The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
112 巻, 4 号
選択された号の論文の25件中1~25を表示しています
  • Takeshi Endo, Susumu Goto
    1992 年 112 巻 4 号 p. 427-430
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    The cell growth suppression activity of the retinoblastoma gene (RB) product Rb is considered to be regulated by phosphorylation, whereas no positive correlation has been demonstrated between the activity and the amount of Rb protein in numbers of cell types examined. Both the RB mRNA and the protein were dramatically induced during terminal differentiation of the mouse skeletal muscle cell line C2 and its transfectant C2SVTts11, which stably harbors the SV40 T antigen gene linked to an inducible promoter. They were gradually deinduced when the terminally differentiated C2SVTts11 myotubes reentered the cell cycle through the induction of the large T antigen. Thus, the accumulation of Rb protein is likely to be required for growth arrest during differentiation of at least these myogenic cells, which have low basal levels of the protein.
  • Michitoshi Watanabe, Yasushi Hasegawa, Tsuyoshi Katoh, Fumi Morita
    1992 年 112 巻 4 号 p. 431-432
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    The amino acid sequence of the 20-kDa regulatory light chain (LC20) of myosin from porcine aorta media smooth muscle was determined. The LC20 consisted of 171 amino acid residues and its N-terminal Ser residue was blocked by an acetyl group. The amino acid sequence was identical with that of chicken gizzard myosin LC20 except that the 60th residue, Met in chicken gizzard LC20, was substituted for Leu in porcine aorta LC20.
  • Mikiharu Yoshida, Atsushi Suzuki, Teruo Shimizu, Eijiro Ozawa
    1992 年 112 巻 4 号 p. 433-439
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Dystrophin was isolated from the purified large oligomeric dystrophin complex with its associated proteins (DC) of rabbit skeletal muscle by alkaline dissociation followed by gel filtration to remove the associated proteins. Isolated dystrophin and DC were subjected to digestion with calpain or α-chymotrypsin, and the generated polypeptide fragments were studied by immunoblot analysis using seven kinds of antibodies raised against antigens corresponding to various regions from the N- to the C-terminal of human dystrophin. For some fragments, the amino acid sequences at the N-termini were determined. Two proteinases, which bear distinct specificities, generated very similar fragments from purified dystrophin with or without the associated proteins. The cleavage sites found by mapping the fragments onto the dystrophin molecule were similar to those found in a previous study using crude mouse muscle cell membrane fraction [Koenig, M. & Kunkel, L. M. (1990) J. Biol. Chem. 265, 4560-4566]. On the basis of these results, we concluded that dystrophin has several unique proteinase-sensitive sites.
  • Shuhei Yamada, Keiichi Yoshida, Makiko Sugiura, Kazuyuki Sugahara
    1992 年 112 巻 4 号 p. 440-447
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    The 1H-NMR spectra of eight unsaturated disaccharides obtained by bacterial eliminase digestion of chondroitin sulfate and of heparan sulfate/heparin were recorded in order to construct an NMR data base of sulfated oligosaccharides and to investigate the effects of sulfation on the proton chemical shifts. These shifts were assigned by two-dimensional HOHAHA (homonuclear Hartmann-Hahn) and COSY (correlation spectroscopy) methods. The results indicated the following. (1) Two sets of proton signals were observed, corresponding to the α and β anomers of these disaccharides, except those containing N-sulfated G1cN (2-deoxy-2-amino-D-glucose), in which only one set of signals appeared, corresponding to the α anomer. (2) Signals of protons bound to an O-sulfated carbon atom and those bound to the immediately neighboring carbon atoms were shifted downfield by 0.4-0.7 and 0.07-0.3ppm, respectively. (3) For the disaccharides containing the N-sulfated GlcN, the signals of the protons bound to C-2 and C-3 were shifted upfield by 0.6 and 0.15ppm, respectively, but that of C-1 was shifted downfield by 0.25ppm when compared with those of the corresponding N-acetylated disaccharides. (4) For the chondroitin sulfate disaccharides sulfated on the C-4 position of GalNAc (2-deoxy-2-N-acetylamino-D-galactose) or the C-2 position of ΔGlcA (D-glueo-4-ene-pyranosyluronic acid), the signal of the H-3 proton of ΔGlcA or the H-4 proton of GalNAc was shifted upfield by 0.1-0.15ppm, indicating the steric interaction of the two sugar components. (5) These effects of sulfation on chemical shifts are additive.
  • Yumiko Saito, Satoshi Tsubuki, Hisashi Ito, Shinobu Ohmi-Imajo, Seiich ...
    1992 年 112 巻 4 号 p. 448-455
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Our previous reports showed that benzyloxycarbonyl (Z)-Leu-Leu-Leu-al (ZLLLal) induces neurite outgrowth in PC12 cells, and that 33-, 35-, and 180-kDa proteins from PC12 cells elute specifically from a Leu-Leu-Leu-al (LLLa1)-coupled affinity column. Several lines of evidence suggest that the 33-, 35-, and 180-kDa proteins are components of clathrin, well-known for its role in endocytosis. Separation of clathrin into its heavy and light chains showed that the clathrin heavy chains have the ability to bind to a LLLa1 affinity column directly. Furthermore, ZLLLal enhances the rate of polymerization of clathrin triskelion to the coat structure. ZLLL-COOH does not cause neurite outgrowth in PC12 cells, and has no effect on the rate of clathrin polymerization. On immunocytochemical analysis of PC12 cells with an anti-clathrin heavy chain antibody, enhanced staining of the clathrin heavy chain was observed concomitantly with neurite outgrowth initiated by ZLLLal, but not by NGF. This study provides new insights into both the role of the clathrin molecule and the regulatory mechanism of neurite outgrowth.
  • Dong-Hyun Kim, Hyung-Soo Kim, Kyoichi Kobashi
    1992 年 112 巻 4 号 p. 456-460
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    A novel type of sulfotransferase was purified from Klebsiella K-36, an intestinal bacterium of rat. The enzyme (Mr 160, 000) is composed of two subunits (Mr 73, 000) with pI and optimal pH values of 5.3 and 10-10.5, respectively. The apparent Km for PNS (p-nitrophenyl sulfate) using phenol as an acceptor and that for phenol using PNS as a donor substrate were determined to be 0.11 and 0.66mM, respectively. The enzyme is activated by magnesium ion and inhibited by EDTA.
  • Shigeko Araki, Sachiko Abe, Shoji Yamada, Mei Satake, Naoshi Fujiwara, ...
    1992 年 112 巻 4 号 p. 461-469
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Two novel acidic glycosphingolipids containing pyruvylated galactose were purified from the nervous tissue of Aplysia kurodai by successive latrobeads column chromatographies. By component analysis, sugar analysis, permethylation studies, fast atom bombardmentmass spectrometry, and proton magnetic resonance spectrometry, the structures of these acidic glycosphingolipids, named F-9 and FGL-I, were determined to be: [3, 4-O-(S-1-carboxyethylidene)] Galβ1→3GalNAcαl→3 [6'-O-(2-aminoethylphosphonyl) Galα1→2] (2-aminoethylphosphoryl→6) Galβ1→4Glcβ1→1ceramide and [3, 4-O-(S-1-carboxyethylidene)] Galβ1→3GalNAcα1→3 (Fucα1→2) (2-aminoethylphosphoryl→6) Galβ1→4Glcβ1→lceramide, respectively. Their major aliphatic components are palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, pyruvylated glycosphingolipids containing phosphoethanolamine in addition to or in place of 2-aminoethyiphosphonate are present in the nervous system of Aplysia.
  • Hiroyuki Arata, Minoru Shimizu, Ken-ichiro Takamiya
    1992 年 112 巻 4 号 p. 470-475
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Trimethylamine N-oxide (TMAO) reductase was purified from an aerobic photosynthetic bacterium Roseobacter denitrificans. The enzyme was purified from cell-free extract by ammonium sulfate fractionation, DEAE ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme was composed of two identical subunits with molecular weight of 90, 000, as identified by SDS-polyacrylamide gel electrophoresis, containing heme c and a molybdenum cofactor. The molecular weight of the native enzyme determined by gel filtration was 172, 000. The midpoint redox potential of heme c was +200mV at pH 7.5. Absorption maxima appeared at 418, 524, and 554nm in the reduced state and 410nm in the oxidized state. The enzyme reduced TMAO, nicotine acid N-oxide, picoline N-oxide, hydroxylamine, and bromate, but not dimethyl sulfoxide, methionine sulfoxide, chlorate, nitrate, or thiosulfate. Cytochrome c2 served as a direct electron donor. It probably catalyzes the electron transfer from cytochrome b-c1 complex to TMAO reductase. Cytochrome c552, another soluble low-molecular-weight cytochrome of this bacterium, also donated electrons directly to TMAO reductase.
  • Yoshiki Miura, Fuminori Tokunaga, Toshiyuki Miyata, Matsuko Moriyasu, ...
    1992 年 112 巻 4 号 p. 476-481
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor _??_ activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and α-chymotrypsin-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not α-chymotrypsin-mediated activation of factor C or factor _??_ activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through intermolecular interaction between the LPS-bound factor C molecules. The dissociation constants (Kd) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9×10-9, 0.6×10-10, and 1.8×10-10M, respectively. By using 2C12 and polyclonal antibody against factor _??_, an enzyme-linked immunosorbent assay for quantitative determination of factor C was established.
  • Hidenori Arai, Yutaka Nagano, Shuh Narumiya, Toru Kita
    1992 年 112 巻 4 号 p. 482-487
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Oxidized low density lipoproteins (LDL) are now considered to be one of the atherogenic lipoproteins in vivo and to play an important role in the pathogenesis of atherosclerosis. We previously demonstrated in mouse peritoneal macrophages that oxidized LDL stimulated prostaglandin (PG) E2 synthesis when incorporated into the cells [Yokode, M. et al. (1988) J. Clin. Invest. 81, 720-729]. In this study, we investigated arachidonate metabolism in macrophages after foam cell transformation. The cells were incubated with 100μg/ml of oxidized LDL for 18h, then stimulated with zymosan. Lipid-enriched macrophages which had taken up oxidized LDL produced much less eicosanoids, such as PGE2, 6-keto-PGF, and leukotriene C4 than control cells. After labeling of the cells with [14C] arachidonic acid, they were stimulated with zymosan and the phospholipase activity was determined. The activity of lipid-enriched cells was about two-thirds of that of control cells. Then we investigated the fatty acid composition of their phospholipid fraction to clarify arachidonic acid content and mobilization. Percent of arachidonic acid of lipid-enriched cells decreased and less arachidonic acid mobilization was observed after stimulation with zymosan. These data suggest that impaired arachidonate metabolism in lipid-enriched macrophages can be explained by their decreased phospholipase activity and changes in their fatty acid composition.
  • Hiro-omi Tamura, Kaori Tameishi, Hideo Yamagata, Shigezo Udaka, Tomoko ...
    1992 年 112 巻 4 号 p. 488-491
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Sphingomyelinase (sphingomyelin cholinephosphohydrolase) [EC 3. 1. 4. 12] of Bacillus cereus was overproduced in a protein-hyperproducing strain, B. brevis 47, by cloning the gene into an expression vector pNU211, which has been developed to express a foreign gene utilizing a promoter and a signal sequence of an outer cell wall protein gene. From 1 liter of culture, about 10mg of protein was purified to near-homogeneity by two steps of column chromatography; this is almost 500 times higher production compared to the conventional preparation from the original strain, B. cereus IAM 1208. The N-terminal amino acid sequence of the secreted enzyme was identical to that of the authentic enzyme, indicating that the signal sequence for secretion of B. cereus was processed properly in B. brevis 47.
  • Mikio Kato, Nobuyoshi Shimizu
    1992 年 112 巻 4 号 p. 492-494
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    The effect of a pyrimidine/purine-biased stretch which has the potential to form an unusual triplex DNA structure on gene expression has been analyzed by measuring the activity of β-lactamase as a reporter gene in recombinant plasmids. The Escherichia coli transformant carrying the plasmid p7ERS which has a potential triplex DNA region expressed about twofold more β-lactamase activity than that carrying the plasmid pUC19. Since the expression of β-lactamase has been shown to be affected by template topology in vitro, this in vivo observation suggests that the inserted pyrimidine/purine-biased stretch modulates the topology of flanking regions by forming unusual DNA structure to keep the template at the superhelicity favorable for the expression of β-lactamase.
  • Hitoshi Aoshima, Yuichi Inoue, Kenzi Hori
    1992 年 112 巻 4 号 p. 495-502
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Since binding of an agonist to an ionotropic neurotransmitter receptor causes not only channel opening, but also desensitization of the receptor, inhibition of the receptor by the antagonist sometimes becomes very complicated. The transient state kinetics of ligand association and dissociation, and desensitization of the receptor were considered on the basis of the minimal model proposed by Hess' group, and the following possibilities were proposed. 1) When an agonist is simultaneously applied to the receptor with an antagonist whose affinity to the receptor is extremely strong and different from that of the agonist, it is usually impossible to estimate the real inhibition constant exactly from the responses because desensitization of the receptor proceeds before the equilibrium of the ligand binding. Simultaneous addition of the antagonist with strong affinity to the receptor may apparently accelerate inactivation (desensitization) of the receptor. The association rate constant of the antagonist can be estimated by analyses of the rate of the inactivation in the presence and the absence of the antagonist. 2) A preincubated antagonist with a slow dissociation rate constant, i.e., a very effective inhibitor, may cause apparent noncompetitive inhibition of the receptor, since the receptor is desensitized by an agonist as soon as the antagonist dissociates from the receptor and the dissociation of the antagonist from the receptor becomes the rate-determining step. A nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus oocytes by injecting mRNA prepared from Electrophorus electricus electroplax and used for the experiments on inhibition by an antagonist. When an antagonist such as gallamine (Gal), (+)-tubocurarine (Tub), or pancuronium (Pan) was applied simultaneously with acetylcholine (ACh), it inhibited the nAChR competitively and apparently accelerated the rate of inactivation of nAChR. On the other hand, it inhibited the receptor in an apparently noncompetitive manner, and very effectively, when it was preapplied to the receptor. These results were analyzed on the basis of our model. Both the association and the dissociation rate constant of the antagonists were estimated from the inhibition experiments on the basis of the model. Moreover, our results suggested the presence of not only competitive binding sites with a high affinity in the nAChR but also a noncompetitive inhibitory site with a low affinity toward the antagonists. Strychnine (Str) also inhibited glycine receptor (GlyR) competitively when applied simultaneously with glycine (Gly), but inhibited GlyR in a noncompetitive manner when it was preapplied.
  • Yuziro Namba, Masaya Ito, Youli Zu, Katsuya Shigesada, Koscak Maruyama
    1992 年 112 巻 4 号 p. 503-507
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    The amino acid sequences deduced from cDNA analyses revealed that human leucocyte L-plastin phosphorylated in response to interleukin 1, 2 closely resembles a chicken intestinal microvilli protein, fimbrin, that bundles actin filaments [de Arruda et al. (1990) J. Cell Biol. 111, 1069-1079]. In the present work, it was observed that unphosphorylated L-plastin isolated from human T cells bundled F-actin just as fimbrin does. L-Plastin acted on T cell β-actin, but hardly acted on muscle α-actin or chicken gizzard γ-actin, whereas fimbrin bundled muscle α-actin. Unlike fimbrin, L-plastin's actin-bundling action was strictly calcium-dependent: the bundles were formed at pCa 7, but not at pCa 6. Under suitable conditions, approximately one molecule of L-plastin bound to 8 molecules of actin monomer in the actin filament.
  • Toshiaki Imagawa, Junichi Nakai, Hiroshi Takeshima, Yasuaki Nakasaki, ...
    1992 年 112 巻 4 号 p. 508-513
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    We constructed an expression plasmid (pMAMCRR51) that carried the entire proteincoding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and Escherichia coli xanthineguanine phosphoribosyltransferase (gpt). Chinese hamster ovary (CHO) cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca2+ transients were selected. Immunoprecipitation with a monoclonal antibody against the canine cardiac ryanodine receptor revealed that the cell clones thus selected exhibited Ca2+-dependent [3H]ryanodine binding activity, which was stimulated by 5mM ATP or 1M KCl. The apparent dissociation constant (Kd) for [3H] ryanodine was 6.6nM in 1M KCl, which was similar to the Kd obtained with cardiac microsomes. Immunoprecipitation also demonstrated that these cell clones expressed a protein indistinguishable in Mr from the ryanodine receptor in canine cardiac microsomes. The ryanodine binding activity expressed in CHO cells increased significantly after dexamethasone induction. In saponin-skinned CHO cells transfected with pMAMCRR51, micromolar Ca2+ or millimolar caffeine evoked rapid Ca2+ release from the intracellular Ca2+ stores. In skinned control CHO cells, we did not observe such Ca2+ release activity. These results clearly demonstrate that the cardiac ryanodine receptor is stably expressed in internal membranes of CHO cells and functions as Ca2+-induced Cal2+ release channels.
  • Takashi Murayama, Yasuo Ogawa
    1992 年 112 巻 4 号 p. 514-522
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    The two ryanodine-binding proteins (RyBPs) have been purified from sarcoplasmic reticulum of bullfrog skeletal muscle by Mono Q column chromatography following solubilization of SR by CHAPS and sucrose density gradient centrifugation. We conclude that the two RyBPs (α- and β-RyBP) are isoforms on the basis (i) that each RyBP is distinguished by a specific polyclonal antibody and (ii) that distinct polypeptides are generated by limited tryptic digestion of the two RyBPs. Monomeric molecular weights for α- and β-RyBP are estimated to be (690±10) and (570±10) kDa, respectively, as determined from mobilities on disc SDS-PAGE using the Weber-Osborn buffer system without 6M urea, which gives an estimate of (590±10) kDa for RyBP of rabbit skeletal muscle. Similar determination in the presence of 6M urea gave 630 kDa for α-RyBP and unchanged estimates for the other RyBPs. Both RyBPs show [3H] ryanodine-binding activities which are activated by Ca2+, AMPOPCP, and caffeine, and inhibited by ruthenium red, MgCl2, and procaine. β-RyBP, however, has higher affinity for Ca2+. In the presence of Ca2+ and AMPOPCP, both RyBPs show single homogeneous binding sites for [3H] ryanodine with Kd=2-5nM. The values of Bmax for α- and β-RyBP were 320-340 and 320-375pmol/mg protein, respectively. These results are consistent with the conclusion that a homo-tetramer of each RyBP binds one ryanodine molecule, taking account of the estimated molecular weight. Corresponding to ryanodine binding, α -and β-RyBP show Ca2+-dependent channel currents which are activated by ATP and inhibited by ruthenium red on planar lipid bilayers. These results demonstrate that the two RyBP isoforms, which occur in approximately equal amounts in bullfrog skeletal muscle, constitute Ca2+-induced Ca2+ release channels.
  • Tadashi Mizoguchi, Hirofumi Nanjo, Toshifumi Umemura, Tohru Nishinaka, ...
    1992 年 112 巻 4 号 p. 523-529
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Three enzymes (DD1, DD2, and DD3) having dihydrodiol dehydrogenase activity were purified to homogeneity from bovine liver cytosol. DD1 and DD2 were identified as 3α-hydroxysteroid dehydrogenase and high-Km aldehyde reductase, respectively, as judged from their molecular weights, substrate specificities and inhibitor sensitivities. DD3 was a unique enzyme which could specifically catalyze the dehydrogenation of trans-benzenedihydrodiol and trans-naphthalenedihydrodiol without any activity toward the other tested alcohols, aldehydes, ketones, and quinones. The Km value of DD3 (0.18mM) for benzenedihydrodiol was lower than those of other dihydrodiol dehydrogenases so far reported. DD3 immunologically crossreacted with DD1, but showed no crossreactivity with DD2. Additionally, DD3 was inhibited in a competitive manner, with a low K1 value of 1μM, by androsterone, which was a good substrate for DDl. It was assumed that DD3 is a novel enzyme which is specific to dihydrodiols, exhibiting similarity to DD1 in immunological and structural properties.
  • Tomohiro Tamura, Naoki Shimbara, Masashi Aki, Naruhiro Ishida, Faycal ...
    1992 年 112 巻 4 号 p. 530-534
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Proteasomes (multi-protease complexes) are composed of approximately 15 non-identical subunits of similar sizes (molecular weight=21-32 kDa), but different charges (isoelectric point=4-9). Previously, we deduced the primary structures of 6 subunits of rat proteasomes by recombinant DNA techniques. In this paper we report the nucleotide sequences of 4 other subunits, rIOTA, rZETA, rDELTA, and rRING12, determined from cDNA clones isolated by screening a rat H4TG hepatoma cell cDNA library with the cDNAs of their human counterparts as probes. The polypeptides deduced from their nucleotide sequences consisted of 246, 241, 202, and 219 amino acid residues with calculated molecular weights of 27, 399, 26, 391, 21, 649, and 23, 324, and calculated isoelectric points of 6.37, 4.65, 4.84, and 4.70, respectively. These results and previous findings indicate that the primary structures of the subunits of rat proteasomes show considerably high inter-subunit homology, but can be classified into apparently distinct sub-groups, suggesting that rat proteasome genes form a multi-gene family with the same evolutionary origin, but have diverged during evolution to acquire possibly subunit-specific functions.
  • Koichi Inano, Takehiko Ishida, Saburo Omata, Tsuneyoshi Horigome
    1992 年 112 巻 4 号 p. 535-540
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    We previously showed that the 9S estrogen receptor can be reconstituted from purified vero ER (estradiol binding subunit) and purified hsp 90 (heat shock protein 90) in vitro [Inano, K. et al. (1990) FEBS Lett. 267, 157-159]. In this study, we further characterized our reconstitution system to investigate the mechanism underlying the formation of 9S ER. When a vero ER preparation stored at 4°C for more than 20h after affinity chromatography was used for the reconstitution of 9S ER, 0.5M NaSCN was essential, but not Na2MoO4 or other reagents. When, however, vero ER was used within 3h after dissociation from an affinity resin, 9S ER could be reconstituted in a relatively high yield without NaSCN. Moreover, if such a fresh vero ER preparation was used, 9S ER could be reconstituted in the absence of NaSCN from not only unoccupied vero ER but also the occupied form. From these results it was suggested that the conformation of purified vero ER tends to change quickly in a time dependent manner, and so a chemical perturbant, NaSCN, is generally necessary for the reconstitution of 9S ER from purified vero ER and purified hsp 90. The concentration of hsp 90 required for the reconstitution was only about 1.0μM, which was lower than its physiological concentration. Based on these results, the mechanism underlying the forma tion of 9S ER was discussed.
  • Bunzo Mikami, Mamoru Sato, Takumi Shibata, Masaaki Hirose, Shigeo Aiba ...
    1992 年 112 巻 4 号 p. 541-546
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    The three-dimensional structure of a complex of soybean β-amylase [EC 3. 2. 1. 2] with an inhibitor, α-cyclodextrin, has been determined at 3.0 Å resolution by X-ray diffraction analysis. Preliminary chain tracing showed that the enzyme folded into large and small domains. The large domain has a (βα)8 super-secondary structure, while the smaller one is formed from two long loops extending from the β3 and β4 strands of the (βα)8 structure. The interface of the two domains together with shorter loops from the (βα)8 structure form a deep cleft, in which α-cyclodextrin binds slightly away from the center. Two maltose molecules also bind in the cleft. One shares a binding site with α-cyclodextrin and the other is situated more deeply in the cleft.
  • Kimikazu Hashino, Tomoko Shimojo, Fusao Kimizuka, Ikunoshin Kato, Tosh ...
    1992 年 112 巻 4 号 p. 547-551
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    A new artificial cell adhesive protein was engineered by grafting the Arg-Gly-Asp (RGD) sequence, the minimal recognition signal of fibronectin for interaction with integrins, to a calpastatin segment by in vitro mutagenesis. The mutagenized protein showed cell adhesive activity in addition to calpain inhibitory activity. The RGD signal grafted to the calpastatin segment was recognized by the vitronectin receptor but not by the fibronectin receptor.
  • Satoshi Shibata, Hirokazu Sato, Masahiro Maki
    1992 年 112 巻 4 号 p. 552-556
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Calphobindins (CPBs, placental annexins) are intracellular Ca2+- and phospholipid-dependent proteins like protein kinase C [EC 2. 7. 1. 37]. We investigated the inhibitory effects of calphobindins on the protein kinase C activity in vitro. CPB I inhibited the protein kinase C activity for both histone phosphorylation and lipocortin phosphorylation, but CPB II and CPB III inhibited only the protein kinase C activity for histone phosphorylation. In the case of histone phosphorylation, all CPBs inhibited the protein kinase C activity in a concentration-dependent manner, and the IC50 (concentration required for 50% inhibition) value of CPB I was 70nM. The inhibition of protein kinase C by CPB I was Ca2+-dependent, and did not disappear upon increasing the concentration of phosphatidylserine. Kinetic analysis by double-reciprocal plots indicated that CPB I interacted not only with phosphatidylserine but also with protein kinase C. Although CPB I partially interacts with phospholipid, it is conceivable that the inhibitory action of CPB I on protein kinase C results from direct interaction of CPB I with protein kinase C. Since CPBs are mainly present under the plasma membrane, it is presumed that CPB I is an endogenous inhibitor of protein kinase C, and according to intracellular circumstances, CPB II and CPB III may also be endogenous inhibitors.
  • Nam Ho Choi-Miura, Yoshiyuki Takahashi, Yasuko Nakano, Takashi Tobe, M ...
    1992 年 112 巻 4 号 p. 557-561
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    SP-40, 40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40, 40 were determined. SP-40, 40 was purified from human seminal plasma by affinity chromatography using an anti-SP-40, 40 monoclonal antibody and reversed-phase, high-performance liquid chromatography (HPLC). The protein was digested with trypsin and the fragments were separated by reversed-phase HPLC. The peptides containing disulfide bonds were fluorophotometrically detected with 4-(aminosulfonyl)-7-fluoro-2, 1, 3-benzoxadiazole (ABD-F). The peptides containing more than two disulfide bonds were further digested with Staphylococcus aureus V8 protease and lysylendopeptidase, and the fragments were isolated by HPLC. The amino acid compositions and the amino acid sequences of the peptides containing only a disulfide bond were determined. Disulfide bonds thus determined were between Cys58(α)-CyslO7(β), Cys68(α)-Cys99(β), Cys75(α)-Cys94(β), and Cys86(α)-Cys8O(β). Since there was no free sulfhydryl groups in the SP-40, 40 molecule, Cys78(α) and Cys91(β) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in SP-40, 40 are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of SP-40, 40.
  • Francisco J. G. Muriana, Carmen M. Vazquez, Valentina Ruiz-Gutierrez
    1992 年 112 巻 4 号 p. 562-567
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Male rats were fed diets containing olive (00) or evening primrose (EPO) oil (10%w/w), with or without added cholesterol (1%w/w). After 6-week feeding, the lipid and fatty acid compositions, fluidity, and fatty acid desaturating and cholesterol biosynthesis/esterification related enzymes of liver microsomes were determined. Both the OO and EPO diets, without added cholesterol, increased the contents of oleic and arachidonic acids, respectively, of rat liver microsomes. The results were consistent with the increases in Δ9 and Δ6 desaturation of n-6 essential fatty acids and the lower microviscosity in the EPO group. Dietary cholesterol led to an increase in the cholesterol content of liver microsomes as well as that of phosphatidylcholine (PC). The cholesterol/phospholipid and PC/PE (phosphatidylethanolamine) ratios were also elevated. Fatty acid composition changes were expressed as the accumulation of monounsaturated fatty acids, with accompanying milder depletion of saturated fatty acids in rat liver microsomes. In addition, the arachidonic acid content was lowered, with a concomitant increase in linoleic acid, which led to a significant decrease in the 20:4/18:2 ratio in comparison to in animals fed the cholesterol-free diets. Cholesterol feeding also increased Δ9 desaturase activity as well as membrane microviscosity, whereas it decreased Δ6 and Δ5 desaturase activities. There was a very strong correlation between fluidity and the unsaturation index reduction in the membrane. Furthermore, the activity of hydroxymethylglutaryl-CoA reductase increased and the activity of acyl-CoA: cholesterol acyltransferase decreased in liver microsomes from both cholesterol-fed groups. This seems to indicate that the responses of these enzyme activities to the different diets take place in order to maintain the homeostasis of membranes.
  • Sugie Higashi-Fujime, Masami Suzuki, Koiti Titani, Tetsu Hozumi
    1992 年 112 巻 4 号 p. 568-572
    発行日: 1992年
    公開日: 2008/11/18
    ジャーナル フリー
    Skeletal muscle actin was lightly digested by proteinase K, which cleaved the peptide bond between Met-47 and Gly-48, producing a C-terminal 35 kDa fragment. Proteinase K-cleaved actin (proK-actin) did not polymerize into F-actin upon addition of salt. In the presence of phalloidin, however, it polymerized slowly into F-actin (proK-F-actin), indicating that the cleaved actin did not dissociate into the individual cleaved fragments but retained the global structure of actin. Electron microscopy showed that proK-F-actin had the typical double-stranded structure of a normal actin filament and formed the arrowhead structure when decorated with HMM. Heavy meromyosin ATPase was weakly activated by proK-F-actin: Vmax=0.24s-1, and Kapp=2.8μM, while Vmax=7.6s-1, and Kapp=13μM by F-actin. Correspondingly, in vitro this proK-F-actin slid very slowly on HMM attached to a glass surface at an average velocity of 0.47μm/s, or 1/12 of that of intact F-actin. The fraction of sliding filaments was less than 50%. Assuming that the nonmotile filaments attached to HMM were not involved in ATPase activation, the sliding velocity correlated with the ATPase activity activated by proK-F-actin.
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