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Volume 116 (1994)
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Volume 116, Issue 6
Displaying 1-32 of 32 articles from this issue
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Keiichi Uyemura, Yasuo Takeda, Hiroaki Asou, Kiyoshi Hayasaka1994 Volume 116 Issue 6 Pages 1187-1192
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNeural cell adhesion proteins play important roles in neural development and are involved in various neurological diseases. PO, a major protein in mammalian peripheral myelin, mediates not only homophilic cell adhesion but also neurite outgrowth. The PO glycopeptide inhibits the cell adhesion, but not the neurite outgrowth. Several point mutations of the PO gene in human chromosome 1q22-23 were found in Charcot-Marie-Tooth (CMT) disease type 1B and Dejerine-Sottas (DS) disease. PASII/PMP22 and connexin 32 were also reported as target proteins of similar hereditary neuropathies. L1 is a large multifunctional protein involved in cell adhesion, neurite outgrowth, fasciculation, and neuronal cell migration. A short isoform of L1 localizes in non-neuronal cells in contrast to the complete L1 exclusively expressed in neurons. Recently various L1 mutations have been reported in X-linked hydrocephalus, MASA syndrome with mental retardation and spastic paraplegia type 1. Further studies on the mutations and disease phenotypes are important and interesting.View full abstractDownload PDF (769K) -
Tsuyoshi Inoue, Naoki Shibata, Hisao Nakanishi, Satoshi Koyama, Hideak ...1994 Volume 116 Issue 6 Pages 1193-1197
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of azurin from a denitrifying bacterium, Achromobacter xylosoxidans NCIB11015, has been refined at 2.5 Å resolution using diffraction data obtained by means of synchrotron radiation at KEK. Crystals suitable for X-ray experiment were obtained by the macro-seeding method and an intensity data were obtained on imaging plates mounted on a Weissenberg camera (Rmerge=0.09). The initial model was obtained by the molecular replacement method using the structure of azurin from Alcaligenes denitrificans NCTC8582 as a starting model. The structure was refined by molecular dynamics optimization and the restrained least-squares method to a crystallographic R-value of 0.205. However, the current model gave an electron-density of the side-chain regions of several residues close to the N-terminus quite different from those expected from the amino acid sequences reported. Very recently, two kinds of azurins (Az-I and Az-II) were isolated from this bacterium by a slightly modified purification method and have been characterized and found to have different CD spectra. On analysis of amino acid sequences around the N-terminus, the second azurin (Az-II) was proved to be a new type of azurin in this bacterium. It was consequently revealed that the current model corresponds to a new type of azurin because of the complete agreement between the electron-density and the amino acid sequence of the newly determined 20 residues from the N-terminus. Determination of the whole amino acid sequence of this azurin and further refinement are in progress.View full abstractDownload PDF (1481K) -
Makoto Sato, Mutsuhiko Mizobuchi, Koji Murao, Miho Tamaki, Jiro Takaha ...1994 Volume 116 Issue 6 Pages 1198-1201
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and efficient method for preparing rcRNA through competitive RT-PCR has been developed. The basis of this method is the use of a false-priming PCR product including the same primers as a specific product. A 290 bp fragment obtained by two-step PCR was subcloned into a plasmid vector and then the cloned DNA was transcribed into rcRNA. After competitive RT-PCR using sample RNA and rcRNA had been carried out, Southern blot hybridization was performed. The method was applied to determine the amounts of PIMT mRNAs in the rat pituitary. The quantitative analysis indicated that an at least 2-fold difference in PIMT mRNA level can be accurately determined with our method.View full abstractDownload PDF (3116K) -
Kazuko Hori, Fumihiko Saito, Kazuhiko Tokita, Toshitsugu Kurotsu, Masa ...1994 Volume 116 Issue 6 Pages 1202-1204
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMutant grs1 genes were cloned and sequenced from the Bacillus brevis Nagano BI-4, C-3, E-1, and E-2 strains, which produce defective gramicidin S synthetase 1 (GS1), lacking racemase activity. Surprisingly, these mutant genes had entirely the same sequence as that of the wild type gene. These mutant strains also produce defective gramicidin S synthetase 2 (GS2), lacking 4'-phosphopantetheine, a prosthetic group of this enzyme. The participation of this group in phenylalanine racemization is suggested.View full abstractDownload PDF (1573K) -
Tsutomu Hitotsumatsu, Tetsuyuki Kitamoto, Toru Iwaki, Masashi Fukui, J ...1994 Volume 116 Issue 6 Pages 1205-1207
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe succeeded in cloning the exon 8-spliced out transcript of neurofibromatosis 2 (NF2) gene in human glioma cell lines. We then investigated the expression of the spliced out form in various human tissues by PCR analysis followed by Southern blot hybridization and sequencing to evaluate whether it was generated by normal alternative splicing or by a splicing mutation. The analysis revealed the splicing out of exon 8 in the various tissues, and the transcript missing exon 8 should thus be considered as a product of normal alternative splicing. Our results further support the possibility that the NF2 gene expresses multiple alternatively-spliced transcripts variantly in different tissue types.View full abstractDownload PDF (1495K) -
Takuya Saiki, Kyoko Kohno, Naoki Goshima, Yasunobu Kano, Fumio Imamoto1994 Volume 116 Issue 6 Pages 1208-1211
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA plasmid carrying both gyrA and gyrB genes (pTS7) and a plasmid without the gyr gene (pTS4) were constructed. Introduction of pTS7 into the YK1100 (wild-type) cells resulted in an increase in the level of gyrA and gyrB mRNA by 5-to 6-fold over the level of the control transformant with pTS4. In the transformant cells carrying pTS7, the reporter plasmid pGP241, which is compatible with pTS plasmids, was significantly more highly negatively supercoiled than in the transformant with pTS4. The activity of DNA gyrase to supercoil the relaxed pGP241 DNA was 4-8 times higher with the S-30 extract from the transformant carrying pTS7 than with the extract from the transformant with pTS4.View full abstractDownload PDF (1436K) -
Hisae Hori, Tetsuko Kanamori, Toshihiko Mizuta, Noriko Yamaguchi, Ying ...1994 Volume 116 Issue 6 Pages 1212-1219
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have produced four monoclonal antibodies (mAbs) against human placenta laminin purified by immunoaffinity chromatography. Three clones, 2D9, 3DM, and 4F1, recognized 320 kDa (M) chain of the laminin and the other one, 3DB, recognized B2 chain. One cDNA clone (HLM-1, 3.5 kb) was immunoscreened from human placenta cDNA library using 2D9, and three further overlapping cDNA clones (HLM-2, HLM-3, and HLM-4) covering 2.0 kb were isolated. Nucleotide sequencing and fusion protein analysis demonstrated that the amino acid sequence deduced from HLM-1 coincided with that of the G-domain of human merosin chain, and HLM-2, HLM-3, and HLM-4 encoded the long-arm and EGF-like domain of M chain. The binding regions of 2D9 and 3DM to M chain were defined as homologous repeating sequences of G2-G3 region of G-domain and the carboxy-terminal region of the long-arm, respectively. The extents of identity of amino acid sequences of the long-arm and EGF-like domains between human M chain and A chain were about 37% and 52%, respectively, which were lower than between mouse and human A chains. Northern blot analysis revealed that M chain mRNA, 8.6 kb, was highly expressed in heart and placenta, but less highly expressed in skeletal muscle, brain, and lung. Immunostaining showed selective distributions of M chain in nerve fibers in the dermis and mesangial matrix of the kidney, and B2 chain in subepidermal and kidney glomerular basement membranes.View full abstractDownload PDF (5871K) -
Yoshifumi Watanabe, Hirokazu Nomoto, Ryuichi Takezawa, Naoki Miyoshi, ...1994 Volume 116 Issue 6 Pages 1220-1226
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTransfection methods for primary cultured mouse hepatocytes were examined. Of four conventional transfection methods examined, involving use of DEAE-dextran, calcium phosphate, cation-liposomes (lipofection), and cation-multilamellar liposomes, only cation-liposomes induced highly efficient transfection into primary cultured mouse hepatocytes. The highest transfection rate reached more than 60% of the total cells. Three other commonly used cell types (CHO-K1, COS-1, 3T3-L1) were also tested as target cells, but highly efficient transfection was observed specifically in primary cultured mouse hepatocytes. The transfection remained at a high level from 6 to 48h after the start of incubation with the cation-liposome-DNA complex in the absence of serum, and the transfection rate decreased in inverse relation to the increase in cell density. The transfection was inhibited by free low density lipoprotein (LDL), EDTA, and an endocytosis inhibitor, cytochalasin B. These data suggest that the transfection is mediated not only by membrane fusion, as is generally accepted, but also by endocytosis. This information should be useful for research in hepatocyte biology and the development of gene therapy. As one of the applications, simple and successful immunization was achieved by administration of hepatocytes transfected with murine adhesion molecule, integrin VLAβ1 subunit, genes into a Syrian hamster.View full abstractDownload PDF (2957K) -
Kou-Wha Kuo, Long-Sen Chang, Pai-Mei Lin, Robert J. Leaber, Chun-Chang ...1994 Volume 116 Issue 6 Pages 1227-1232
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnti-cobrotoxin antibodies can be separated into precipitin and non-precipitin antibodies. The precipitin antibody possesses the same binding affinity to cobrotoxin as non-precipitin antibody, but the neutralizing capability of the latter is superior to that of the former in blocking cobrotoxin binding to nicotinic acetylcholine receptor (nAChR). After preincubation with antibodies, cobrotoxin completely lost its binding activity to nAChR. The dissociation of cobrotoxin-nAChR complex by the antibodies was low, and 60% of the complex formation appeared to be irreversible. These results indicate that the neutralization of cobrotoxin by the antibody may predominantly involve unbound, receptor-free cobrotoxin. The relationships of neutralization capacity and binding affinity as well as bond strength between cobrotoxin and its antibodies are incongruous. Different local conformational changes of a unique Trp in cobrotoxin on binding with the precipitin and non-precipitin antibodies seem to lead to different accessibility for fluorescence quenchers. Characterization of the binding domains by immunoprecipitation with the antibodies correlated with the quenching results. Thus, the binding topography of cobrotoxin may play an important role over the binding affinity and bond strength in neutralization by cobrotoxin antibody.View full abstractDownload PDF (707K) -
Mitsuo Yamashita, Takuya Kinoshita, Michiko Ihara, Tomomi Mikawa, Yosh ...1994 Volume 116 Issue 6 Pages 1233-1240
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo study the structure and function of pullulanase from Klebsiella aerogenes, a method involving random mutagenesis of the entire gene for pullulanase was used. Out of 50, 000 clones screened at high temperature, seven genes for mutant proteins were identified by DNA sequencing. The amino acid substitutions in the seven mutant proteins were clustered on the NH2-terminal side of the four conserved regions found in α-amylases. These mutant pullulanases were classified into two types: those whose catalytic activity was altered and those whose thermal stability was increased. The results presented here and in previous reports suggest that pullulanase from K. aerogenes has similar active sites to those of α-amylases with the four conserved regions, as well as another substrate-binding site closer to the NH2-terminus. The plate assay method used for isolation of thermostable variants may be applicable to the generation of useful variants of other enzymes.View full abstractDownload PDF (928K) -
Tsutomu Uchida1994 Volume 116 Issue 6 Pages 1241-1250
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe purification and characterization of choline kinase R1 expressed in Escherichia coli, using expression plasmid pKCK(H) constructed with rat liver choline kinase cDNA [Uchida, T. and Yamashita, S. (1992) J. Biol. Chem. 267, 10156-10162], were carried out. Choline kinase R1 was chromatographically, enzymatically, and immunologically distinct from rat brain choline kinase [Uchida, T. and Yamashita, S. (1990) Biochim. Biophys. Acta 1043, 281-288] . This report, for the first time, definitively demonstrates the presence of two types of choline kinase isozymes. Rat choline kinase was immunologically classified into choline kinases P and R. Choline kinase P was purified from rat brain as described previously. Choline kinase R was designated as the choline kinase generated from the choline kinase R gene for choline kinase R1. Choline kinases from cytosol were chromatographically separated into two fractions, that is, one passed through a Blue-Toyopearl column and the other was retained on it. The choline kinase retained on the Blue-Toyopearl column was highly purified from rat liver cytosol and characterized in this study, being found to comprise isoforms of choline kinase R, and a choline kinase which was immunologically similar to choline kinase P. Choline kinases P and R are co-distributed in rat tissues. The carcinogen, 3-methylcholanthrene, and hepatotoxic carbon tetrachloride increased the enzyme activity, and the transcript and protein levels of choline kinase R in rat liver. The relation of choline kinase isozymes to previously purified enzymes was discussed.View full abstractDownload PDF (4629K) -
Tomoko Takahashi, Sae Kawauchi, Kenji Suzuki, Etsuko Nakao1994 Volume 116 Issue 6 Pages 1251-1256
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of hydrostatic pressure on the bindability and digestibility of raw corn starch with two types of glucoamylase [EC 3. 2. 1. 3] from Rhizopus sp., Glue1 and Glue2, was investigated in the range of 100 to 600 MPa. Pressurization of raw starch at 100 to 300 MPa for 1h had no detectable effect, whereas pressurization at higher pressures than 400 MPa markedly enhanced the bindability and digestibility with Glue, and Glue, , especially with Glue2, which scarcely binds to raw starch and has much lower activity than Glue1 towards raw starch. The binding constants Ks of Glue1 and Glue2 for the 400-MPa-pressurized starch reached 16 and 1.6×105M-1, respectively, as compared with the Ks, of 2.1 and 0.082×105M-1 for raw starch. The 500- to 600-MPa-pressurized starch was digested by Glue1 and Glue2 at about 4.2 and 80 times higher rates, respectively, than raw starch. Thus, the high-pressure-treated starch was hardly distinguishable from high temperature (75 to 100°C)-treated starch at least with respect to behavior with the enzymes, although some difference was observed between these starches by scanning electron microscopy and differential scanning calorimetry.View full abstractDownload PDF (1732K) -
Kazunori Mizuno, Toshihiko Hayashi1994 Volume 116 Issue 6 Pages 1257-1263
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSType I collagen in phosphate-buffered saline (PBS) bound to a heparin-Sepharose column, while heat-treated type I collagen, denatured chains of α1(I) and α2(I), did not. Conformation-dependent association of type I collagen with heparin was further examined in various urea concentrations. The relative amount of bound fraction decreased in proportion to the concentration of added urea; from over 90% in the absence of urea to about 30% in 3M urea, although circular dichroism spectrum of type I collagen was not changed by the presence of 4M urea at 25°C. In 2M urea, the relative amount of bound fraction was about 50%. Rechromatography of the flow-through fraction or bound fraction of type I Collagen in 2M urea after lyophilization showed a similar pattern to that of the initial type I collagen solution in that about a half of either sample was recovered as bound fraction. This result indicated that the association potential of type I collagen with heparin appeared to have changed reversibly. The relative amount of bound fraction was little affected by the initial protein concentration, suggesting that intermolecular interaction between type I collagen molecules or the aggregate possibly resulting from the interaction is not important in the affinity with heparin. From these results, we suggest that triple-helical type I collagen molecules undergo reversible changes of conformation in urea solution between the conformation with heparin affinity and that without the affinity. In contrast, type V collagen or α1(V) chain binds to heparin under the same conditions and therefore the conformational change of type V collagen in urea solution would not be discernible in terms of heparin affinity, even if the type V collagen molecules have altered conformation in urea, as is suggested for type I collagen.View full abstractDownload PDF (1787K) -
Hiroyuki Iwamoto, Masahiro Ohno, Masayuki Ohmori, Junzo Hirose, Akiyos ...1994 Volume 116 Issue 6 Pages 1264-1268
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe change in fluorescence spectra of crystalline pullulanase from Klebsiella pneumoniae caused by the addition of α-, β-, and γ-cyclodextrins and 6-O-α-glucosyl-α-cyclodextrin and 6-O-α-glucosyl-β-cyclodextrin was investigated at 25°C and pH 5.6. The fluorescence intensity at around 325 nm (excitation at 280 nm) was increased by the addition of all the cyclodextrins studied. The dissociation constant, Kd, of the enzyme-cyclodextrin complex was evaluated by fluorometric titration for each cyclodextrin, and was consistent with the inhibitor constant, K1, obtained previously [Iwamoto et al. (1993) J. Biochem. 113, 93-96]. The Kd, values of β-cyclodextrin and 6-O-α-glucosyl-β-cyclodextrin were approximately two orders of magnitude smaller than those of α- and γ-cyclodextrins. Fluorescence titration of a cyclodextrin in the presence of another cyclodextrin revealed competition among α-, β-, and γ-cyclodextrins for binding with the enzyme, which indicates that the binding region of β-cyclodextrin overlaps those of α- and γ-cyclodextrins. On the other hand, with excitation at 295 nm, a fluorescence spectral change similar to that excited at 280 nm was observed for α- and γ-cyclodextrins and 6-O-α-glucosyl-α-cyclodextrin, whereas β-cyclodextrin and 6-O-α-glucosyl-β-cyclodextrin did not show any such change. These results suggest that the binding site or the binding mode of β-cyclodextrin is slightly different from those of α- and γ-cyclodextrins. Preliminary kinetic studies were done on the binding of the enzyme with α-, β-, and γ-cyclodextrins by following the increase in fluorescence intensity (excitation at 280 nm) with a micro-stopped-flow apparatus. A progress curve of single exponential type was observed for every cyclodextrin studied. The apparent first-order rate constants, kapp, for α- and γ-cyclodextrins were independent of cyclodextrin concentration in the range studied (0.05-1.25mM). In contrast, β-cyclodextrin showed a hyperbolic concentration dependence of kapp, increasing asymptotically to the maximum value of about 2.9 s-1. Judged from these results, the rate-limiting step of the enzyme-cyclodextrin binding is considered to be a unimolecular process, probably a conformational isomerization.View full abstractDownload PDF (544K) -
Boel Lanne, Birgitta Schierbeck, Karl-Anders Karlsson1994 Volume 116 Issue 6 Pages 1269-1274
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe carboxyl group of the natural cholera toxin receptor, the ganglioside GM1, Galβ-3GalNAcβl-4(NeuAcα2-3)Galβ1-4G1cβ1-Cer, has been converted to a number of C(1)-amides of NeuAc. The binding of cholera toxin B-subunit to these derivatives was monitored by exposing the modified glycolipids, on solid phases, to radiolabeled toxin. Binding was obtained, although substantially reduced, with the amide and to a lesser extent with the benzylamide and also the C(1)-alcohol. In the assay system used, the methyl-, ethyl-, or propylamides did not bind. It was concluded that the hydrogen bonding capacity of a carboxyl or amide group is needed for strong binding. This is in agreement with the recently published crystal structure of the B-subunit in complex with the GM1 pentasaccharide.View full abstractDownload PDF (3254K) -
Yoshio Inokuchi, Masayuki Kajitani, Akikazu Hirashimat1994 Volume 116 Issue 6 Pages 1275-1280
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSQβreplicases in which the Gly residue of the β-subunit in the motif sequence, YGDD, was replaced with Ala, Ser, Pro, Met, or Val lost their replicase activity in vivo. In an in vitro Mg2+-dependent RNA-synthesizing system using poly(rC) or MDV-poly(+) RNA (a derivative of the naturally occurring small RNA that accumulates in the cells during Qβ phage infection) as templates, the lysates from the cells expressing such defective replicases exhibited only 2-6% of the enzyme activity of the lysate from those expressing wild-type replicase. However, the defective replicases, especially A357, with Ala substituted for the Gly, recovered enzyme activity when Mn2+ was added to the reaction mixture. Furthermore, the characteristics of the MDV-poly(+) RNA-dependent RNA synthesis by A357 replicase were similar to those by wild-type replicase in the presence of Mn2+. Gel retardation assay showed that all of the defective replicases could bind MDV-poly(+) RNA. These results suggest that the Gly residue in this motif of Qβ replicase is involved in Mg2+-catalyzed polymerization. In the Mn2+-catalyzed polymerization, A357 and S357 replicases can act as well as the wild-type replicase.View full abstractDownload PDF (3831K) -
Yozo Takasaki1994 Volume 116 Issue 6 Pages 1281-1286
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRestriction endonuclease HindIII was purified from Haemophilus influenzae Rd. Two active fractions, P1 and P2, were obtained in phosphocellulose chromatography. HindIII could be purified completely from the first fraction, P1, by subsequent DEAE-cellulose chromatography. The second fraction, P2, showed HindIII activity higher than that of P1, though it was still contaminated with some minor proteins. The HindIII in P2 fraction showed differences in stability, binding to substrate DNA, electrophoretic mobility, etc., from the HindIII in P1 fraction. It is likely that there are two forms of HindIII in the bacterial cell. The endonuclease HindIII in P2 fraction was finally purified by DNA-cellulose chromatography, though considerable loss of enzymatic activity resulted. Upon infection of the cells with phage T4, the P2 fraction in phosphocellulose chromatography almost disappeared. The presence of two forms of HindIII may be related to bacterial defense against viral infection.View full abstractDownload PDF (5191K) -
Hiromu Takamatsu, Akitaka Nakane, Akihiro Oguro, Yoshito Sadaie, Kouji ...1994 Volume 116 Issue 6 Pages 1287-1294
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlthough wild-type Bacillus subtilis SecA barely complements the growth and protein translocation defect of Escherichia coli secA51(ts) at the non-permissive temperature, an N-terminal peptide of B. subtilis SecA complements the defects. To elucidate the mechanism of this complementation, a series of plasmids encoding truncated SecA proteins was constructed and their products were analyzed in E. coli cells. The truncated B. subtilis SecA protein consisting of the N-terminal 234 amino acid residues (BN234) complemented the growth and protein translocation defects of E. coli secA51 but not those of another secA amber mutant, E. coli secA13(ts). BN234 existed in both a soluble form, possibly as a homodimer, and a higher-molecular-weight complex in E. coli strain MM52 (secA51). The purified complex, consisting of at least BN234, SecA51, and ATP-dependent protease La, was held together by a cross-linking reagent, EDAC. The other truncated proteins consisting of the N-terminal 584 or 396 amino acid residues and the C-terminal 607 residues of B. subtilis SecA did not complement the two E. coli mutants or form a complex with SecA51. These results suggest that BN234 and SecA51 proteins form a functional complex in vivo and complement the defects of E. coli MM52.View full abstractDownload PDF (6226K) -
Yasushi Kamisaka, Toro Nakahara1994 Volume 116 Issue 6 Pages 1295-1301
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDiacylglycerol acyltransferase (DGAT) was examined as a key enzyme for tiacylglycerol (TG) accumulation of an oleaginous fungus, Mortierella ramanniana var. angulispora. Subcellular fractionation of the fungus showed that DGAT activity was highest in the lipid body fraction, which occupied 77% of the recovered DGAT activity. DGAT activity in the lipid body fraction was much higher than that in the membrane fraction in terms of both total activity and specific activity. Similar results were obtained with another homogenization method. After repeated washing of the lipid body fraction, DGAT activity in the lipid body fraction was still larger than those in other fractions. The lipid body fraction contained larger amounts of lipids, especially TG and diacylglycerol. Moreover, the lipid body fraction had a specific set of polypeptides at 24, 29, and 59 kDa. These analyses of lipid and polypeptide composition suggested that the lipid body fraction represented a specific intracellular structure, presumably the lipid body. DGAT activity in the lipid body fraction had a similar characteristics to that in the membrane fraction, although some differences in sensitivity to SH-reagents were observed. Increase in DGAT activity in the lipid body fraction was observed when lipids were accumulated in the fungus. On the other hand, DGAT activity in the lipid body fraction decreased when lipids were accumulated with an increase in carbon to nitrogen ratio in media.View full abstractDownload PDF (2134K) -
Yoshimi Kakinumaz, Kazuei Igarashi1994 Volume 116 Issue 6 Pages 1302-1308
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously reported the molecular cloning and sequences of the ntp genes for Enterococcus hirae Na+-translocating ATPase [Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K., and Kakinuma, Y. (1994) J. Biol. Chem. 269, 11037-11044]; the expected structure of this enzyme complex resembles those of the vacuolar H+-ATPase complexes in eukaryotes. In this paper we report purification and characterization of the catalytic moiety of Na+-ATPase, whose molecular size was about 400 kDa, consisting of polypeptides of 69 kDa (NtpA), 52 kDa (NtpB), and 29 kDa (NtpD) with a probable stoichiometry of 3:3:1. Purified enzyme hydrolyzed GTP as the best substrate (GTP>CTP> UTP> ATP), and the activity was maximal at around pH 6.0. The activity was not stimulated by sodium ions, and was selectively inhibited by nitrate. These properties were different from those of membrane-bound Na+-ATPase, suggesting that a significant conformational change of the catalytic moiety may take place upon dissociation from the membraneembedded moiety and probably also loss of other hydrophilic subunits. Antiserum against purified enzyme inhibited the Na+-stimulated ATPase activity of the membranes. Immunoblotting analysis revealed that the change in the amounts of A and B subunits of the membranes paralleled that of the Na+-ATPase activity. Furthermore, the A subunit was missing in the membranes of a Na+-ATPase mutant, and recovered in those of its revertant. These immunochemical data are consistent with the notion that this enzyme is the hydrophilic catalytic moiety of the V-type Na+-ATPase in E. hirae.View full abstractDownload PDF (3514K) -
Naoe Kotomura, Masahiro Okada, Yasuharu Ninomiya, Toshio Tsukiyama, Ka ...1994 Volume 116 Issue 6 Pages 1309-1316
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of repression of transcription by ELP, the embryonal long terminal repeat binding protein, was investigated. ELP represses the Moloney murine leukemia virus long terminal repeat by binding to a site which overlaps with a sequence element for retinoic acid receptor binding. This suggests possible competition of ELP with retinoic acid receptor for the same sequence elements. Oligonucleotides corresponding to ELP and/or retinoic acid receptor binding elements were placed upstream of the SV40 promoter and their effect on gene expression was analyzed by CAT assay. Elements which have affinity to both ELP and retinoic acid receptor were activated by retinoic acid receptor and these activations were repressed by ELP. An ELP binding element without affinity to retinoic acid receptor was insensitive to both activation by retinoic acid receptor and repression by ELP. Furthermore, cellular ELP binding elements and the Moloney leukemia virus long terminal repeat were activated by retinoic acid. These data suggest that one of the mechanism of transcriptional repression by ELP is competition for binding sites with transactivators such as retinoic acid receptors.View full abstractDownload PDF (1945K) -
Kohei Hosaka, Jun-ichi Nikawa, Tsutomu Kodaki, Hideki Ishizu, Satoshi ...1994 Volume 116 Issue 6 Pages 1317-1321
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe SCS3 gene of Saccharomyces cerevisiae was cloned by functional complementation, using a conditional mutant exhibiting myo-inositol auxotrophy in the presence of choline, and sequenced. The sequence contained an open reading frame capable of encoding 380 amino acids with a calculated molecular weight of 42, 734. Disruption of the SCS3 locus caused myo-inositol auxotrophy. The gene appeared to be involved in the synthesis of inositol phospholipids from inositol but not in the control of inositol synthesis.View full abstractDownload PDF (2905K) -
Yohtaro Uchiyama, Shigenori Iwai, Yoshio Ueno, Morio Ikehara, Eiko Oht ...1994 Volume 116 Issue 6 Pages 1322-1329
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo study the interaction and the role of the metal ion in the reaction catalyzed by Escherichia coli ribonuclease HI (E. coli RNase HI), substrate analogues containing a phosphorothioate linkage or 2'-modified nucleosides at the cleavage site were used. In the presence of Mg2+, Mu2+, Co2+, Zn2+, or Cd2+, the phosphorothioate linkage with the Rp-configuration was cleaved, while the Sp-isomer was not. Kinetic studies showed that Mn2+ and Cd2+ facilitated the cleavage of the phosphorothioate to only a small extent, which indicated the absence of an interaction between the metal ion and this phosphate residue. The interaction of the metal ion with the 2'-functional group was analyzed by Mg2+-titration experiments using the -OH, -NH2, and -F substrates. From Hill plots, it was found that the KMg values were almost the same. These results are evidence of an interaction between Mg2+ and the 2'-functional group by the formation of an outer-sphere complex with a water molecule. The Hill coefficient of 1.0 for the -OH substrate indicated that a single Mg2+ ion is required for the catalysis.View full abstractDownload PDF (2343K) -
Yoshimi Yamamoto, Koichi Takimoto, Susumu Izumi, Mayumi Toriyama-Sakur ...1994 Volume 116 Issue 6 Pages 1330-1335
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have isolated and sequenced a 1, 486-base-pair near full-length cDNA coding for Bombyx egg cysteine proteinase. The cDNA encodes 344 amino acid residues containing a typical signal peptide sequence (16 residues), pro-peptide (104 residues), and the sequence for mature enzyme (224 residues). Sequence alignments show that the egg cysteine proteinase is similar to lobster cysteine proteinase (61% identity), barley cysteine proteinase, Aleurain (52%), rice cysteine proteinase, Oryzain (54%), and rat cathepsin L (59%). The amino-terminal sequencing of the egg cysteine proteinase indicates that the enzyme purified as an inactive form from eggs is a pro-enzyme. Pro-egg cysteine proteinase was detected in other silkmoth tissues such as ovary, fat body, hemocyte, and hemolymph by immunoblotting.View full abstractDownload PDF (3255K) -
Ahmed Ajzannay, Carol Minassian, Jean-Paul Riou, Gilles Mithieux1994 Volume 116 Issue 6 Pages 1336-1340
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlucose-6-phosphatase (Glc6Pase) is a liver microsomal protein exhibiting a high specificity for glucose-6-phosphate (Glc6P) when present in the intact membrane. On the contrary, Glc6Pase of detergent-treated liver microsomes is able to hydrolyze both Glc6P and mannose-6-phosphate (Man6P) with the same affinities and rates (Km and Vmax) at pH 6.5 [Ajzannay, A., Minassian, C., Riou, J. P., and Mithieux, G. (1993) Eur. J. Biochem. 212, 335-338]. We have carried out competition experiments between constant micromolar concentrations of [U-14C]Glc6P and [U-14C]Man6P and increasing millimolar concentrations of unlabeled Glc6P and Man6P. The rate of hydrolysis of [U-14C]Glc6P was progressively inhibited as the competitor concentration increased, but it was significantly higher in the presence of Man6P than in the presence of Glc6P (twofold higher at 20mM). In the same manner, the rate of hydrolysis of [U-14C]Man6P was inhibited by increasing concentrations of competitor, but it was significantly higher when the competitor was Man6P than when the competitor was Glc6P. These data provide direct evidence that, in the presence of both Glc6P and Man6P, Glc6Pase is able to exhibit a specific, albeit limited, kinetic behavior towards the former after detergent treatment of the membrane. They also emphasize the role of the membrane in the acquisition of the final specificity of Glc6Pase under physiological conditions.View full abstractDownload PDF (621K) -
Dae-Hyun Seog, Masahiro Kito, Koji Yoda, Makari Yamasaki1994 Volume 116 Issue 6 Pages 1341-1345
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously shown that the Saccharomyces cerevisiae USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a 200-kDa protein (1, 790 amino acids) which is present in a nonglobular high molecular mass complex. Antibodies against an N-terminal portion of Usol protein recognized a 100-kDa protein in Western blot of the temperature-sensitive uso1-1 mutant cell lysate. The nucleotide sequence of uso1-1 indicated the 951st codon was UAG (amber) in place of CAG (glutamine) in USO1. Deletion study of USO1 gene indicated that such truncated Usol polypeptides are sufficiently functional at 25°C but not at 37°C. Mutant Uso1-1 protein displayed an apparent molecular mass of 400-500 kDa in gel filtration while it cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa), in sucrose density gradient centrifugation. These results indicated that truncated Uso1-1 protein is still present in a nonglobular high molecular mass complex, similar to the wild-type Usol protein.View full abstractDownload PDF (3000K) -
Yuji Ito, Masaki Hirashima, Hidenori Yamada, Taiji Imoto1994 Volume 116 Issue 6 Pages 1346-1353
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLysozyme was extracted from the feces of rabbit (Japanese White) with 2.5% acetic acid and purified by ion-exchange chromatography. Subsequent ion-exchange HPLC at pH 4.0 revealed the presence of two isozymes, namely rabbit colonic lysozymes 1 and 2. The amino acid sequences of these lysozymes were determined. The colonic lysozymes 1 and 2 showed 98% identity with each other and 94 and 95% identities with rabbit kidney lysozyme, respectively. The very high identities between kidney and colonic lysozymes indicate that the colonic isozymes diverged from the conventional kidney lysozyme very recently, probably after the divergence of rabbit from other rodents, accompanying the gene duplication. Despite the small changes in the sequences, the enzymatic properties of colonic lysozyme differ from those of the kidney lysozyme. The activity of the colonic lysozyme against Micrococcus luteus cells showed a narrow and acidic pH optimum, in contrast to the wide and high pH optimum of the kidney lysozyme. Changes in the enzymatic properties are analogous to those of the ruminant stomach lysozymes and may implicate adaptive evolution in the functional conversion of rabbit colonic lysozymes in gut.View full abstractDownload PDF (855K) -
Kazuyo Ohashi, Mari Nishimura, Asako Goi Terasaki, Hiroyuki Nakagawa1994 Volume 116 Issue 6 Pages 1354-1359
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe purified a 36-kDa protein from a low-salt, alkaline extract of chicken gizzard smooth muscle by sequential column chromatography using DEAE-Cellurofine A-800m, hydroxylapatite, and CM-Cellurofine C-500. This protein decreased the low-shear viscosity of actin filaments and coprecipitated with them by centrifugation at 18, 500×g. Electron microscopy showed that the 36-kDa peptide bundled actin filaments. Immunoblot analysis revealed that an affinity-purified antibody against the 36-kDa protein reacted exclusively with the 36-kDa protein band of smooth muscle. In indirect immunofluorescence microscopy, the affinity-purified anti-36-kDa protein antibody stained the dotty structures of isolated smooth muscle cells, while in post-embedding immunoelectron microscopy, most of the colloidal gold particles representing the 36-kDa protein were found on the dense bodies of ultrathin sections of chicken gizzard smooth muscle cells. The antibody did not stain the dense plaques of isolated smooth muscle cells. Judging from its molecular weight, effect on actin assembly, immunological reactivity, and localization in smooth muscle cells, the 36-kDa protein is concluded to be a new component of the dense bodies of smooth muscle.View full abstractDownload PDF (5166K) -
Akimasa Yamazaki, Shunji Kaya, Takeo Tsuda, Yoshio Araki, Yutaro Hayas ...1994 Volume 116 Issue 6 Pages 1360-1369
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSParanitrophenylphosphate (pNPP) induced fluorescence changes in fluorescence isothiocyanate (FITC)-labeled Na+, K+-ATPase preparations. The extents of changes were similar to those induced by acetylphosphate (AcP) accompanying accumulation of a K+-sensitive phosphoenzyme (E2P) and an ouabain bound phosphoenzyme in the presence of Mg2+ and 16mM Na+. Phosphoenzymes formed from [32P] pNPP were shown to turn over. The ratio of the maximum amount of the phosphoenzyme formed from pNPP to that of the phosphoenzyme formed from ATP and that of the ouabain-enzyme complex under steadystate conditions was shown to be close to 1:0.5:1. Such extra phosphorylation has hitherto only been observed in a transient state with the additions of high concentrations of ATP [Peluffo, R. D., Garrahan, P. J., and Rega, A. (1992) J. Biol. Chem. 267, 6596-6601]. Our data are compatible with the simultaneous presence of high and low affinity ATP-binding sites in Na+, K+-ATPase [Hamer, E. and Schoner, W. (1993) Eur. J. Biochem. 213, 743-748]. The maximum amount of paranitrophenol-sensitive fraction to synthesize [32P]pNPP in fully accumulated ADP-sensitive phosphoenzyme (E1P) from [32P] ATP was around 1/4 of the amount of ouabain-enzyme complex. These data and others indicate that a much higher degree of oligomerization, rather than (αβ)2, may be the functional unit of the enzyme in the membranes.View full abstractDownload PDF (1360K) -
Kunishige Kataoka, Katsuyuki Tanizawa, Toshio Fukui, Hiroshi Ueno, Toh ...1994 Volume 116 Issue 6 Pages 1370-1376
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase.View full abstractDownload PDF (892K) -
Li-Hong Ye, Kohichi Hayakawa, Yuan Lin, Tsuyoshi Okagaki, Koichiro Fuj ...1994 Volume 116 Issue 6 Pages 1377-1382
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin light chain kinase (MLCK) is present in muscle cells including those of smooth muscle as an actin-binding protein. By avoiding complication introduced as a result of kinase activity of MLCK, we have demonstrated regulatory role of MLCK through its actin-binding activity [Kohama et al. (1992) Biochem. Biophys. Res. Commun. 184, 1204-1211]. To analyze such a regulatory role of MLCK, we compared the effects of MLCK on the velocity of the movement of actin filaments on a surface coated with smooth muscle myosin with those of another actin-binding proteins in smooth muscle, namely, caldesmon (CaD) and calponin (CaP). Both CaD and CaP stimulated movement when their concentrations were low, but they inhibited movement as their concentrations were increased. Calmodulin (CaM) in the presence of Ca2+ (Ca-CaM) antagonized the inhibition but hardly affected the stimulation. The effect of MLCK, by contrast, was simply inhibitory when Ca-CaM was not present. No stimulation was observed until Ca-CaM was added. The inhibitory ability of these actin-binding proteins increased in the following order: CaD<CaP<MLCK. The effect of MLCK and CaD on movement was further examined on surfaces coated with skeletal muscle myosin. The basic effect was similar to that observed with smooth muscle myosin. However, 10-fold greater concentrations of MLCK and CaD were required for a comparable effect. Such an increase in the required concentration was also observed when the velocity of movement was increased by elevation of the temperature during the assay with smooth muscle myosin. Thus, it is the velocity of movement itself that determines the required concentrations of MLCK and CaD.View full abstractDownload PDF (2083K) -
Héctor H. Valdivia, Brian M. Martin, Angelina N. Ramíire ...1994 Volume 116 Issue 6 Pages 1383-1391
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour novel Na+ channel-blocking toxins (numbered 6 to 9) were purified from the venom of the scorpion Centruroides noxius Hoffmann by gel filtration and high-performance liquid chromatography and their chemical and pharmacological properties were characterized. Amino acid analysis and SDS-PAGE of the pure toxins showed them to be composed of approximately 65 amino acid residues with a molecular mass of _??_7, 500 Da. The amino acid sequences of the newly isolated toxins displayed substantial similarity to those of previously isolated and characterized C. noxius toxins. Toxin 7, the most active toxin from this venom, selectively blocked the whole-cell inward Na+ current (INa) from guinea pig ventricular myocytes without altering the K+ or Cat+ currents. Using rat brain synaptosomes, the specific binding parameters of 125I-Toxin 7 were determined: KD=40 pM and Bmax, =1.8 pmol/mg protein. The binding was independent of membrane potential and was displaced by all Na+ channel-toxins from C. noxius venom, with K0.5s ranging from 60 pM to 60 nM. Tityus γ-toxin, a toxin representative of β-scorpion toxins, totally displaced 125I-Toxin 7 binding, but AaH II and Lqq V toxins, representative of α-scorpion toxins, had no effect. All four C. noxius toxins inhibited [3H]GABA uptake by synaptosomes with IC50s similar to KDs. The toxin effect was not synergistic with veratridine. From these results, it was concluded that the newly purified toxins exert their effects by binding to Site 4 of the voltage-sensitive Na+ channel and must, therefore, be classified as β-scorpion toxins.View full abstractDownload PDF (2114K)
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