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Volume 102 (1987)
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- Issue 5 Pages 963-
- Issue 4 Pages 673-
- Issue 3 Pages 445-
- Issue 2 Pages 243-
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Volume 102, Issue 3
Displaying 1-30 of 30 articles from this issue
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Shigeru HIRANO, Yoshiki MATSUURA, Masami KUSUNOKI, Yasuyuki KITAGAWA, ...1987 Volume 102 Issue 3 Pages 445-446
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA lectin from Flammulina veltipes (Enoki-dake) has been crystallized in a form suitablefor crystallographic structure analysis. Two types of crystals were grown: one from polyethylene glycol 6000 solution at pH 7 and the other from ammonium sulfate solution at pH 7. The latter type is more suitable for the crystallographic investigations because of its high resolution X-ray diffraction and smaller number of asymmetric molecules.View full abstractDownload PDF (491K) -
Reiko SUZUKI, Tadaomi TAKENAWA1987 Volume 102 Issue 3 Pages 447-450
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThyrotropin releasing hormone (TRH) caused significant breakdown of phospha-tidylinositol 4, 5-bisphosphate (PIP2) in GH3 cells, but vasoactive intestinalpeptide (VIP) did not. However, VIP enhanced the TRH-induced hydrolysis of PIP, the conversion of phosphatidylinositol 4-phosphate (PIP) to PIP2, and the accumulation of phosphatidic acid (PA). On the other hand, the tumor promoter, tetradecanoyl phorbol acetate (TPA), suppressed the TRH-induced hydrolysis of PIP. In the membrane fraction, the addition of cAMP inhibited the PI kinase activity in a dose-dependent manner, but stimulated the PIP kinase activity. TPA did not affect the PI and PIP kinase activities at all. VIP enhanced the first spike phase of the TRH-induced increase in the intracellular Ca2+ level, while TPA inhibited such Ca2+ mobilization. These results suggested that cAMP-increasing agents enhanced inositol phospholipid metabolism and Ca2+ mobilization induced by TRH in GH3 cells but that TPA inhibited them.View full abstractDownload PDF (284K) -
Ken KARASAWA, Kagari FUJITA, Noriko SATOH, Toshio HONGO, Morio SETAKA, ...1987 Volume 102 Issue 3 Pages 451-453
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpecific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As thehapten weused the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Anti-body activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycero-phosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.View full abstractDownload PDF (206K) -
Hisami YAMADA, Noriko OSHIMA, Takeshi MIZUNO, Hiroshi MATSUI, Yoshiyuk ...1987 Volume 102 Issue 3 Pages 455-464
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method is presented for the efficient location of antigenic determinants using a series of chimeric proteins. By means of in vivo homologous recombination between the ompC and ompF genes coding for OmpC and OmpF, homologous proteins of the Escherichia coli outer membrane, a series of ompF-ompC chimeric genes was constructed (Nogami, T., Mizuno, T., & Mizushima, S. (1985) J. Bacteriol. 164, 797-801, and this work). The OmpF-OmpC chimeric proteins expressed by these genes were successfully used to locate antigenic determinants recognized by monoclonal antibodies, which specifically react with either the OmpC or OmpF protein. Interaction between monoclonal antibodies and the chimeric proteins was examined by means of either enzyme-linked immunosorbent assay or immu-noblot analysis. The antigenic determinants recognized by three anti-OmpC anti-bodies and one anti-OmpF antibody were thus located. Finally, the polypeptides covering these regions were chemically synthesized for two of them and then tested as to their reactivity with the antibodies. The peptides reacted with the corre-sponding antibodies when the former were chemically coupled with bovine serum albumin. Most of the monoclonal antibodies isolated in this work were highly specific to the unfolded monomer of the protein against which the antibody was raised. But they did not react with the trimer, the native form. These results are discussed in relation to the structures and functions of the OmpC and OmpF pro-teins. The use of a series of monoclonal antibodies for studying the mechanism of nrntein tranclnratinn art-ricc the rytnnlacmir membrane is also discussed.View full abstractDownload PDF (1854K) -
Takayoshi IIO1987 Volume 102 Issue 3 Pages 465-470
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rate constant of the conformational change of skeletal troponin C (TnC) induced by the ca2+-binding reaction with the high-affinity Ca2+-binding sites was determined in the presence of Mg2+ by the fluorescence stopped-flow method in 0.1 M KCl, 50 mM Na-cacodylate-HCl pH 7.0 at 20°C. The [MgCl2] dependence of the rate constants of the observed biphasic conformational change leveled off at the high [MgCl2] region: the rate constants were 60 ± 9 s-1 and 8 ± 2 s-1, respectively. These values are larger than the rate constants of the biphasic fluorescence intensity change of TnC induced by Mg2+ removal reaction at the high-affinity Ca2+-binding sites (37±7 s-1 and 3.0 ± 0.6 s-1) under the same experimental conditions. These results suggest that the Ca2+-Mg2+ exchange reaction at the high-affinity Ca2+-binding sites is faster than the resultant conformational change accompanying the fluo-rescence intensity change. Based on these results, we also reexamine the molecular kinetic mechanism of the conformational change of the protein induced by the Mg2+ binding or removal reaction with the high affinity Ca2+-binding sites of skeletal TnC.View full abstractDownload PDF (514K) -
Mikio SUDA, Toshiyuki WATANABE, Mikihiko KOBAYASHI, Kazuo MATSUDA1987 Volume 102 Issue 3 Pages 471-479
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo types of phosphorylase [EC 2.4.1.1] from the etiolated soybean (Glycine max) cotyledons were separated by column chromatography on DEAE-Sephacel and further purified to apparent homogeneities. Molecular weights of the subunits were 100, 000 and 113, 000 for phosphorylases I and II, respectively. The native enzymes I and II were a dimer (200, 000) and tetramer (450, 000), respectively. Electrophoretic analysis by the Hedrick and Smith method indicated that the two phos-phorylases were distinct proteins with no correlation. The apparent Km values for glucose 1-phosphate, glycogen, and maltoheptaose of enzyme I were 4.00 mM, 0.18 mg/ml, and 10.3 mM, respectively, while those values of enzyme II were 5.43 mM, 23.8 mg/ml, and 0.30 mM, respectively. The relative activity of enzyme I increased with increasing chain length of the substrate glucans, and waxy maize amylopectin was the best substrate among the 11 saccharides examined. For enzyme II, maltooligosaccharides with degree of polymerization (DP) 5-7 were the better substrates than amylose (DP 38) and glycogen. These results indicated that the soybean phosphorylases I and II have similar properties to a cytoplasmic and chloroplastic type of plant leaf enzyme, respectively.View full abstractDownload PDF (1984K) -
Nobuhito SONE, Mihoko SEKIMACHI, Yoshihiro FUKUMORI, Tateo YAMANAKA1987 Volume 102 Issue 3 Pages 481-486
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteoliposomes reconstituted from purified cytochrome c oxidase of Pseudomonas AM1 and from a heptyl β-D-thioglucoside-extract of its membranes showed respiratory control but did not show H+ pumping upon a pulse with reduced cytochrome c. The stoichiometries of respiration-dependent H+ translocation in the resting cells respiring ascorbate via N, N, N', N'-tetramethyl-p-phenylenediamine were measured by the oxygen-pulse and initial rate methods. The apparent H+/O ratio of about 2 was due to 2H+ release from the hydrogen-donating substrate. These results strongly suggested that Pseudomonas AM1 does not pump H+ intrinsically, although the enzyme catalyzes electron transfer across the membranes.View full abstractDownload PDF (492K) -
Dong-Hyun KIM, Kyoichi KOBASHI1987 Volume 102 Issue 3 Pages 487-491
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSArylsulfotransferase was stabilized for storage more markedly by covalent immobilization onto AH-Sepharose-4B or CH-Sepharose -4B with EDC than by adsorptive immobilization onto DEAE-cellulose or DEAE-Sephadex. The optimal pH, Km for sulfate donor and thermostability of covalently immobilized arylsulfotransferase were similar to those of the free enzyme. Tyrosine-containing peptides such as cholecytokinin-8-nonsulfate, tyrosine methylester and (Leu)enkephalin as acceptor substrates were effectively sulfated by the immobilized enzyme.View full abstractDownload PDF (329K) -
Sumie KAWANO, Tetsuya KAMATAKI, Toshio YASIMORI, Yasushi YAMAZOE, Ryui ...1987 Volume 102 Issue 3 Pages 493-501
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo forms of cytochrome P-450 (P-450 human-1 and P-450 human-2) have been purified from human liver microsomes to electrophoretic homogeneity. P-450 human-1 and P-450 human-2 differ in their apparent molecular weights (52, 000 and 56, 000, respectively) and Soret peak maxima in the CO-binding reduced difference spectrum (447.6 and 450.3 nm, respectively). In the reconstituted system using rat liver NADPH-cytochrome c (P-450) reductase, P-450 human-2 more effectively oxidized benzo(a)pyrene (80-fold), ethylmorphine (2-fold), and 7-ethoxy-coumarin (2-fold) than did P-450 human-1. However, P-450 human-1 showed higher testosterone 6β -hydroxylase activity, and the activity was markedly increased by the inclusion of cytochrome b5 or spermine in the reconstituted system. Anti-bodies raised against P-450 human-1 inhibited more than 80% of microsomal testosterone 6β -hydroxylase activity in human liver. Immunoblotting analysis using anti-P-450 human-1 IgG revealed a single immuno-staining band near Mr 52, 000 in all human liver samples examined. The amount of immunochemically determined P-450 human-1 varied in parallel with the testosterone 6β -hydroxylase activity in human liver. These results indicate that P-450 human-1 is a major form of cytochrome P-450 responsible for microsomal testosterone 6β -hydroxylation. Thus, this paper is the first report on human cytochrome P-450 responsible for testo-sterone 6β -hydroxylation, which is the major hydroxylation pathway in human liver microsomes.View full abstractDownload PDF (1778K) -
Masato HOSHINO, David J. CLANTON, Thomas Y. SHIH, Masao KAWAKITA, Seis ...1987 Volume 102 Issue 3 Pages 503-511
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide exchange reaction was observed with purified ras oncogene product p21 overproduced in Escherichia coli (Hattori, S. et al. (1985) Mol. Cell Biol. 5, 1449-1455) under various conditions. (NH4)2SO4 increased the rate of dissociation of bound GDP from c-rasH and v-rasH p21. The dissociation kinetics were those of a first order reaction, and there was a linear relationship between the rate constant and the (NH4)2SO4 concentration. At any concentration of (NH4)2SO4, the exchange rate was faster with v-rasH p21 than that with c-rasH p21. EDTA and (NH4)2SO4 synergetically stimulated the dissociation reaction. Nucleotide-free p21 was prepared by gel filtration on Sephadex G-25 in the presence of 5 mM EDTA and 200 mM (NH4)2SO4 at room temperature. The free p21 was quite thermolabile, but the addition of GDP or GTP completely protected p21 from thermal inactivation. The dissociation constants for GDP and GTP were determined with free p21 to be 8.9 and 8.2 nM, respectively, for v-rasH p21, and 1.0 and 2.6 nM for c-rasH p21. In the presence of 200 mM (NH4)2SO4, these dissociation constants increased 3- to 12-fold.View full abstractDownload PDF (678K) -
Takayuki NEMOTO, Yuko OHARA-NEMOTO, Minoru OTA1987 Volume 102 Issue 3 Pages 513-523
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nontransformed glucocorticoid receptor (GR) and an 88-kDa protein in rat liver cytosol were selectively adsorbed on protamine- and arginine-Sepharose. The 88-kDa protein was purified from rat liver cytosol to homogeneity by precipitation with protamine sulfate, followed by DEAE-ion exchange chromatography, gel chromatography, and DEAE ion-exchange high-performance liquid chromatography. The 88-kDa protein appeared to be present as a dimer at both low and high salt concentrations. The physicochemical properties and the amino acid composition of the 88-kDa protein were almost the same as those of heat-shock protein 90 of HeLa cells and yeast. Preincubation of the GR with the polyclonal antibody raised against the 88-kDa protein increased the sedimentation coefficient of the nontransformed GR, but did not change that of the transformed GR. These results indicate that heat-shock protein 90 is associated with rat liver nontransformed GR and is responsible for the interaction of GR with protamine.View full abstractDownload PDF (2652K) -
Masami FUKUOKA, Yoshihiro FUKUMORI, Tateo YAMANAKA1987 Volume 102 Issue 3 Pages 525-530
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome a1c1 (nitrite-cytochrome c oxidoreductase) purified from Nitrobacter winogradskyi (formerly N. agilis) contained molybdenum, non-heme iron, and acid-labile sulfur in addition to hemes a and c; it contained 1 mol of heme a, 4-5g atoms of non-heme iron, 2-5g atoms of acid-labile sulfur, and 1-2g atoms of molybdenum per mol of heme c, but did not contain copper. The fluorescence spectra of the molybdenum cofactor derivative prepared from cytochrome a1c1 were very similar to those of the cofactor derivative from xanthine oxidase, and the aponitrate reductase of nit-1 mutant of Neurospora crassa was complemented by addition of the molybdenum cofactor derived from the cytochrome. Further, the ESR spectrum of cytochrome a1c1 was similar to that of liver sulfite oxidase. The content of cytochrome a1 in the cells cultivated with the medium in which tungsten was substituted for molybdenum markedly decreased as compared with that in the cells cultivated in the molybdenum-supplemented medium. These results indicate that cytochrome a1c1 is an iron-sulfur molybdoenzyme which contains hemes a and c.View full abstractDownload PDF (482K) -
Shin-ichi NAKAYAMA, Tokuko WATANABE, Kenji TAKAHASHI, Masamatsu HOSHIN ...1987 Volume 102 Issue 3 Pages 531-535
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFicin was alkylated with a series of haloacetamide spin labels with various distances between the spin probes and reactive groups. From the relation of these distances to the τc values of the labels incorporated into protein, it was estimated that the depth of the active site hole of ficin is ca. 8 Å. The results are somewhat different from those reported previously for papain (S. Nakayama et al. (1981) Biochem. Biophys. Res. Commun. 98, 471-475). Examination of the pH dependence of the ESR spectra for ficin and papain alkylated with an iodoacetamide or a maleimide spin label suggested that these enzymes have an amino acid residue of pKa 4 (probably a histidine residue) around the active site cysteine and that the active site conformations change at around pH 5.View full abstractDownload PDF (357K) -
Masafumi HIRONO, Masahiro NAKAMURA, Minoru TSUNEMOTO, Tomoyoshi YASUDA ...1987 Volume 102 Issue 3 Pages 537-545
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThough actin is ubiquitous in eukaryotes, its existence has not been clearly proven in Tetrahymena. Recently, we have succeeded in cloning and sequencing the Tetrahymena actin gene using a Dictyostelium actin probe (Hirono, M. et al. (1987) J. Mol. Biol. 194, 181-192). The primary structure of the Tetrahymena actin deduced from the nucleotide sequence of its gene is greatly divergent from those of other known actins, making it necessary to ascertain whether the predicted Tetrahymena actin is indeed an actin. In this paper, we investigated the localization of the predicted Tetrahymena actin by an immunofluorescence technique using antibody against its synthetic N-terminal peptide, in order to elucidate its possible biological roles. The results showed that immunofluorescence was localized in the division furrow of the dividing cell, and in the intranuclear filament bundles formed in cells exposed to heat shock or DMSO. In addition, the oral apparatus and the proximity of the cytoproct, which are organellesinvolved in endocytosis and exocytosis, respectively, also fluoresced. Thus, we conclude that the Tetrahymena actin we identified is indeed an actin and plays the same biological roles as ubiquitous actins do, although it is considerably divergent in its amino acid sequence.View full abstractDownload PDF (4200K) -
Takashi HIROTA, Yuko NISHIKAWA, Minoru TANAKA, Kuniaki FUKUDA, Takashi ...1987 Volume 102 Issue 3 Pages 547-550
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn antibody prepared against dehydropeptidase-I, one of the glutathione processing enzymes, from the renal membrane fraction of rats was employed to localize at the light microscopic level the enzyme in the kidney using the avidin/biotin-peroxidase complex method. Dehydropeptidase-I was found to be present on both the brush border and the basolateral membranes of proximal tubular cells. Furthermore, the enzyme activity in the isolated brush border and basolateral membrane fractions was also determined. Distribution profiles of the enzyme activity showed good agreement with the results of the immunohistochemical observations.View full abstractDownload PDF (1048K) -
Kumiko KAWAGUCHI-NAGATA, Takehiko WATANABE, Atsushi YAMATODANI, Masato ...1987 Volume 102 Issue 3 Pages 551-557
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInjection of Staphylococcal enterotoxin A (SEA) into WBB6F1-W/WV mice genet-ically deficient in mast cells resulted in a 10-fold increase in the histidine decar-boxylase [HDC, L-histidine carboxylyase, EC 4.1.1.22] activity of their spleen. The nature of the spleen cells responsible for this increased HDC activity was studied. The HDC induction by SEA was abolished on day 1 after X-ray irradiation of the mice at 400 rad and restored by transplantation of bone marrow cells from normal WBB6F1-+/+ littermates into the X-ray irradiated WBB6F1-W/WV mice. Trans-plantation of cells from other organs of the normal mice, such as the thymus, mesen-teric lymph node and spleen, did not restore the HDC increase significantly. Trans-plantation of cultured mast cells also did not restore the increase. Moreover, the high HDC activity of spleen cells induced by SEA was not affected by their treat-ment with anti-Thy-1, 2 antibody and complement. Depletion of phagocytes from the spleen by treatment with carbonyl iron resulted in decrease in HDC activity. These results suggested that phagocytic cells derived from haemopoietic stem cells of the bone marrow were responsible for the increase in HDC activity induced by SEA.View full abstractDownload PDF (574K) -
Ken-ichirou MOROHASHI, Hidefumi YOSHIOKA, Osamu GOTOH, Yoshiie OKADA, ...1987 Volume 102 Issue 3 Pages 559-568
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESScDNA clones of the mRNA for bovine adrenal cytochrome P-450(11β) were iso-lated. Sequence analysis of a 4 kb long cDNA revealed the primary structure of P-450(11β), which consisted of 503 amino acids (Mr: 57, 924) and contained an extension peptide of 24 amino acids at the NH2-terminus of the mature P-450(11β) molecule. A bovine genomic DNA containing the 1st exon and its leader sequence of P-450(11β) gene was also isolated from a bovine gene library. Determination of the transcription initiation site by S1 nuclease analysis using the cloned genomic DNA confirmed that the methionine codon near the 5' side of the 4 kb long cDNA was the initiation codon. Comparisons of the primary structures among P-450(11β) and other forms of cytochrome P-450 including P-450(SCC) indicated that the two mitochondrial P-450s, P-450(11β) and P-450(SCC), were significantly different from microsomal forms of cytochrome P-450. The homology between P-450(11β) and P-450(SCC) was 36%, which is higher than the values between P-450(11β) and various microsomal P-450s. An alignment of P-450(11β) and P-450(SCC) to give maximum matching showed four highly conserved regions (C-1, C-2, C-3, and C-4). The homology values of these regions were 58-70%, considerably higher than the overall homology between these two mitochondrial P-450s. A putative heme binding site and a steroid binding site were located in the conserved regions. Hy-dropathy profiles of P-450(11β) and P-450(SCC) were very similar. A definite difference was noticed at the NHrterminal portion between mitochondrial and microsomal types of P-450. Microsomal type of cytochrome P-450 had a hydro-phobic sequence consisting of about 20 amino acids, whereas mitochondria' type had an extension peptide containing many positively changed amino acids.View full abstractDownload PDF (1621K) -
Kaoru MIYAZAKI, Yoshiyuki ASHIDA, Yasunori KIHIRA, Keisuke MASHIMA, Ji ...1987 Volume 102 Issue 3 Pages 569-582
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibro-nectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by ρ-chloromercuribenzoate and cal-cium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen acti-vator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.View full abstractDownload PDF (4222K) -
Yoshiharu INOUE, Hae-ik RHEE, Kunihiko WATANABE, Kousaku MURATA, Akira ...1987 Volume 102 Issue 3 Pages 583-589
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlyoxalase I catalyzing the conversion of methylglyoxal into S-lactoylglutathione in the presence of glutathione was purified approximately 1, 400-fold with 2.9% activity yield from mold, Aspergillus niger. The enzyme consisted of a single polypeptide chain with a relative molecular weight of 36, 000 on both SDS-polyacrylamide gel electrophoresis and Sephadex G-150 gel filtration. The enzyme was most active at pH 7.0, 35-37°C. Among the various aldehydes tested, the enzyme was active on methylglyoxal and 4, 5-dioxovalerate with. Km values of 1.25 and 0.87mM, respectively. The activity of the enzyme was completely inhibited by Zn2+. at 0.5mM. An equimolar amount of EDTA (0.5mM) protected the enzyme from inactivation by Zn2+. EDTA competitively (K1=1.3mM) inhibited the activity of the enzyme. Fe2+ was a potent activator for the enzyme, the activation being approximately 2.4-fold at 0.5mM.View full abstractDownload PDF (1035K) -
Toshihiko NANBA, Yasuo MUKOHATA1987 Volume 102 Issue 3 Pages 591-598
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn ATPase was newly identified on the inner face of the plasma membrane of the extremely halophilic archaebacterium Halobacterium halobium. The enzyme was released into an alkaline EDTA solution and purified by several chromatographic steps in the presence of sulfate at 1M or over. The molecular weight of the native enzyme was around 320, 000; it is most likely composed of two pairs (α2β2) of 86, 000 (α) and 64, 000 (β) subunits. The enzyme hydrolyzed ATP and other nucleoside triphosphates but neither ADP nor AMP. The enzyme required divalent cations, among which Mn2+ was most effective (Mg2+ activated 35% of Mn2+). The ATPase activity was optimum at pH between 5.5 and 6, particularly in a nearly saturated Na2SO4 (or Na2SO3) solution, while it was very low in a chloride salt solution even at 4M at any pH. The Km value for ATP was 1.4mM and the K1 value for ADP (competitive to ATP) was 0.08mM. Neither azide (a specific inhibitor for F0F1- and Fl-ATPase) nor vanadate (for E1E2-ATPase) inhibited the enzyme. The ATPase was stable at high concentrations of sulfate. At low concentrations of salts, or at low temperatures even in high NaCI concentrations, the enzyme was inactivated. Although the ATPase isolated here from halobacterial membrane has such unusual characteristics, it is the most probable candidate for the (catalytic part of) halobac-terial ATP synthase, which differs from F0F1-ATPase/synthase (Mukohata et al. (1986) J. Biochem. 99, 1-8; Mukohata and Yoshida (1987) J. Biochem. 101, 311-318).View full abstractDownload PDF (627K) -
Mamoru NUKATSUKA, Shigeharu NAGASAWA1987 Volume 102 Issue 3 Pages 599-605
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSC4b-binding protein, C4bp, is a regulatory factor of the complement system and is also known to be a binding protein of vitamin K-dependent coagulation factor, protein S. Whereas the C4b-binding site is known to be located in the middle part of the subunit chains of C4bp, the location and properties of protein S-binding site are uncertain. Therefore, we have examined the characteristics of the interaction between human protein S and C4bp. Proteolysis of C4bp-protein S complex with chymotrypsin yielded N-terminal-derived 48-kDa fragments of C4bp subunit chains and a C-terminal-derived 160-kDa core fragment of C4bp, to which protein S was still bound. This result suggested that the protein S-binding site is located in the core domain of C4bp. Gel filtration of guanidine-treated C4bp-protein S complex in the absence of guanidine resulted in the separation of C4bp and protein S. Binding assay with 125I-labeled protein S showed that the guanidine-treated C4bp lacked the protein S-binding activity. This result suggests that the protein S-binding site in C4bp is denatured irreversibly by guanidine treatment and therefore seems to be dependent on a specific conformation of C4bp. The C4bp-binding site of protein S was lost upon thrombin treatment, suggesting that the N-terminal thrombinsensitive region of protein S may be related to the C4bp-binding site. Although free protein S was susceptible to chymotrypsin, leukocyte elastase, and cathepsin G, C4bp-bound protein S was found to be resistant to these proteases. This result suggests that binding of protein S to C4bp may be significant for the protection of protein S from proteolytic inactivation during inflammatory responses.View full abstractDownload PDF (2530K) -
Norihisa KIKUCHI, Kiyoshi NAGATA, Toshihiko HORII, Sadao MIYAZAKI, Mas ...1987 Volume 102 Issue 3 Pages 607-612
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA synthetic gene for human pancreatic secretory trypsin inhibitor (PSTI) was fused to the coding sequence for the amino-terminal 135 amino acid residues of human interferon-γ (IFN-γ) by interposing a methionine codon sequence, and the result-ing hybrid gene was efficiently expressed in Escherichia coil cells. Recombinant human PSTI (rHu-PSTI) was separated from the IFN-γ/PSTI fused protein by cleavage at the methionine residue with cyanogen bromide. Finally, rHu-PSTI was purified by affinity chromatography on a bovine trypsin-CH-Sepharose 4B column. The amino acid composition, partial amino-terminal sequence, disulfide formation, human trypsin inhibitory activity, and immunoreactivity against rabbit anti-human PSTI serum of rHu-PSTI corresponded to those of the natural form.View full abstractDownload PDF (391K) -
Taiko AKAC, Teruaki AKAO, Kyoichi KOBASHI1987 Volume 102 Issue 3 Pages 613-619
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS7β-Hydroxysteroid dehydrogenase (7β-HSD) was produced by Ruminococcus sp. P01-3 obtained from among human intestinal bacteria. The enzyme was purified from a crude extract by ammonium sulfate fractionation, and Butyl-Toyopearl 650M, Sephadex G-150, Mãtrex Red A and Octyl-Sepharose chromatographies. The purified enzyme was obtained as a single band on polyacrylamide gel electro-phoresis with enzyme activity staining and as one band corresponding to a molecular weight of 30, 000 on SDS-polyacrylamide gel electrophoresis. On gel filtration, its apparent molecular weight was estimated to be 60, 000. The enzyme had a sulf-hydryl group(s) in its active site. Substrate specificity studies revealed that the enzyme showed absolute specificity for the β-configuration of a hydroxyl group at the 7 position of bile acids, and required NADP+ and NADPH as cosubstrates. The Km values for ursodeoxycholic acid, 7-k etolithocholic acid, NADP+, and NADPH were 5.0, 8.5, 7.7, and 24 μM, respectively.View full abstractDownload PDF (1122K) -
Tomofumi KUROKAWA, Saburo HARA, Tadashi TESHIMA, Tokuji IKENAKA1987 Volume 102 Issue 3 Pages 621-626
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe P1' Ser(50) at the second reactive site of soybean protease inhibitor C-II was replaced with arginine to confirm the contribution of this residue to the inhibition. The Arg derivative had less trypsin inhibitory activity (K1=1 × 10-7 M) than the Ser derivative (K1=2 × 10-8 M), in contrast to the results obtained from studies on peanut protease inhibitor B-III reported in the previous paper (J. Biochem. 101, 723-728 (1987)). These results suggest that each Bowman-Birk type inhibitor has an amino acid at the P1' position inherently best suited to maintaining its inhibitory activity, and that serine is not unique for the P1' amino acid in Bowman-Birk type inhibitors.View full abstractDownload PDF (401K) -
Akira MURASUGI, Nobuyuki TAKEMORI, Shuichi HASHIMOTO1987 Volume 102 Issue 3 Pages 627-633
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOne of the cytocidal (cyt) mutants of adenovirus 2 (Ad2), cyt15, has been shown to have two point mutations separated by 36 bases in its early region 1B (E1B) gene that encodes the 19K (Mr, 19, 000) protein. A part of the cyt15 E1B gene, including one of the point mutations, was probed with a 23-base long oligodeoxyribonucleotide (MEIB23). Another oligonucleotide probe of the same length was used for the corresponding region of the wild type (wt) E1B gene (E1B23). Over the 32P-end labeled oligonucleotide probe, a 25-fold molar excess of a competitor (1) (the un-labeled oligonucleotide of each counterpart) was added to the hybridization mixture. The E1B gene with or without a point mutation could easily be distinguished from its counterpart and quantitated in the presence of its counterpart under the hybridi-zation conditions employed. Under the stringent wash conditions, the E1B gene of wt Ad2 could be completely discriminated from even a 100-fold molar excess of cyt15 E1B gene with the 32P-labeled ElB23 probe. The wt E1B gene could also be quantitated in the presence of a 100-fold molar excess of the mutant E1B gene.View full abstractDownload PDF (3014K) -
Yutaka MUTO, Yuji FUKUMOTO, Yoji ARATA1987 Volume 102 Issue 3 Pages 635-641
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA proton nuclear magnetic resonance (NMR) study is reported of the solution con-formation of human C3a, that is released on activation of C3, the third component of complement. The intact C3a was used along with des-Arg-C3a, which is formed on cleavage of Arg-77 at the C terminal of C3a, and C3a Arg69, which is a 69-residue fragment produced on tryptic digestion of C3. A method of carboxypeptidase digestion/difference spectroscopy (Endo & Arata (1985) Biochemistry 24, 1561-1568) was extensively used for the spectral assignments of Ile-43, Ile-60, Leu-63, Tyr-15, and Tyr-59. On the basis of the results of nuclear Overhauser effect (NOE) mea-surements, we discuss the solution conformation of the C3a molecule. It has been concluded that removal of Arg-77, which is essential for expression of the biological activity of C3a, does not induce any significant change in the solution conformation of the C3a molecule. The C3a molecule is known to consist of a core region that comprises segment Tyr-15-Tyr-59. We conclude that in solution the C terminal segment sticks out of the core and takes on a helix-like conformation. Possible roles of the core region and the N terminal segment in maintaining the conformation of the C terminal segment are briefly discussed.View full abstractDownload PDF (560K) -
Wataru ITO, Miwa NISHIMURA, Nobuo SAKATO, Hajime FUJIO, Yoji ARATA1987 Volume 102 Issue 3 Pages 643-649
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA proton nuclear magnetic resonance (NMR) study is reported of the molecular structural basis of antigen-antibody interactions. An immunologically reactive proteolytic fragment corresponding to one of the antigenic regions on hen egg-white lysozyme (HEL) was used in combination with a monoclonal antibody that recognizes this site. Using spin diffusion, we prepared an antibody in which the magnetization of the antigen binding site was saturated by non-specific nuclear Overhauser effect. Under these conditions the effect of the saturation of the antibody was observed to spread over the peptide fragment through the antigen binding site. On the basis of the results obtained for the intermolecular nuclear Overhauser effect, we discuss how the peptide fragment interacts with the antibody. The side chains of aromatic residues, Trp, Tyr, and His, and of ionic residues, especially Arg, Lys, and Glu, are suggested to be important in the antigen-antibody interaction.View full abstractDownload PDF (852K) -
Hirohiko KIKUCHI, Yuji GOTO, Kozo HAMAGUCHI, Shinzaburo ITO, Masahide ...1987 Volume 102 Issue 3 Pages 651-656
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe constant (CL) domain of the type-λ immunoglobulin light chain contains two tryptophyl residues. The fluorescence lifetimes of the CL fragment and its two modified forms, the reduced CL, and reduced and alkylated CL fragments, were measured at pH 7.5 and 25°C. The fluorescence decay kinetics of these fragments were described in terms of two lifetimes. The static and dynamic quenching reactions by acrylamide of the tryptophyl residues of these fragments were measured and their dynamic properties were compared with the static properties reported previously (Goto, Y. & Hamaguchi, K. (1979) J. Biochem. 86, 1433-1441; Goto, Y. & Hamaguchi, K. (1986) Biochemistry 25, 2821-2828; Ashikari, Y., Arata, Y., & Hamaguchi, K. (1985) J. Biochem. 97, 517-528). It was found that although the static conformation of the reduced CL fragment is very similar to that of the intact CL fragment, the dynamic conformations of these two fragments differ considerably.View full abstractDownload PDF (442K) -
Nobuo SHIMODA, Hisako MURAMATSU, Tatsushi OSAWA, Toshihide YAMADA, Mas ...1987 Volume 102 Issue 3 Pages 657-664
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSReceptors for peanut agglutinin (PNA) were isolated from Kato III human gastric cancer cells by affinity chromatography on PNA agarose, and were labeled by the galactose oxidase-NaB3H4, method. Alkaline NaBH4 treatment of the labeled receptors released two small oligosaccharide alcohols, which were identified as Galβ1→3GalNAc-ol and Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAc-ol. Higher oligosaccharides and glycopeptides of both N- and O-linked type were also detected, but they did not appear to bear PNA binding sites. The presence of oligo-N-acetyllactosamine units in the N-linked type sugars was indicated by endo-β-galactosidase digestion.View full abstractDownload PDF (595K) -
Tatsuo SUGANUMA, Hisako MURAMATSU, Fusayoshi MURATA, Takashi MURAMATSU1987 Volume 102 Issue 3 Pages 665-671
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN-Acetylglucosaminide β1→4 galactosyltransferase was chromatographically purified about 1, 700-fold from F9 embryonal carcinoma cells after solubilization with Triton X-100, using N-acetylglucosamine as the acceptor. As the last step of the purification, affinity chromatography was performed either on N-acetylglucosamine-Sepharose or on α-lactalbumin-Sepharose: in both cases, two protein bands with molecular weights of around 68, 000 and 59, 000 were detected by SDS-polyacrylamide gel electrophoresis of the purified preparations. The enzymological properties including behavior toward α-lactalbumin were very similar to those of the enzyme from other sources. The specificity of the enzyme was confirmed by determining the structure of the product; it was mostly Galβ1→4G1cNAc. β-Galactosidase-treated embryoglycan (poly-N-acetyllactosamine) and asialo-agalactofetuin could serve as acceptors with the purified enzyme. Thus, the embryonic enzyme, apparently involved in the synthesis of poly-N-acetyllactosamines, has properties similar in several respects to those of the β-galactosyltransferases so far studied.View full abstractDownload PDF (1061K)
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