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Volume 121 (1997)
- Issue 6 Pages 997-
- Issue 5 Pages 811-
- Issue 4 Pages 633-
- Issue 3 Pages 399-
- Issue 2 Pages 179-
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Volume 121, Issue 6
Displaying 1-29 of 29 articles from this issue
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Keun-Hong Park, Ryotaro Takei, Mitsuaki Goto, Atsushi Maruyama, Akira ...1997 Volume 121 Issue 6 Pages 997-1001
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA reducing glucose-carrying polymer, called poly [3-O-(4'-vinylbenzyl)-n-glucose] (PVG), interacted with erythrocytes carrying the type-1 glucose transporter (GLUT-1) on the cell membrane. The cooperative interaction between a number of GLUT-1s and a number of reducing 3-O-methyl-n-glucose moieties on a PVG polymer chain is responsible for the increase in the interaction with erythrocytes. In contrast to the PVG homopolymer, other sugar-carrying polymers showed lower interaction with erythrocytes. The affinity of erythrocytes and PVG was studied using FITC-labeled glycopolymers. The fluorescence intensity significantly changed, whereas a small change in fluorescence intensity was observed for other homopolymers. The specific interaction between GLUT-1 on erythrocytes and the PVG polymer carrying reducing glucose was suppressed by the inhibitors, phloretin, phloridzin, and cytochalasin B, and a monoclonal antibody to GLUT-1. Direct observation by confocal laser microscopy with the use of FITC-labeled PVG demonstrated that erythrocytes interacted with the soluble form of the PVG polymer via GLUT-1, while fluorescence labeling of the cell surface was prevented on pretreatment with the monoclonal antibody to GLUT-1.View full abstractDownload PDF (1722K) -
Yoichiro Arata, Jun Hirabayashi, Ken-ichi Kasai1997 Volume 121 Issue 6 Pages 1002-1009
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSome properties of recombinant proteins derived from the 32-kDa galectin isolated from the nematode Caenorhabditis elegans, which lectin is composed of two tandemly repeated homologous domains [Hirabayashi et al. (1992) J. Biol. Chem. 267, 15485], were studied in order to elucidate the function of this unique polypeptide architecture. We expressed the whole molecule (N32), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) in Escherichia coli using the expression vector pET21a. All of the recombinant proteins were bound by asialofetuin-Sepharose. CD spectra of the recombinant proteins indicated all of them to be rich in β-structure and properly refolded. Gel filtration on an HPLC column suggested that all of them existed as monomers. Neither Nh nor Ch seemed to form dimers, in contrast to vertebrate proto-type galectins. Only N32 showed hemagglu-tination activity towards trypsinized rabbit erythrocytes. Comparison of the affinity of N32, Nh, and Ch for asialofetuin-Sepharose by frontal affinity chromatography [Kasai et al. (1986) J. Chromatogr. 376, 33] showed that Ch has 7-fold weaker affinity than N32, and Nh proved to have still weaker affinity. Since the Asn residue in the CRD (carbohydrate recognition domain), which is conserved in all other galectins, is substituted by Ser in the case of Nh, these data suggest that the two CRDs in this tandem-repeat galectin have different sugar binding properties and that the 32-kDa galectin may serve as a heterobifunc-tional crosslinker.View full abstractDownload PDF (2527K) -
Yuichi Narita, Sen-ichi Oda, Akihiko Moriyama, Osamu Takenaka, Takashi ...1997 Volume 121 Issue 6 Pages 1010-1017
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree pepsinogens, namely, pepsinogens A, C-1, and C-2, were purified from gastric mucosa of adult house musk shrew (Suncus murinus) by conventional chromatographic and gel filtration procedures. The molecular masses were 40, 39, and 41 kDa for pepsinogens A, C-1, and C-2, respectively. Pepsinogen C-2 contains an Asn-linked carbohydrate chain(s) of about 2 kDa. Each pepsinogen was converted to pepsin through an intermediate form under acidic conditions. By NH2-terminal sequence analysis of these protein species, the amino acid sequences of activation segments (proparts) of pepsinogen A and C-1 were determined to be LYKVPLVKKKSLRQNLIENGLLKDFLAKHNVNPASKYFPTE and KVTKVTLKKF-KSIRENLREQGLLEDFLKTNHYDPAQKYHFGDF, respectively. The similarity of these two sequences is nearly 50%. Each pepsin cleaved preferentially peptide bonds between hydrophobic and aromatic amino acids, or bonds on either side of these amino acids. Although each activation segment had several sites susceptible to pepsin action, activation proceeded by limited cleavages of the segment, presumably due to the steric inflexibility of the segment in native pepsinogen. The activity of pepsin A was inhibited completely in the presence of a more than equimolar amount of pepstatin, while a hundred-molar excess amount of pepstatin was needed for the complete inhibition of the activity of pepsins C-1 and C-2.View full abstractDownload PDF (2133K) -
Taishin Takuma, Tokuro Ichida1997 Volume 121 Issue 6 Pages 1018-1024
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe evaluated the role of cytosolic phospholipase A2 (PLA2) in the exocytosis of amylase from parotid acinar cells. The exocytosis stimulated by isoproterenol was dose-depen-dently inhibited by bromoenol lactone (BEL), a potent suicide inhibitor of Ca2+-indepen-dent cytosolic PLA2. The IC50, value of BEL was approximately 7 μM. AACOCF3, a selective inhibitor of Ca2+ -dependent cytosolic PLA2, did not inhibit the exocytosis at least up to 30μM. BEL also inhibited amylase release evoked by forskolin and membrane-permeable cAMP, but it did not inhibit cAMP-dependent protein kinase activity. PLA2 activity in parotid acinar cells was found to be predominantly Ca'-independent, and was strongly inhibited by BEL, whose IC50 value was approximately 2 pM when it was applied to intact acini. Although isoproterenol scarcely enhanced [3H] arachidonic acid release from intact acinar cells, BEL dose-dependently decreased the basal arachidonic acid release to approximately one half of the control value. These results suggest that thecytosolic Ca2+- independent PLA2 activity plays a role in the membrane fusion process ofexocytosis in parotid acinar cells.View full abstractDownload PDF (803K) -
Satoko Isemura, Eiichi Saitoh1997 Volume 121 Issue 6 Pages 1025-1030
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide sequence of gene PBI, the putative translational product of which is homologous to salivary proline-rich protein P-B, has been determined. PBI is 6.4 kb long and contains 3 exons. PBI appears to code for the precursor of a proline-rich protein which we have designated P-B1. The P-B1 precursor is composed of 134 amino acid residues including 22 residues of signal sequence, 23 residues of N-terminal sequence, five repeating units of 13 to 14 residues and 22 residues of C-terminal sequence. The signal sequence of this precursor is identical with that of the P-B precursor. The N-terminal 61 residue sequence of P-B1 has homology of 75% with the whole sequence of P-B (57 residues), when deletion of 4 residues is taken into account. P-B1 is 55 residues longer than P-B. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession number D89501.View full abstractDownload PDF (1543K) -
Toshiharu Suzuki, Yuji Tanaka, Masami Ishida, Morio Ishizuka, Akihiko ...1997 Volume 121 Issue 6 Pages 1031-1034
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mutant plasmid with elevated expression efficiency of GC-rich Thermusthermophilus leuB gene was screened in Escherichia coli. A wild-type plasmid pHB2 carrying T. thermophilus leuB gene was introduced into leuB-deficient E. coli C600 cells. During successive cultures of the transformant in leucine-free medium, the original plasmid was spontaneously replaced by a mutant plasmid. The expression efficiency of the leuB gene on the mutant plasmid was 4.8-fold higher than that of the wild-type plasmid. Sequencing of the mutant plasmid revealed that the open reading frame (ORF1) in front of the leuB gene was shortened from 822 to 306 bp. Several expression vectors were constructed to investigate the effect of the length of ORF1, and the optimal length for the expression of the following leuB gene was determined. It was also shown that the stop codon of ORF1 should be overlapped with the initiation codon of leuB gene for the highest efficiencyView full abstractDownload PDF (417K) -
Hiroyuki Toda, Toshiyuki Takeuchi, Naohiro Hori, Keizo Ito, Kenzo Sato1997 Volume 121 Issue 6 Pages 1035-1040
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rat catalase gene promoter lacks a TATA box, and has eight initiation sites of transcription as well as several GT and CCAAT boxes. To elucidate the mechanism of transcription in this TATA-less promoter, we analyzed nuclear factors binding to this regulatory region of the catalase gene. Functional analysis of the promoter region revealed that a pair of inverted repeat motifs located on both sides of the transcription initiation sites played an important role in the gene expression. The core sequence of this element is GYCMGGCCCKCTCYKG (M=A/C, K=G/T, Y=T/C), and four species of complex were observed to bind to this sequence. Furthermore, this element in the chimeric promoter negatively interacted with the initiator element of the terminal deoxynucleotidyl transferase gene. These results suggested that the rat catalase gene is regulated by a novel mechanism involving the inverted repeated structure of the promoter and characteristic binding factors.View full abstractDownload PDF (3651K) -
Motoi Itoga, Mikako Tsuchiya, Hiroshi Ishino, Makoto Shimoyama1997 Volume 121 Issue 6 Pages 1041-1046
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOne biological effect of nitric oxide (NO) has been believed to be exerted through induction of the ADP-ribosyltransferase activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Though this notion is based on the finding that NO increases the auto-ADP-ribosylation of GAPDH, controversial data have also been reported. To determine whether or not NO really activates ADP-ribosylation, we re-examined the NO-induced modification of GAPDH with NAD+. GAPDH was modified equally with [adenosine-14C]NAD+ and [carbonyl-14C]NAD+, indicating that the glycoside bond of NAD+ between ADP-ribose and nicotinamide is intact. The release of nicotinamide from NAD+ was not evident during incubation of GAPDH with [carbonyl-14C]NAD+. Thus, the modification of GAPDH is apparently not ADP-ribosylation. In addition, we found that basal and glyceraldehyde-3-phosphate-induced modifications of GAPDH, both of which have also been explained as ADP-ribosylation, were not ADP-ribosylation, and that the modification of GAPDH in the absence and presence of NO or GA3P wadistinct in the dithiothreitol effect or resistance to HgCl2.View full abstractDownload PDF (3286K) -
Makoto Kadotani, Teruaki Nishiuma, Masakazu Nanahoshi, Yosuke Tsujishi ...1997 Volume 121 Issue 6 Pages 1047-1053
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhorbol ester treatment of Chinese hamster ovary cells stably overexpressing the δ isoform of protein kinase C induced the association of the isoform with the particulate fraction and the tyrosine phosphorylation of a small portion of the δisoform. The δ isoform without tyrosine phosphorylation was recovered as an enzyme dependent on phospholipid and diacylglycerol, whereas the tyrosine-phosphorylated δ isoform was recovered in two fractions, one dependent on, and the other independent of, phospholipid and diacylglycerol. The tyrosine-phosphorylated δisoform independent of lipid activators might be associated with phorbol ester and phospholipids. Immunoblot analysis revealed that the c isoform is a doublet protein of 76 and 78 kDa, and that the δ isoform fraction without tyrosine phosphorylation contained 76- and 78-kDa proteins, whereas the tyrosine-phosphorylated δ isoform contained the 78-kDa protein but not the 76-kDa protein. In vitro analysis showed that the 78-kDa protein of the δ isoform without tyrosine phosphorylation is an efficient substrate of tyrosine kinase only when phosphatidylserine and either diacylglycerol or phorbol ester are present; however, the 76-kDa protein can not be tyrosine-phosphorylated even in the presence of these lipid activators. The phospholipid and diacylglycerol-dependent form of the tyrosine-phosphorylated enzyme isolated from the cell line required lower concentrations of phosphatidylserine and phorbol ester for its activity in vitro as compared with the enzyme without tyrosine phosphorylation. These results suggest that the tyrosine-phosphorylated enzyme generated upon stimulation of the cells may associate with membranes and exert its full activity even with the lower concentrations of the lipid activators.View full abstractDownload PDF (4544K) -
Pham Hùng, Koji Yamada, Beong Ou Lim, Mitsuo Mori, Takeshi Yuki ...1997 Volume 121 Issue 6 Pages 1054-1060
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMesenteric lymph node (MLN) and spleen lymphocytes of Sprague-Dawley rats were cultured with 1 mM unsaturated fatty acids (UFAs) with or without 100 μM α-tocopherol (Toc), and the immunoglobulin content and thiobarbituric acid (TBA) value of the culture media were measured to clarify the relationship between lipid peroxidation and the IgE level in the culture medium. The increase in the IgE content and TBA value induced by UFAs was alleviated in the presence of Toc in both lymphocytes, and was correlated well with their oxidation rates in most cases. γ-Linolenic acid enhanced the IgE level much more than would be expected from its oxidation rate in both lymphocytes, and linoleic acid showed similarly high activity only in splenocytes. These results suggest that lipid peroxidation is partly responsible for the enhancement of IgE level induced by UFAs.View full abstractDownload PDF (776K) -
Masahiro Tomita, Takashi Kitajima, Katsutoshi Yoshizato1997 Volume 121 Issue 6 Pages 1061-1069
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe present study describes the production of human procollagen I in a baculovirus expression system. Recombinant baculovirus carrying proα1(I) or proα2(I) cDNA was constructed and infected to Sf9 cells. Full-length proα1(I) or proα2(I) chains were synthesized by the cells infected with either of the recombinant viruses. The proα1(I) chains formed pepsin-resistant homotrimers stabilized by interchain disulfide bonds, a small proportion of which was secreted into the culture medium. The proα2(I) chains were not linked into trimers by disulfide bonds and failed to form stable triple helices, although some chains were suggested to exist as dimers or unstable trimers in which only two chains were linked by disulfide bonds. In spite of their non-helicity, the proα2(I) chains were secreted at a higher rate than the proα1(I) chains. Sf9 cells simultaneously synthesized both proα1(I) and proα2(I) chains when the cells were co-infected with the two recombinant viruses. Pepsin-treatment of the product clearly demonstrated the production of procollagen I heterotrimers composed of two proα1(I) chains andone proα2(I) chain, homotrimers of the proα1(I) chains being negligible. This expression system appears to offer a unique means of studying the mechanism of chain association and secretion during procollagen biosynthesis.View full abstractDownload PDF (6532K) -
Christine Ellouze, Bengt Norden, Masayuki Takahashi1997 Volume 121 Issue 6 Pages 1070-1075
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecA protein catalyzes the DNA annealing and mimics the DNA strand exchange reaction in vitro in the presence of ATP or its non-hydrolyzable analog, adenosine 5'- O-3-thiotri-phosphate (ATP γS). For these activities RecA coordinates two DNA molecules [Takahashi, M. and Norden, B. (1994) Adv. Biophys. 30, 1-35]. In order to get a better understanding of how RecA performs the search for sequence complementarity or homology between two DNA molecules, the association and dissociation kinetics of a second DNA molecule to and from RecA in the presence of ATPγS have been investigated. The kinetics were monitored by fluorescence measurements of partly etheno-modified poly(dA) assisted by linear dichroism measurements of the flow-oriented complex. The association of the second DNA is fast, regardless of whether the sequence is complementary or not. By contrast, the dissociation kinetics is strongly dependent on sequence complementarity. If the second DNA is complementary to the first, dissociation is extremely slow, whilst that of non-com-plementary second DNA is fast. In no case does the first DNA leave the RecA fiber. Our findings indicate that the dissociation step is important in the search for homology by RecA.View full abstractDownload PDF (708K) -
Hiroyuki Motoshima, Shouhei Mine, Kiyonari Masumoto, Yoshito Abe, Hiro ...1997 Volume 121 Issue 6 Pages 1076-1081
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the N-terminal region of the α-helix of the c-type lysozymes, two Asx residues exist at the 18th and 27th positions. Hen lysozyme has Asp18/Asn27 (18D/27N), and we prepared three mutant lysozymes, Asn18/Asn27 (18N/27N), Asn18/Asp27 (18N/27D), and Asp18/Asp27 (18D/27D). The stability of the wild-type (18D/27N) lysozyme supported the existence of a hydrogen bond between the side chain of Asp18 and the amide group at the N1 position in the α-helix, while the stability of the 18N/27D lysozyme supported the presence of the capping box between the Ser24 (N-cap) and Asp27 residues. Although electrostatic repulsion was observed between Asp18 and Asp27 residues in 18D/27D lysozyme, the dissociation of each residue contributed to stabilizing the B-helix in 18D/27D lysozyme through hydrogen bonding and charge-helix macrodipole interaction. This is the first evidence that two neighboring negative charges at the N-terminus of the helix both increased the stability of the proteinView full abstractDownload PDF (741K) -
Hideo Satsu, Hirohito Watanabe, Soichi Arai, Makoto Shimizu1997 Volume 121 Issue 6 Pages 1082-1087
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe characterized the taurine transport system in human intestinal Caco-2 cells and showed that it is subject to adaptive regulation. The activity of taurine transport in Caco-2 cells was evaluated by means of an Na+-and Cl--dependent high-affinity transport system, the characteristics of which were similar to those of the β-amino acid-specific taurine transport system previously described for various tissues. The activity of taurine transport was down-regulated on culturing in taurine-containing medium. This taurine-induced downregulation was dependent on both the incubation time with taurine and the concentration of taurine. Hypotaurine and β-alanine were also capable of inducing this adaptive regulation, whereas α-amino acids and γ-aminoisobutyric acid were not. Kinetic analysis of control and taurine-treated cells suggested that the down-regulation was associated with a decrease in the maximal velocity of taurine transport and also with a decrease in the affinity of the taurine transporter. Cycloheximide treatment weakened the taurine-induced down-regulation. The mRNA level of the taurine transporter (HTAU type) in taurine-treated cells was markedly decreased compared with in control cells. These results indicate that a complex regulatory mechanism is involved in this down-regulation.View full abstractDownload PDF (1777K) -
Sailaja S. Seeram, Kazumi Hiraga, Atsushi Saji, Misao Tashiro, Kohei O ...1997 Volume 121 Issue 6 Pages 1088-1095
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStreptomyces metalloproteinase inhibitor (SMPI), isolated from Streptomyces nigrescens TK-23, is a small proteinaceous metalloproteinase inhibitor consisting of 102 amino acidresidues and two disulfide bridges. SMPI specifically inhibits metalloproteinases such asthermolysin. After prolonged incubation with a catalytic amount of thermolysin, it iscleaved at Cys64-Val65 [Murai, H., Hara, S., Ikenaka, T., Oda, K., and Murao, S. (1985) J. Biochem. 97, 173-180]. Hence, for identification of the reactive site, mutants were constructed by substituting Val65 with various amino acid residues (Leu, Ile, Phe, Tyr, Gly, Ser, Lys, and Glu). The mutants were analyzed for inhibitory activity. Among them, V65I, V65L, V65F, and V65Y retained strong inhibitory activity, whereas V65S, V65G, V65K, and V65Eshowed very weak inhibitory activity against thermolysin. The K1 values were found to beof the order of 10-10M by using a fluorogenic substrate, MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. In addition, susceptibility to enzyme degradation was analyzedby means of limited proteolysis with thermolysin. Mutants which retained strong inhibi-tory activity were cleaved by thermolysin only at the reactive site, in the same way asnative SMPI. The mutants which showed weak inhibitory activity underwent rapiddegradation. These results were consistent with the substrate specificity of thermolysin. Based on these results, the reactive site of SMPI was identified as Cys64-Val65.View full abstractDownload PDF (4096K) -
Claudio Napoli, Francesco Paolo Mancini, Gaetano Corso, Antonio Malorn ...1997 Volume 121 Issue 6 Pages 1096-1101
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsual purification procedures of LDL and Lp(a) require numerous, extensive and prolonged sample handlings: this greatly increases the possibility of spontaneous oxidation. We have developed a method which, making use of two short-run ultracentrifugations in vertical rotors alternated by two rapid column-chromatography steps (SRUC), significantly shortens the preparation time to 3.5h (LDL) and does not demand additional instrumentation or particular accuracy. Purification of Lp(a) requires a further wheat germ agglutinin chromatographic step, which can be accomplished within 30min. More importantly, the method significantly reduces spontaneous oxidation as compared with classical isolation procedures. LDL isolated by the standard sequential method exhibits more extensive apolipoprotein B100 degradation, lipid peroxi dation, and endogenous antioxidant (vitamin E) loss than the same lipoproteins obtained by means of the SRUC. This procedure may have be particularly valuable in experiments evaluating the effects of oxygen radical-induced modifications, especially in vitroView full abstractDownload PDF (3532K) -
Yi Xiong, Binggen Rue1997 Volume 121 Issue 6 Pages 1102-1106
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mouse metallothionein-I (mMT-I) cDNA was amplified by polymerase chain reaction (PCR), inserted into vector pGEX-4T-1, and expressed in Escherichia coli as a carboxyl terminal extension of the 26-kDa glutathione-S-transferase (GST). Analyzed by SDS-PAGE, the amount of the expressed fusion protein GST-MT was over 50% of total cellular proteins. After the fusion protein had been digested with thrombin on a Glutathione-Sepharose 4B affinity chromatography column, recombinant mMT-I was purified by gel filtration on Sephadex G50. The results of molecular mass, amino acid composition and sequence of 10 amino acids at the N-terminus of the recombinant mMT-I demonstrate that the purified protein is the one we desired. The ratios of metal:protein and thiol :protein are the same as those of wild-type MT. The half-dissociation pHs of Cd, Cu, and Zn from recombinant mMT-I were 3. 57, 1. 40, and 5. 20, respectively, which are in agreement with those from native rabbit MT-I. The ultraviolet absorbance and circular dichroism (CD) spectra at pH 8. 0 and pH 2. 0 were all similar to those of native MT, indicating that they have the same metal-thiolate structure even though six amino acid residues have been added at the N-terminus of the recombinant protein.View full abstractDownload PDF (1625K) -
Yasushi Kamisaka, Sanjay Mishra, Toro Nakahara1997 Volume 121 Issue 6 Pages 1107-1114
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDiacylglycerol acyltransferase (DGAT) [EC 2.3.1.20] was purified to apparent homogeneity from the lipid body fraction of an oleaginous fungus, Mortierella ramanniana var. angulispora. The enzyme was solubilized from the lipid body fraction with 0.1% Triton X-100, and purified by subsequent column chromatography on Yellow 86 agarose, Superdex-200, Heparin-Sepharose, second Superdex-200, and second Yellow 86 agarose. The enzyme activity was finally enriched 4, 802-fold over that of the starting 1, 500×g supernatant. The apparent molecular mass of the enzyme was 53 kDa on SDS-polyacrylamide gel electrophoresis. The purified enzyme did not exhibit glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, lipase, transacylase, or acyl -CoA bydrolase activities, although 2-monoolein was acylated with about a half of the enzyme activity toward 1, 2-diolein. The purified DGAT depended on exogenous sn-1, 2-diolein and oleoyl-CoA, with the highest activity at about 200 and 20 μM, respectively. Purified DGAT utilized a broad range of molecular species of both diacylglycerol and acyl-CoA as substrates. The highest activity was observed with sn-1, 2-diolein and lauroyl-CoA. Anionic phospholipids such as phosphatidic acid (PA) activated the purified enzyme, as found for the Triton X-100 extract. Sphingosine dose-dependently inhibited DGAT activity activated by PA and basal activity without PA. These results provide a basis for further studies on the molecular mechanism of triacylglycerol biosynthesis and lipid body formation, in which DGAT plays an important role.View full abstractDownload PDF (2152K) -
Ikuko Hayashi, Takashi Yokogawa, Gota Kawai, Takuya Ueda, Kazuya Nishi ...1997 Volume 121 Issue 6 Pages 1115-1122
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mammalian mitochondrial tRNASerGCU (mt tRNASerGCU) has a unique structure in that it lacks the whole D arm. To elucidate its higher-order structure, we synthesized unmodified bovine mt tRNASerGCU using T7 RNA polymerase and measured its 1H-NMR spectrum in the imino proton region. Although the imino proton signals heavily overlapped, we succeeded in assigning all the seven imino proton signals of the G-C base pairs by a combination of base replacement and 15N-labeling of the G residues of a whole tRNA molecule or of the 3'-half fragment. The results indicate that the tRNA possesses the secondary structure that has been supposed on the basis of biochemical studies. Analysis of the effect of the magnesium concentration on the G-C pairs suggests that the acceptor and T stems do not form a co-axial helix, and that the core region of the tRNA does not interact with magnesium ions. These features are significantly different from those of canonical tRNAs. Despite this, it is very likely that the tRNA as a whole takes a nearly L-shape tertiary structure.View full abstractDownload PDF (2128K) -
Taiichi Sakamoto, Yoichiro Tanaka, Tomoko Kuwabara, Mi Hee Kim, Yasuyu ...1997 Volume 121 Issue 6 Pages 1123-1128
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProperties of a hepatitis delta virus (HDV) RNA ribozyme system, which consists of three RNA oligomer strands (substrate 8-mer ; enzyme 16-mer plus 35-mer) and contains a hybrid sequence of genomic and antigenomic RNA cores, are reported. Effects of Mg2+ concentration, divalent metal ion species, pH, and temperature on the cleavage activity were examined. The substrate cleavage activity increased with increasing Mg2+ concentration (0-100 mM). Ca2+ and Mn2+ ions were the most effective divalent cations and Mg2+ was less effective. The cleavage activity increased with increasing pH (5-7.5). The optimum temperature for the cleavage activity was 25-40°C. The Mg2+ concentration, pH and temperature dependencies are different from those reported for the single-strand ribozymes (about 90-mer) although the divalent metal ion preference is very similar. Conformational change induced by Mg2+ ion titration was monitored by CD. The CD data and the activity-Mg2+ concentration data were analyzed by curve-fitting analysis using equations derived for multiple metal ion binding mechanisms. The data can be explained by a model in which three Mg2+ ions bind to one ribozyme unitView full abstractDownload PDF (740K) -
Yasuo Suda, Tomohiko Ogawa, Wataru Kashihara, Masato Oikawa, Takashi S ...1997 Volume 121 Issue 6 Pages 1129-1133
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chemical structure of a novel lipid A, which was obtained as a major component from lipopolysaccharide of Helicobacter pylori strain 206-1, was determined to be a glucosamine β(1-6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyocta-decanoic acid and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2- and 2'-position, respectively. The absence of a phosphoryl group at the 4'-position and fatty acyl groups at the 3- and 3'-position, and the stoichiometric presence of 2-aminoethyl phosphate at the 1-position are unique features, distinguishing it from the lipid A of enterobacteria.View full abstractDownload PDF (568K) -
Kyoko Hiramatsu, Hiroko Miura, Kayo Sugimoto, Sachiko Kamei, Kentaro I ...1997 Volume 121 Issue 6 Pages 1134-1138
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN8-Acetylspermidine was coupled to mercaptosuccinylated BSA using a bifunctionalcross-linker, N-(4-maleimidobutyryloxy)succinimide, and the resulting conjugate was used to raise N1, N8-diacetylspermidine (DiAcSpd)-specific antibodies in rabbits. DiAc-Spd-specific antibodies were enriched from crude sera through a series of affinity-based fractionations using ligands with structures mimicking thoseof DiAcSpd and monoacetyl-spermidines. With the N8-acetylspermidine-BSA conjugate as a solid phase antigen in a competitive ELISA system, the selectivity for DiAcSpd over other polyamine species was high, but competition by DiAcSpd added to the fluid phase was too weak for the system to be applicable to measurement of the concentration of DiAcSpd in human urine. In contrast, with the N1-acetylspermidine BSA conjugate adsorbed on the ELISA plate, DiAcSpd efficiently competed for the same antibody, thus yielding a sensitive competitive ELISA system for measuring DiAcSpd. The K1 value for DiAcSpd with the latter competitive ELISA system was 54 nM, and the cross-reactivity with DiAcSpd, N1, N12-diacetylsper-mine, N8-acetylspermidine, N1-acetylspermidine, and acetylputrescine was 100, 1. 2, 0.74, 0. 12, and 0. 08%, respectively. The DiAcSpd-specific antibodies and the competitive ELISA system developed in this study will prove to be useful for analyzing the urinary level of DiAcSpd, that was recently shown to be a promising diagnostic and prognostic indicator of malignant disorders.View full abstractDownload PDF (693K) -
Haruhiko Tamaoki, Chiaki Setoyama, Retsu Miura, Yasuzo Nishina, Iwaho ...1997 Volume 121 Issue 6 Pages 1139-1146
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo forms of rat peroxisomal acyl-CoA oxidase (ACO-I and -II) interact with the substrate analogs, 3-ketoacyl-CoAs, forming a complex characterized by the so-called charge-transfer (CT) band around 575 nm in the absorption spectra. The CT band of ACO-I exhibited a broad dependency on the acyl chain-length from C4 to C16, whereas that of ACO-II showed increased intensity with a longer acyl chain to reach a maximum with a chain-length of C12. These chain-length dependencies of the CT bands were compared with those of the enzymatic activities reported previously [Setoyama et al. (1995) Biochem. Biophys. Res. Commun. 217, 482-487] . The differences in spectroscopic and enzymatic properties between ACO-I and -II suggest that the amino acid stretch corresponding to the third exon in the ACO sequence affects the binding of the ligand and substrate, since the difference in the primary structure between ACO-I and -II lies in the short amino acid stretch corresponding to the third of the total of 14 exons. On the other hand, resonance Raman spectra of the complexes of ACO-I and -II with 3-ketoacyl-CoAs excited in the CT band showed similar features. The two prominent FAD bands II and III, associated with the C(4a)=N(5) moiety of FAD, were observed at 1, 577 and 1, 545cm-1, respectively. In contrast, the bands at 1, 615 and 1, 493cm-1 in the ACO-I•3-keto-C8-CoA complex were assigned to the stretching modes of C=O at positions 3 and 1 of the ligand, respectively, by using the isotopically labeled ligands. Both C=O stretching bands were shifted to lower wave numbers upon complex formation with ACO-I, implying that the C=O bond involves the single bond (C-O-) character in the active site cavity. The downshift of the C(1)=O stretching band was larger than that of the C(3)=O stretching band. Therefore, the ligand lies in the active site as the anionic form with a major contribution from C(1)-O-. These observations demonstrate that the CT band around 575 nm arises from the charge-transfer interaction between the oxidized FAD and the enolate transformed after the elimination of the a-proton. The band II of FAD in the complexes reveals a significant decrease in the frequency in comparison with the complexes of medium-chain acyl-CoA dehydrogenase (MCAD) with 3-ketoacyl-CoA. This observation suggests a difference between ACO and MCAD in the hydrogen-bonding network associated with enzyme-bound FAD.View full abstractDownload PDF (967K) -
Iwaho Hazekawa, Yasuzo Nishina, Kyosuke Sato, Motoaki Shichiri, Retsu ...1997 Volume 121 Issue 6 Pages 1147-1154
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRaman spectroscopy was used to investigate the hydrogen bonding at the C(4)=O moiety ofthe isoalloxazine nucleus in a series of flavins and flavoproteins. Isotope effects of Ramanbands confirmed that the band observed around 1, 710 cm-1 is mainly derived from C(4)=Ostretching vibrational mode. A linear correlation was observed between the frequency ofC(4)=O stretching and the chemical shift of 13C(4), suggesting that the data from both Ramanand NMR spectroscopies reflect a common perturbation, i. e., hydrogen bonding. Themaximum difference of C(4)=O frequency among flavins and flavoproteins examined is 36cm-1 [1, 723 cm-1 for riboflavin-binding protein (Kim, M. and Carey, P. C. (1993) J. Am. Chem. Soc. 115, 7015-7016) and 1, 687 cm-1 for the complex of medium-chain acyl-CoAdehydrogenase with acetoacetyl-CoA] ; the maximum difference of 40-70 kJ/mol in thehydrogen bonding strength at the C(4)=O exists among flavoproteins. By use of an empiricallinear correlation between the frequency of C=O stretching and the bond length of the C=0, it is estimated that the maximum difference in the bond length among flavoproteins treatedhere is ca. 0.017 Å. The hydrogen bonding at the C(4)=O in medium-chain and short-chainacyl-CoA dehydrogenases becomes stronger upon complexation with substrate analogs. Since the hydrogen bonding at the C(4)=O is expected to enhance the electron-acceptingcapacity of the N(5) position, substrate-binding itself probably raises the reactivity offlavin, through enhancing the hydrogen bonding.View full abstractDownload PDF (1048K) -
Lidong Liu, Tohru Yoshimura, Keiji Endo, Nobuyoshi Esaki, Kenji Soda1997 Volume 121 Issue 6 Pages 1155-1161
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA glutamate racemase gene (murI) was found in Bacillus pumilus cells and cloned into Escherichia coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B. pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG. The deduced amino acid sequence shows significant similarity with those of glutamate racemases from E. coli (ratio of identical residues, 28%), Pediococcus pentosaceus (44%), and Staphylococcus haemolyticus (49%). B. pumilus MurI was expressed as a fusion protein connected to the N-terminal 12 residues of β-galactosidase; the fusion protein showed glutamate racemase activity, and resembled the enzyme of P. pentosaceus in physicochemical and enzymological properties.View full abstractDownload PDF (3519K) -
Kazuhiro Takahashi, Eishi Hara, Kazuhiro Ogawa, Dai Kimura, Hiroyoshi ...1997 Volume 121 Issue 6 Pages 1162-1168
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo explore the involvement of nitric oxide (NO) in the induction of heme oxygenase-1, an essential enzyme in heme catabolism, we studied the effects of NO donors on the expression of heme oxygenase-1 mRNA in HeLa human cervical cancer cells. Treatment with each of three NO donors, sodium nitroprusside, 3-morpholinosydnonimine, and S-nitroso-L-glu-tathione, caused noticeable increases in the expression levels of heme oxygenase-1 mRNA, but not heme oxygenase-2 mRNA. On the other hand, nitrite or 8-bromo cGMP exerted no noticeable effect on the levels of heme oxygenase-1 mRNA. We showed that sodium nitroprusside also increased the levels of heme oxygenase-1 protein. The sodium nitroprus-side-mediated increase in heme oxygenase-1 mRNA levels was abolished by treatment with actinomycin D. The expression levels of heme oxygenase-1 mRNA were also increased by NO donors in human melanoma and neuroblastoma cell lines. Thus, the observed induction of heme oxygenase-1 may represent an important response to NO or NO-related oxidative stress. The half lives of heme oxygenase-1 and heme oxygenase-2 mRNAs were estimated to be about 3.2h and more than 5h, respectively.View full abstractDownload PDF (5717K) -
Yoshihiro Sambongi, Tokumitsu Wakabayashi, Takao Yoshimizu, Hiroshi Om ...1997 Volume 121 Issue 6 Pages 1169-1175
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe full-length cDNA coding for a putative copper transporting P-type ATPase (Cu2+- ATPase) was cloned from Caenorhabditis elegans. The putative Cu2+-ATPase is a 1, 238-amino acid protein, and highly homologous to the Menkes and Wilson disease gene products mutations of which are responsible for human defects of copper metabolism. The Saccharomyces cerevisiae mutant with a disrupted CCC2 gene (yeast Menkes/Wilson disease gene homologue) was rescued by the cDNA for the C. elegans Cu2+-ATPase but not by the cDNA with an Asp-786 (an invariant phosphorylation site) to Asn mutation, suggesting that the C. elegans Cu2+-ATPase functions as a copper transporter in yeast. The expressed C. elegans protein was detected in yeast vacuolar membranes by immunofluorescence microscopy. The yeast expression system may facilitate further studies on copper transporting P-type ATPasesView full abstractDownload PDF (3096K) -
Sumio Ohtsuki, Ko-ichi Homma, Shoichiro Kurata, Shunji Natori1997 Volume 121 Issue 6 Pages 1176-1181
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA specific prolyl endopeptidase (PEP) inhibitor, ZTTA, selectively inhibited DNA synthesis by imaginal discs and cultured embryonic cells of Sarcophaga peregrina (flesh fly). PEP was found to be localized in restricted nuclear regions. Unfertilized eggs were shown to contain a maternal message for PEP and analysis of Sarcophaga embryos at blastdermal stage revealed PEP was localized exclusively in the nuclei. These results suggest that PEP participates in DNA synthesis by, and therefore cell proliferation, of insect cells. This is the first demonstration of a biological function of PEP.View full abstractDownload PDF (3796K) -
Jose Paolo V. Magbanua, Nobuo Ogawa, Satoshi Harashima, Yasuji Oshima1997 Volume 121 Issue 6 Pages 1182-1189
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe transcriptional regulators Pho4p and Pho2p are involved in transcription of several genes in the PHO regulon of Saccharomyces cerevisiae. Genetic evidence with temperaturesensitive pho4 and pho2 mutants suggested that Pho4p and Pho2p interact with each other. Immunoprecipitation experiments showed that Pho4p and Pho2p form a complex on a 36-bp sequence bearing an upstream activation site (UAS) and protein binding assays indicated that these proteins interact directly. DNA-binding experiments with crude extracts prepared from yeast strains expressing T7-PHO4, encoding Pho4p tagged with the T7 epitope, indicated that Pho2p interacts with T7-Pho4p and enhances the binding affinity of T7-Pho4p to the UAS. Protein binding experiments also showed that both Pho4p and Pho2p could bind with the general transcription factors, TBP, TFIIB, and TFIIEβ suggesting that the Pho4p-Pho2p complex bound to the UAS activates transcription of the PHO genes by direct interaction with the general transcription factors.View full abstractDownload PDF (3750K)
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