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Volume 103 (1988)
- Issue 6 Pages 901-
- Issue 5 Pages 741-
- Issue 4 Pages 581-
- Issue 3 Pages 393-
- Issue 2 Pages 209-
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Volume 103, Issue 4
Displaying 1-29 of 29 articles from this issue
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Tomoyuki Kawase, Akitoshi Suzuki1988 Volume 103 Issue 4 Pages 581-582
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPhosphatidic acid (PA) evoked a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]1) in osteoblasts isolated from neonatal mouse calvaria. This increase was observed in both low (below 150 μM) and high (1.26mM) Ca2+-containing medium. In contrast, other phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, failed to increase [Ca2+]1 in osteoblasts. In high Ca2+-containing medium, A 23187 also increased [Ca2+], in the cells, but the mode of the change was different from that in the case of PA. These results suggest that PA may induce Ca2+-mediated cellular responses through Ca2+ release from intracellular stores in osteoblasts.View full abstractDownload PDF (265K) -
Sachiko Seki, Atsushi Ikeda, Makoto Ishimoto1988 Volume 103 Issue 4 Pages 583-584
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe NAD (P) H-dependent nitrate reductase system in Clostridium perfringens was reconstituted with rubredoxin (Rd), nitrate reductase (NaR), and an unadsorbed fraction, on a DEAE-cellulose column, of the extract (designated as fraction A), under nitrogen gas. Ferredoxin in place of Rd was not effective as an electron carrier in this reconstituted system. NAD (P) H-dependent nitrate reducing activity was also obtained by replacing fraction A with ferredoxin-NADP+ reductase from spinach. We propose the following scheme for the electron transfer in this NAD (P) H dependent nitrate reduction system. NAD (P) H→NAD (P) H-Rd reductase→Rd→NaR→NO3-View full abstractDownload PDF (240K) -
Keiko Kitagishi, Keitaro Hiromi, Sumio Tanase, Fujio Nagashima, Yoshim ...1988 Volume 103 Issue 4 Pages 585-588
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe reaction of pig heart mitochondrial and cytosolic aspartate aminotransferases (ab-breviated to mAspAT and cAspAT, respectively) with an enzyme-suicide substrate (mechanism-based inhibitor), gostatin (5-amino-2-carboxyl-4-oxo-1, 4, 5, 6-tetrahydropyridine-3-acetic acid) was studied kinetically, by following the spectral change with a micro-stopped-flow apparatus, as well as the inactivation of the enzyme activity. No significant difference in kinetic behavior was observed between mAspAT and cAspAT. From the analysis of time-dependent spectral change, no positive evidence for the existence of spectrophotometrically distinguishable intermediates was obtained. Both the spectral change and the inactivation followed, at least in appearance, simple bimolecular association kinetics, under the conditions studied. However, the second-order rate constant of the spectral change was found to be 1.5 to 2 times as large as that of the inactivation. The effects of pH and temperature on kon (the second-order rate constant of the spectral change) were also studied.View full abstractDownload PDF (453K) -
Wei-Jia Ou, Akio Ito, Masato Umeda, Keizo Inoue, Tsuneo Omura1988 Volume 103 Issue 4 Pages 589-595
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIn vitro synthesized precursors of several mitochondrial proteins, including P-450 (SCC), adrenodoxin, and malate dehydrogenase, bound to liposomes prepared from mitochondrial phospholipids, but not to those from microsomal phospholipids. When liposomes were prepared from various pure phospholipids, adrenodoxin precursor was bound only to the liposomes that contained cardiolipin. The liposomes containing other phospholipids did not show the binding affinity for the precursor. The binding was observed only with the precursor peptides of adrenodoxin and malate dehydrogenase, and their mature forms were not bound to the liposomes. The binding of the precursors was dependent on the concentration of cardiolipin in the liposomes. Liposomes containing various cardiolipin derivatives with modified polar head groups showed very different binding affinity for adrenodoxin precursor, suggesting the importance of the structure of the polar head of the cardiolipin molecule. Two or three positively charged amino acid residues in the extension peptide of P-450 (SCC) precursor were replaced by neutral amino acid residues by site-directed mutagenesis. The mutated P-450 (SCC) precursors did not bind to the liposomes containing cardiolipin. The results indicated that mitochondrial protein precursors have specific affinity for cardiolipin, and the affinity was due to the interaction between the extension peptides of the precursors and the polar head of the cardiolipin molecule.View full abstractDownload PDF (3381K) -
Tiee-Cherng Shieh, Shun-ichiro Kawabata, Hiroshi Kihara, Motonori Ohno ...1988 Volume 103 Issue 4 Pages 596-605
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe amino acid sequence of a coagulant enzyme, named flavoxobin, isolated from the venom of Trimeresurus flavoviridis (the habu snake) was determined by sequencing the Spyridylethylated derivative of the protein and its peptides generated by chemical (cyanogen bromide and hydroxylamine) and enzymatic (clostripain, Staphylococcus aureus V 8 protease, Achromobacter protease I, and elastase) cleavages. Hydrazinolysis was also employed to determine the C-terminal amino acid. The enzyme consisted of 236 amino acids and had a calculated molecular weight of 25, 744. Flavoxobin was found to be highly (69%) homologous in sequence to batroxobin, a coagulant enzyme from the venom of Bothrops atrox, and 27, 39, and 31% homologous to bovine thrombin, bovine trypsin, and human kallikrein, respectively. The sequence around the active site serine residue deduced from the homology relationship was Phe-Asp-Ser-Gly-Thr, which is different from the common sequence, Gly-Asp-Ser-Gly-Gly, for most serine proteases. Flavoxobin appears to be similar in secondary structure composition to batroxobin.View full abstractDownload PDF (900K) -
Nobuhito Sone, Fumiaki Yokoi, Tao Fu, Shigeo Ohta, Tuula Metso, Mirja ...1988 Volume 103 Issue 4 Pages 606-610
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe gene coding for cytochrome oxidase subunit I (COI) was isolated from a genomic DNA library of the thermophilic bacterium PS 3 and sequenced. The N-terminal of the COI protein was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequence of COI protein is composed of 536 amino acid residues and its molecular mass is 59, 510. The protein is clearly homologous to the corresponding subunit in the mitochondrial cytochrome oxidase and similarly appears to have 12 trans-membrane segments. The proposed ligands to two hemes (cytochrome aa3) and a copper atom (CuB) in this protein (Holm et al. (1987) EMBO J. 6, 2819-2823) are conserved in the sequence.View full abstractDownload PDF (588K) -
Yoshinori Suminami, Fumio Kishi, Tadashi Torigoe, Atsushi Nakazawa1988 Volume 103 Issue 4 Pages 611-617
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe gene encoding cytosolic adenylate kinase (AK1) was isolated from a chicken genomic DNA library by using its cDNA as a hybridization probe. The chicken AK1 gene spanned about 6 kilobase pairs and consisted of 7 exons. Analyses of the 5'-flanking region sequence and Sl nuclease mapping revealed that the transcription initiation site is located at 84 base pairs upstream from the ATG initiation codon. The TATA box and the putative CAT box were located 29 base pairs and 97 base pairs upstream from the transcription initiation site, respectively. A total of 7 GC boxes were found in the 5'-flanking region, the exon 1, and the intron 1. The GC boxes were surrounded by the sequences with extremely high G+C contents. When projected on the three-dimensional structure of the AK1 protein molecule, introns fell either between or near the ends of α-helices and β-strands, and most of the coding exons encoded at least one α-helix and one β-strand. The dot matrix plot analysis between chicken AK1 and bovine mitochondrial adenylate kinase (AK2) suggested that the AK1 gene might have evolved from the AK2 gene by deletion of one or more exon (s).View full abstractDownload PDF (1803K) -
Masato Nagaoka, Masami Muto, Teruo Yokokura, Masahiko Mutai1988 Volume 103 Issue 4 Pages 618-621
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe isolation and analysis of the cell wall and the polysaccharide-glycopeptide complexes of Bifidobacterium adolescentis YIT4011 are presented. Polysaccharide-glycopeptide complexes, PS-GP1 and PS-GP2, were solubilized from the cell wall by treatment with N-acetylmuramidase. PS-GP1 and PS-GP2 were found to be composed of glucose, 6-deoxytalose and a small amount of glycopeptide. The products of Smith degradation of the PS-GPs had no glucose-containing fraction, but were composed of 1, 2/1, 3-linked 6-deoxytalose. Furthermore, a second Smith degradation of this fraction yielded trisaccharide-glyceraldehyde. These results and methylation analysis led to the conclu-sion that PS-GP 1 or 2 has a repeating unit of →3) 6dTal (β1→3) 6dTal(β1→3) 6dTal (β1→2) 6dTal (α1→2) 6dTal (α1→2) 6dTal (α1-, and that glucose residues are linked to position C-3 of the 2-O-substituted 6-deoxytalose residues.View full abstractDownload PDF (435K) -
Hong-Yon Cho, Katsuyuki Tanizawa, Hidehiko Tanaka, Kenji Soda1988 Volume 103 Issue 4 Pages 622-628
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSDipeptidase (dipeptide hydrolase [EC 3. 4. 13. 11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothernzophilus IFO 12983. The enzyme has a molecular weight of about 86, 000, and is composed of two subunits identical in molecular weight (43, 000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmas values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3. 5. 1. 14] : their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.View full abstractDownload PDF (2775K) -
Norio Muto, Makoto Yamamoto, Satoru Tani, Satoshi Yonezawa1988 Volume 103 Issue 4 Pages 629-632
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe antiserum raised against the high-molecular-weight acid proteinase from rat gastric mucosa, termed 86-kDa acid proteinase, has been shown to recognize rat cathepsin E, but not cathepsin D (Muto, N. et al. (1987) J. Biochem. 101, 1069-1075). Using this specific antiserum, characteristic distribution of cathepsin E in rats was demonstrated. The enzyme was detected in a limited number of tissues, such as stomach, thymus, spleen, bladder, and erythrocyte membranes. Among them, the highest activity was observed in the stomach. In contrast, cathepsin D immunoreactive with the antiserum specific to rat gastric cathepsin D was demonstrated in all the tissues examined. Cathepsin E-type enzymes partially purified from these five tissues were precipitated in the same manner by the specific antiserum, and they had the same molecular weight, electrophoretic mobility, and resistance against denaturation by 4M urea. These results indicate that they could be exactly classified as cathepsin E. This type of enzyme was also detectable in mice and guinea pigs, but they showed relatively weak immunoreactivities with the antiserum. Thus, it is concluded that the distribution of cathepsin E is intrinsically different from ordinary cathepsin D, suggesting that it has a different physiological role from cathepsin D.View full abstractDownload PDF (1341K) -
Tsuyoshi Katoh, Hitomi Katoh, Fumi Morita1988 Volume 103 Issue 4 Pages 633-635
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe binding of one of the alkali light chains of myosin, A1, with the isolated renatured 20-kDa fragment of myosin subfragment-1 heavy chain was demonstrated by means of difference UV absorption spectroscopy. The difference spectrum with either rabbit or chicken A1 showed two characteristic peaks at 279 and 287nm indicating a perturbation of tyrosyl chromophores by the association with the 20-kDa fragment. The Δε at 287nm increased with an increase in the molar ratio of A1/20-kDa fragment and reached a maximum value at around equimolar ratio. The maximum Δε value was approximately three times larger with rabbit A1 than with chicken A1. Based on the positions of Tyr residues in the amino acid sequences, the contact surface of A1 with myosin heavy chain was concluded to be spread over a large area of A1. The binding of 20-kDa fragment with F-actin was measured by following the increase in turbidity. The affinity appeared to increase several times in the presence of A1. A1 may possibly control the affinity of myosin for actin.View full abstractDownload PDF (364K) -
Yasuhiko Shinoda, Akiko Yamada, Koichi Yagi1988 Volume 103 Issue 4 Pages 636-640
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCrayfish (Procambarus clarkii) myofibrils contain two basic proteins of molecular weights of 25, 000 and 23, 000. Both of the two proteins inhibit actomyosin ATPase as the vertebrate troponin-I does. These results differ from the previous one that troponin-I of crayfish (Astacus leptodactylus) showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).View full abstractDownload PDF (3650K) -
Isao Takahashi, Hidehiko Saito1988 Volume 103 Issue 4 Pages 641-643
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA rapid purification procedure with high recovery of blood coagulation factor XII (Hageman factor, HF) was established. Homogeneous HF was isolated in 6 days on a monoclonal antibody-immunoaffinity column chromatography followed by gel filtration. Approximately 4, 300-fold purification of HF was attained with 31% yield on average (n=4). Using this method, an abnormal HF was purified from plasma of a patient with cross-reacting material (CRM)-positive Hageman trait (factor XII TORONTO). The abnormal HF was found to be a single chain polypeptide with the same molecular weight (80, 000) as the normal HE Both abnormal and normal HF had similar amino acid compositions.View full abstractDownload PDF (1245K) -
Toshiyuki Tanaka, Takashi Masuko, Yoshiyuki Hashimoto1988 Volume 103 Issue 4 Pages 644-649
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA murine monoclonal antibody, B 3, to rat cells and monoclonal antibody HBJ127 to human cells were found previously to recognize the homologous cell surface antigen systems (gp125) which are predominantly expressed on proliferating cells in the respective species. Biochemical signals required for the induction of gp125 antigen, and the kinetics of the antigen appearance were examined by use of lymphocytes. Costimulation with a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and a calcium ionophore, A23187, resulted in strong induction of the gp125 antigen and subsequent DNA synthesis. The effect of TPA plus A23187 on rat T cells was diminished by EGTA, but recovered when CaCl2 was added. Human lymphocytes were also stimulated with TPA plus A23187 for the human gp125 induction. Dibutyryl cAMP and forskolin showed inhibitory effect on both gp125 antigen appearance and DNA synthesis in TPA/A23187-stimulated rat T cells whereas dibutyryl cGMP did not affect the gp125 antigen induction. Kinetic studies revealed that the appearance of the gp125 antigen on TPA/A23187-stimulated lymphocytes was faster than that of transferrin receptor in both rat and human systems. These results suggest regulatory roles of protein kinase C and Ca2+ in the induction of the gp125 antigen in the early phase of lymphocyte activation.View full abstractDownload PDF (790K) -
Hajime Tokuda, Makoto Asano, Yoshiki Shimamura, Tsutomu Unemoto, Shige ...1988 Volume 103 Issue 4 Pages 650-655
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSBioenergetic characteristics of Na+ pump-defective mutants of a marine bacterium Vibrio alginolyticus were compared with those of the wild type and revertant. Generation of membrane potential and motility at pH 8.5 in the mutants were completely inhibited by a proton conductor, carbonylcyanide m-chlorophenylhydrazone, whereas those in the wild type or revertant were resistant to the inhibitor. Motility and amino acid transport were driven by the electrochemical potential of Na+ not only in the wild type or revertant but also in the mutants. In the absence of the proton conductor, motility and amino acid transport of the mutants did not significantly differ from those of the wild type or revertant even at pH 8.5, where the Na+ pump has maximum activity. Therefore, the electrochemical potential of Na+ in the mutants seemed to be maintained at a normal level by a respirationdependent H+ pump and Na+/H+ antiporter. On the other hand, growth of the mutants became defective as the medium pH increased, especially on minimal medium. These results indicate that the Na+ pump is an important energy-generating mechanism when nutrients are limited at alkaline pH.View full abstractDownload PDF (683K) -
Koichiro Takanaka, Peter J. O'Brien1988 Volume 103 Issue 4 Pages 656-660
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe stimulation of polymorphonuclear leukocytes (PMNs) by phorbol-12-myristate-13-acetate in the presence of sodium fluoride caused the release of protons into the reaction medium concomitant with the generation of superoxide anions. The rates of oxygen consumption and proton release due to the metabolic burst were 16.3±3.5 and 10.2±1.1 nmol/min/107 cells respectively. When the superoxide anions were trapped with cytochrome c, the proton release was increased (35.8±0.5 nmol/min/107 cells) until the cytochrome c was reduced. Since the protons released from the activated cells would be consumed by the generated superoxide anions in the extracellular medium, the net amount of the protons released was 3-4-fold greater than that observed in the absence of extracellular cytochrome c. The increased proton release may be coupled to increased cellular respiration, since the inhibition of the respiratory burst with deoxyglucose, p-chloromercuribenzoic acid or chlorpromazine decreased the proton release. Amiloride (2mM) inhibited the proton release by up to 40%. These observations suggest that some mechanisms other than a Na+/H+ antiport and carbon dioxide diffusion could be transporting the H+ generated in the cytosol of the activated PMNs.View full abstractDownload PDF (519K) -
Hiroshi Kutsukake, Katsuyuki Imai, Takayuki Harada, Shigeru Morikawa, ...1988 Volume 103 Issue 4 Pages 661-666
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCulture supernatants from several human leukemic T cell lines were found to contain a macrophage activating factor which enhanced hydrogen peroxide release from human peripheral blood monocyte-derived macrophages. The macrophage activating factor from a T cell line, CCRF-CEM, was characterized biochemically and compared with interferon-γ, which is also an immunological product of T cells and has a potent macrophage activating activity. In contrast to interferon-γ, the macrophage activating factor in the culture supernatants bound to an anion exchanger and did not adsorb onto concanavalin A gel. Culture supernatants and active fractions from chromatographies were essentially devoid of anti-viral activity. Anti-human interferon-γ monoclonal antibody also failed to neutralize the macrophage activating factor from CCRF-CEM. MAF was eluted in the fractions with molecular weight of 40, 000 to 60, 000 on gel filtration in the presence of a detergent and a salt. MAF was partially purified to about 1, 300-fold by the methods described above: chromatography with anion exchangers and gel filtration. It was concluded that MAF from CCRF-CEM was biochemically and immunologically different from interferon-γ.View full abstractDownload PDF (654K) -
Tetsuya Asakawa, Naomi Azuma1988 Volume 103 Issue 4 Pages 667-671
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMyosin subfragment-1 (S1), which has one heavy chain (HC) (93 kDa) and two light chains (LC1 and LC2), was prepared by papain digestion of myosin from abalone smooth muscle in the presence of Ca2+. The Ca-sensitivity of abalone S1 itself was not lost completely (about 30%). The tryptic digestion of Si showed that in the presence of EDTA, S1 HC was split into 68, 55, and 23 kDa fragments, as in the presence of Call, but 23 kDa was further degraded into 19 kDa. In contrast to the result in the presence of Ca2+, LCs disappeared in the early stage of reaction and Ca-ATPase activity decreased rapidly to about 70% of that of intact S1. This rapid decrease of Ca-ATPase activity seemed to be accompanied with the digestion of LCs. Therefore, LCs contribute to the protection of 23 kDa fragment from further digestion, and to the maintenance of Ca-ATPase activity by stabilizing the structure of Sl to some extent in the presence of Ca2+. Since F-actin suppressed the cleavage of S1 HC to 68 and 23 kDa during tryptic digestion, it might be that 23 and 68 kDa corresponded to 20 kDa (C-terminal fragment) and to 50+25 kDa (N-terminal fragment) of skeletal myosin S1, respectively.View full abstractDownload PDF (3670K) -
Shinji Asano, Toshiaki Iino, Yoshiaki Tabuchi, Noriaki Takeguchi1988 Volume 103 Issue 4 Pages 672-677
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe obtained two kinds of vesicle preparations which were of different density from the same gastric mucosae of hogs stimulated with food before slaughter. Both kinds contained H+, K+-ATPase. The light vesicle preparation differed from the heavy vesicle preparation as follows: the KCl permeability across the membrane of heavy vesicles was larger than that of light vesicles, the actin (46-kDa peptide on SDS-polyacrylamide gel) content of heavy vesicles was much higher than that of light vesicles, and the H+, K+-ATPase activity of heavy vesicles was less sensitive to a monoclonal antibody raised against light vesicles (HK2032) than that of light vesicles. Furthermore, there was a drastic difference in reactivity to SCH 28080, which is an H+, K+-ATPase-specific inhibitor and reacts competitively with the K+-high affinity site. SCH 28080 is more potent in light vesicles than in heavy vesicles. These results suggest that the conformation of H+, K+-ATPase changed during the translocation from tubulovesicles to the apical plasma membrane. On the other hand, H+, K+-ATPase activities in both vesicles had similar pH and [K+] dependences.View full abstractDownload PDF (3206K) -
Shin-ichiro Sano, Yoshihiro Matsuda, Hachiro Nakagawa1988 Volume 103 Issue 4 Pages 678-681
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.View full abstractDownload PDF (2436K) -
Tadashi Tai, Ikuo Kawashima, Nobuhiko Tada, Kazuo Dairiki1988 Volume 103 Issue 4 Pages 682-687
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe fine structural specificities of six monoclonal antibodies (MAbs) to ganglioside GD2, GalNAcβ1→4 (NeuAcα2→8NeuAcα2→3) Galβ1→4Glc-Cer, were studied. The binding specificities of these MAbs were found to differ from each other by virtue of their binding to structurally related authentic standard glycolipids as revealed by three different assay systems, including enzyme immunostaining on thin-layer chromatography, enzymelinked immunosorbent assay, and immune adherence inhibition assay. The MAbs examined could be divided into three binding types. MAbs A1-201, A1-410, and A1-425 bound specifically to ganglioside GD2 and none of the other gangliosides tested. Two other MAbs (A1-245 and A1-267) reacted not only with GD2, but also with several other gangliosides having the sequence NeuAcα2→8NeuAcα2→3Gal (GD3, GD1b, GT1a, GT1b, and GQ1b). The reactivities with these gangliosides varied to some degree. In addition, these MAbs were found to react with both GD3 (NeuAc-NeuAc) and GD3 (NeuGc-NeuAc), but not with GD3 (NeuAc-NeuGe) or GD3 (NeuGc-NeuAc). The last MAb (A1-287) also reacted with several other gangliosides but with lower avidity than A1-245 and A1-267. These findings suggest that each MAb to ganglioside GD2 may have an individual binding specificity and avidity. These MAbs represent potentially useful reagents for analyzing the function of GD2 on cell surface membranes, and provide a system for precisely studying the interactions between an anti-ganglioside antibody and the binding epitope of the antigenic determinant.View full abstractDownload PDF (3409K) -
Hiroyuki Iwamoto, Yuhei Morita, Takashi Kobayashi, Eiichi Hasegawa1988 Volume 103 Issue 4 Pages 688-692
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe heavy and the light subunits of human myeloperoxidase (donor: H2O2 oxidoreductase [EC 1. 11. 1. 7]) I, II, and III were isolated from the reduced and S-carboxymethylated enzymes. These three enzymes have the same terminal amino acid sequences and similar chemical compositions in both subunits. The NH2-terminal sequences of the heavy and light subunits were determined to be Val-Asn-Cys-Glu-Thr- and Thr-Cys-Pro-Glu-Gln-, respectively; a heterogeneity was observed in the NH2-termini of the latter subunits for the three enzymes. As for COOH-termini, the sequences -(Asn, 2 Leu, Ala, Ser, Trp)-Arg-Glu-Ala and -Ala-Arg were obtained for the heavy and the light subunits, respectively. The heavy subunits contained 8-10 mol/mol of glucosamine. On the basis of these results and the amino acid sequence deduced from cDNA clones, the heavy subunits probably correspond to amino acids 279-744 and the light subunits to amino acids (164-167)-272. For the heavy subunits, Ser-745, which was predicted as the COOH-terminal amino acid from the nucleotide sequence, was removed. The light subunits were also processed at their COOH-termini by 6 residues. Four or five high mannose type carbohydrate chains were attached to the heavy subunits.View full abstractDownload PDF (1918K) -
Yuhei Morita, Honami Yamashita, Bunzo Mikami, Hiroyuki Iwamoto, Shigeo ...1988 Volume 103 Issue 4 Pages 693-699
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPeroxidase (donor: H2O2 oxidoreductase [EC 1. 11. 1. 7]) was purified from a culture broth of an inkcap Basidiomycete, Coprinus cinereus S. F. Gray. A single component containing a low amount of carbohydrate was isolated by affinity chromatography on concanavalin A-Sepharose and crystallized from ammonium sulfate solution. The enzyme is an acidic protein (pI 3.5) and consists of a single polypeptide chain having the molecular weight of 41, 600 daltons. The enzyme contains one protohemin per molecule and exhibits the characteristic absorption, circular dichroism, and magnetic circular dichroism spectra of a heme-protein. The Coprinus peroxidase forms two characteristic intermediate compounds, I and II, and the rate constants for hydrogen peroxide and guaiacol had similar values to those for higher plant peroxidases. The ferric enzyme formed a cyanide compound with a dissociation constant similar to those for higher plant enzyme, but the dissociation constant of the ferrous enzyme-cyanide was large. The chemical composition of Coprinus peroxidase showed 381 amino acid residues, 1 glucosamine, 3 true sugars, 3 calcium, and 1 non-heme iron other than 1 protohemin. The secondary structure of the fungal enzyme was very similar to that of horseradish peroxidase.View full abstractDownload PDF (1769K) -
Toshikazu Nakamura, Takeru Fujii, Akira Ichihara1988 Volume 103 Issue 4 Pages 700-706
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHepatocytes from neonatal rats of 0 to 3 days old grew actively in primary culture without added serum or growth factors. In these culture conditions, growth of hepatocytes decreased progressively with increase in age of the rats from which they were isolated, and hepatocytes from rats of 2 weeks old showed scarcely any growth. Actively growing hepatocytes were found to secrete a growth factor that promoted their growth and that of Swiss 3T3 cells, but not that of adult hepatocytes. This growth factor in conditioned medium of growing hepatocytes was heat- and acid-stable, but sensitive to trypsin, and had a molecular weight of over 10, 000. It did not inhibit the binding of [125I] epidermal growth factor to its receptor, and its growth promoting activity was not inhibited by monoclonal antibody against insulin-like growth factor II. Therefore, it seems to be a new growth factor. These results, together with previous findings (Nakamura, T., Nagao, M., & Ichihara, A. (1987) Exp. Cell Res. 169, 1-14) demonstrated a reciprocal relation between growth and maturation of neonatal hepatocytes during development, like that of adult cells, but indicated that unlike growth of the latter, growth of neonatal cells is induced by an autocrine mechanism.View full abstractDownload PDF (2028K) -
Shun'ichiro Taniguchi, Junji Sagara, Takeo Kakunaga1988 Volume 103 Issue 4 Pages 707-713
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSHUT-14 cells, tumorigenic human fibroblasts, express a mutant β-actin which has a single amino acid substitution at position 244 (glycine to aspartic acid), in addition to normal β- and γ-actin. In order to characterize the biochemical function of the mutant β-actin, actins were extracted and purified from HUT-14 cells. The partially purified actin fraction contained β-, γ-, and mutant β-actin in the ratio of 1:1:1, the same ratio as in the cells. When the actin of this fraction was purified through a polymerization step, mutant β-actin was always less incorporated into actin filaments than β- and γ-actin. When the polymer-ization ability of purified HUT-14 actins was examined by sedimentation technique, it. was lower than those of muscle and of cytoplasmic actins from another human cell line (HUT-11) which expresses only normal β- and γ-actin, in the ratio of 2:1. The deficient polymerization of mutant β-actin was also observed by examining the ratio of β-, γ-, and mutant β-actins incorporated into actin filaments. The ratio of mutant β-actin in polymer-ized actins under all conditions examined was always less than that before polymerization. These results indicate that the single amino acid substitution at position 244 caused the reduction of incorporation of the mutant β-actin into actin filaments in vitro.View full abstractDownload PDF (2880K) -
Kiyofumi Maruyama1988 Volume 103 Issue 4 Pages 714-721
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSSteady-state kinetic analyses suggest that Pseudomonas ochraceae 4-carboxy-2-hydroxy-muconate-6-semialdehyde dehydrogenase (4-carboxy-2-hydroxy-cis, cis-muconate-6-semialdehyde: NADP+ oxidoreductase [EC 1. 2. 1. 45]) functions through an ordered BiBi mechanism. The enzyme binds one NADP+ molecule per subunit with a Kd of 4.8±0.8 μM. The enzyme is adsorbed to a Blue Sepharose CL-6B column and can be eluted therefrom with reagents having high affinity for the enzyme such as NADP+, NAD+, ATP, and Reactive Blue 2. Equilibrium dialysis and difference spectral titration show the binding of four molecules of Reactive Blue 2 per enzyme subunit. Two of these dye molecules show high-affinity binding with a Kd of 0.03±0.02 μM. The resulting 1:2 enzyme-dye complex can be isolated by gel filtration on Bio-Gel P-6. The kinetic, spectroscopic, and chromatographic properties of the complex indicate that the dye-binding sites are different from the coenzyme binding site. The other two dye molecules, in contrast, bind loosely with a Kd of 0.8±0.5 μM to a site overlapping the coenzyme binding site. This is confirmed by the following findings: NADP+ effectively abolishes the difference spectrum associated with the enzyme-dye binding, and the slope of the double reciprocal plot showing the competitive inhibition of the dye (K1=0.20±0.02 μM) with respect to NADP+ linearly depends on the square of the dye concentration. Essentially similar results are also obtained with methoxy Reactive Blue 2 and Reactive Blue 4. The binding of Blue Dextran-2000 to the enzyme is somewhat unlike that of Reactive Blue 2, possibly due to insufficient contact of the dye chromophore with the enzyme.View full abstractDownload PDF (995K) -
Michiko Sekine, Kyoko Nakamura, Minoru Suzuki, Fuyuhiko Inagaki, Tamio ...1988 Volume 103 Issue 4 Pages 722-729
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTwo extended globogangliosides, designated as Z1 and Z2, were purified from the kidney of DBA/2 mice. By means of GLC, 1H-NMR spectroscopy, negative-ion fast atom bombard-ment mass spectrometry, methylation analysis, and enzymatic digestion, the structures of Z1 and Z2 were determined to be NeuGcα2-3Galβ1-3GalNAcβ1-3Galαl-4Galβ1-4Glcβ1-Cer and NeuGcα2-8NeuGcα2-3Galβ1-3GalNAcβ1-3Galαl-4Galβ1-4Glcβ1-Cer, respectively. Since Z1 and Z2 were not detectable in the kidney of C57BL/10 and 6, BALB/c, and WHT/Ht mice, the mode of genetic control on ZI and Z 2 expression was examined by mating experiments between C57BL/10 or BALB/c and DBA/2. The results indicated that the expression of Z1 and Z2 is a recessive phenotype and that DBA/2 mice carry a single autosomal recessive gene. In the previous paper, we reported that DBA/2 mice do not express GL-Y (Galβ1-4(Fucαl-3)GlcNAcβ1-6(Galβ1-3)Gb4Cer) but express GL-X (Galβ1-3Gb4Cer) in the kidney (J. Biochem. 101, 553-562 (1987)), and that a single autosomal defective gene responsible for the defective GL-Y expression was identified by genetic analysis (J. Biochem. 101, 563-568 (1987)). In the present study, the results of analysis of the expression of GL-Y, Z1, and Z2 in the kidney of backcross mice indicated that 42/83 mice did not express GL-Y but expressed GL-X, Z1, and Z2, and the other 41 mice expressed GL-Y but neither Z 1 nor Z 2. The absence of a single exception in 83 mice, i.e., mice having either one of the following two phenotypes, GL-Y (-), Z1 (-), and Z2 (-) or GL-Y (+), Z1 (+), and Z2 (+), indicates that either a single recessive gene controls the expression of GL-Y, Z1, and Z2, or possibly two genes linked at a distance of less than 1.2 (=1/84) centimorgan are responsible. We propose a hypothesis to explain the mode of genetic control of the expression of these glycolipids.View full abstractDownload PDF (4375K) -
Fukuichi Ohsawa, Shunji Natori1988 Volume 103 Issue 4 Pages 730-734
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe mode of action of tumor necrosis factor (TNF) was studied. On treatment of TNFsensitive L929 cells with radioiodinated TNF, the TNF molecule was found to be internalized into the cells and extensively degraded. On treatment of TNF-insensitive embryonic fibroblast cells with TNF, less TNF was internalized and it was not degraded appreciably. The L929 cells excreted the degradation products of TNF into the culture medium, and this medium showed activity for degradation of liposomes composed of phosphatidylserine and phosphatidylcholine. The sensitive cells may contain some specific proteinase that cleaves TNF molecules.View full abstractDownload PDF (2705K) -
Keiichi Ando, Shunji Natori1988 Volume 103 Issue 4 Pages 735-739
Published: April 01, 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe effect of sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina (flesh fly), on Escherichia coli was investigated. Sarcotoxin IIA was found to have a bacterial effect on growing bacteria, but little on non-growing bacteria. At a concentration of 25 μg/ml, it induced significant morphological change of growing E. coli cells. In its presence, growing cells became greatly elongated, and spheroplast-like bulges and projections appeared on their surface. A rough mutant strain of E. coli with a defect in the structure of lipopolysaccharide was more sensitive than the parent strain to sarcotoxin IIA. These results suggest that the main effect of sarcotoxin IIA is to inhibit cell wall synthesis, including septum formation.View full abstractDownload PDF (3537K)
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