The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 106 , Issue 2
Showing 1-29 articles out of 29 articles from the selected issue
  • Wolfram Saenger, Flogel Rainer, Piotr Zielenkiewicz, Masatoshi Inoue, ...
    1989 Volume 106 Issue 2 Pages 189-191
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    In the cocrystal formed by 7-methylguanosine-5'-phosphate-phenylalanine-6H2O, the interactions between guanine and phenylalanine are similar to those observed in the complex of ribonuclease T1 with 2'-guanylic acids, and those of the two G-proteins, Elongation Factor-Tu and ras oncogene p21, with GDP. They are similar in the following three points:(a) guanine N (1) H and N (2) H donate cyclic N-H…O hydrogen bonds to the carboxylate group of phenylalanine in the former cocrystal and to the side chain carboxylate group of Asp or Glu in the latter proteins, (b) O (6) of guanine accepts hydrogen bond (s) from main-chain NH group (s), and (c) the purine moiety is sandwiched between aromatic (or hydrophobic) amino acid side chains.
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  • Yoshihiko Sumi, Yukiya Koike, Yataro Ichikawa, Nobuo Aoki
    1989 Volume 106 Issue 2 Pages 192-193
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    α2-Plasmin inhibitor (α2PI) was purified directly from human plasma by using a monoclonal antibody affinity column, which recognizes the reactive site of α2PI.α2PI was eluted from the column under mild conditions with 50% v/v ethyleneglycol containing 0.05% Tween 80 in phosphate-buffered saline, pH 7.4. The yield was around 50% and the specific activity of the purified protein was the same as that of the best product of preparation conventionally purified α2PI.
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  • Emi Kusunose, Arata Sawamura, Hidenori Kawashima, Ichiro Kubota, Masam ...
    1989 Volume 106 Issue 2 Pages 194-196
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    Two different forms of cytochrome P-450, highly active in the ω-hydroxylation of prostaglandin A, and the ω-and (ω-1)-hydroxylation of fatty acids (P-450ka-1 and P-450ka-2), have been purified from kidney cortex microsomes of rabbits treated with di (2-ethylhexyl)-phthalate. On the basis of the peptide map patterns and NH2-terminal amino acid sequence, P-450ka-1 was determined to be a new form of ω-hydroxylase cytochrome P-450, whereas P-450ka-2 is identical to P-450ka reported earlier. The first 20 NH2-terminal amino acid sequence (ALNPTRLPGSLSGLLQVAGL) and (ALSPTRLPGSFSGFLQAAGL) of P-450ka-1 and P-450, showed 90 and 80% homology with that of the lung prostaglandin whydroxylase, respectively, suggesting that these three cytochromes P-450 are members of the same ω-hydroxylase cytochrome P-450 gene family.
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  • Toshiyuki Fukao, Keiju Kamijo, Takashi Osumi, Yukio Fujiki, Seiji Yama ...
    1989 Volume 106 Issue 2 Pages 197-204
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    cDNA clones for rat mitochondrial acetoacetyl-CoA thiolase were isolated and sequenced. The most 5'-extended clone (RT2-6) consisted of 1, 460 bases and contained a 1, 272-base open reading frame encoding a polypeptide of 424 amino acid residues. A coupled in vitro transcription/translation analysis of RT2-6 revealed that RT2-6 encodes the entire precursor of this enzyme. The amino-terminal sequence and amino acid composition of the purified enzyme agreed with the primary structure deduced from the cDNA. The calculated molecular masses of the precursor and the subunit of the mature enzyme are 44, 694 and 41, 364 Da, respectively. The primary structure of this enzyme was compared with those of four other thiolases (rat mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, acetoacetyl-CoA thiolase of Zoogloea ramigera, and cytosolic acetoacetyl-CoA thiolase of Saccharomyces uvarum). Marked homology between any two of them (34-51% identity) indicates that the genes of thiolases have evolved from a common ancestral gene. It has been reported that this enzyme has two isoenzymes A and B. However, the purified isoenzymes were indistinguishable from each other in some analyses. Though 17 independent cDNA clones were isolated, no definite evidence indicating the presence of different cDNAs was found.
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  • Kyosuke Nagata, Kaoru Takeuchi, Akira Ishihama
    1989 Volume 106 Issue 2 Pages 205-208
    Published: August 01, 1989
    Released: January 25, 2011
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    Using isolated nuclei prepared from influenza virus-infected HeLa cells, factors affecting the synthesis of two species of positive-sense RNA transcripts, i.e., mRNA and cRNA (complementary RNA to vRNA) were analyzed. In the presence of low concentrations of salt, both mRNA and cRNA were synthesized, whereas in the presence of high concentrations of salt, mRNA was synthesized predominantly. Salt-extracts of nuclei (NE) mainly produced cRNA while mRNA was a major product synthesized by salt-treated nuclei (ΔN). In the presence of high concentrations of salt, the NE produced mRNA instead of cRNA. After centrifugation of the NE, the precipitates (NEP) predominantly produced mRNA while the supernatant (NES) alone exhibited a low level of cRNA synthesis activity. With the addition of the NES fraction, mRNA synthesis by the NEP was switched to cRNA synthesis. Glycerol gradient centrifugation of the NES fraction in the presence of high salt yielded vRNA-RNA polymerase complexes that catalyzed mRNA synthesis. These observations indicate that a regulatory factor (s) that can be dissociated from vRNA-RNA polymerase complexes upon exposure to high ionic strength is involved in the switch from mRNA to cRNA synthesis. This activity was not detected in nuclear extracts prepared from uninfected cells, suggesting that such a factor (s) is either encoded by the virus genome or induced by virus infection.
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  • Yoshio Nakano, Chiaki Kato, Eiichi Tanaka, Kinuko Kimura, Koki Horikos ...
    1989 Volume 106 Issue 2 Pages 209-215
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    We have determined the complete nucleotide sequence of a 2.4kb chromosomal EcoT22INspV fragment, containing the Bacillus cereus glnA gene (structural gene of glutamine synthetase). The deduced amino acid sequence indicates that the glutamine synthetase subunit consists of 444 amino acid residues (50, 063 Da). Comparisons are made with reported amino acid sequences of glutamine synthetases from other bacteria. Upstrem of glnA we found an open reading frame of 129 codons (ORF129) preceded by the consensus sequence for a typical promoter. Maxicell experiments showed two polypeptide bands, with molecular weights in good agreement with that of glutamine synthetase and that of ORF129, in addition to vector-coded protein. It is possible that the product of this open reading frame upstream of glnA has a regulatory role in glutamine synthetase expression.
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  • Joseph Vamecq, Jean-Pierre Draye
    1989 Volume 106 Issue 2 Pages 216-222
    Published: August 01, 1989
    Released: January 25, 2011
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    In control rats, long-chain monocarboxylyl-CoA, ω-hydroxymonocarboxylyl-CoA, and dicarboxylyl-CoA esters were substrates for hepatic, renal, and myocardial peroxisomal β-oxidation. The latter enzyme system could not be detected in skeletal muscle. Clofibrate treatment resulted in an enhancement of peroxisomal β-oxidizing capacity in various tissues. Intact mitochondria from control rat liver and kidney cortex incubated in the presence of L-camitine were capable of oxidizing long-chain monocarboxylyl-CoAs and ω-hydroxymonocarboxylyl-CoAs but not dicarboxylyl-CoAs. However, control rat liver mitochondria permeabilized by digitonin oxidized dodecanedioyl-CoA indicating that the liver mitochondrial β-oxidation system can act on dicarboxylyl-CoA esters even if the overall intact mitochondrial system is inactive on these substrates. Intact liver mitochondria ffom clofibrate-treated animals rapidly oxidized lauroyl-CoA and 12-hydroxylauroyl-CoA but not dodecanedioyl-CoA. These mitochondria were active on hexadecanedioyl-CoA and this activity amounted to 20-25%of that measured with palmitoyl-CoA and 16-hydroxypalmitoyl-CoA as substrates. No mitochondrial dicarboxylyl-CoA oxidation could be detected in kidney cortex from animals receiving clofibrate in their diet. Heart and skeletal muscle illtact mitochondria from untreated and clofibrate-treated rats were capable of oxidizing each type of acyl-CoA as a substrate. Dicarboxylyl-CoA synthetase and camitine dicarboxylyltmnsfbrase activities were detected in various tissues from untreated and clo6brate-treated rats with the exception of carnitine dodecanedioyltransfbrase reaction in livers ffom untreated and ciofibrate-treated rats. In skeletal muscle, the acyl-CoA synthetase activities could be detected only in the presence of detergents.
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  • Kazuhiro Mitsui, Takuzo Nakagawa, Kunio Tsurugi
    1989 Volume 106 Issue 2 Pages 223-227
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes. By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1, 614 nucleotides including about 500 nucleotides in the 5'-flanking region. The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region. The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD. In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA. On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue.
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  • Masaya Takamoto, Tamao Endo, Mamoru Isemura, Yu Yamaguchi, Kunihiro Ok ...
    1989 Volume 106 Issue 2 Pages 228-235
    Published: August 01, 1989
    Released: January 25, 2011
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    Fibronectin purified from human term amniotic fluid contains 10 asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction and fractionated by anion-exchange column chromatography and serial lectin affinity chromatography. The structures of these sugar chains were determined by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bisected and non-bisected bi-and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Galf β1→GlcNAcβ1rarr;, GlcNAcβ1→, Neu5Acα2→3Galβ1→4GlcNAβ1→, and Neu5Acα2→6Galβ1→4GlcNAcβ1→groups in their outer chain moieties.
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  • Takahiko Hara, Tamao Endo, Kiyoshi Furukawa, Masao Kawakita, Akira Kob ...
    1989 Volume 106 Issue 2 Pages 236-247
    Published: August 01, 1989
    Released: January 25, 2011
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    Had-1, which was isolated from mouse FM3A carcinoma cells, was a non-permissive mutant cell line to Newcastle disease virus infection. Comparative study of the asparaginelinked sugar chains of the surface glycoproteins of the mutant and its parental cells revealed that galactosylation of the complex-type sugar chains is extensively reduced in the mutant. Assay of galactosyltransferase in the two cell lines, however, showed that the enzymatic activity in Had-1 cells is virtually identical to that in FM3A cells. Somatic cell hybridization analysis indicated that the mutant has the same defect as Chinese hamster ovary cell mutant Lec 8, which is deficient in UDP-galactose transport into Golgi vesicles.
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  • Hiroshi Yokota, Nobuyuki Ohgiya, Goshi Ishihara, Kazuhide Ohta, Akira ...
    1989 Volume 106 Issue 2 Pages 248-252
    Published: August 01, 1989
    Released: January 25, 2011
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    Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5-fold by treatment of the animal with β-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of β-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography: FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed “GT-2, ” was highly purified as judged from the single protein band (Mr 54, 000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50, 000 by endo-β-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50, 000 Da polypeptide carrying “high mannose” type oligosaccharide chain (s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu-Ile-Val. It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators. The notion that the NH2-terminal region to the 25th amino acid resiue of newly synthesized transferase contains the signal peptide was thus confirmed by this study.
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  • Morio Fukuhara, Takehiko Nohmi, Keinosuke Mizokami, Momoko Sunouchi, M ...
    1989 Volume 106 Issue 2 Pages 253-258
    Published: August 01, 1989
    Released: January 25, 2011
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    Three forms of cytochrome P-450 of liver microsomes of 3-methylcholanthrene-treated Golden hamsters were purified and characterized as regards their catalytic activity toward aflatoxin B1-related hepatocarcinogenic mycotoxins. These include two major forms, designated as cytochrome P-450-AFB (P-450-I) and P-450-II, and one minor form, P-450-III. Cytochromes P-450-AFB, P-450-II, and P-450-III have their absorption maximum in the carbon monoxide-complex of the reduced form at 448.5, 447.0, and 448.0nm, have apparent molecular weights of 56, 000, 58, 000, and 59, 500, and are in the low spin, high spin, and low spin state, respectively. Of these, cytochrome P-450-AFB was shown to be highly active in the mutagenic activation of aflatoxin B1-related hepatocarcinogens such as sterigmatocystin and O-methylsterigmatocystin. Activation of aflatoxin B1 by hepatic microsomes of 3-methylcholanthrene-treated hamsters was inhibited almost completely by the antibody against P-450-AFB but not by the antibody against P-450-II, indicating that P-450-AFB is the major component responsible for the activation of aflatoxin B1 by hamster liver. Western blot analysis demonstrated that no protein cross-reacted with the antibody to P-450-AFB in the liver microsomes from guinea pig, rat, mouse, and house musk shrew (Suncus murinus) treated with 3-methylcholanthrene, while one or two proteins cross-reacted with the antibody to P-450-II in the liver microsomes of these animals.
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  • Toshiaki Ohtsuka, Masaki Ozawa, Naoki Okamura, Sadahiko Ishibashi
    1989 Volume 106 Issue 2 Pages 259-263
    Published: August 01, 1989
    Released: January 25, 2011
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    Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with a phosphatidate containing short-chain fatty acids, 1, 2-didecanoyl-3-sn-phosphatidate (PA10), induced substantial superoxide anion (O2-) production in a dose-dependent manner, whereas phosphatidates prepared from egg lecithin and 1, 2-dioleoyl-3-sn-phosphatidate (PA18:1) had no such effect. Calcium was not involved in PA10-induced O2- production, since the production was also observed in the case of addition of EGTA prior to PA10 or pretreatment of PMNL with quin-2 and EGTA to eliminate contributions of both extracellular and intracellular calcium. We have reported in previous papers that the phosphorylation of 46K protein (s), which was commonly observed in parallel with an activation of NADPH oxidase in PMNL, was increased by treatment with 10μM 1-oleoyl-2-acetylglycerol (OAG) with little change in the O2- production (Okamura et al.(1984) Arch. Biochem. Biophys. 228, 270-277; Ohtsuka et al.(1988) Arch. Biochem. Biophys. 260, 226-231). Treatment of PMNL with a combination of PA10, which slightly increased 46K protein phosphorylation, and such a low concentration of OAG induced a marked increase in the O2 production with the increase in 46K protein phosphorylation, which was probably due to OAG action. Thus, it is likely that this protein phosphorylation plays a significant role in the stimulation of the O2- production by phosphatidate in PMNL.
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  • Akira Hayashi, Toshiko Matsubara, Masanori Morita, Takeshi Kinoshita, ...
    1989 Volume 106 Issue 2 Pages 264-269
    Published: August 01, 1989
    Released: January 25, 2011
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    The structures of intact choline phospholipids were determined by positive and negative ion mode fast atom bombardment mass spectrometry, tandem mass spectrometry, and B2/E and B/E constant linked scan mass spectrometry. The molecular weight of the choline lipid could be clearly determined by the appearance of [M+H]+ or [M+Na]+ in the positive ion mode and triplet ions, e. g., [M-15]-, [M-60]-, and [M-86]-, in the negative ion mode. The structures of the triplet ions were assigned to [M-CH3]-, [M-HN (CH3)3]-, and [M-CH2=CHN (CH3)3]-, respectively, by the MS/MS of each triplet ion, and the origin of the triplet ions was found as the matrix-ion adduct to the target molecule by using the B2/E linked scan technique. The polar group could be identified by the existence of ions indicating glycerophosphocholine and its cleavage products and by the presence of the triplet ions in the negative ion mode. Positional determination of the distribution of constituent fatty acyl groups was carried out by comparing the intensity of deacylated ions from positions 1 and 2 in the positive ion mode and of the ions produced by MS/MS of the triplet ions. From the mass number of the [RCOO] -ion which appeared in the negative ion mode, the molecular weight and degree of unsaturation of the fatty acyl group were determined. The position of double bond (s) in the acyl group was determined from the MS/MS of the [RCOO] -ion.
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  • Tsutomu Nishihara, Yoshihiro Takubo, Etsuko Kawamata, Tomihiko Koshika ...
    1989 Volume 106 Issue 2 Pages 270-273
    Published: August 01, 1989
    Released: January 25, 2011
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    The outer coat fraction (OC-Fr) of Bacillus megaterium ATCC 12872 spore was isolated as a resistant residue after alkali extraction, sonic treatment, and pronase digestion of the spore coat preparation, and its backbone structure was determined by chemical analysis to be composed ofgalactosamine-6-phosphate (Ga1N-P) polymers with polypeptides and calcium.OC-Fr was not fully solubilized after ordinary acid hydrolysis. OC-Fr was insensitive to all hexosaminidases tested, and moreover, an isolated fragment, a pentamer ofGa1N-P, was also resistant to lysozyme and hexosaminidases even after N-acetylation, being sensitive to them to some extent after dephosphorylation. Molecular sieving experimentsrevealed that the outer coat limited the entry of compounds with a molecular weight of more than 2, 000. Exchange of the metal on the spore surface also influenced the heat resistance. Spores of0C-Fr-deficient mutants were less resistant but were still much more resistant than the vegetative cells. These results suggest that the outer coat protects the contents of the spore against chemical, physical and enzymatic treatments owing to the chemical structure itself, composed mainlyofGa1N-P polymers, and the molecular sieving effect.
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  • Hiroshi Kawasaki, Yasufumi Emori, Shinobu Imajoh-Ohmi, Yasufumi Minami ...
    1989 Volume 106 Issue 2 Pages 274-281
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    We reported previously the cDNA cloning of the endogenous inhibitor for calciumdependent protease (CANP inhibitor, calpastatin) and the expression of its fragments in Escherichia coli. The CANP inhibitor has four internal repeating domains each spanning about 140 amino acid residues. The inhibitory activity arises from these domains which have a well-conserved sequence, TIPPXYR, in their central positions. The inhibitory activities of various fragments expressed in E. coli suggest the involvement of the regions around the well-conserved sequences. In this report, we describe further detailed investigation on the interaction site of the CANP inhibitor with CANP by truncating inhibitor fragments and by using chemically synthesized peptides. The results clearly indicate that the sequence around the well-conserved sequence, TIPPXYR, is an interaction site. A peptide as short as 23 amino acid residues retained inhibitory activity, but a 9-residue peptide corresponding to the conserved sequence, VTIPPKYRE had none. The inhibitory sequence is suggested as LGXKD/REXTIPPXYRXLL. The analysis of the competition between an inhibitor peptide and an irreversible inhibitor, E-64 for the reaction with the active site suggests no involvement of the active site cysteine residue of CANP in the inhibitory interaction between CANP and the CANP inhibitor. The high specificity of the CANP inhibitor to CANP arises from its interaction with residues other than the active site cysteine residue, possibly the subsite for substrate-binding of CANP.
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  • Fuminori Yoshizaki, Tatsuya Fukazawa, Yukari Mishina, Yasutomo Sugimur ...
    1989 Volume 106 Issue 2 Pages 282-288
    Published: August 01, 1989
    Released: January 25, 2011
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    Plastocyanin was purified from a multicellular, marine green alga, Ulva arasakii, by conventional methods to homogeneity. The oxidized plastocyanin showed absorption maxima at 252, 276.8, 460, 595.3, and 775nm, and shoulders at 259, 265, 269, and 282.5nm; the ratio A276.8/A595.3 was 1.5. The midpoint redox potential was determined to be 0.356V at pH7.0 with a ferni-and ferrocyanide system. The molecular weight was estimated to be 10, 200 and 11, 000 by SDS-PAGE and by gel filtration, respectively. U. arasakii also has a small amount of cytochrome c6, like Enteromorpha prolifera. The amino acid sequence of U. arasakii plastocyanin was determined by Edman degradation and by carboxypeptidase digestion of the plastocyanin, six tryptic peptides, and five staphylococcal protease peptides. The plastocyanin contained 98 amino acid residues, giving a molecular weight of 10, 236 including one copper atom. The complete sequence is as follows: AQIVKLGGDD GALAFVPSKISVAAGEAIEFVNNAGFPHNIVFDEDAVPAGVDADAISYDDYLNSKGETVVRKLSTPGVYGVYCEPHAGAGMKMTITVQ. The sequence of U. arasakii plastocyanin is closest to that of the E. prolifera protein (85% homology). A phylogenetic tree of five algal and two higher plant plastocyanins was constructed by comparing the amino acid differences. The branching order is considered to be as follows: a blue-green alga, unicellular green algae, multicellular green algae, and higher plants.
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  • Naoya Kenmochi, Yoshiaki Takahashi, Kikuo Ogata
    1989 Volume 106 Issue 2 Pages 289-293
    Published: August 01, 1989
    Released: January 25, 2011
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    Ribosomal proteins from cysts and nauplii of Artemia salina were analyzed by three kinds of two-dimensional polyacrylamide gel electrophoreis. The basic-acidic and basic-SDS gel systems were used to compare the basic ribosomal proteins, and some changes were observed between the cysts and nauplii in proteins S6, S14, and L24. The phosphorylation of protein S6 was increased in the nauplii. Basic proteins S14 and L24 in the cysts changed and none of the corresponding proteins in the nauplii were detected at the same positions on two-dimensional gels as in the cysts. The acidic-SDS gel system was used to compare the acidic proteins in ribosomes and it was revealed that an acidic protein, AX (Mr=24, 000), in the cysts was not present in the ribosomes from the nauplii. The ribosomal activities as to the formation of an 80S initiation complex with globin mRNA and poly (U)-directed polyphenylalanine synthesis were compared. There was no significant difference between the cyst and nauplius ribosomes.
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  • Kazuei Igarashi, Keiko Kashiwagi, Hiroshi Kobayashi, Reiko Ohnishi, To ...
    1989 Volume 106 Issue 2 Pages 294-298
    Published: August 01, 1989
    Released: January 25, 2011
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    The effect of polyamines on F1-ATPase catalyzed reactions has been studied through the use of submitochondrial particles and F1-ATPase. ATP degradation catalyzed by submitochondrial particles and F1-ATPase was inhibited by spermine and spermidine. Spermine's inhibition was much greater than spermidine's effect. In contrast, P1-ATP exchange and succinate dependent ATP synthesis catalyzed by submitochondrial particles were both stimulated by spermine. The inhibition of ATPase activity by polyamines probably occurs through polyamine's replacement of Mg2+ on ATP, for the following reasons.(a) The ATPase activity inhibited by spermine was partially recovered when Mg2+ was added.(b) Spermine bound to ATP and phospholipids but not to F1-ATPase; yet spermine inhibited the ATPase reaction catalyzed by F1-ATPase, a protein free of phospholipid.(c) The binding of spermine to ATP was inhibited by Mg2+. The ATP content in polyamine-deficient cells definitely was lower than that in normal cells. On the basis of these results, the possible role of spermine in keeping the ATP concentration at a high level is discussed.
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  • Shin-ichi Miyoshi, Sumio Shinoda
    1989 Volume 106 Issue 2 Pages 299-303
    Published: August 01, 1989
    Released: January 25, 2011
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    Vibrio vulnificus, an etiologic agent of wound infections and septicemia in humans, elaborates a metalloprotease which is known to be an important virulence factor of the Vibrio. The proteolytic activity of V. vulnificus metalloprotease (VVP) toward casein and elastin was inhibited by α2-macroglobulin (α2 M) at the molar ratio of 1:1, although partial activity was maintained. Permeability-enhancing and hemorrhagic activities were also inhibited, but the peptidase activity toward Z-Gly-Phe-NH2 was not reduced, even by an excess amount of α2 M. VVP formed a complex with α2 M through cleavage of the bait regions of all four α2 M subunits and elicitation of conformational change of the α2M molecule, which resulted in entrapment of VVP in the α2 M molecule. The peptidase activity of α2 M-VVP complex was inhibited by low-molecular-weight inhibitors such as phosphoramidon, but IgG antibody against VVP failed to neutralize its peptidase activity. Of human plasma proteins, α2 M was the only inhibitor for VVP. These findings indicate that VVP produced during V. vulnificus infection is inactivated by plasma α2 M that leaks from the vascular system.
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  • Fumio Okada, Kazuo Yamaguchi, Akira Ichihara, Toshikazu Nakamura
    1989 Volume 106 Issue 2 Pages 304-310
    Published: August 01, 1989
    Released: January 25, 2011
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    A latent form of transforming growth factor type-β(TGF-β) with a high molecular weight was purified to homogeneity from rat platelets by a six-step procedure. The yield of the purified latent TGF-β from platelets of 2, 500 rats was 1.4mg. The purified latent TGF-β was activated by treatment with urea at concentrations of over 4M or acidic solutions of below pH4. SDS-PAGE and gel filtration chromatography showed that the latent TGF-β consisted of active TGF-β and glycoproteins of about 200kDa as masking components, and that under physiological conditions, these components formed a high molecular weight complex of about 400kDa linked by non-covalent bonds. Here, we found that the masking protein was composed of one large subunit of about 110kDa and two small subunits of 39kDa linked by disulfide bridges. The N-terminal amino acid sequence of the small subunit was identical to the N-terminal region of the TGF-β precursor lacking a signal peptide. From these findings, we proposed a structural model for the latent TGF-β from rat platelets.
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  • Kiyoshi Furuhashi, Sadashi Hatano
    1989 Volume 106 Issue 2 Pages 311-318
    Published: August 01, 1989
    Released: January 25, 2011
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    Many protein factors regulating actin polymerization can be extracted from plasmodia of Physarum polycephalum in the presence of a high EGTA concentration (30mM). A protein factor with the molecular weight of 60, 000 (60kDa protein) was especially interesting because of its fragmin-like properties. We purified and characterized this 60kDa protein in the present study. The purified 60kDa protein enhanced the initial rate of G-actin polymerization, severed F-actin, and capped the barbed end of F-actin in a Ca2+-dependent way. The threshold concentration for Ca2+ was around 10-6M. The flow birefringence measurement showed that the length of F-actin decreased from 2.8 to 1.0μm depending on the concentration of 60kDa protein added to F-actin. These properties were identical to those of fragmin (Mr 42, 000) isolated from plasmodia (Hasegawa et al.(1980) Biochemistry 19, 2677-2683). However, the molecular weight, the tryptic peptide map, and the cross-reactivities with polyclonal anti-fragmin antibodies were different from those of fragmin. We concluded from these results that 60kDa protein is a new Ca2+-sensitive F-actin-severing protein. Considering its similarity to fragmin, we termed the 60kDa protein fragmin 60.
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  • Yasuhiro Hashimoto, Mitsuru Sakaizumi, Yuri Nakamura, Kazuo Moriwaki, ...
    1989 Volume 106 Issue 2 Pages 319-322
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    C57BL/10 (H-2b) and A/J (H-2a) mice have different phenotypes as to the age dependent changes in the expression of GM1 and GDla in the liver, i. e., the contents of these two components in total gangliosides in C57BL/10 and A/J mice were 1 and 20% at 4 wk and had decreased to almost 0 and 10%, respectively, at 8 wk [Y. Nakamura et al.(1988) J. Biochem. 103, 393-396]. To analyze the mode of inheritance of these phenotypes, C57BL/10 mice were mated with A/J or A/Wy (H-2a) mice. The phenotype of A/J or A/Wy mice was found to be inherited by the F1 hybrids, indicating that the phenotype of A mice is dominant over that of C57BL/10 mice. The (C57BL/10×A/WY) Fi hybrids were then backcrossed with one of the parent strains, C57BL/10 mice. The phenotype of A/Wy mice was demonstrated to be inherited by 23 out of 35 mice and all of the 23 had the H-2a haplotype. These results suggest that the two phenotypes of C57BL/10 and A mice are controlled by a pair of allelic genes on a locus which is closely linked to the H-2 complex on chromosome 17. Unexpectedly, B10. A (H-2a) mice were found to have significantly higher amounts of GM1 and GDla than both C57BL/10 and A mice, although they were a H-2 congenic strain carrying the H-2 genes transferred from A/Wy mice and should have the same genes on loci next to H-2 as one of the two strains. It remains to be determined how B10.A mice have higher amounts of GM1 and GDla than C57BL/10 and A/Wy mice.
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  • Akeo Shinkai, Hisami Yamada, Takeshi Mizuno, Shoji Mizushima
    1989 Volume 106 Issue 2 Pages 323-330
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied. Both the intact OmpC and central domain-deleted OmpC were examined. The hydrophobic segment was derived from the signal peptide of OmpF. Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment. Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments. Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane. Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane. It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain. The secretion was dependent on the amino-terminal signal peptide. Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation. Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.
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  • Yuji Iwasaki, Akihiko Tsuji, Kiyoshi Omura, Yoshiyuki Suzuki
    1989 Volume 106 Issue 2 Pages 331-335
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    Lysosomal β-mannosidase was purified almost 10, 000-fold from human placenta. The final preparation showed several protein bands on polyacrylamide gel electrophoresis. Its molecular mass was estimated to be 110 kDa, the optimal pH was 4.5, the Km was 0.56 mM, and the isoelectric point was 4.7. The enzyme was found to bind completely to Con A-Sepharose, and the pI was not changed after neuraminidase treatment. These results indicate that the purified enzyme represents a lysosomal form which contains high mannose type oligosaccharide chains and only a few sialic acids, if any.
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  • Yasuhide Seki, Sachiko Seki, Masayuki Satoh, Atsushi Ikeda, Makoto Ish ...
    1989 Volume 106 Issue 2 Pages 336-341
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    The complete primary structure of rubredoxin (Rd) isolated from Clostridium perfringens was sequenced to be: MICKFICDVCGYIYDPAVGDPDNGVEPGTEFKDIPDDWVCPLCGVDKSQFSETEE. The sequence was highly homologous to that of C. pasteurianum Rd but was different at 13 sites out of the total 54 amino acid residues (76% homology). It contained 1Fe atom, 4 cysteine residues, and no labile sulfur, had a molecular weight of 6, 056, and shared the general properties of classical anaerobic Rds. The pI was 4.4. The Rd was reduced with NADH in the presence of a specific NAD (P) H oxidoreductase preparation from the bacterium. The Km value of nitrate reductase for Rd as an electron-donor was 12 μM, a value comparable to that of the 13 μM for ferredoxin (Fd). These results taken together provide additional support for its role as the electron carrier in the nitrate reductase system [Seki, S., Ikeda, A., and Ishimoto, M.(1988) J. Biochem. 103, 583-584].
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  • Toshiaki Imagawa, Toshiyuki Takasago, Munekazu Shigekawa
    1989 Volume 106 Issue 2 Pages 342-348
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    Cardiac ryanodine receptor was purified from canine ventricle as a single polypeptide of Mr 400, 000 by a stepwise sucrose density gradient centrifugation and heparin-Sepharose CL-4B column chromatography. The [3H] ryanodine binding capacity (Bmax) was 60-fold enriched from cardiac microsomes without a change in affinity for [3H] ryanodine. The purity of the final preparation was determined to be>95% by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Using this purified preparation as an antigen, we produced six monoclonal antibodies which immunoprecipitated the cardiac ryanodine receptor. Three of these antibodies recognized the cardiac receptor on immunoblot analysis. In contrast, no protein in the microsomes isolated from Type I (slow) or Type II (fast) skeletal muscles was recognized by these antibodies. The [3H] ryanodine binding to cardiac and skeletal muscle microsomes was dependent on free Ca2+ concentration. In skeletal muscle microsomes, the [3H] ryanodine binding was remarkably enhanced by the addition of ATP or KCl and inhibited by high free Ca2+, whereas it was less sensitive to these agents in cardiac microsomes. All of these results clearly demonstrate that the cardiac ryanodine receptor is different from the skeletal muscle receptors and is not present even in Type I (slow) skeletal muscle fibers, in which cardiac isoforms of some of the muscle proteins are constitutively expressed.
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  • Kazuo Inaba, Hideo Mohri
    1989 Volume 106 Issue 2 Pages 349-354
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    Tryptic digestion of 21S outer arm dynein from sea urchin sperm flagella in the presence of ATP (or ADP) and vanadate produced quite different polypeptides from those obtained in the absence of ATP (ADP) and/or vanadate (Inaba and Mohri (1989) J. Biol. Chem. 264, 8384-8388). The 21S dynein heavy chains were consistently digested into 165-and 135-kDa polypeptides in the absence of both ATP (ADP) and vanadate. In the presence of 2mM ADP and 100μM vanadate, 300-kDa polypeptide, which appeared to be a precursor of 165-and 135-kDa polypeptides, became less accessible to trypsin, and 165- and 135-kDa polypeptides were digested into 150-/148-kDa and 96-kDa polypeptides, respectively. Quantitative analysis of the degradation of 165-and 135-kDa polypeptides showed that the conformations of these polypeptides change remarkably in the presence of ATP (ADP) and vanadate, and slightly in the presence of ATP γs. Photoaffinity labeling with 8-azidoadenosine 5'-triphosphate and vanadate-mediated photocleavage of dynein heavy chains revealed that both adenine-and γ-P1-binding sites were located on 165-and 150-/148-kDa polypeptides, but not on 135-kDa polypeptide. These results suggest that the conformational change occurring in the 165-kDa region on binding ATP spreads to the 135-kDa region and causes the conformational change of the 135-kDa region.
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  • Makoto Suematsu, Iwao Kurose, Hiroshi Asako, Soichiro Miura, Masaharu ...
    1989 Volume 106 Issue 2 Pages 355-360
    Published: August 01, 1989
    Released: January 25, 2011
    JOURNALS FREE ACCESS
    Oxyradical-dependent chemiluminescence from granulocytes sticking to venular endothelium was successfully visualized in rat mesenteric microvascular beds treated with platelet-activating factor (PAF). Intravital photonic image intensifier microscopy revealed that topical application of PAF-acether (100nM) caused remarkable granulocyte adherence on endothelial walls and the subsequent activation of a luminol-dependent photonic burst. Chemilumigenic sites clearly corresponded to the spatial distribution of sticking cells in post-capillary venules. The present findings thus serve as the first demonstration of an intravital oxidative burst of granulocytes on venular endothelium in PAF-induced microcirculatory disturbances.
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