巻号一覧
113 巻, 3 号
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Keiichi Ozaki, Nobuyuki Itoh, Toshisuke Kawasaki1993 年 113 巻 3 号 p. 271-276
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーA macrophage asialoglycoprotein binding protein (M-ASGP-BP), which is an endocytic receptor specific for Gal/GalNAc-terminated glycoproteins, was shown to be functionally active as a homooligomer (hexamer or octamer) of a single polypeptide chain of 42 kDa [Ozaki, K., Ii, M., Itoh, N., & Kawasaki, T. (1992) J. Biol. Chem. 267, 9229-9235]. In various endocytic receptors, a four-amino-acid sequence, Tyr-X-Y-Phe, in the cytoplasmic domain has been identified as an internalization signal [Pearse, B. M. F. & Robinson, M. S. (1990) Annu. Rev. Cell Biol. 265, 151-171]. The amino-terminus of the M-ASGP-BP deduced from its cDNA sequence was found to contain the sequence, Tyr 5-Glu 6-Asn 7-Phe 8, in its cytoplasmic tail. This was confirmed by the fact that the recombinant M-ASGP-BP isolated from transfected COS-1 cells was found to have the amino-terminal sequence, Thr 2-Met 3-Ala 4-Tyr 5-Glu 6-Asn 7-Phe 8. The role of this presumptive internalization signal in the cytoplasmic tail was studied by measuring the endocytic activity of the wild-type and mutant M-ASGP-BPs expressed on COS-1 cells through transfection with the wild-type and mutant cDNAs prepared by oligonucleotide-directed mutagenesis, respectively. On the deletion of Tyr 5 or replacement of it with alanine, the internalization of asialoorosomucoid (ASOR) decreased to approximately one-fourth that in the case of the wild-type molecule. On replacement of Tyr 5 with phenylalanine, the internalization proceeded at a rate similar to that in the case of the wild-type molecule. On the other hand, the levels of the receptor on the surface of the cells were essentially the same for the wild-type and mutant receptors. These results suggest that Tyr 5 in the recombinant M-ASGP-BP molecule is necessary for its rapid internalization. Co-transfection with the wild-type and Tyr 5-deleted mutant cDNAs in various ratios resulted in the expression of the surface receptor in essentially the same numbers. However, the internalization rate of the receptor decreased roughly in proportion to the decrease in the amount of the wild-type lectin cDNA. These results suggest that the presence of multiple (three or more) Tyr 5 residues on a receptor molecule is required for its rapid internalization.抄録全体を表示PDF形式でダウンロード (1741K) -
Nadina Rubio, Paul M. Sharp, Miriam Rits, Kamyar Zahedi, Alexander S. ...1993 年 113 巻 3 号 p. 277-284
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe structure and expression of the pentraxins, serum amyloid P component (SAP), and C-reactive protein (CRP), have been investigated in the guinea pig. Northern blot analysis of hepatic RNA from animals in which acute inflammation had been induced by intraperitoneal injection of thioglycollate established that neither SAP or CRP is a major acute phase reactant in the guinea pig. Genomic clones of SAP and CRP were isolated and sequenced, and the gene and the derived protein sequences were compared with other mammalian homologues. Both genes have organizations typical of the pentraxin genes of other species, but some differences were defined in the regions that potentially determine the capacity of the pentraxin gene to be induced during acute inflammation. Nucleotide substitutions in coding regions have occurred at similar rates in the two pentraxin genes. Nonsynonymous substitution rates indicate that SAP and CRP are subject to similar, relatively low levels of constraint; at the amino acid sequence level the rate of evolution is approximately two replacements per site per 109 years. An estimate of the phylogenetic relationship among the pentraxin genes suggests that SAP and CRP arose as the result of a gene duplication event that occurred very early in mammalian evolution, but subsequent to the divergence of the reptilian ancestors of the mammalian and avian lineages. This raises doubts about the identity of proteins from fish, which have been previously characterized as CRP and SAP.抄録全体を表示PDF形式でダウンロード (2708K) -
Kengo Fujimoto, Ken-ichiro Masuda, Nobuhiko Asada, Eiji Ohnishi1993 年 113 巻 3 号 p. 285-291
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーTwo isoforms of prophenoloxidase were isolated from pupae of Oregon-R strain of Drosophila melanogaster. The purification procedure included ammonium sulfate fractionation, Sephacryl S-200 gel chromatography, DEAE-cellulose, and hydroxylapatite column chromatography. The two isoforms, A1 and A3, could be separated by ammonium sulfate fractionation. The isoelectric points of A1 and A3 were determined to be pH 5.8 and 6.7, respectively. The molecular weights of the monomers of A1 and A3 were estimated by SDS-PAGE to be 78 and 77 kDa, respectively. The native states of A1 and A3 are considered to be homodimeric, as judged by gel-filtration chromatography.抄録全体を表示PDF形式でダウンロード (2014K) -
Hideyuki Mukai, Takayoshi Kuno, Chang-Duk Chang, Ben Lane, Jay R. Luly ...1993 年 113 巻 3 号 p. 292-298
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe interaction of calcineurin (Ca2+/calmodulin-dependent protein phosphatase) with the potent immunosuppressive agent FK 506 and its 12 kDa isoform binding protein (FKBP 12) was investigated. The FKBP 12-FK 506 complex inhibited the Ca2+/calmodulin-stimulated phosphatase activity of each of two calcineurin isoforms, which contain either the catalytic subunit Aα or Aβ (calcineurin Aα or Aβ) of bovine calcineurin. Calcineurin phosphatase activity was inhibited by the FKBP 12-FK 506 complex irrespective of the substrate or the enzyme activation mechanism. FK 506 and FKBP-12 inhibited calcineurin in a concentration-dependent manner, and complete inhibition of the phosphatase activity appeared to require a molar excess of FKBP 12-FK 506 complex. Immunochemical measurements revealed tissue differences in the concentration of calcineurin, which may be of importance to the selectivity for immunosuppression of all of the biological effects. Direct binding studies with [3H] dihydro-FK 506 suggest that the ratio of FKBP 12-FK 506 complex to calcineurin in vivo when IL 2 production is inhibited is well correlated with the ratio when calcineurin phosphatase activity is inhibited in vitro. These results suggest that calcineurin is a relevant cellular target of FK 506 when bound to FKBP-12.抄録全体を表示PDF形式でダウンロード (2187K) -
Toru Yamaguchi, Hiroshi Kido, Riko Kitazawa, Sohei Kitazawa, Masaaki F ...1993 年 113 巻 3 号 p. 299-303
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe structure and location of a membrane-bound metallo-endopeptidase, previously purified from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563-571], were examined by immunochemical and immunohistochemical methods with a rabbit polyclonal antibody against the purified enzyme. On treatment with endoglycosidase F, the subunit of the purified enzyme (molecular mass=88 kDa) was converted to a smaller form (78.5 kDa), indicating that the enzyme contained at least 11% N-linked carbohydrate. Treatment of kidney membranes with papain resulted in release of the enzyme, as shown by Western blotting analysis of the solubilized fraction. Immunoassays of rat tissues showed that only the kidney, and small and large intestine expressed significant amounts of the antigen. Moreover, immunohistochemical studies showed that the antigen was confined to the luminal surfaces of the proximal renal tubules and the intestinal villi. Thus, like another kidney membrane metallo-endopeptidase, meprin [Kounnas et al. (1991) J. Biol. Chem. 266, 17350-17357], the purified enzyme is shown to be a glycoprotein that is probably anchored in the plasma membrane, and located in the luminal surface of microvillar membranes of the kidney and intestine. These results indicate that our enzyme and meprin have clear structural and topological similarities.抄録全体を表示PDF形式でダウンロード (1693K) -
Guiyun Xu, Takashi Suzuki, Gorou Hanagata, Eiki Deya, Makoto Kiso, Aki ...1993 年 113 巻 3 号 p. 304-307
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーSialyl-linkage specificity of the sialidase of influenza B viruses isolated in different years from 1940 through 1990 (B/Lee/40, B/Setagaya/3/56, B/Tokyo/7/66, B/Kagoshima/l/68, B/Gifu/2/73, B/Kanagawa/3/76, B/Ibaraki/2/85, B/Yamagata/16/88, and B/Bangkok/163/90) was studied with N-acetylneuraminyl (α2-3)- and (α2-6)-lactoses, GM 3 gangliosides containing the same sialyl-oligosaccharide sequences as sialyllactose, and also with type I and type II lacto-series gangliosides carrying Neu 5 Acα2-3 Gal and Neu 5 Acα2-6 Gal linkages as substrates. From an examination of up to nine strains, the sialidases of all viruses preferentially hydrolyze substrates with Neu 5 Acα2-3 Gal linkage rather than the Neu 5 Acα2-6 Gal linkage. It was found that the sialidase activity toward Neu 5 Acα2-6 Gal linkage relative to Neu 5 Acα2-3 Gal linkage is increased in later strains, whether sialyllactose or ganglioside is used as the substrate. These results suggested that the sialidase of influenza B virus isolates has shown a drift in linkage specificity which correlates with the year of isolation.抄録全体を表示PDF形式でダウンロード (527K) -
Makoto Haino, Shigeru Kawahire, Sahuro Omata, Tsunevoshi Horigome1993 年 113 巻 3 号 p. 308-313
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーA nuclear localization signal binding protein in nuclear envelope was studied as the first step to determine the mechanism of nuclear protein recognition by nuclear envelope. The rat liver nuclear envelope extract was resolved by SDS-PAGE and ligand blotted with 125I-labeled nucleoplasmin bearing a strong nuclear localization signal. A nuclear localization signal binding protein with molecular mass of 60 kDa (NBP 60) was detected in the extract. NBP 60 could be extracted with 2% Triton X-100-1 M KCl but not with 1M KCl, 2M urea, or 2% Triton X-100. The protein was partitioned to the lower layer in a two phase system using Triton X-114. These results suggested that the protein is an intrinsic membrane protein and has a hydrophobic surface. This protein was bound to not only nucleoplasmin but also the nuclear localization signal peptide of SV 40 large T-antigen (T-peptide) conjugated to human serum albumin. The binding of NBP 60 to nucleoplasmin-Sepharose was inhibited by 50% in the presence of 0.12mM T-peptide. However, a high concentration of 2.1mM was necessary, when mutant T-peptide in which the essential amino acid lysine was substituted with threonine was used. These results suggested that NBP 60 binds specifically to nuclear localization signals. NBP 60 extracted from the nuclear envelope was purified by nucleoplasmin-Sepharose affinity chromatography following hydroxyapatite high performance liquid chromatography.抄録全体を表示PDF形式でダウンロード (2677K) -
Akemi Ota, Jiro Seki, Xiao Liang Liu, Yu Zhao, Xiaohui Wang, Kiyoshi K ...1993 年 113 巻 3 号 p. 314-320
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe specificity of hen egg-white lysozyme (HEL)-reactive rabbit antibodies induced by the peptide loop I.II (sequences 57-107 containing Cys 64-Cys 80 and Cys 76-Cys 94) of HEL was clarified by analyzing their cross-reactions with various avian lysozymes and their reaction with synthetic peptides (sequences 59-82) in which alanine was substituted for the amino acid at certain positions. The Arg-68 residue of HEL plays a dominant role in the binding, while Gly-71, Ser-72, Arg-73, and Pro-79 also contribute to the binding of two anti-Ploop I.II antibodies (rabbit number 125 and 126). These residues, although remote in sequence, are grouped together in the crystal structure of HEL and may form an area of contact with the antibody. Contributions by Trp-63, Ile-78, and Asn-77 to the binding of the two antibodies to HEL were excluded. These results support the idea that the anti-Ploop I.II antibodies recognize a conformational type of epitope which is similar to that of native HEL. The immunogenicity of the reduced and alkylated form of Ploop I.II was also tested, but it failed to induce an HEL-reactive antibody.抄録全体を表示PDF形式でダウンロード (1021K) -
Takashi Takagi, Hideaki Yasunaga, Akio Nakamura1993 年 113 巻 3 号 p. 321-326
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーPreparations of thin filament from ascidian (Halocynthia roretzi) body wall muscle were found to be contaminated with large amounts of an unknown protein which, when purified, migrated to 29 kDa on SDS-PAGE and was thus named HR-29. To elucidate its physiological function, we have determined the primary structure of HR-29 by peptide and eDNA sequence analysis. Genomic sequence analysis showed that HR-29 is divided to four exons by three introns. It is composed of 251 amino acid residues and the N-terminal Ser is blocked by an acetyl group. The N-terminal domain of 150 residues is hydrophilic, and from residue 36 to 92 there are three similar repeated sequences of 19 residues. The N-terminal domain showed no significant sequence homology with other proteins. On the other hand, the C-terminal domain of 100 residues has homology with those of small heat-shock proteins and α-crystallin of lens protein. HR-29 is thought to be a fusion protein of two different origins. Recently αB-crystallin was identified in skeletal muscle and suggested to be a myofibril-stabilizing protein [Atomi, T. et al. (1991) J. Biochem. 110, 2360-2364]. Thus, HR-29 may also be a myofibril-stabilizing protein, as suggested by the sequence similarity of the C-terminal domain with α-crystallin, although the origin and role of the N-terminal domain are not clear.抄録全体を表示PDF形式でダウンロード (592K) -
Michiki Kasai, Takashi Kawasaki1993 年 113 巻 3 号 p. 327-333
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe effect of ryanodine on the permeation of glucose, choline, and Ca2+ through the Ca2+ channel in sarcoplasmic reticulum vesicles was studied by means of the light scattering and/or the tracer method. Ryanodine had the same effect on the permeabilities of choline and Ca2+ low concentrations of ryanodine (10 μM) locked the channel in the open state, and high concentrations (100 μM) opened the channel transiently, which closed finally. Glucose became permeable on the ryanodine treatment even in the absence of KCl, although it was not permeable before the treatment when the channel was open. Submolar concentrations of KCl enhanced the glucose and choline permeability of the ryanodine-treated channel as well as the untreated one. Activators such as ATP and caffeine did not enhance the permeability of the treated channel in the open locked state, but inhibitors such as Mg2+ and ruthenium red closed the channel, although much higher concentrations were required than for the untreated channel. The maximal rates of choline and glucose permeation of the ryanodine-treated channel were lower than those of the untreated one attained in the presence of ATP and/or caffeine. This result is consistent with the fact that the single channel conductance after the ryanodine treatment decreased to 40-50% of the maximal conductance of the untreated one. This result suggests that the conformation of the channel opened by ryanodine was different from that with ATP or caffeine.抄録全体を表示PDF形式でダウンロード (745K) -
Kenzo Ohtsuki, Masahiro Matsumoto, Hitoshi Saito, Toyoki Kato1993 年 113 巻 3 号 p. 334-342
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーCK-II has been partially purified from a 1.5M KCl extract of unfertilized sea urchin eggs by means of DEAE-cellulose column chromatography, gel filtration on Sephacryl S 300, and heparin-agarose column chromatography, successively. This partially purified CK-II was associated with a 98 kDa polypeptide (designated as p 98), which was then separated from the kinase by Mono Q column chromatography (HPLC). The biochemical characteristics of CK-II purified from unfertilized eggs were similar to those of CK-IIs from various mammalian cells: requirements of divalent cations (Mg2+ and Mu2+) and phosphate acceptors (casein and p 98), response to basic polypeptides and heparin, subunit structure and molecular size. Moreover, it was found that (i) p 98 (apparent pI 10.0) has DNA-binding ability and functions as an effective phosphate acceptor for CK-II in vitro; (ii) phosphorylation of p 98 by the kinase was highly stimulated by histone-like sperm protein from sea urchin sperm, protamine (salmon sperm), and poly Arg; and (iii) selective phosphorylation of p 98 by CK-II was detected when the DEAE-cellulose fraction from unfertilized eggs was incubated with [γ-32P] ATP in the presence of protamine. Some biochemical characteristics of p 98 from the eggs were similar to those of transcriptional factor Spl. The evidence obtained suggests that (i) arginine-rich sperm proteins function as potent activators for CK-II in unfertilized eggs and (ii) specific phosphorylation of p 98 by the activated kinase may play an important role in the transcriptional regulation in the eggs accompanying fertilization.抄録全体を表示PDF形式でダウンロード (5709K) -
Hitoshi Niwa, Kimi Araki, Shigemi Kimura, Shin-ichi Taniguchi, Shoji W ...1993 年 113 巻 3 号 p. 343-349
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーNew trap vectors (U 1 and U 2) have been developed to trap genes in murine embryonic stem (ES) cells. The polyA addition signal of the neomycin phosphotransferase II (neo) gene was removed from these vectors so that they needed to trap an endogenous polyA signal for expression of the neo gene. The frequency of gene-trap events of these vectors was about five times higher than with the vector containing the polyA signal, and only one copy of the trap vector was integrated in most cases. Four out of five 5'-flanking regions of the integrated vector in ES cell lines were found to be novel endogenous promoters, suggesting that this method is efficient for trapping genes in ES cells. In two cases analyzed, large deletions or rearrangements spanning more than 10 kb were found in the 3'-flanking region of the trap vector introduced by electroporation. This result suggests that phenotypes observed in homozygotes with a mutated allele could be due to the disruption of a gene adjacent to the trapped gene, but not of the trapped gene.抄録全体を表示PDF形式でダウンロード (3200K) -
Haruo Mimura, Tadao Hashimoto, Yukuo Yoshida, Naoki Ichikawa, Kunio Ta ...1993 年 113 巻 3 号 p. 350-354
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーYeast mitochondrial F1FoATPase has three regulatory subunit proteins: ATPase inhibitor, 9 K protein, and 15 K protein. Mutant yeasts lacking one or more of these protein factors were constructed by gene disruption [Ichikawa, N. et al. (1990) J. Biol. Chem. 265, 6274-6278; Yoshida, Y. et al. (1990) Eur. J. Biochem. 193, 49-53]. Dissipation of the electrochemical potential of protons of the mitochondrial inner membrane by an uncoupler or by a combination of valinomycin and potassium ions induced ATP-hydrolyzing activity of F1FoATPase in mitochondria of all the mutants, as in those of wild-type cells. However, the ATPase activity was inactivated within a minute in normal mitochondria, but was not suppressed in inhibitor-deficient mitochondria, and in mitochondria lacking either 9 K or 15 K protein, the inactivation of ATPase was slow and incomplete. Covalent binding of inhibitor protein to the enzyme was achieved with a zero length cross-linker, EEDQ, in uncoupled normal mitochondria, in which the inhibitor linked directly to both the α- and β-subunits. This result strongly suggests that the binding site of the inhibitor protein is located at the interface between the two subunits.抄録全体を表示PDF形式でダウンロード (2826K) -
Tanetoshi Koyama, Shusei Obata, Masami Osabe, Ayumi Takeshita, Ken Yok ...1993 年 113 巻 3 号 p. 355-363
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe structural gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli cells. A 1, 260-nucleotide sequence of the cloned fragment was determined. This sequence specifies an open reading frame of 891 nucleotides for farnesyl diphosphate synthase. The deduced amino acid sequence shows a 42% similarity with that of E. coli FPP synthase [Fujisaki et al. (1990) J. Biochem. 108, 995-1000]. Comparison with prenyltransferases from a wide range of organisms, from bacteria to human, revealed the presence of seven highly conserved regions. In contrast to thermolabile prenyltransferases, which have four to six cysteine residues, the thermostable farnesyl diphosphate synthase carries only two cysteine residues. This enzyme is also unique in that some of the amino acids that are fully conserved in equivalents from other sources are replaced by functionally different amino acids. Construction of an overproducing strain provided a sufficient supply of this enzyme and it was purified to homogeneity. The purified recombinant enzyme is immunochemically identical with the native B. stearothermophilus enzyme, and it is not inactivated even after treatment at 65°C for 70min.抄録全体を表示PDF形式でダウンロード (1785K) -
Kentaro Yomogida, Masayuki Yamamoto, Takashi Yamagami, Hiroyoshi Fujit ...1993 年 113 巻 3 号 p. 364-371
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーTo understand the regulatory mechanisms controlling the heme biosynthetic pathway in animal liver, RNA blot hybridization analysis was used to examine the developmental stage-specific transcription of the gene encoding nonspecific form δ-aminolevulinate synthase (ALAS-N). The expression of the erythroid-specific δ-aminolevulinate synthase (ALAS-E) mRNA was also studied. The results demonstrated that, while ALAS-E is the key enzyme which supplies large quantities of heme for hemoglobin synthesis in fetal rat liver, ALAS-N functions to supply heme for the cytochrome P-450 system in fetal, newborn, and adult rat liver. ALAS-N was also suggested to work as a housekeeper gene to supply heme for respiratory cytochromes and other hemoproteins in various tissues. The structure and organization of the rat ALAS-N gene were next analyzed to study the molecular mechanisms regulating ALAS-N gene transcription. The ALAS-N gene was found to span more than 14 kb in the rat genome, encompassing eleven exons. The promoter region of the gene was found to contain several potential cis-acting regulatory elements, including motifs matching the TATA box sequence and the nuclear respiratory factor 1 binding sequence. The organization of the rat ALAS-N gene was determined to be quite similar to that of the ALAS-E gene in mouse; the mouse ALAS-E gene consists of eleven exons. This observation suggested that the ancestral gene for ALA synthase in animals was probably composed of eleven exons, and both the ALAS-N and ALAS-E genes were derived from this ancestral gene.抄録全体を表示PDF形式でダウンロード (2883K) -
Koji Kigawa, Kayo Kihara, Yasuko Miyake, Shoji Tajima, Tohru Funahashi ...1993 年 113 巻 3 号 p. 372-376
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーA deletion mutant in the low density lipoprotein receptor gene of a Japanese patient with heterozygous familial hypercholesterolemia was analyzed. Genomic Southern blotting showed abnormal size restriction fragments with BamHI (7.8 kb), EcoRI (3.8 kb), BglII (17 kb), KpnI (>23 kb), EcoRV (13 kb), and XbaI (14 kb). The abnormal EcoRI fragment, 3.8 kb, was cloned into λ phage vector, and the deleted region of 10 kb including exons 2 and 3 was identified. The nucleotide sequence around the deletion joint was determined. The sequence of the eight nucleotides in the deletion-joint region of the mutant gene was identical to the corresponding sequences of both introns 1 and 3 of the normal gene. The deletion seemed to occur by an unequal recombination between the Alu-like sequences in the same direction in introns 1 and 3.抄録全体を表示PDF形式でダウンロード (2302K) -
Konami Kita, Saburo Omata, Tsuneyoshi Horigome1993 年 113 巻 3 号 p. 377-382
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーIt is known that nucleoporins, a family of glycoproteins with N-acetylglucosamine that are found in nuclear pore complexes, are essential for nuclear import and export. A major component of the family, p 62, was purified from a salt extract of rat liver nuclear envelopes by wheat germ agglutinin-Sepharose affinity chromatography and DEAE-anion exchange HPLC. p 62 was purified as a complex with two glycoproteins of 60 and 54 kDa. The presence of the complex was confirmed by gel filtration, glycerol density gradient centrifugation, and cross-linking experiments. The molecular ratio of the 62-, 60-, and 54-kDa components of the complex was estimated to be 1:1.1±0.2:1.7±0.3 from the intensity of Coomassie Blue staining of SDS-PAGE gels. The complex was stable against 1M NaCl, 1% Triton X-100, and 2M urea. The Stokes' radius and sedimentation coefficient of the complex are 8.0 nm and 6.7 S. The molecular mass and frictional ratio of the complex were estimated to be about 231 kDa and 2.0, respectively. p 62 and p 54 were acidic and neutral proteins, respectively, exhibiting charge heterogeneities, and p 60 was assumed to be a basic protein. p 60 tended to undergo proteolytic degradation to a 47-kDa fragment.抄録全体を表示PDF形式でダウンロード (3898K) -
Dong Hyun Ahn, Akihito Hattori, Koui Takahashi1993 年 113 巻 3 号 p. 383-388
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーPostnatal structural changes in Z-disks of skeletal muscles of chicken from 2 to 35 wk after hatching were examined to elucidate how Z-disks develop from the embryonic to the mature stage. The mechanical strength of Z-disks of breast muscle (i. e. the ratio of the number of myofibrils to the total number of myofibrils and myofibrillar fragments composed of 1-4 sarcomeres, which were formed under the mechanical forces exerted during homogenization) increased from 66% at 2 wk of age to 75% at 10 wk, and leveled off at 76% at 25 wk. The Z-disks of leg muscle were stronger than those of breast muscle throughout growth. Measurements by electron microscope showed that the width of Z-disks increased during growth from 26 to 33 nm, 48 to 53 nm, and 89 to 101 nm, in white, intermediate, and red muscle fibers, respectively. It was proved that the configuration of Z-filaments, the structural backbone of Z-disks, takes its final shape within 2 wk after hatching; the immature Z-disks are then reinforced by the accumulation of amorphous matrix materials. These results confirm that the maturation of Z-disks is brought about by physical motion in muscle tissue and the accompanying development of tension.抄録全体を表示PDF形式でダウンロード (2317K) -
Takahiro Kawabata, Yukio Nishimura, Masahide Higaki, Keitaro Kato1993 年 113 巻 3 号 p. 389-394
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーIn order to characterize the intracellular processing event of lysosomal cathepsin B, the proenzyme was purified from the rat liver microsomal contents using a Con A-Sepharose column, a Sepharose-Gly-Phe-GlySe column, and an anti-cathepsin B IgG column. The purified proenzyme gave a single protein band of 39 kDa on SDS/polyacrylamide gel electrophoresis. The proenzyme showed no appreciable enzymatic activity. When the purified proenzyme was incubated with the cathepsin B-free tritosomal contents, prepared by treatment of the tritosomal contents with anti-cathepsin B IgG Sepharose, at pH 3.0, 30°C, a remarkable increase of enzymatic activity was observed. Immunoblot analysis showed that the proenzyme was completely converted to the active intermediate form of 31 kDa after 1 h incubation. These processing and activation events were blocked in the presence of pepstatin. When the proenzyme was incubated with the cathepsins B- and D-free tritosomal contents, prepared by treatment of the cathepsin B-free tritosomal contents with anti-cathepsin D IgG Sepharose, the processing and activation did not occur. These results indicate that cathepsin D is involved in the processing and activation of procathepsin B in rat liver lysosome. In the NH2-terminal sequence analysis of the 31 kDa form, the terminal was assigned as proline (66 th residue). Since the NH2-terminus of the mature single-chain form of cathepsin B (29 kDa) ends at leucine (80 th residue), the NH2-terminus of the 31 kDa form is 14 amino acid residues longer than that of the single-chain form. Therefore, we presume that procathepsin B is first processed by cathepsin D, splitting the bond between the 65 th and 66 th amino acid residues, and then lysosomal aminopeptidase (s) hydrolyzes the 14 amino acid residues from 66 to 79.抄録全体を表示PDF形式でダウンロード (2657K) -
Terumasa Tsuchiya, Masatsugu Ueda, Hirokazu Ochiai, Shinobu Imajoh-Ohm ...1993 年 113 巻 3 号 p. 395-400
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe 5'-flanking region of the human erythropoietin (Epo) gene contains a 0.14-kb sequence that is conserved in the Epo gene from mouse and located within a promoter that is activated under hypoxic conditions such as anemia. Using a fragment containing this sequence in DNA mobility shift assays, we found that specific DNA-binding proteins were induced in mouse kidney nuclei under anemic hypoxia. Using synthetic double-stranded oligonucleotides that contain this sequence, the essential binding site was defined to be the -40 to -20 region upstream of the transcription initiation site in the human Epo gene. By DNA affinity chromatography using a column with the immobilized 5'-flanking sequence, two inducible binding proteins with apparent molecular masses of 55 and 45 kDa were identified in the nuclei of mouse kidney and liver under anemic hypoxia. These binding proteins were also induced during cobalt exposure.抄録全体を表示PDF形式でダウンロード (2824K) -
Takashi Uemori, Yoshizumi Ishino, Kayo Fujita, Kiyozo Asada, Ikunoshin ...1993 年 113 巻 3 号 p. 401-410
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe pot gene of the thermophilic eubacterium Bacillus caldotenax was cloned in a plasmid and expressed in Escherichia coli. The PCR method was used to clone the gene with no previous knowledge of the gene or protein sequence. The 3, 329-bp DNA fragment containing the structural gene for DNA polymerase was sequenced. DNA polymerase, as deduced from the DNA sequence, consisted of 877 amino acids, had a molecular weight of 99, 452, and was structurally homologous to the DNA polymerases of the Pol I family (family A), which includes E. coli DNA polymerase I and T 7 DNA polymerase. B. caldotenax DNA polymerase (Bca polymerase) purified from the recombinant E. coli strain was characterized. Like E. coli Pol I, Bca polymerase had 5'→3' exonuclease activity. The degraded product with the molecular weight of 65, 000 was also purified and found to have polymerase activity. To overproduce this Klenow-type fragment of Bca polymerase, a recombinant expression plasmid pUI 205 with a deletion in the 5'-region of the pol structural gene was constructed. The DNA polymerase produced by pUI 205 is more suitable for use in the dideoxy sequencing method than the other DNA polymerases that have been used for sequencing.抄録全体を表示PDF形式でダウンロード (4678K)
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