巻号一覧
113 巻, 6 号
選択された号の論文の25件中1～25を表示しています
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Yuan Lin, Li-Hong Ye, Ryoki Ishikawa, Koichiro Fujita, Kazuhiro Kohama1993 年 113 巻 6 号 p. 643-645
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーCalponin, a calmodulin-binding protein of smooth muscle that inhibits the actin-myosin interaction by binding to actin, was shown to bind to myosin and to stimulate the ATPase activity of myosin to some extent. Actin abolished this myosin-linked, stimulatory effect of calponin. Ca2+-calmodulin affected neither the myosin-binding activity nor the stimulatory effect of calponin. We further presented a few data which suggest that calponin may exert regulatory activity toward myosin in quite a different way from caldesmon, another smooth muscle protein that binds to myosin, actin, and calmodulin.抄録全体を表示PDF形式でダウンロード (343K) -
Hidekazu Kizaki, Yasuo Hata, Kunihiko Watanabe, Yukiteru Katsube, Yuzu ...1993 年 113 巻 6 号 p. 646-649
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe crystal structure of anoligo-1, 6-glucosidase from Bacillus cereus ATCC7064 was determined by the X-ray diffraction method at 3.0 Å resolution. The structure was solved by the multiple isomorphous replacement method and refined to a crystallographic R-factor of 0.208, using the molecular dynamics refinement program, X-PLOR. The electron density map revealed the folding of a polypeptide chain consisting of 558 amino acid residues. The molecule can be subdivided into three domains (N-terminal domain, subdomain, and C-terminal domain). The N-terminal domain has an (α/β)8-barrel structure called the TIM-barrel structure. The C-terminal domain is characterized by a β-barrel structure composed of eight antiparallel β-strands, while the subdomain has a loop-rich structure with a small α-helix and a β-sheet. The enzyme has a large cleft between the N-terminal domain and the subdomain. The cleft leads from the molecular surface to the molecular center. The bottom of the cleft is at the C-terminal end of the parallel β-strands of the (α/β)8-barrel. These structural features closely resemble those of α-amylases from Aspergillus oryzae and pig pancreas.抄録全体を表示PDF形式でダウンロード (1595K) -
Michihito Inagaki, Mamoru Tamura1993 年 113 巻 6 号 p. 650-657
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーUsing a new cannulation method, which completely prevented interruption of circulation to brain tissue during surgical operations, we could prepare a functionally intact hemoglobin-free isolated perfused rat head in situ. The frequency distribution (8-25 Hz) and amplitude of a spontaneous electroencephalogram, EEG (50 μV), were kept within the normal ranges for up to 4 h after the perfusion was started. Our preparation gave characteristic flash-evoked EEG responses through the eyes. The administration of bicuculline elicited an epileptic seizure similar to that in normal rats. Near-infrared difference spectra of the rat head obtained on an aerobic to anaerobic transition were similar to those of cytochrome oxidase in isolated mitochondria as well as in the purified state. Under the aerobic perfusion conditions, both heme a+a3, and copper in cytochrome oxidase were in the fully oxidized states, showing that normoxia was maintained in the brain tissue. The redox behaviors of these two chromophores in the brain were identical to those observed in isolated mitochondria. The usefulness of brain perfusion for bridging in vitro and in vivo studies is well documented.抄録全体を表示PDF形式でダウンロード (820K) -
Yoshiko Okamoto, Kiyoshi Ogawa, Terushige Motohiro, Norio Nishi, Eiko ...1993 年 113 巻 6 号 p. 658-664
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーSpotted mackerel protamine, scombrine, was isolated from the sperm of a spotted mackerel (Scomber australasicus) by extraction with sulfuric acid and fractionated into one major (scombrine II) and one minor (scombrine I) components by chromatography on CM-Sephadex C-25. Scombrine II gave a single band, whereas scombrine I gave three bands upon PAGE. Scombrine II (scombrine γ) consists of 34 amino acid residues, and its sequence is: Pro-Arg-Arg-Arg-Arg-Arg-Ala-Ser-Arg-Pro-Val-Arg-Arg-Arg-Arg-Arg-Ala-Arg-Arg-Ser-Thr-Ala-Val-Arg-Arg-Arg-Arg-Arg-Val-Val-Arg-Arg-Arg-Arg. The ion spray mass spectrum shows that scombrine γ has a molecular mass of 4, 532.13 Da. The other minor component (scombrine I) is considered to be a mixture of degradation products of scombrine γ based on the results of PAGE and ion spray mass spectrometry. Scombrine γ has a similar sequence to sardaine Z2 from striped bonito except for two positions.抄録全体を表示PDF形式でダウンロード (2304K) -
Yasuhiko Miwa, Yasutaro Fujita1993 年 113 巻 6 号 p. 665-671
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe mechanism underlying catabolite repression in Bacillus species remains unknown. A recent study of the promoter-independent catabolite repression of the gnt operon implicated a consensus sequence (ATTGAAAG) in catabolite repression in the genus Bacillus. The introduction of base-substitutions into the ATTGAAAG sequence in the chromosomal gnt operon affected catabolite repression of the gnt operon. Deletion analysis indicated that the ATTGAAAG sequence is probably part of a cis sequence necessary for the promoter-inde-pendent catabolite repression of the gnt operon. Furthermore, we subjected gnt transcripts synthesized with and without glucose to S1 nuclease and slot blotting analyses. The results indicated that the gnt transcripts decreased in the region (+93 to +203; +1, the transcription initiation nucleotide) only in the presence of glucose. Mechanisms underlying this promoter-independent catabolite repression are discussed.抄録全体を表示PDF形式でダウンロード (2872K) -
Shoji Watabe, Takayuki Hara, Hiroyuki Kohno, Tomoko Hiroi, Nagasumi Ya ...1993 年 113 巻 6 号 p. 672-676
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe have examined in vitro degradation of mitochondrial proteins by ATP-dependent protease in bovine adrenal cortex. Purified ATP-dependent protease degraded at least five proteins in vitro in the presence of ATP and Triton X-100 using mitochondrial fraction as a substrate. Two of the degraded proteins were identified as P-450scc and adrenodoxin reductase by immunoblotting. No degradation of P-45011β or adrenodoxin was detected. Purified P-450scc and adrenodoxin reductase were also degraded. By analyzing degradation products of P-450scc we identified 49 peptides and 59 cleavage sites. The size of the products was between 3 and 18 amino acid residues. The protease preferentially cleaved the carboxy-side of Leu, Phe, Ala, Val, Met, and some other amino acids. Since the protease (a matrix enzyme) is accessible to P-450scc and adrenodoxin reductase localized on the matrix side of inner membrane, the present results suggest that the protease might participate in the turnover of these two proteins and some other mitochondrial proteins.抄録全体を表示PDF形式でダウンロード (2895K) -
Miho Tarutani, Naoko Norioka, Tomohiro Mega, Sumihiro Hase, Tokuji Ike ...1993 年 113 巻 6 号 p. 677-682
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe structures of the sugar chains of hen yolk riboflavin-binding protein (RBP) were established. Asparagine-linked sugar chains of yolk-RBP were liberated by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. Fluorescent pyridylamino (PA-) derivatives of the sugar chains were purified by gel-filtration and reversed-phase HPLC. Seven PA-sugar chains were isolated, and the structure of each was determined by composition analysis, sequential exoglycosidase digestion, methylation analysis, and 500-MHz1H-NMR spectroscopy. These analyses showed that the main sugar chains had sialylbiantenna and sialyltriantenna structures. PA-sugar chains of plasma-RBP were also isolated, and the structures of the PA-sugar chains of yolk- and plasma-RBPs were compared as to their elution patterns on anion-exchange chromatography and reversed-phase HPLC. The plasma RBP had almost the same sugar chains as the yolk RBP did, indicating that sugar chains are not modified during incorporation into the oocyte.抄録全体を表示PDF形式でダウンロード (639K) -
Jun Aketagawa, Shigenori Tanaka, Hiroshi Tamura, Yuko Shibata, Hazime ...1993 年 113 巻 6 号 p. 683-686
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーIt has been demonstrated that both linear and branched (6-O-β-D-glucosyl) (1→3)-β-D-glucans taking a single helical conformation are more effective than those taking a triple helical conformation for the activation of factor G from horseshoe crab amebocytes, as revealed by high-resolution solid-state 13C-NMR spectroscopy [Saitô, H. et al. (1991) Carbohydr. Res. 217, 181-190]. Annealing the linear glucan at 180°C was essential to convert the conformation from the single helix to the triple helix. We found that heating of the glucan at such a high temperature resulted in depolymerization of the sample to molecular weight smaller than 10, 000, which may influence the conformation and the abovementioned biological activity. To eliminate ambiguity arising from the depolymerization of the glucan during annealing, we aimed to relate the biological activity to the conformation of samples whose chain lengths are identical, because the potency is known to depend on the molecular weight of the glucans. This molecular weight dependency of the potency, however, was found to be not the dominant factor, provided that the molecular weight is large enough to allow formation of the single helix conformation. Therefore, the single helical conformation of (1→3)-β-D-glucans is clearly demonstrated to be the dominant contributor to the activation of limulus coagulation factor G.抄録全体を表示PDF形式でダウンロード (452K) -
Hiroyuki Seimiya, Mikihiko Naito, Tetsuo Mashima, Hisashi Yasui, Yoshi ...1993 年 113 巻 6 号 p. 687-691
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe T cell receptor α and β chains are covalently linked via a disulfide bond in their extracellular constant regions. To use these domains as specific hetero-cross-linkers of two different polypeptides, we created genetic constructs encoding a chimeric antibody Fab fragment in which mouse immunoglobulin constant regions from a phosphorylcholine-specific antibody were substituted for human αβ-T cell receptor (TCR) extracellular constant regions (for solubilization, the transmembrane- and cytoplasmic-regions of the receptor were deleted). These constructs, i.e., chimeric heavy (VHCβCκ) and light (VLCα) chains, were cotransfected into murine SP2/0 myeloma cells for expression. Cells transfected with the genes expressed mRNAs for chimeric heavy and light chains. Without CD3 molecules, the two chimeric chains specifically associated via a disulfide bond to form a chimeric Fab fragment in the cells. These data indicate that the TCR Cα- and Cβ-regions might be used as potent specific hetero-cross-linkers for protein engineering.抄録全体を表示PDF形式でダウンロード (1713K) -
Yoshito Ihara, Atsushi Nishikawa, Takaya Tohma, Hidenobu Soejima, Nori ...1993 年 113 巻 6 号 p. 692-698
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーUDP-N-acetylglucosamine: β-D-mannoside β1, 4 N-acetylglucosaminyltransferase III (GnT-III) [EC 2. 4. 1. 144] catalyzes the addition of N-acetylglucosamine in β1-4 linkage to the β-linked mannose of the trimannosyl core of N-linked sugar chains to produce a bisecting GlcNAc residue. We have isolated six independent cDNA clones of human GnT-III from a fetal liver cDNA library. The cDNA sequence has an open reading frame that predicts a protein of 531 amino acids. The homology to rat GnT-III is 86% at the nucleotide level and is 91% at the amino acid level. The amino-terminal transmembrane domain and the catalytic domain are well conserved in the two species. Human GnT-III has a deletion of four amino acids in the “neck” region and several differences in the COON-terminal region compared with the rat sequence. Using one of the human cDNA clones as the probe, two overlapping genomic clones have been isolated from a human cosmid library. The GnT-III gene has been mapped to chromosome 22 q. 13. 1 using fluorescence in situ hybridization.抄録全体を表示PDF形式でダウンロード (1688K) -
Ludwig Mauch, Jochen Seißler, Heinz Haubruck, Neil J. Cook, Char ...1993 年 113 巻 6 号 p. 699-704
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーcDNAs coding for the full-length human 65 and 67 kDa glutamic acid decarboxylases (GAD65 and GAD67) were amplified from pancreas and hippocampus cDNA libraries by polymerase chain reaction, respectively. Both cDNAs were inserted into a baculovirus vector which mediated highly efficient expression of the human GAD65 and GAD67 with histidine-hexapeptides as affinity ligands at their C-termini in Spodoptera frugiperda (Sf9) cells. The recombinant GAD proteins were purified to homogeneity by affinity chromatography using a metal-chelating matrix. The infected Sf9 insect cells expressed the recombinant human GAD65 and GAD67 with natural-like conformations, as confirmed by measurement of their enzyme activities as well as their fully restored autoantigenicities. Immunoprecipitation of metabolically labeled infected Sf9 cells demonstrated the autoantigenic potential of the recombinant GAD proteins. The practicability of using recombinant GAD65 and GAD67 derived from the baculovirus expression system for the development of an immunoassay for the diagnosis of insulin-dependent diabetes mellitus is discussed.抄録全体を表示PDF形式でダウンロード (2797K) -
Shin-ichi Hisanaga, Setsuko Yasugawa, Takashi Yamakawa, Eishichi Miyam ...1993 年 113 巻 6 号 p. 705-709
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatase. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2 A, 2 B, and 2 C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.抄録全体を表示PDF形式でダウンロード (3310K) -
Satoru Harumiya, Sang-Kee Jung, Yoshiyuki Sakano, Daisaburo Fujimoto1993 年 113 巻 6 号 p. 710-714
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーBecause there are contradictory reports about the interaction of plasma fibronectin with elastin, we investigated the interaction in vitro. When human plasma was applied to an α-elastin-Sepharose column at 4°C, the column-binding fraction contained fibronectin. When isolated plasma fibronectin was applied to the same column at 4°C, most of the fibronectin bound to the column and was eluted with 1M KBr. However, the binding affinity of plasma fibronectin to the α-elastin-Sepharose column was much weaker at 25°C than at 4°C. The elastin-plasma fibronectin interaction was further confirmed by demonstrating the binding of α-elastin to fibronectin on polyvinylidene difluoride membranes using an α-elastin specific antibody. The elevation of the surface hydrophobicity of plasma fibronectin at 4°C was observed by hydrophobic chromatography, using alkyl-Sepharose columns. It seems that the binding of plasma fibronectin to α-elastin involves hydrophobic interaction, which is affected by temperature and possibly by other factors.抄録全体を表示PDF形式でダウンロード (1451K) -
Nobuaki Higashi, Masahiro Higuchi, Nobutsugu Hanada, Jun-ichi Oeda, Yo ...1993 年 113 巻 6 号 p. 715-720
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーMacrophages are activated by a two-step mechanism involving at least two kinds of factors, a priming and a triggering factor, to become cytotoxic to various tumor cells. In the present study, we purified macrophage-activating factor for cytotoxicity I (MAF-C I), defined as a priming macrophage activating factor (MAF), by about 1, 600-fold from the culture supernatant of a human T cell hybridoma, H3-E9-6, by a series of chromatographic procedures. We identified MAF-C I activity released from H3-E9-6 cells as interleukin-2 (IL-2) from the following findings. (i) The physicochemical properties of MAF-C I and IL-2 were almost identical. (ii) Purified MAF-C I active fraction also showed T cell proliferating activity. (iii) MAF-C I activity in the purified fraction was completely neutralized by anti-IL-2 antibodies. (iv) Human recombinant IL-2 (rIL-2), at a suboptimal dose, and lipopolysaccharide (LPS) synergistically induced monocyte-mediated cytotoxicity.抄録全体を表示PDF形式でダウンロード (628K) -
Yuri E. Bukhtiyarov, Yuri A. Shabalin, Igor S. Kulaev1993 年 113 巻 6 号 p. 721-728
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWhen the membrane fraction of Saccharomyces carlsbergensis was incubated with radiolabeled isopentenyl diphosphate in the presence of farnesyl diphosphate and Mg2+, phosphorylated and free long-chain polyprenols were formed. The reaction was inhibited by EDTA and heavy metal cations. A series of non-ionic detergents were studied for their efficacy to solubilize the prenyltransferase. The enzyme completely lost its activity in the presence of 0.1% of Triton X-100. n-Octyl-β-(D) glucopyranoside at the concentration of 0.25-0.5% (10-15mM) was used to solubilize the prenyltransferase. Both the membranebound enzyme and the solubilizate possessed a broad pH optimum shifted to alkaline pH values. The temperature optimum of the solubilizate was somewhat lower than that of the membrane preparation, owing to the significantly lower thermostability of the solubilized enzyme in comparison with the membrane-bound one. The phosphorylated reaction products formed in the presence of the membrane preparation had the same composition as the yeast dolichol synthesized in vivo. Non-phosphorylated polyprenols were formed during the incubation with membranes but not the solubilized enzyme. The composition of the polyprenols was also coincident with that of yeast dolichol, and the individual C80-homolog of the mixture was polyprenol but not dolichol as judged by adsorption HPLC. The results are discussed in relation to the terminal stages in the biosynthesis of dolichol derivatives.抄録全体を表示PDF形式でダウンロード (2583K) -
Tapan K. Chaudhuri, Kali P. Das, Nirmal K. Sinha1993 年 113 巻 6 号 p. 729-733
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーSurface hydrophobicity has recently been emphasized as an important parameter for functional correlation of proteins. However, evaluations of the parameter by different experimental techniques often do not correlate well with each other. In this paper we have compared surface hydrophobicity of a basic protein with those of β-lactoglobulin, ovalbumin and lysozyme by fluorescence probe method using ANS as an external probe. Two different fluorimetric approaches to determining the surface hydrophobicity parameter, namely, the slope method and the binding parameter method, follow the same relative order. Denaturants, urea, and guanidine hydrochloride disrupted the hydrophobic clefts of the inhibitor on the surface, causing a drastic reduction of surface hydrophobicity.抄録全体を表示PDF形式でダウンロード (481K) -
Fritz Märki, Valérie Hanulak1993 年 113 巻 6 号 p. 734-737
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーTo further explore the role of the N-terminus of group II phospholipase A2 (PLA2) for enzymatic activity, kinetics of recombinant human synovial fluid PLA2 (rPLA2) and a variant with an extra N-terminal methionine (Met-rPLA2) were analyzed with the substrates [1-14C] oleate-labeled Escherichia coli, vesicular phosphatidylethanolamine, and micellar phosphatidylcholine. While N-terminal variation did not affect assay parameters such as pH-optimum, or optimal concentration of calcium and E. coli substrate, it drastically reduced maximum velocities with all three substrates, without any decrease of substrate affinities, suggesting a role of the N-terminus in the catalytic step (s) subsequent to binding of the substrate. Eleven compounds from various structural classes were found to potently inhibit both enzymes. Several of these compounds were equipotent on rPLA2 and Met-rPLA2; others had significantly lower potency on Met-rPLA2 than on rPLA2. This suggests participation of the N- terminal region of the enzyme in the mechanism of inhibition by the latter compounds. The study provides new evidence for the role of the N-terminus of group II PLA2 for catalytic activity.抄録全体を表示PDF形式でダウンロード (434K) -
Francisco J. G. Muriana, Angel M. Relimpio1993 年 113 巻 6 号 p. 738-741
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーPartially purified glutamine synthetase from rat liver underwent rapid inactivation upon incubation with NADH and benzyl viologen, under aerobic conditions. This in vitro inactivation was prevented by catalase or chelating-agents, which suggests that hydrogen peroxide and metal ions are involved in the process. Similar inactivation was observed when the rat liver glutamine synthetase was preincubated, under anaerobic conditions, with NADH and benzyl viologen, and hydrogen peroxide was added to the reaction mixture. A radical scavenger, histidine, partially prevents the inactivation, while hydrogen peroxide shows a low inactivation capacity when incubated without NADH. Furthermore, the fact that the inactivation can also be catalyzed by a model consisting of ferrous ions and hydrogen peroxide leads to the conclusion that hydroxyl radicals, or something with similar reactivity, are most likely produced through a Fenton reaction.抄録全体を表示PDF形式でダウンロード (547K) -
Senarath B. P. Athauda, Eiji Ido, Hideo Arakawa, Masaaki Nishigai, Hir ...1993 年 113 巻 6 号 p. 742-746
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe inhibitory effect of α2-macroglobulin (α2M), a major plasma proteinase inhibitor, on human immunodeficiency virus (HIV) proteinase was investigated. The activity of HIV proteinase toward the Moloney murine sarcoma virus-derived gag protein (a highmolecular-mass substrate) was found to be inhibited by α2M at pH 5.5-7.4. On the other hand, the activity toward the B chain of oxidized insulin (a low- molecular-mass substrate) was scarcely inhibited. The complex of α2M and HIV proteinase was isolated by gel filtration and the enzyme was shown to be significantly protected by the complex formation from autoinactivation under nonreducing conditions. The stoichiometry of the complex formation was found to be 2:1 (enzyme:α2M, mol/mol). These results demonstrate the entrapment and concomitant inhibition of HIV proteinase by α2M.抄録全体を表示PDF形式でダウンロード (2761K) -
Kazutoshi Nakazawa, Kiyoshi Furukawa, Hisashi Narimatsu, Akira Kobata1993 年 113 巻 6 号 p. 747-753
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーRecombinant β-1, 4-galactosyltransferase which synthesizes the Gal β1→4GlcNAc group of glycoprotein sugar chains was obtained as a soluble form from Escherichia coli by transfection of the human cDNA lacking the transmembrane segment. Kinetic study revealed that the soluble transferase has the same apparent Km, values toward sugar nucleotide and sugar acceptors as those of mouse membrane-bound β-1, 4-galactosyltransferase previously characterized [Nakazawa et al. (1991) Eur. J. Biochem. 196, 363-368]. However, the Vmax value of this transferase was low when compared to that of the mammalian transferase, probably due to the instability of the transferase caused by the lack of protein glycosylation. The soluble transferase was purified from the E. coli lysates almost to homogeneity by chromatography on DEAE-Sepharose and α-lactalbumin-Sepharose columns. Using this purified transferase, the acceptor specificity of the transferase has been studied. The results showed that the transferase has apparent Km values of 170, 190, and 830 μM for agalacto-poly-N-acetyllactosamine, lacto-N-triose II, and lacto-N-triaosylceramide, respectively, but has apparently no activity toward glucosylceramide. These results suggest that the β-1, 4-galactosyltransferase may be involved in the synthesis of poly-N-acetyllactosamine, lacto-N-neotetraose, and probably lacto-N-neotetraosylceramide in addition to the formation of the Gal β1→4GlcNAc group of glycoprotein sugar chains and lactose.抄録全体を表示PDF形式でダウンロード (1835K) -
Shin-ichi Ugai, Tomohiro Tamura, Nobuyuki Tanahashi, Shigeharu Takai, ...1993 年 113 巻 6 号 p. 754-768
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーAn ATP/ubiquitin-dependent proteasome complex with an apparent sedimentation coefficient of 26S was purified from rat liver to near homogeneity by an improved method based on procedures reported previously. Two electrophoretically distinct forms of the 26S complex, named 26Sα and 26Sα, with very similar subunit compositions were found not only in purified preparations but also in crude extracts, indicating that the 26S proteasome is present as two isoforms. The 26S proteasome was shown to degrade multi-ubiquitinated, but not unmodified, lysozymes in an ATP-dependent fashion, to have ATPase activity supplying energy for proteolysis, and to contain isopeptidase activity to generate free ubiquitin Mg2+/ATP-dependently. The 26S proteasome also catalyzed the ATP-independent hydrolyses of three types of fluorogenic peptides with basic, neutral, and acidic amino acids at their cleavage sites, respectively. These peptides are also good substrates for the 20S proteasome, but their degradation by the free 20S proteasome and by its assembled form in the 26S complex differ markedly, suggesting a functional difference between the two forms of proteasomes. Electrophoretic and immunochemical analyses showed that the large 26S complex was composed grossly of two different structures: a core 20S proteasome with multicatalytic proteinase functions and an associated part possibly with a regulatory role. These two structures both consisted of multiple polypeptides with molecular masses of 21-31 and 35-110 kDa, respectively. The subunit multiplicity of the rat 26S proteasome closely resembled that of the human counterpart, showing only minor species-specific differences in certain components. The assembly of this multi-component complex was found not to involve a sulfhydryl bond. Electrophoretic peptide mapping with lysyl-endopeptidase indicated the non-identity of the multiple subunits of the 26S proteasome. From these structural and functional characteristics, the 26S proteasome, which is widely distributed in mammals, is suggested to be a new type of multi-molecular complex catalyzing the soluble energy- and ubiquitin-dependent proteolytic pathway.抄録全体を表示PDF形式でダウンロード (6019K) -
Takayuki Nemoto, Yuko Ohara-Nemoto, Nobuko Sato, Minoru Ota1993 年 113 巻 6 号 p. 769-775
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーAssociation of the 90-kDa heat shock protein (HSP90) is required for the high-affinity ligand-binding of the glucocorticoid receptor (GR), but not for that of the androgen receptor [Ohara-Nemoto, Y., Nemoto, T., & Ota, M. (1991) J. Biochem. 109, 113-119]. In the present study, we investigated the ligand- and HSP90-binding characteristics of the mineralocorticoid receptor (MR), which shares to some degree the ligand-binding specificity of the GR. A truncated human MR (designated MR351) starting from Gly-351 was translated in vitro with rabbit reticulocyte lysate. Scatchard analysis revealed the presence of a single class of high-affinity binding sites for [3H] aldosterone (Kd=0.35±0.2nM), comparable to those of the native receptor in target tissues. Glycerol gradient centrifugation and immunoadsorption analyses showed that MR351 associated with rabbit HSP90. Exposure to 0.4M NaCl induced the dissociation of HSP90 from MR351 and simultaneously enhanced binding of MR351 to DNA-cellulose. Moreover, when measured at 10 nM, HSP90-free MR351 showed only 9% of the [3H] aldosterone-binding found in the presence of HSP90. On the other hand, when MR351 was expressed in Escherichia coli as a protein tagged with a histidine hexamer at the N-terminus (designated H6MR351), specific binding of [3H] aldosterone was detected. The binding affinity (Kd=338±45 nM) was, however, 1, 000-fold lower than that of MR351 translated in vitro. Gel retardation analysis revealed that H6MR351 specifically interacted with the glucocorticoid response element of the rat tyrosine aminotransferase gene, irrespective of aldosterone-binding. These results indicate that HSP90 has dual roles in the function of the MR, as in that of the GR, i.e., suppression of DNA-binding and maintenance of the high-affinity, steroid-binding state.抄録全体を表示PDF形式でダウンロード (4432K) -
Tomoko Ohama, Norio Matsuki, Chao-Hsiung Wang, Hiroshi Saito, Makoto K ...1993 年 113 巻 6 号 p. 786-789
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーWe have previously reported that fatty liver was induced in a novel experimental animal, Suncus murinus (suncus), by 24-h fasting and that apolipoprotein B (apo B) was not actively synthesized in the liver. However, a faintsignal of apo B mRNA was detected in the liver, suggesting possible synthesis of apo B. Small amounts of VLDL and LDL have been separated from suncus serum by ultracentrifugation. Electron microscopic study of the lipoproteins revealed the existence of small particles in VLDL. High performance liquid chromatographic analysis of the lipoproteins showed that the peaks of TG and cholesterol were mainly at the HDL fraction. These results indicate the existence of lipoproteins as small as HDL which were rich in TG and floated at the density of VLDL upon ultracentrifugation. Apolipoprotein analysis showed two bands of 500- and 200-kDa proteins in VLDL and LDL. Western blot analysis using antibody against the 500-kDa protein revealed reaction not only with suncus 500- and 200-kDa proteins but also with human apo B-100. In conclusion, a small amount of apo B is transported in the suncus serum as VLDL and LDL, although almost all lipid is packed in HDL-size particles.抄録全体を表示PDF形式でダウンロード (1540K) -
Akio Kanatani, Tadashi Yoshimoto, Ana Kitazono, Toshio Kokubo, Daisuke ...1993 年 113 巻 6 号 p. 790-796
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーA strain of Aeromonas hydrophila was found to show prolyl endopeptidase activity. The enzyme gene was cloned and expressed in Escherichia coli JM83. A 12 kbp EcoRI fragment containing the enzyme gene was subcloned at the HincII site of pUC19 to construct plasmid pAPEP-3 with a 3.5 kbp insert. E. coli JM83 transformed with this plasmid showed about 100-fold higher activity than the parent Aeromonas. Analysis of the nucleotide sequence of the insert revealed that the mature enzyme-encoding sequence starts just after the ATG initiation codon of the open reading frame. The enzyme was a single polypeptide composed of 689 amino acid residues with a molecular weight of 76, 383. It showed properties very similar to those of Flavobacterium prolyl endopeptidase, except that the isoelectric point was 5.5. The amino acid sequence was 56 and 41% homologous to those of Flavobacterium and porcine brain prolyl endopeptidases, respectively. From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-537, His-656, and Asp-512 (or Asp-621).抄録全体を表示PDF形式でダウンロード (886K) -
Ryuichi Tatsumi, Akihito Hattori, Koui Takahashi1993 年 113 巻 6 号 p. 797-804
発行日: 1993年
公開日: 2008/11/18
ジャーナル フリーThe locations of five kinds of nebulin subfragments in chicken myofibrils were studied by indirect immunofluorescence microscopy and immunoelectron microscopy. The subfragments were produced by treatment of the myofibrils with a solution containing 0.1mM CaCl2. Antinebulin-subfragment antibodies displayed five stripes from the Z-disk to the distal end of thin filaments in each half sarcomere. Anti-40-kDa subfragment antibodies provided a wide stripe near the Z-disk. Anti-33-kDa subfragment antibodies displayed three stripes in the I-band. Anti-23-kDa subfragment antibodies displayed three stripes, whose positions could not be distinguished from those of three stripes provided by anti-33-kDa subfragment antibodies. Anti-180-kDa subfragment antibodies provided fluorescence at the A-I junction region. Anti-200-kDa subfragment antibodies displayed a single stripe atthe distal end region of thin filaments. The location of these stripes corresponded well to that of the mother protein, nebulin. On the basis of these results, we propose a model for the substructure of chicken nebulin filaments. All the nebulin subfragments possessed the property of binding to F-actin, indicating that nebulin filaments bind to thin filaments along their entire length in situ. There is a possibility that nebulin filaments are anchored at the Z-disk through interaction with some other Z-disk constituents than α-actinin, because the binding site for α-actinin exists on the distal end region of nebulin filaments.抄録全体を表示PDF形式でダウンロード (8185K)
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