巻号一覧
113 巻, 2 号
選択された号の論文の26件中1～26を表示しています
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Kaoru Nagai, Haruki Yamaguchi1993 年 113 巻 2 号 p. 123-125
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe investigated the role of the intramolecular high-mannose oligosaccharide chains in the folding and assembly of soybean lectin polypeptides. Soybean lectin, dissociated into subunits and completely denatured in 6 M guanidine hydrochloride, was quantitatively reconstituted to the active tetrameric structure by simple dilution. However, neither the activity nor the tetrameric structure was regained in the presence of 100 μM of an asparagine-linked oligosaccharide, Man9GlcNAc2Asn, having the same structure as that of the sugar chains of soybean lectin. Besides, the same concentration of this glycopeptide even dissociated, although only gradually, the native lectin into subunits. On the other hand, the deglycosylated subunits had no ability to regain the activity or the tetrameric structure. The present study provided for the first time direct evidence of the essential role of the intramolecular high-mannose oligosaccharide chains in the proper folding and assembly of glycopolypeptides.抄録全体を表示PDF形式でダウンロード (399K) -
Kazufumi Kuroda, Ritsuko Kagiyama-Takahashi, Asuko Okuda, Akira Omori1993 年 113 巻 2 号 p. 126-128
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe immunoaffinity-purified DNA polymerase α-primase complex from Drosophila melanogaster Kc cells contains three high molecular weight polypeptides besides the 180 kDa catalytic polypeptide. These polypeptides are immunologically cross-reactive with the 180 kDa polypeptide. When the immunoaffinity-purified complex was kept at 4°C for about four weeks, the amounts of the three polypeptides increased, while the 180 kDa polypeptide completely disappeared. Sodium bisulfite inhibited the decrease in the 180 kDa polypeptide. The N-terminal amino acid sequences of all the polypeptides were all assigned to ones present in a portion close to the N-terminus of the 180 kDa polypeptide. The N-terminal residue of all the three polypeptides was Ser. The cleavage sites were Phe130-Ser131, Thr180-Ser181, and Phe237-Ser238. These results show that the three polypeptides are cleavage products of the 180 kDa catalytic polypeptide, the cleavage occurring at specific labile sites including a Ser residue. The amino acid residues at the sites are quite different from those (Lys-Lys) in the human 180 kDa catalytic polypeptide.抄録全体を表示PDF形式でダウンロード (1171K) -
Juan F. Santarén, Agustín Alconada, José M. Cuezv ...1993 年 113 巻 2 号 p. 129-131
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe report the one-step processing of the rat liver β-F1-ATPase precursor protein, as examined by high resolution 2D-gel electrophoresis. Proteolytic cleavage of the positively charged mitochondrial targeting signal of the precursor promotes decreases in both the molecular weight (_??_3 kDa) and the isoelectric point (_??_0.2 pH unit) of the protein. The results obtained illustrate the usefulness of this technique, since it takes advantage of both results of the maturation process, for molecular characterization of the processing of mitochondrial precursor proteins.抄録全体を表示PDF形式でダウンロード (1454K) -
Tsutomu Nakagawa, Masahiro Hosaka, Seiji Torii, Toshio Watanabe, Kazuo ...1993 年 113 巻 2 号 p. 132-135
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe used the polymerase chain reaction to identify a mouse cDNA which represented a new member of a growing class of mammalian endoproteases homologous to the yeast Kex2 protease involved in the processing of precursor proteins. This cDNA encoded a 915-residue protein, designated as PC6, containing a subtilisin-like catalytic domain closely related to those of other Kex2-like members (furin, PC2, PC1/3, PC4, and PACE4). It exhibited striking sequence similarity to PACE4 and contained similar protein domains, such as the COOH-terminal Cys-rich region. Northern blot analysis revealed that PC6 mRNA, as with furin and PACE4 mRNAs, was expressed in various tissues and cell lines, with the highest level in the intestine. Transfection experiments revealed that PC6 was capable of cleaving precursors at dibasic sites. These observations suggest that PC6 is a candidate for a processing endoprotease responsible for the maturation of gastrointestinal peptides.抄録全体を表示PDF形式でダウンロード (1540K) -
Tetsuro Yamashita, Masao Kawakita1993 年 113 巻 2 号 p. 136-140
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーIn order to analyze the localization and pharmacological characteristics of the Na+/H+-antiporter of bovine kidney, renal cortex membranes were fractionated by 35-48% continuous sucrose density gradient centrifugation. Both the Na+/H+-antiporting activity and the 110-kDa peptide cross-reactive with a polyclonal antibody against a peptide representing the C-terminal 22 amino acid residues of human Na+/H+-antiporter (NHE1) were found in the same fractions as alkaline phosphatase, a marker enzyme of the brush-border membrane. Most of the Na+/H+-antiporter distributed among the fractions was found to be of the low amiloride-sensitive type. Brush-border membranes were solubilized with sodium cholate and the solubilized proteins were applied to an immunoaffinity matrix (Protein A Sepharose CL-4B coupled with the above antibody). The 110 kDa protein was adsorbed to the affinity matrix and then recovered in the fraction eluted from the column at pH 11.5. After reconstitution into proteoliposome, the fraction containing the 110 kDa protein was highly active in Na+/H+-antiport. A 125-fold increase in the specific activity of the Na+/H+ antiport reaction was attained with the present immunoaffinity purification procedure. This represents the highest degree of purification of the native Na+/ H+-antiporter so far attained.抄録全体を表示PDF形式でダウンロード (1570K) -
Mizuho Une, Noboru Izumi, Takahiko Hoshita1993 年 113 巻 2 号 p. 141-143
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe have investigated the stereochemistry of the side chain of the intermediates, 3α, 7α, 12α-trihydroxy-5β-cholest-24-enoic acid and 3α, 7α, 12α, 24-tetrahydroxy-5β-chole-stanoic acid, in the conversion of 3α, 7α, 12α-trihydroxy-5β-cholestanoic acid to cholic acid by rat liver peroxisomes. The intermediates formed were converted to the p-bromophenacyl ester derivatives and analyzed by reversed-phase high-performance liquid chro-matography. Only the (24E) form of the two isomers of the Δ24-unsaturated acid and the (24R, 25S) form of the four isomers at C-24 and C-25 of the 24-hydroxy acid were found to be formed stereospecifically from either (25R)- or (25S)-3α, 7α, 12α-trihydroxy-5β-cholestanoic acid. Formation of the other isomers of the aβ-unsaturated bile acid or the β-hydroxy bile acid was not detected. The findings support the proposed pathway for the side-chain cleavage in cholic acid biosynthesis, which is thought to be similar to that of peroxisomal fatty acid β-oxidation.抄録全体を表示PDF形式でダウンロード (278K) -
Masatoshi Nomura, Ken-ichirou Morohashi, Sirou Kirita, Yasuki Nonaka, ...1993 年 113 巻 2 号 p. 144-152
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe isolated three genomic clones of rat P-450(11β) genes (CYP11B). Two of them corresponded to 11β-hydroxylase gene (CYP11B1) and aldosterone synthase gene (CYP11B2), respectively. The third one was a novel gene resembling both CYP11B1 and CYP11B2, and was named CYP11B3 gene (CYP11B3). CYP11B2 and CYP11B3 are located tandemly in the genome in the same direction approximately 24 kb apart. These three genes were highly homologous in their amino acid coding regions, with 88% (CYP11B1 to CYP11B2), 89% (CYP11B2 to CYP11B3), 96% (CYP11B1 to CYP11B3) nucleotide identity. The numbers and the locations of the exons of these three genes also exactly corresponded to each other. However, the nucleotide sequences of the 5' upstream regions of CYP11B1 and CYP11B2 were significantly different, suggesting different transcriptional regulations. CYP11B3 had almost the same sequence as CYP11B1 gene in the 5' upstream region. A putative Ad4 site, a cis-acting element present in the promoter regions of all the steroidogenic P-450s so far reported [Morohashi, K., Honda, S., Inomata, Y., Handa, H., Omura, T. (1992) J. Biol. Chem. 267, 17913-17919], was found in the promoter regions of both CYP11B1 and CYP11B2. Gel retardation analysis showed the binding of Ad4BP purified from bovine adrenal cortex to these two Ad4 sites. We analyzed the relative abundance of the mRNAs corresponding to these three genes by the generation of RT-PCR libraries from rat adrenal total RNAs. In the RT-PCR library from normal-diet rats, the recombinants generated from CYP11B1 were dominant, but the relative abundance of the mRNA corresponding to CYP11B2 was elevated by 12-fold when the rats were treated with low Na+-high K+-diet. The expression of CYP11B3 could not be detected even by the RT-PCR method.抄録全体を表示PDF形式でダウンロード (4638K) -
Wen-Feng Xu, Wei-Kang Tao, Zhen-Zhen Gong, Cheng-Wu Chi1993 年 113 巻 2 号 p. 153-158
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーArrowhead double-headed proteinase inhibitors A and B consist of 150 amino acid residues with three disulfide bonds. Based on their primary structures, three cDNA fragments of the inhibitors were amplified in vitro by the PCR method using a constructed arrowhead cDNA library as a template. With the overlapping sequences, the full-length cDNA sequences of the inhibitors were then ascertained. The open reading frame encodes a pre-inhibitor, including a 24 residue signal peptide. The deduced amino acid sequences are consistent in principle with those determined by primary structure analysis, except that there are seven extra residues in the C-terminal part of the inhibitors, which might be cleaved off by proteinase post-processing immediately after protein synthesis. It is worth pointing out that cDNAs of both inhibitors A and B contain an 87 by intron in the AAG codon of residue Lys-97. According to the elucidated cDNA sequences, the structural genes of inhibitors A and B were amplified using the total cDNA or genomic DNA of arrowhead as a PCR template. It was indicated that both the cDNA and genomic structures of inhibitors A and B have the same sequences.抄録全体を表示PDF形式でダウンロード (2545K) -
Hideko Atoda, Takashi Morita1993 年 113 巻 2 号 p. 159-163
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーBlood coagulation factor IX/factor X-binding protein (IX/X-bp) is a two-chain anticoagulant protein that was isolated from the venom of Trimeresurus flavoviridis. The amino acid sequence of IX/X-bp is homologous to the sequences of C-type lectin-like proteins, such as asialoglycoprotein receptor, tetranectin, and the low-affinity Fcε receptor of immunoglobulin E. The amino acid composition and amino acid sequence of cystinecontaining peptides, formed as a result of enzymatic digestion of CNBr-generated fragments of IX/X-bp, were analyzed to determine the location of the seven disulfide bridges in the protein. Three disulfide bridges in the A chain link Cy2 to Cys13, Cys30 to Cys127, and Cys102 to Cys19. Three disulfide bridges in the B chain link Cys2 to Cys13, Cys30 to Cys119, and Cys96 to Cys111 An interchain disulfide bond links Cys79 of the A chain and Cys75 of the B chain. The intrachain disulfide-bonding patterns of both the A and B chains of IX/X-bp are similar to those found in other C-type lectin-like proteins. We discuss in this report the sequence homology between IX/X-bp and other two-chain, C-type lectin-like proteins that have been isolated from snake venoms and we compare the S-S bonding patterns of proteins that are homologous to IX/X-bp.抄録全体を表示PDF形式でダウンロード (442K) -
Eleonora Cordella-Miele, Lucio Miele, Simone Beninati, Anil B. Mukherj ...1993 年 113 巻 2 号 p. 164-173
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe have previously demonstrated that when phospholipase A2 is treated with either tissue transglutaminase or human plasma Factor XIIIa, a striking increase of its catalytic activity is observed, due to the formation of an intramolecular ε-(γ-glutamyl)-lysine crosslink [Cordella-Miele et al. (1990) J. Biol. Chem. 265, 17180-17188]. Here, we report the effect of transglutaminase substrates such as mono-, di-, and polyamines on this transglutaminase-catalyzed post-translational modification of phospholipase A2. Incorporation of radioactively labeled polyamines into phospholipase A2 was demonstrated by using porcine pancreatic phospholipase A2 as a substrate in a conventional transglutaminase assay. These results were further confirmed by SDS-polyacrylamide gel electrophoresis followed by fluorography. Additionally, γ-glutamyl-polyamine was detected and unequivocally identified in proteolytic digests of polyaminated phospholipase A2. When phospholipase A2 was incubated with transglutaminase in the presence of putrescine, spermine, spermidine, dansylcadaverine, or methylamine, a 2-3-fold increase in phospholipase A2 activity was observed. The increase of phospholipase A2 activity was found to be dependent upon the concentration of phospholipase A2, preincubation time, and the duration of the reaction. Increase in phospholipase A2 activity after transglutaminase treatment in the presence of polyamines was demonstrated using two different assay systems. Kinetic studies on phospholipase A2 pretreated with spermidine and transglutaminase demonstrated a significant increase of the apparent Vmax but no significant change in the apparent Km. Unlike phospholipase A2 pretreated with transglutaminase alone, polyaminated phospholipase A2 does not undergo non-covalent dimerization in solution. Polyaminated phospholipase A2 was further purified by chromatofocusing and was found to contain N-mono (γ-glutamyl)-spermidine in a molar ratio of about 1:1 to phospholipase A2. Freshly purified, polyaminated phospholipase A2 had a specific activity approximately 3-fold higher than that of control phospholipase A2 treated in an identical way except for the absence of transglutaminase. To our knowledge, this is the first demonstration that transglutaminase catalyzes the incorporation of amines into a phospholipase, and that this post-translational modification increases phospholipase A2 activity.抄録全体を表示PDF形式でダウンロード (2662K) -
Tadashi Ueda, Yoshito Abe, Kenji Akasaki, Yasunori Yamaguchi, Hiroshi ...1993 年 113 巻 2 号 p. 174-179
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーA method was developed to evaluate the association constant at physiological pH (pH 7.5) between a lysozyme and the Fab fragment derived from anti-lysozyme monoclonal antibody 37-7, which was immobilized to the adsorbent for HPLC. Comparison of the association constants between lysozymes and the immobilized Fab fragment indicated that mAb 37-7 recognized the prominently exposed regions (hills and ridges) around His15 of hen lysozyme, but His15 itself was not directly involved in the binding with mAb 37-7. Moreover, the epitope was confirmed by the reactivity of His15 with monoiodoacetic acid in the presence of mAb 37-7. The association constant of 15-carboxymethylated histidine lysozyme (15CM lysozyme) with the immobilized Fab fragment was smaller by oneseventh than that of 15-carboxamidated histidine lysozyme, though the side chains introduced were almost identical in size. From the pH titration of 15CM lysozyme with 13C-enriched carboxyl group by use of 13C-NMR, the pKa of the introduced carboxyl group was evaluated to be 5.06. Since the carboxyl group was fully ionized under the conditions of measurement (pH 7.5), electrostatic repulsion was found to disturb severely the association between mAb 37-7 and hen lysozyme. Moreover, it was demonstrated that, because of the high reproducibility of measurement, the immobilized Fab fragment could detect subtle differences in the surface structure of lysozymes.抄録全体を表示PDF形式でダウンロード (676K) -
Akira Hiraga, Keiko Hata, Yoichi Suzuki, Shigeru Tsuiki1993 年 113 巻 2 号 p. 180-188
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーErythrocyte Band 3 protein (Band 3), brain microtubule associated protein 2 (MAP2), and tubulin were phosphorylated to high stoichiometries (1-6 mol P1/mol protein) on tyrosine residues using a rat spleen protein-tyrosine kinase in the presence of polylysine. After total removal of polylysine, the quantitatively phosphorylated proteins as well as tyrosineglutamate copolymer [Poly(Glu4 Tyr1)], which was also phosphorylated (1.5 mol/mol) by the kinase, were employed to assay rat liver protein-tyrosine phosphatases (PTPases). Of the four partially purified PTPases termed L1, L2, L3, and L4, PTPase L1 was previously purified to homogeneity and demonstrated to be a novel enzyme with sequence similarity to src-homology region 2 [Hiraga, A. et al. (1992) Eur. J. Biochem. 209, 195-206]. In the present work PTPase L2 was purified to near homogeneity by a procedure involving chromatography on DEAE-cellulose, Blue Sepharose CL-6B, hydroxylapatite, Phenyl Sepharose CL-4B, and TSKgel Heparin-5PW. PTPase L2 was purified 20, 000-fold with a recovery of 0.9% from the extract and 0.005 mg was isolated from 300 g of liver. The highly purified PTPase L2 showed a major protein band of 36 kDa on SDS/polyacrylamide gel electrophoresis. PTPase L2 had a specific activity of about 6, 000 nmol of P1 released min-1 mg-1 toward either Band 3 or poly(G1u4, Tyr1), the values being within the range of those obtained for PTPases purified thus far. PTPase L2 dephosphorylated Band-3 9-fold and 5-fold faster than tubulin and MAP2, respectively, under the assay conditions employed. PTPase L2 was highly sensitive to PTPase inhibitors such as zinc acetate, sodium vanadate, and non-phosphorylated poly(Glu4 Tyr1) as compared with rat liver PTPase L1 and L3. PTPase L2 was most similar to human placental PTPase 1B in molecular mass and sensitivity to the three inhibitors but appeared to be significantly different in sensitivity to heparin and affinity for DEAE-cellulose from the placental enzyme.抄録全体を表示PDF形式でダウンロード (2179K) -
Shigeki Fujiwara, Hiroyuki Kawahara, Kazuhiro W. Makabe, Noriyuki Sato ...1993 年 113 巻 2 号 p. 189-195
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe have isolated and sequenced a cDNA clone encoding an ascidian embryonic nuclear protein, Hgv2. An insert about 2 kb long covered almost the entire length of 2.3-kb Hgv2 mRNA. The amino acid sequence of Hgv2 deduced from the cDNA sequence showed that this protein is related to the amphibian karyophilic histone-binding protein N1, which is thought to be involved in nucleosome assembly. Homology between these two proteins is evident from their extremely similar amino acid compositions and hydropathy profiles. In addition, Hgv2 protein has sequences strikingly similar to the nuclear targeting signal of N1. This is therefore the first report of molecular cloning of a homologue of N1 in non-amphibian species. Putative histone-binding domains of N1 are composed of two acidic residue-rich clusters. Hgv2 polypeptide contains two highly acidic regions, but amino acid sequences of the regions are not conserved. Since Hgv2 protein exists in nuclei of every embryonic cell but disappears from nuclei of metamorphosed juvenile tissues, this protein may function as a nucleosome assembly factor during rapid embryonic cell divisions.抄録全体を表示PDF形式でダウンロード (2851K) -
Takayoshi Iio, Sho Takahashi, Seiji Sawada1993 年 113 巻 2 号 p. 196-199
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリー1-(2-Hydroxyethyl)-6-[(2, 2-dicyano)vinyl]-2, 3, 4-trihydroquinoline (DCQ) is a fluorescent dye and its fluorescence yield is determined by a freedom of intramolecular rotation. When DCQ was added to an actin solution, its fluorescence intensity increased as the actin molecule transformed from G-actin to F-actin. This fluorescence intensity increase was induced both by the higher binding constant of the dye to F-actin than to G-actin and by the higher fluorescence yield of the dye bound to F-actin than to G-actin. DCQ bound more strongly to Mg2+-F-actin than to Ca2+-F-actin, resulting in higher fluorescence intensity of DCQ-Mg2+-F-actin solution than of DCQ-Ca2+-F-actin solution. DCQ did not distinguish Mg2+-G-actin and Ca2+-G-actin. Thus, DCQ is a good probe for polymerization of actin and polymorphism of F-actin.抄録全体を表示PDF形式でダウンロード (473K) -
Tsuyoshi Tanabe, Mamoru Yamada, Takafumi Noma, Tadashi Kajii, Atsushi ...1993 年 113 巻 2 号 p. 200-207
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーAdenylate kinase (AK) is known to play an important role in homeostasis of adenine nucleotide metabolism. We isolated cDNAs for rat AK isozymes (AK1, AK2, and AK3), determined their mRNAs in rat tissues by Northern blot analysis, and measured the isozyme activities. Tissue-dependent activities of AK1 and AK2 paralleled the contents of mRNAs. Tissues with high AK1 levels showed low AK2 levels and vice versa, suggesting that tissue-specific expressions of the AK1 and AK2 genes are inversely regulated. AK3 mRNA was detected in most tissues examined, suggesting that AK3 gene expression is constitutive. We further examined developmental changes in mRNAs and enzyme activities of AK isozymes in rat skeletal muscle and liver. In the skeletal muscle, AK1 and AK3 activities started to increase at around the weaning period. AK1 mRNA accumulated at the prenatal stage and further increased during development, while AK3 mRNA was at high levels during the fetal stage and remained fairly constant during development. In the liver, AK2 and AK3 activities started to increase after birth and were further elevated during growth, whereas their mRNAs were present at relatively high levels throughout development. The physiological meanings of the tissue-specific expression of the AK isozyme genes are discussed.抄録全体を表示PDF形式でダウンロード (1922K) -
Sumiko Gomi, Lan Lee, Tomonori Iwama, Yasuo Imae1993 年 113 巻 2 号 p. 208-213
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThr-154 of the chemoreceptor Tar in Escherichia coli is important for aspartate sensing. Taking advantage of the fact that Tar has no Cys residues, we have further investigated the role of Thr-154 by replacing it with Cys in order to subject it to SH modification. Tar-T154C retained the abilities of aspartate sensing and repellent sensing. However, when cells with Tar-T154C were treated with an SH-modifying reagent, 5, 5'-dithiobis-2-nitrobenzoic acid (DTNB), they specifically lost the ability to sense aspartate; the ability was restored by the reducing reagent, 1, 4-dithiothreitol. DTNB showed no detectable effect on the function of wild-type Tar or serine-replaced Tar, Tar-T154S. Thus, DTNB modifies Cys-154 of Tar-T154C in intact cells and causes a specific defect in the aspartate-sensing ability of Tar. The addition of 1 mM or higher concentrations of aspartate resulted in protection of Cys-154 from the modification; serine had no effect in this regard. These results suggest that not only is Thr-154 important for aspartate sensing but also, it may be located at the actual aspartate-binding site.抄録全体を表示PDF形式でダウンロード (2085K) -
Shigeki Shibahara, Miki Yoshizawa, Hiroyuki Suzuki, Kazuhisa Takeda, K ...1993 年 113 巻 2 号 p. 214-218
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe cloned a cDNA coding for a putative human heme oxygenase isozyme, designated type 2 (HO-2), and analyzed its function by transient expression assays. HeLa cells transfected with either HO-2 cDNAs or a cDNA coding for authentic heme oxygenase (HO-1) expressed the activity of heme oxygenase, although no activity was detected in the mock transfected cells. Using specific anti-HO-1 antibody, we showed that expression of a HO-1 cDNA resulted in the increase in its protein levels, but HO-1 protein was not detectable in the cells expressing HO-2 cDNAs. We thus confirmed the functional identity of HO-1 and HO-2. Then, we analyzed their expression in an erythroid cell line, YN-1-0-A. Treatment with hemin or by heat shock (42°C) led to a remarkable increase in the HO-1 mRNA levels, while HO-2 mRNA expression was not induced at all, suggesting that they are under separate regulation.抄録全体を表示PDF形式でダウンロード (1441K) -
Kazuko Ohgi, Hiroyuki Horiuchi, Hideaki Watanabe, Masanori Iwama, Masa ...1993 年 113 巻 2 号 p. 219-224
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe active site of a base non-specific RNase from Rhizopus niveus (RNase Rh), consists of three histidine residues and one carboxyl group [Ohgi, K. et al. (1992) J. Biochem. 111, 132-138]. In order to identify this acidic amino acid residue, we chose Asp51 and G1u105 as candidates based on a comparison of the primary structures of four fungal RNases and self-incompatibility factors of Nicotiana alata which belong to the RNase T2 family. We substituted these amino acid residues with other amino acids by site-directed mutagenesis, and determined the enzymatic properties of the mutated enzymes. The enzymatic activities of E105Q, E105D, and E105A mutant enzymes were decreased markedly, but those of D51N, D51E, and D51A were decreased only slightly when RNA was used as a substrate. Therefore we concluded that G1u105 is related to the catalytic function. Kinetic constants for the enzymatic activity of E105Q and E105D toward ApU suggest that the proper size and negative charge of side chain groups are important for the catalysis of RNase Rh. However, the enzymatic activity of D51N toward ApU, but not toward UpU, decreased markedly. Therefore, we suggest that Asp51 is one of the amino acid residues forming the base recognition site. The substitution of Asp51 by Asn causes the enzyme to be more guanine nucleotide-preferential.抄録全体を表示PDF形式でダウンロード (1824K) -
Takayuki Takahashi, Toshinobu Tokumoto, Katsutoshi Ishikawa, Kenji Tak ...1993 年 113 巻 2 号 p. 225-228
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe specificity of action of the proteasome purified from the cytosol of Xenopus oocyte was investigated using oxidized insulin B chain as the substrate. HPLC analyses of the produced peptides followed by amino acid analyses showed that it cleaved four peptide bonds, Leu6-Cya7, Glu13-Ala14, Leu15-Tyr16, and Leu17-Va118, of the peptide. Cleavage at Leu6-Cya7 was found to be specific to the Xenopus enzyme. The enzyme did not cleave Gln4-His5 and Cya19-Gly20, which are commonly hydrolyzed by proteasomes from rat and mouse liver, and human erythrocyte. In contrast to previous results obtained with the mammalian proteasome, the cleavage by the Xenopus enzyme was inhibited selectively by chymostatin. These results demonstrate distinct species difference in cleavage specificity and inhibition profile among proteasomes of different origins.抄録全体を表示PDF形式でダウンロード (445K) -
Yoshiaki Tamanoue, Takavuki Takahashi, Kenji Takahashi1993 年 113 巻 2 号 p. 229-235
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーAn extensive purification of microsomal serine proteinase was achieved from rat liver microsome membranes solubilized with 1% sodium cholate by a series of column chromatographic steps on hydroxylapatite, DEAE-cellulose, and benzamidine-Sepharose 6B, and polyacrylamide gel electrophoresis (PAGE). In the final step of PAGE, the enzyme was separated into two fractions with slightly different mobilities, designated microsomal serine proteinases 1 (MSPl) and 2 (MSP2). The former was purified about 8, 000-fold to apparent homogeneity, and the latter was purified about 800-fold, starting from the sample which had been spontaneously activated after elution from the hydroxylapatite column. Both enzyme fractions showed essentially the same properties, including molecular weight, susceptibility to various proteinase inhibitors and metal ions, specificity of action toward protein substrates with special preference for a basic protein histone as well as toward synthetic substrates, and pH dependence of activity toward a synthetic substrate and two neuropeptides. Taken together with the same behavior on a series of chromatographic steps, the two isoforms are thought to be essentially the same enzyme. Further, the detailed kinetic studies of the enzyme activity, especially of MSP1, toward various synthetic and naturally occurring peptide substrates showed that it was highly specific for basic amino acid pairs, strictly hydrolyzing at the COOH side of arginine residue. These results are consistent with those obtained previously with a partially purified enzyme and establish more definitely and in detail the specificity as well as other molecular and enzymatic properties of the enzyme.抄録全体を表示PDF形式でダウンロード (839K) -
Kyoko Yokoyama, Hitoshi Ohmori, Akira Kumon1993 年 113 巻 2 号 p. 236-240
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーNω-Phosphoarginine hydrolase from rat liver cytosol was purified to apparent homogeneity on SDS-PAGE, by employing column chromatographies on Sephadex G-75, DEAE-cellulose, QAE-Toyopearl, and glutathione-2-pyridyl-disulfide-Superose. One milligram protein of the final preparation released 4 μmol/min of inorganic phosphate from Nω-Phosphoarginine. The molecular mass on SDS-PAGE, the Stokes' radius and the sedimentation coefficient were estimated to be 17.3 kDa, 1.63 nm, and 2.0 s, respectively, indicating that this enzyme consists of a single peptide. The stability of the enzyme to heat depended on the buffers employed and treatment of the enzyme preparation in 50 mM Tris-HC1, pH 7.0 at 50°C, reduced the hydrolytic activity with a decay constant of 0.099 per min.抄録全体を表示PDF形式でダウンロード (1420K) -
Hiroyasu Nakata1993 年 113 巻 2 号 p. 241-244
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーAntisera against the non-selective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) have been raised by immunizing rabbits with NECA-coupled bovine serum albumin. The antisera which bind [3H]NECA with high affinity were purified by affinity chromatography using NECA-coupled Sepharose as the affinity gel without signifi-cant changes in [3H]NECA-binding properties. The order of the affinity for various adenosine receptor ligands of the purified or unpurified antisera was 5'-N-cyclopropyl-carboxamidoadenosine ≥ NECA > 2', 5'-dideoxyadenosine > 2-chloroadenosine > theophyl-line > isobutylmethylxanthine > (R)-N6-phenylisopropyladenosine=N6-cyclohexyladenosine. This specificity was found to be similar to that of the non-receptor NECA-binding sites, which had been found in various tissues such as human placentas and mouse P815 mastocytoma cells, rather than to that of adenosine receptors. These anti-NECA antibodies will be useful as immunochemical agents in the search for endogenous ligand(s) to the non-receptor NECA-binding sites.抄録全体を表示PDF形式でダウンロード (480K) -
Noriyuki Ishii, Hideyuki Yoshimura, Kuniaki Nagayama, Yasuo Kagawa, Ma ...1993 年 113 巻 2 号 p. 245-250
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe three-dimensional molecular structure of the F1-ATPase from a thermophilic bacterium PS3 (TF1) at 3 nm resolution was reconstructed from a series of tilted, negatively stained electron microscopic images of two-dimensional crystals which were formed on a clean surface of mercury. It was shown that six ellipsoidal columns, each γ7 nm in length and -3 nm in diameter, corresponding to the a and β subunits, surrounded a central hollow cavity of _??_2.8 nm in diameter. The cavity, however, did not penetrate the molecule, and a mass, most likely the γ subunit, existed in the cavity at a depth of γ3.4 nm from the top.抄録全体を表示PDF形式でダウンロード (3379K) -
Noriyuki Kasai, Akiko Kamimura, Ichiro Miyoshi, Toshio Ariga1993 年 113 巻 2 号 p. 251-257
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe gangliosides in the livers of various inbred strains of rats and hepatoma of LEC rats were purified and analyzed by thin-layer chromatography. The patterns of ganglioside distribution in these rat livers were classified into three phenotypes depending on the strain, that is, a-type (ACI, LEA, LEW, BUF), b-type (WKAH, SHR/SP), and LEC type, which are characterized by dominance of a- or b-series of gangliosides, or a variation of a-type, respectively. A sex difference was also recognized in the molar ratio of GM3 which was much higher in males (60-75%) than in females (33-56%) except in LEC rats. In addition, the content of a-series gangliosides was lower and the content of b-series gangliosides was higher in a-type male rats than in a-type female rats. The opposite was true in b-type rats. LEC rats were an exception, characterized by no sex difference and a quite low content of b-series gangliosides. The LEC rat is a mutant strain that spontaneously develops fulminant hepatitis around 14 to 20 weeks of age and hepatoma at 1 to 1.5 years old. The gangliosides of the hepatoma were characterized by the appearance of the newly synthesized gangliosides, fucosyl-GM1 and α-galactosyl α-fucosyl GM1 (BGM1). In particular, BGM1 ganglioside accumulated in the hepatoma of female rats.抄録全体を表示PDF形式でダウンロード (3692K) -
Han-Chung Wu, Jung-Yaw Lin1993 年 113 巻 2 号 p. 258-263
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe complete amino acid sequence of a Kunitz-type two-chain trypsin inhibitor was determined for the first time. The sequence of the inhibitor from Acacia confusa (ACTI) was determined by analysis of peptides obtained from the reduced and S-carboxymethylated protein by digestion with endopeptidase Lys-C, endopeptidase Arg-C, and V8 endopeptidase. ACTI is comprised of two chains, namely A and B chains linked by the disulfide bridge between Cys(133) and Cys(141), and the inhibitor consists of 175 amino acid residues, 136 residues in the A-chain and 39 residues in the B-chain. The N-terminal amino acid sequence of ACTI shows extensive homology to the trypsin inhibitors from Acacia elata and Albizzia julibrissin, while the whole amino acid sequence of ACTI has a high degree of homology to the other Kunitz-type trypsin inhibitors from soybeans, winged bean seeds [Psophocarpus tetragonolobus (L) DC.], and seeds of Erythrina species.抄録全体を表示PDF形式でダウンロード (492K) -
Takahiro Tsujita, Hiromichi Okuda1993 年 113 巻 2 号 p. 264-269
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーCholesteryl oleate liquid crystals were prepared by sonication as a model of lipid droplets accumulated in foam cells derived from macrophages. These liquid crystals were spherical and showed an anisotropic cross image. Hormone-sensitive lipase from bovine adipose tissue hydrolyzed these liquid crystals optimally at pH 6.8. The Km for the liquid crystals was about 8 times that for vesicles or emulsified cholesteryl oleate. The Vmax for the liquid crystals was the same as that for the emulsified substrate and 6 times that for the vesicle substrate. Phospholipid inhibited cholesteryl oleate hydrolysis in a concentration-depen-dent fashion, phosphatidylserine being especially inhibitory. The effect of phospholipids on the activity changed upon their incorporation into the cholesteryl oleate liquid crystals, phosphatidylethanolamine increasing the activity to about twice that of the control. These results suggest that hormone-sensitive lipase hydrolyzes cholesteryl oleate liquid crystals as a model of endogenous lipid droplets and its activities are affected by phospholipids.抄録全体を表示PDF形式でダウンロード (1282K)
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