-smooth muscle type actin and α-tropomyosin transcripts in smooth muscle cells.抄録全体を表示PDF形式でダウンロード (4109K) -
Jean Guillot, Michel Giollant, Mireille Damez, Martine Dusser1991 年 109 巻 6 号 p. 840-845
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーA lectin (LDL) has been isolated from carpophores of the edible mushroom, Lactarius deliciosus, using a combination of affinity chromatography on stromas of group 0 erythrocytes embedded in polyacrylamide gel and hydroxylapatite, and gel filtration chromatography. Its molecular weight, as determined by gel filtration, is about 37, 000 and its structure is dimeric, with two distinct types of subunits (about 19, 000 and 18, 000). It appeared homogeneous on HPLC gel filtration, but exhibited microheterogeneity on isoelectric focusing. Amino acid analysis revealed that it contains a large amount of glycine. Hapten inhibition assaying indicated that the Lactarius lectin is most specific for D-Galβ1→3D-GalNAc. The lectin was found in the mycelium and its possible role in the fungus is discussed.抄録全体を表示PDF形式でダウンロード (2471K) -
Nobuyuki Takahashi, Taihei Koseki, Etsushiro Doi, Masaaki Hirose1991 年 109 巻 6 号 p. 846-851
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーOvalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the nonreactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6. 8°C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation.抄録全体を表示PDF形式でダウンロード (2860K) -
Hiromi Kirino, Tairo Oshima1991 年 109 巻 6 号 p. 852-857
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーA gene (leuB) coding for 3-isopropylmalate dehydrogenase [EC 1.1.1.85] from an extreme thermophile, Thermus aquaticus YT-1 was cloned in Escherichia coli and the nucleotide sequence was determined. It contains an open reading frame of 1, 035 by encoding 344 amino acid residues. The homology with that from T. thermophilus HB8 is 87.0% in nucleotide and 91.3% in amino acid sequences. No overlapped gene was found in the present leuB gene, in contrast to the previous prediction that Thermus leuD gene is overlapped with leuB [Croft et al. (1987) Mol. Gen. Genet. 210, 490-497] . Substitutions in the primary structure which are unique for the thermophile sequences are discussed in relation to the unusual stability of the thermophile dehydrogenase based on amino acid sequence comparison of 9 micro-organisms including thermophiles and mesophiles.抄録全体を表示PDF形式でダウンロード (662K) -
Tsuyoshi Okagaki, Sugie Higashi-Fujime, Ryoki Ishikawa, Hiromi Takano- ...1991 年 109 巻 6 号 p. 858-866
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 μm/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 μm/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.抄録全体を表示PDF形式でダウンロード (5034K) -
Ichiro Masai, Yoshiki Hotta1991 年 109 巻 6 号 p. 867-871
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーThe Drosophila mutant no receptor potential A (norpA) is the phototransduction-defective mutant which lacks phosphatidylinositol-specific phospholipase C (PI-PLC) activity. Recently, norpA cDNA was isolated and its homology to a bovine PI-PLC was demonstrated [Bloomquist, B. T. et al. (1988) Cell 54, 723-733] . On the basis of its cDNA, we determined the genomic organization of the norpA gene and revealed that the norpA gene consists of 13 coding exons spreading over a 15 kb genomic area. Furthermore, we identified the mutational sites of two temperature-sensitive (ts) mutants. The analysis of norpAH52 revealed that the single amino acid change from Ser-551 to Tyr causes the PI-PLC activity to become temperature-sensitive. The other allele, norpAKO52, has two substitutions from Ser-406 to Phe and from Gly-451 to Ser. These three mutations are located in regions highly conserved in other mammalian PI-PLC molecules. This suggests that these regions are important for PI-PLC catalytic activity.抄録全体を表示PDF形式でダウンロード (1335K) -
Lin Xie, Jun-Ichi Miyazaki, Tamio Hirabayashi1991 年 109 巻 6 号 p. 872-878
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーTwo-dimensional gel electrophoresis of eight digestive organ extracts from 60-day-old chicks was performed. Judging from the similarity of their protein maps, the organs were classified into the following four types: 1) esophagus type, 2) proventriculus type, 3) gizzard type, and 4) intestine type. In four representative organs of these types, the distribution of tropomyosin isoforms was examined, and four high- and five low-Mr-type isoforms in addition to α and β isoforms were detected in the embryonic organs. In the adult organs, however, there were three high- and four low-Mr-type isoforms, which were restricted to the mucous membrane, in addition to α and β isoforms. Immunoblot analysis indicated that the high- and low-Mr-type isoforms in the embryo corresponded with those in the adult mucous membrane, but differences in the number and amount of the isoforms were found between the embryo and the adult mucous membrane.抄録全体を表示PDF形式でダウンロード (5853K) -
Kenji Kihira, Akira Okamoto, Seiichiro Ikawa, Takahiro Mikami, Michiko ...1991 年 109 巻 6 号 p. 879-881
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーMetabolism of sodium 3α, 7α-dihydroxy-5β-cholane-24-sulfonate, the sulfonate derivative of chenodeoxycholic acid, was studied in hamsters. In bile fistula hamsters, the sulfonate analogue was efficiently absorbed from the ileum and secreted rapidly into the bile without any modification such as conjugation. However, absorption from the jejunum was smaller than that observed for the ileum. After oral administration, the sulfonate analogue of chenodeoxycholic acid was recovered quantitatively in the feces as the unchanged form in contrast to simultaneously administered chenodeoxycholic acid, which was entirely converted to lithocholic acid during its passage through the intestinal tract. These results demonstrate that the sulfonate analogue is absorbed mainly from the ileum by active transport, enters the enterohepatic circulation like the endogenous conjugated bile acids, and completely resists bacterial degradation.抄録全体を表示PDF形式でダウンロード (290K) -
Hitoshi Ueda, Hitoshi Maruyama, Mitsunobu Doi, Masatoshi Inoue, Toshim ...1991 年 109 巻 6 号 p. 882-889
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーAn artificial gene coding for the human cap binding protein (hCBP: human IF-4E) was chemically synthesized and expressed in Escherichia coli under the control of a trp promoter. The DNA duplex of 662 by was designed and constructed from 44 oligodeoxynu-cleotide fragments of typically 30 nucleotides in length. Although the hCBP gene was not directly expressed in E. coli HB101, we succeeded in its high-level expression as a fusion protein connected with a portion of human growth hormone through a tetradecapeptide (Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) that contains the recognition sequence for a site-specific protease α-thrombin. Upon induction with 3-indoleacrylic acid, the fusion protein accumulated with a yield of about 20% of the total proteins of the host cell. Upon the treatment of the fusion protein with α-thrombin, which recognizes the sequence “Val-Pro-Arg, ” specific proteolysis at the fused junction occurred efficiently. In this system, nonspecific digestion by α-thrombin was not marked. About 15 mg of recombinant hCBP was obtained from a 1-liter culture. Association constants between the recombinant hCBP and mRNA cap structure analogues were determined by fluorescence spectros-copy. The values obtained for the m7GpppA, m7GTP, and m7GMP were almost the same as those reported for the IF-4E isolated from human erythrocyte cells. The results show that the binding constants for nonmethylated analogues GpppA, GTP, and GMP, a 2-amino deletion analogue m7IMP, a nonphosphorylated analogue 7-methylguanosine, and a ribose-deleted analogue 7-methyl-9-ethylguanine, were smaller than those for methylated cap structure analogues by about one order of magnitude. Moreover, m7AVP (an analogue containing the 7-methylguanine base and phosphate group) showed nearly the same association constant as the cap structure. Based on these results, a possible model for mRNA cap structure recognition by eIF-4E has been proposed.抄録全体を表示PDF形式でダウンロード (1400K) -
Kei-ichi Enjyoji, Kaoru Miyazaki, Hisao Kato1991 年 109 巻 6 号 p. 890-898
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーWe have found that rat plasma corrected the non-activated PT of human normal or factor-X deficient plasma, and the factor Xa-like activity being constantly detected in every 1 ml of blood collected via the cannulated carotid artery of rats. The present study was undertaken to characterize the factor Xa-like activity in rat plasma by preparing rat factor X and a monoclonal antibody against it. Factor X was purified from a BaCl2 eluate of rat plasma by chromatographies on columns of DEAE-Sepharose CL-6B and Sulfate Cellulofine or on a column of Affi-Gel 10 conjugated with a monoclonal antibody against rat factor X. Factor Xa-like activity in rat plasma was eliminated by the treatment of rat plasma with a monoclonal antibody which recognized the heavy chain portions of rat factors X and Xa. A kinetical study demonstrated that rat factor Xa was strongly inhibited by rat antithrombin III, with a K1 of 2.2×10-11M, in the presence of heparin. However, in the absence of heparin, the second order rate constant for the inhibition of rat factor Xa by rat antithrombin III was 2.6×104M-1•min-1, which was one forty-third that for the inhibition of human factor Xa by human antithrombin III. Furthermore, rat factor Xa was resistant to the inhibition by rat α-1-antitrypsin and α-2-macroglobulin. These results indicate that the small amount of factor Xa (1.7% of total factor X) in rat plasma was derived through the continuous activation of factor X in the circulation or through the instantaneous activation of factor X through the interaction of blood with the vascular wall during the collection, and remained stable in the plasma after the collection.抄録全体を表示PDF形式でダウンロード (930K) -
Kaoru Kusui, Kazuo Yamamoto, Yukiko Konami, Toshiaki Osawa1991 年 109 巻 6 号 p. 899-903
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーBauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1, 152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.抄録全体を表示PDF形式でダウンロード (2790K) -
Atsushi Hara, Tamotsu Taketomi1991 年 109 巻 6 号 p. 904-908
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーCharacterization and elucidation of the changes of glycosphingolipids in the aorta along with the progression of atherosclerosis were performed in the Watanabe hereditable hyperlipidemic (WHHL) rabbit, an animal model for human familial hypercholesterolemia, as compared with in the normal rabbit. Neutral glycosphingolipids in aortae of both normal and WHHL rabbits were composed of glucosylceramide, galactosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, and galactosylneolactotetraosylceramide. The total amount of neutral glycosphingolipids in the aorta of the WITHL rabbit (557nmol/g tissue) was increased about 5-fold compared to the normal level (107nmol/g tissue). Prominent increases were observed in glucosylceramide (13-fold the normal level) and lactosylceramide (12-fold the normal level). The amount of total gangliosides in the aorta of the WHHL rabbit (207μg NeuAc/g tissue) was markedly increased, being about 12-fold the normal level (17μg NeuAc/g tissue). GM3 ganglioside was increased about 11-fold compared to normal. GD3 ganglioside, which was almost undetectable in normal aorta, also showed a marked increase in that of the WHHL rabbit (51.7 μg NeuAc/g tissue). Sulfatide, which was absent in the aorta of the normal rabbit, was markedly accumulated in that of the WHHL rabbit (280nmol/g tissue). The fatty acid composition of neutral glycosphingolipids of WHHL rabbit was found to include a higher amount of C23:0, which is the major fatty acid of glycolipids in serum lipoproteins. Gangliosides in the aorta of the WHIZ, rabbit contained more C16:0 than in the normal rabbit. Sphingosine of sulfatide in the aorta of the WHHL rabbit was composed of sphingenine (86%), sphinganine (9%), 4-D-hydroxysphinganine (4%), and 4-D-hy-droxyeicosasphinganine (less than 1%). The results of fatty acid analysis of glycosphin-golipids in the aorta of WHHL rabbit suggested that the various glycosphingolipids mostly derived from serum lipoproteins were accumulated in the aorta of the WHEL rabbit along with the progression of atherosclerosis, and that most of these glycolipids were hydrolyzed into less polar glycolipids such as glucosylceramide or lactosylceramide. On the other hand, the moderate increases in globotriaosylceramide, globotetraosylceramide, and galactosylneolactotetraosylceramide, which are ordinary constituents of the normal aorta, indicated the marked intimal thickening of the aorta of the WHITE, rabbit. It is also suggested that GM3 and GD3 gangliosides were derived not only from sera but also from new-type cell populations, such as foam cells or macrophages in the atherosclerotic lesions, because the fatty acids of these gangliosides included more palmitic acid than those of either serum lipoproteins or the normal aorta. The most interesting finding was that the occurrence of sulfatide and GD3 ganglioside in the aorta of the WHHL rabbit could be a useful indicator of the degree of progression of atherosclerosis, since these glycosphingolipids were hardly detected in the normal aorta.抄録全体を表示PDF形式でダウンロード (3932K) -
Haruo Suzuki, Yasuko Kawamura-Konishi1991 年 109 巻 6 号 p. 909-917
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーSarcosine oxidase from Corynebacterium sp. U-96 is inhibited by iodoacetamide (IAM) and the inhibition is prevented by the substrate analog, sodium acetate. To elucidate the mechanism of inhibition of the enzyme by IAM, we determined the amino acid sequences around the IAM-reactive cysteine residues, and the effects of the modification on the enzyme activity and the oxidation-reduction of the FAD moieties of the enzyme. The enzyme was specifically labeled with [14C] IAM, and the labeled subunit B was digested with trypsin and chymotrypsin. The HPLC profiles of the proteolytic digests showed mainly two radioactive peaks. The 14C-labeled peptides were purified, and their N-terminal sequences were determined to be Cys-Gly-Thr-Pro-Gly-Ala-Gly-Tyr (TC-1) and Ala-Gly-Ile-Ala-Cys-Xaa-Asp-Xaa-Val-Ala- - (TC-2). Peptide TC-2 contains a covalent FAD-binding sequence [Asx -His-Val-Ala ; Shiga et al. (1983) Biochem. Int., 6, 737]. [14C]IAM-incorporation into the TC-1 sequence was strongly inhibited by sodium acetate. The N-terminal amino acid sequence of the CNBr fragment containing the TC-1 sequence (65 residues) was determined. According to the secondary structure predictions, Gly-Thr-Pro-Gly-Ala-Gly of the TC-1 sequence is located between the β sheet and α helix of the sequence, indicating the presence of an AMP-binding site in the TC-1 region. The activity of the enzyme treated with IAM in the presence and absence of sodium acetate was not inhibited by sodium sulfite, which is known to react specifically with covalent FAD. The time courses of reduction of the flavin moieties of the IAM-treated enzymes by sarcosine showed fast and slow phases. In the presence and absence of sulfite, the rates of the fast phase were almost the same for the enzymes treated in the presence and absence of sodium acetate. But the fractions for the fast phase for the enzymes treated in the presence and absence of acetate were 55 and 20% of the total absorbance change (the oxidized minus the reduced form) in the case of the native enzyme, respectively. The rates of oxidation of the IAM-treated enzymes were approximately half of that in the case of the native enzyme. These data suggest that at least two cysteine residues in the enzyme are located at different sites on subunit B, one at the sarcosine-binding site and the other at the covalent FAD-binding site, and that IAM-treatment of the enzyme impairs the activity of the covalent FAD and thus the noncovalent FAD mainly functions in the oxidation of sarcosine.抄録全体を表示PDF形式でダウンロード (944K) -
Shinichi Yoshimura, Keiichi Watanabe, Hiroshi Suemizu, Takashi Onozawa ...1991 年 109 巻 6 号 p. 918-923
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーRat plasma glutathione peroxidase (GSH-Px) was purified 1, 400-fold from rat serum by a combination of phenyl Sepharose, DEAE Sephacel, blue Sepharose and Sephacryl S-200 column chromatographies. The purified GSH-Px migrated as a single band corresponding to a molecular weight of 22, 500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was used for the immunization of chickens to obtain a specific antibody and for determination of its amino acid sequence. Two overlapping cDNA clones for rat plasma GSH-Px were isolated from a placental cDNA library. The composite nucleotide sequence is 1, 529 base-pairs long and encodes 226 amino acids. The deduced amino acid sequence completely coincided with the sequences of five individual peptide fragments derived from the purified plasma GSH-Px on digestion with lysyl endopeptidase. In order to identify the tissue(s) generating this plasma GSH-Px, immunoblot analysis was performed on homogenates prepared from 13 tissues. A single immunoreactive band of 22.5kDa, corresponding to plasma GSH-Px, was detected for the kidney homogenate. A much fainter band was observed for the lung preparation, but liver, spleen, bone marrow, and other tissues examined were negative. Northern blot analysis further revealed that the expression level of the plasma GSH-Px gene was high in kidney and low in lung. No transcript was detected in liver or spleen. These results indicate that plasma GSH-Px is predominantly synthesized and secreted by renal cells.抄録全体を表示PDF形式でダウンロード (3118K) -
Sohei Tanabe, Teruko Sugo, Michio Matsuda1991 年 109 巻 6 号 p. 924-928
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーBy monitoring the activation of protein C and the regulation of factor Xa-catalyzed thrombin formation by the activated protein C (APC) on the surface of human umbilical vein endothelial cells (HUVEC), we found that functional protein C was synthesized in cultured HUVEC and expressed thereon in the presence of vitamin K. Furthermore, without exogenously added protein S, time-dependent and saturable accumulation of APC (20fmol APC/105 cells) on the surface of HUVEC was observed. During prothrombin activation by the complex of membrane-bound factor Xa and endogenous factor Va formed on the surface of HUVEC, APC was generated, and the rate of thrombin formation decreased. Treatment of HUVEC with an antibody that inhibits the APC-catalyzed inactivation of endogenous factor Va clearly quenched the activity of surface-associated APC. Immunostaining of HUVEC with a horseradish peroxidase (HRP)-conjugated antibody that solely recognizes human protein C confirmed the presence of protein C on the surface of HUVEC. Northern blot analysis revealed that an about 1.8kb mRNA species derived from HUVEC was hybridized with 32P-labeled protein C cDNA, as in the case of those from HepG2, which are known to synthesize normal protein C. The increase in the amount of protein C mRNA in HUVEC in parallel with cell growth provided supporting evidence for the synthesis of protein C during the culture of HUVEC. These results indicate that blood coagulation is regulated by endogenously generated and activated protein C, together with or without protein S, through inactivation of factor Va on the surface of endothelial cells.抄録全体を表示PDF形式でダウンロード (1459K) -
Seung-Ho Kim, Saburo Hara, Tokuji Ikenaka1991 年 109 巻 6 号 p. 929-932
発行日: 1991年
公開日: 2008/11/18
ジャーナル フリーSingly modified soybean trypsin inhibitors (STIs), Tiea* [Tia cleaved at Arg(63)-Ile(64)] and Tib* [Tib cleaved at Arg(63)-Ile(64)], were prepared by limited proteolysis with trypsin at pH 3.0. These singly modified inhibitors were further modified to yield doubly modified inhibitors, Tia** and Tib**, by limited proteolysis with subtilisin BPN', which cleaved the Met(84)-Leu(85) bonds of Tia* and Tib*, respectively. The doubly modified inhibitors could be separated into two parts: protein moiety A and peptide moiety a (IRFIAEGHPLSLKFDSFAVIM) for Tia**, and protein moiety B and peptide moiety b (IRFIAEGNPLRLKFDSFAVIM) for Tib**. These protein and peptide moieties showed no trypsin inhibitory activities alone. However, the inhibitors can be reconstituted through the mutual exchange of the protein and peptide moieties isolated from STIs.The reconstituted inhibitor which has tyrosine at position 62 and histidine at position 71 shows the highest inhibitory activity. Its K1 value for bovine trypsin is around 10-10M, which is almost the same as that of Tia for bovine trypsin. The inhibitor possessing either tyrosine at position 62 or histidine at position 71 exhibits a K1 value of around 10-9M, which is between those of Tia and Tib. The inhibitor having phenylalanine and asparagine at positions 62 and 71, respectively, shows the weakest inhibitory activity of around 10-8M similar to that of Tib for bovine trypsin.抄録全体を表示PDF形式でダウンロード (452K)
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