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Volume 120 (1996)
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Volume 120, Issue 6
Displaying 1-31 of 31 articles from this issue
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Shiro Iuchi, Lorin Weiner1996 Volume 120 Issue 6 Pages 1055-1063
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUpon exposure to oxygen, Escherichia coli increases the expression of enzymes essential for aerobic respiration, such as components of the TCA cycle and terminal oxidase complexes. This increase requires the elimination of repression mediated by the Arc regulatory system under anaerobic conditions. Coordinately, the synthesis of enzymes that function in anaerobic processes such as fermentation decreases, partly due to the inactivation of the transcription factor Fnr. E. coli is thus able to adjust the levels of respiratory enzymes to fit its environmental circumstances, and in this case, reduces the production of the less energy efficient fermentation enzymes in favor of the aerobic pathways. In contrast to the advantage in energy production, aerobiosis brings a disadvantage to E. coli: the production of reactive oxygen species (ROS), i.e. superoxide anion radical (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (-OH). These byproducts of aerobic respiration damage many biological molecules, including DNA, proteins, and lipids. To alleviate the toxicity of these compounds, E. coli induces the synthesis of protective enzymes, such as Mn-dependent superoxide dismutase (SodA) and catalase I (HP I), and this induction is controlled by the regulatory proteins SoxRS, OxyR, and ArcAB. Thus, ArcAB, Fnr, SoxRS, and OxyR function in concert so that E. coli can optimize its energy production and growth rate. Fnr and SoxRS are cytoplasmic, DNA-binding proteins, and these regulatory systems utilize iron-sulfur clusters as cofactors which may directly sense the redox environment. OxyR is also a cytoplasmic, DNA-binding protein, and appears to respond to redox potential through the oxidation state of a specific cysteine residue. In the ArcAB system (which belongs to the family of two-component regulatory systems), ArcB, a membrane protein, functions as the sensor, and ArcA, a DNA-binding protein, directly controls target gene expression. Under anaerobic conditions, ArcB undergoes autophosphorylation and transphosphorylates ArcA, stimulating ArcA's DNA-binding activity. During aerobic growth, the transphosphorylation of ArcA does not occur. In this signal transduction mechanism, the ArcB C-terminal or “receiver” domain plays a critical role; that is, it stimulates or abolishes the transphosphorylation depending on the metabolic state of the cell, which in turn is influenced by the availability of oxygen. E. coli thus employs at least four global regulatory systems which monitor the cellular oxidative/metabolic conditions, and adjust the expression of more than 70 operons to give the organism a better aerobic life.View full abstractDownload PDF (1171K) -
Naoki Shibata, Hiroki Yamamoto, Tsuyoshi Inoue, Koichi Uemura, Akiho Y ...1996 Volume 120 Issue 6 Pages 1064-1066
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRibulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) from a red alga, Galdieria partita, has been crystallized by the hanging drop vapor diffusion method. Two forms (Forms I and II) of crystals were obtained under distinct conditions. The Form I crystal belongs to monoclinic space group C2 with cell dimensions of a=190.2, b=140.0, c=189.0 Å, and β=102.6°, and diffracts up to 3.0 Å resolution. Diffraction from the Form II crystal was too weak to determine crystal data.View full abstractDownload PDF (1666K) -
Morio Yashiro, Akira Ishikubo, Makoto Komiyama1996 Volume 120 Issue 6 Pages 1067-1069
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA dinuclear La3+ complex hydrolyzes the phosphodiester bond in diribonucleotides efficiently under mild conditions. The rate of hydrolysis is remarkably accelerated on the complex formation between La3+ and TPHP (TPHP=N, N, N', N'-tetrakis[(2-pyridyl)methyl]-2-hydroxy-1, 3-diaminopropane), and the highest activity is attained when the dinuclear structure is formed. The half-life of ApA hydrolysis by the dinuclear La3+ complex (1mM) is 350 s, whereas that by the free La3+ ion (1mM) is 23, 000 s at pH 7.2 and 50°C. The dinuclear La3+ complex is promising as a catalytic center of artificial nucleases.View full abstractDownload PDF (355K) -
Takashi Ohtsuki, Ravi Vinayak, Yoh-ichi Watanabe, Kiyoshi Kita, Gota K ...1996 Volume 120 Issue 6 Pages 1070-1073
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRNA samples corresponding to Ascaris suum mitochondrial tRNAMet were chemically and automatically synthesized in amounts sufficient for NMR measurement. Conventional and rapid deprotection methods gave tRNA samples with the same amino acid-accepting activity as those prepared by other method; enzymatic synthesis, and enzymatic ligation of chemically synthesized fragments. The synthetic tRNA showed the same 1H-NMR spectrum in the iminoproton region as the ligated tRNA. This rapid and reliable preparation method thus provides biologically active tRNA for NMR measurement, and further, it is applicable for synthesis of other large synthetic RNAs, by combining the site-specific isotopic labeling method.View full abstractDownload PDF (1425K) -
Nobuhiro Ishida, Nobuhiko Miura, Shigemi Yoshioka, Masao Kawakita1996 Volume 120 Issue 6 Pages 1074-1078
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe described recently the molecular cloning of human UDP-galactose transporter 1 (bUGT1) [Miura, N. et al. (1996) J. Biochem. 120, 236-241]. Now we have characterized its isoform, hUGT2, that is most likely generated through the alternative splicing of a transcript derived from the UGT genomic gene, that also codes for hUGT1. Introduction of the open reading frame sequence of hUGT2 into a mouse cell line, Had-1, that lacks the UDP-galactose transporter, complemented the genetic defect of the mutant, as judged from the lectin-sensitivity spectra of the transformants and the nucleotide-sugar transporting activity of microsomal vesicles isolated from them. UGT-related genes were found through a BLAST search of dbEST based on their significant similarity with hUGT genes. We report here cDNA clones belonging to two subfamilies of the nucleotide-sugar transporter gene family. One is the human CMP-sialic acid transporter gene, and the other is a group of homologous genes with an undefined function that are distributed in man, mouse, and rat, and show significant similarity to the yeast UDP-N-acetylglucosamine transporter.View full abstractDownload PDF (561K) -
Midori Matsumoto, Fumiko Saito-Ohara, Motonori Hoshi, Shun-ichi Kurata ...1996 Volume 120 Issue 6 Pages 1079-1081
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mouse cDNA encoding a human homologue of heme oxygenase-2 (HO-2) was isolated. The deduced protein contains 315 amino acids and has a calculated molecular mass of 35.8 kDa. The nucleotide sequence is 85.6% identical and the amino acid sequence 87% identical to those of the human protein. The corresponding mRNA is present in brain and testis, but not in ovary, kidney, liver, or spleen.View full abstractDownload PDF (798K) -
Kazuhiko Nakahara, Hirofumi Shoun1996 Volume 120 Issue 6 Pages 1082-1087
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome P450nor (P450nor), a nitric oxide reductase involved in the denitrifying system of the fungus Fusarium oxysporum, revealed molecular multiplicity. Two isoforms of P450nor, termed P450norA (norA) and P450norB (norB), were isolated. They had distinct isoelectric points of 5.1 (norA) and 4.9 (norB). However, their catalytic, spectroscopic, and immunological properties were almost identical. Partial amino acid sequences, involving 263 amino acid residues of norA and 278 residues of norB among 404 and 402 residues, respectively, were determined. Corresponding sequences in the isoforms were identical, and all of the determined partial sequences of norA or norB coincided with the sequence deduced from the CYP 55 gene or its cDNA. The amino acid sequence determination ruled out the possibility that there is a redox center in P450nor derived from amino acid residues, e. g., quinonoid cofactors. The only difference between norA and norB was in their N-termini. The N-terminus of norA was a threonine residue, whereas that of norB was an N-acetylated alanyl residue and norB was shorter by 2 residues than norA. The results suggested that norA and norB may be the products of the same gene, but translated from different initiation codons. The hypothetical precursor of norA would have a presequence containing targeting and sorting signals for transportation to the intermembrane space of mitochondria. This is consistent with the results of a Western-blot analysis which showed that norA was recovered only in particulate fractions, whereas norB was in the soluble fraction. It is therefore likely that the intracellular localizations as well as the N-termini of norA and norB are different, owing to the differences in the translational initiation codons and co/post-translational processings.View full abstractDownload PDF (1616K) -
Kenji Akasaki, Akihiro Michihara, Yoshiteru Fujiwara, Kouichi Mibuka, ...1996 Volume 120 Issue 6 Pages 1088-1094
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLysosomal membranes contain two highly glycosylated proteins, designated as lamp-1 and lamp-2, as major components. Lamp-1 and lamp-2 are similar to each other in the protein structure. Here, we investigated the biosynthetic transport of lamp-2 through the endocytic vacuoles in cultured rat hepatocytes in comparison with that of lamp-1, which has previously been studied [Akasaki et al. (1995) Exp. Cell Res. 220, 464-473]. Newly synthesized lamp-2 (NS-lamp-2) was transported to the trans-Golgi from rough endoplasmic reticulum with a half time (t1/2) of 32min, more slowly than NS-lamp-1 (t1/2=13min). After leaving the trans-Golgi, NS-lamp-2 is transferred to at least three compartments; the cell surface (t1/2=47min), cell peripheral early endosomes (t1/2=38min) and perinuclear late endosomes (t1/2=48min). NS-lamp-2 transported to any compartment is delivered finally to lysosomes (t1/2=90min). A significant fraction of NS-lamp-2 (45% of the total) was transported from the trans-Golgi to early endosomes, and then delivered to dense lysosomes via perinuclear late endosomes, whereas a major portion of NS-lamp-1 follows an intracellular route to late endosomes without passing through the cell periphery. NS-lamp-2 leaves the cell peripheral region more rapidly than NS-lamp-1. The kinetic and quantitative data for biosynthetic transport of NS-lamp-2 to early endosomes and the cell surface indicate that NS-lamp-2 may be transported first to early endosomes, from which a small portion of it (_??_3.5% of the total) moves to the plasma membrane via a recycling system. In contrast, a small fraction of NS-lamp-1 is transported to the plasma membrane directly from the trans-Golgi, since NS-lamp-1 is delivered to the plasma membrane and early endosomes with almost the same half times.View full abstractDownload PDF (4332K) -
Takashi Kamaishi, Tetsuo Hashimoto, Yoshihiro Nakamura, Yutaka Masuda, ...1996 Volume 120 Issue 6 Pages 1095-1103
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComplete nucleotide sequences of the genes putatively encoding translation elongation factors 1α (EF-1α) and 2 (EF-2) from a mitochondrion-lacking protozoan, Glugea plecoglossi, that belongs to microsporidians were determined. The deduced amino acid sequences of the putative EF-1α and EF-2 of Gl. plecoglossi showed very unusual features compared with typical eukaryotic sequences. The degree of divergence was especially great in the EF-1α sequence, although it clearly showed a eukaryotic feature when aligned with homologs from the three primary kingdoms. Phylogenetic analyses of EF-1α and EF-2 on the basis of the maximum likelihood method of protein phylogeny clearly and consistently suggested that among eukaryotic species being analyzed, Gl. plecoglossi and another mitochondrion-lacking protozoan, Giardia lamblia, respectively represent the earliest and the second earliest offshoots of eukaryotes. When the EF-1α and EF-2 phylogenies were totally evaluated, the earliest divergence of Gl. plecoglossi in eukaryotes became more clearly confirmed. If the phylogenetic relationships inferred from the present analysis are correct, microsporidians might be extremely ancient eukaryotes that diverged before the occurrence of mitochondrial symbiosis.View full abstractDownload PDF (2460K) -
Atsushi Ooi, Koshin Mihashi1996 Volume 120 Issue 6 Pages 1104-1110
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetics of ATP exchange on subtilisin-cleaved G-actin was investigated by measuring the fluorescence of 1, N6-ethenoadenosine 5'-triphosphate. The apparent dissociation rate of ATP (k-ATP) was 2.8-fold larger than that of intact G-actin in the presence of 300 μM free Ca2+. Analysis of the dependence of k-ATP on free Ca2+ showed that the dissociation rate constant of tightly bound Ca2+ was not significantly changed by subtilisin cleavage. On the other hand, an equilibrium binding study using 8-amino-2-[(2-amino-5-methylphenoxy)-methyl]-6-methoxyquinoline N, N, N', N'-tetraacetic acid (Quin 2) showed that the affinity of tightly bound Ca2+ for G-actin was reduced by about 13-fold after subtilisin treatment. Consequently, the stabilization by Ca2+ of ATP was weak in cleaved G-actin. Furthermore, the kinetic analysis of ATP exchange revealed that the binding equilibrium between ATP and divalent cation-free cleaved G-actin was much slower than that in the case of intact G-actin.View full abstractDownload PDF (887K) -
Tsutomu Kabashima, Kiyoshi Ito, Tadashi Yoshimoto1996 Volume 120 Issue 6 Pages 1111-1117
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe dipeptidyl peptidase IV [EC 3. 4. 14. 5] gene of Xanthomonas maltophilia, expressed in Escherichia coli, was cloned by the shotgun method. Nucleotide sequence analysis revealed an open reading frame of 2, 223 bp, coding for a protein of 741 amino acids with a predicted molecular weight of 82, 080. The expressed enzyme was extracted with SDS, and the solubilized enzyme was purified about 1, 030-fold on columns of Toyopearl HW65C, DEAE-Toyopearl twice, and hydroxyapatite, with an activity recovery of 50%. The enzyme hydrolyzed a proline-containing peptide at the penultimate position, and was inhibited by diisopropyl phosphofluoridate. The enzyme was most active at pH 8.5, and was stable between at pH 7.0 and 9.0. The molecular weight of the purified enzyme was estimated to be 83, 000 and 165, 000 by SDS-PAGE and gel filtration, respectively.View full abstractDownload PDF (1978K) -
Atsuko Deguchi, Masaya Imoto, Kazuo Umezawa1996 Volume 120 Issue 6 Pages 1118-1122
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that inostamycin, an inhibitor of CDP-DG: inositol transferase, inhibited cell proliferation in normal rat kidney (NRK) cells by blocking cell cycle progression at the G1 phase. In the present paper, we report the effect of inostamycin on the serum-induced activation of Ser/Thr protein kinases that are involved in G1 progression. In quiescent NRK cells mitogen-activated protein kinase (MAP kinase) and casein kinase II were activated within 15min after serum addition. Neither activation was affected by the treatment with inostamycin. However, in the inostamycin-treated cell, cyclin-dependent kinase 2 (CDK2) failed to be activated after serum stimulation. Since serum-induced expression of cyclin E was also suppressed by inostamycin, this inhibitor would appear to block CDK2 activation by inhibiting cyclin E expression. Furthermore, inostamycin also inhibited cyclin D1 expression induced by serum; and consequently, hyperphosphorylation of retinoblastoma protein (pRB) by RB-kinases such as CDK4 and CDK2 was abolished, which would result in elimination of functional inactivation of pRB. Thus, early G1 arrest in NRK cells by inostamycin is due to the inhibition of cyclin D1 and E expressions.View full abstractDownload PDF (3542K) -
Yoshihiko Amano, Masahiro Shiroishi, Kazutosi Nisizawa, Eiichi Hoshino ...1996 Volume 120 Issue 6 Pages 1123-1129
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the fine substrate specificities of four highly purified exo-type cellulases (Exo-A from Aspergillus niger, CBHI and CBHII from Trichoderma reesei, and Ex-1 from Irpex lacteus), water-soluble substrates such as barley glucan, xyloglucan from tamarind (Tamarindus indica L.), and their oligosaccharides were employed. Four exo-type cellulases immediately hydrolyzed 3-O-β-D-cellotriosylglucose to produce cellobiose and laminaribiose. In contrast, CBHII showed no hydrolytic activity towards 32-O-β-D-cellobiosylcellobiose, which was hydrolyzed to cellobiose by the other exo-type cellulases. These cellulases hydrolyzed the internal linkages of barley glucan and lichenan in an endo-type fashion to produce cellobiose and mix-linked oligosaccharides as main products. The DP--lowering activities of the four exo-type cellulases on barley glucan were in the order of Ex-1, CBHII, Exo-A, and CBHI. Based on gel permeation chromatography analysis of the hydrolysates, Ex-1 seemed to attack the internal cellobiosyl unit adjacent to β-1, 3-glucosidic linkages in barley glucan molecule more frequently than did the other cellulases. Xyloglucan was hydrolyzed only by CBHI and CBHII, and produced hepta-, octa-, and nona-saccharides. In addition, a xyloglucan tetradecasaccharide (XG14) was split only to heptasaccharide (XG7) by CBHI and CBHII.View full abstractDownload PDF (3258K) -
Selective Interaction of Synthetic Antimicrobial Peptides Derived from Sapecin B with Lipid BilayersYutaka Hirakura, Juan Alvarez-Bravo, Sho-ichiro Kurata, Shunji Natori, ...1996 Volume 120 Issue 6 Pages 1130-1140
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy measuring carboxyfluorescein leakage from liposomes and the increase in membrane current through planar lipid bilayer membranes, we examined the capacities of a series of low-molecular-weight cationic amphiphilic peptides derived from the α-helix domain of sapecin B for membrane-perturbation and ion-channel formation. Some of these peptides strongly interact with membranes containing acidic phospholipids and phosphatidylethanolamine, with a very negative potential, which are characteristic of the Escherichia coli membrane, in parallel with their antimicrobial activity. In contrast, they do not interact with membranes which predominantly contain choline phospholipids and cholesterol in their outer leaflets, with a slightly negative potential, all of which are characteristic of eukaryotic membranes, thereby providing a molecular basis for their selective toxicity. Membranes doped with these peptides are as permeable to inorganic phosphates as to chloride ions and are far more permeable to cations. The loss of inorganic phosphates may damage bacterial cells due to rapid depletion of cytoplasmic ATP. Examination of the structure-activity relationships of a series of derived peptides in their interaction with a model of the E. coli membrane confirmed the necessity of cationic amphiphilicity for the peptides to attack the bacterial membrane and to exhibit antimicrobial activity.View full abstractDownload PDF (1348K) -
Kayo Ikeda, Tsutomu Masujima, Masanori Sugiyama1996 Volume 120 Issue 6 Pages 1141-1145
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStreptomyces castaneoglobisporus HUT 6202 expresses an enzyme, tyrosinase, responsible for the production of melanin-like pigments. The present study revealed that the tyrosinase synthesis by the microorganism was induced about 80-fold, when young cells cultured for 6h were incubated with methionine (Met) to the mid-log phase of growth, in comparison to without this amino acid. The Met-induced tyrosinase synthesis was inhibited by the addition of rifampicin and chloramphenicol, suggesting that transcriptional and translational events are necessary for the induction. We found that the addition of Cu2+ to the culture medium brings forward the period of expression of Met-induced tyrosinase activity.View full abstractDownload PDF (1643K) -
Kiyohito Shimura, Ken-ichi Kasai1996 Volume 120 Issue 6 Pages 1146-1152
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAffinophoresis is a type of affinity electrophoresis in which an affinophore, a conjugate of an affinity ligand and a multiply charged soluble matrix, causes a change in migration velocity of proteins which have a specific affinity for the ligand. A monoligand affinophore bearing a mannoside was prepared by coupling iodoacetylated p-aminophenyl α-D-mannoside to the free thiol group of N-succinylated glutathione, and used for the affinophoresis of pea lectin in a capillary. The electrophoretic mobility of pea lectin towards the anode increased in the presence of the affinophore as a function of its concentration in a manner that is described by the equation for affinity electrophoresis. Analysis of the suppression of the affinophoresis on the addition of neutral sugars to the system allowed the determination of the dissociation constants of the lectin for these neutral sugars. The dissociation constants obtained on affinophoresis agreed well with the values in the literature. The preparation of a monoligand affinophore for ligands bearing an amino group should facilitate the application of this type of microscale analysis (0.14 ng of protein for each run) to protein ligand interactions.View full abstractDownload PDF (796K) -
Sheng-Fang Tian, Shuji Toda, Hideaki Higashino, Sueo Matsumura1996 Volume 120 Issue 6 Pages 1153-1162
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen fibrous collagen of rat tail tendons was glycated by incubation with ribose, it became highly insoluble in dilute acetic acid and resistant to pepsin digestion at 5°C, since it was cross-linked by advanced glycation end products. Extensively glycated fibrous collagen was found to be much less stable than non-glycated control fibrous collagen against pepsin digestion at 30°C. Under conditions where nearly all of the glycated fibrous collagen was degraded into small peptides by pepsin, approximately 45% of the control collagen was left as large polypeptides having nearly the whole length of its triple-helical region. A soluble collagen, which consisted primarily of the triple-helical region of monomeric collagen, was found to be glycated as efficiently as the fibrous collagen on incubation with ribose at 30°C, while the rate of cross-linking of the soluble collagen was very low, suggesting that the triple-helical strands do not undergo intramolecular cross-linking and that most of the cross-links produced in the glycated fibrous collagen are intermolecular ones. The glycated soluble collagen was as stable as the control collagen against pepsin digestion at 30°C. These results indicate that the triple-helical strands of glycated fibrous collagen are much less stable than those of the non-glycated form against proteolytic digestion by pepsin at a temperature close to but below their melting point. Sugar-derived intermolecular cross-links are supposed to underly the decreased stability of the triple-helical strands.View full abstractDownload PDF (6402K) -
Myeong-Cheol Song, Kunitoshi Shimokata, Sakae Kitada, Tadashi Ogishima ...1996 Volume 120 Issue 6 Pages 1163-1166
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur recent experiments using model peptides of rat malate dehydrogenase (MDH) indicated that a proximal arginine and a distal basic amino acid are important for processing by mitochondrial processing peptidase (MPP). [Niidome, T., Kitada, S., Shimokata, K., Ogishima, T., and Ito, A. (1994) J. Biol. Chem. 269, 24719-24722]. To elucidate if the recognition elements apply to other precursor proteins, we analyzed cleavage of model peptides of human ornithine aminotransferase (OAT). Purified peptidase cleaved peptides that corresponded to N-terminal 1-25 and 3-25 at the correct site (Gly17-Val18) at nearly equal rates. Replacement of Arg16 (-2 position) with lysine or alanine reduced the processing efficiency by 95- and 380-fold, respectively. Either deletion from Met1 to Arg10 or replacement of the basic amino acids between them decreased the processing efficiency considerably. A peptide containing Arg7 in addition to Lys4 and Arg10 was more effective than the control peptide. However, a peptide with one and two consecutive basic amino acids in the distal region had a processing efficiency close to the control peptide. These results indicated that processing of OAT was enhanced by an increase in the number of basic amino acids with a suitable distance between them. In other respects, the processing signal of OAT was essentially the same as that of MDH.View full abstractDownload PDF (541K) -
Hiromitu Iwasaki, Yasuyuki Suzuki, Hyogo Sinohara1996 Volume 120 Issue 6 Pages 1167-1175
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeveral clones encoding plasma α-macroglobulin and murinoglobulin were isolated from guinea pig liver cDNA library and sequenced. The clones for a-macroglobulin contained overlapping sequences which together spanned a stretch of 4, 546 nucleotides with one open reading frame coding for 1, 476 amino acid residues. The clones for murinoglobulin contained overlapping sequences which together spanned a stretch of 4, 578 nucleotides with one open reading frame coding for 1, 464 amino acid residues. The phylogenetic analyses of 11 proteins of the α-macroglobulin family revealed that the mammalian tetrameric α-macroglobulins consist of two main branches: αM-1 subfamily (rat α1- and mouse α-macroglobulins) and αM-2 subfamily (human α2-, rat α2-, and guinea pig α-macroglobulins). This dichotomy is in good accordance with their immunological, chemical, and physicochemical properties, and indicates that guinea pig a-macroglobulin is orthologous to human and rat α2-macroglobulins but paralogous to rat a, - and mouse α-macroglobulins. The divergence of the two subfamilies was a phylogenetically ancient event which occurred around the separation of metatherians and eutherians. The genes of the two subfamilies have been maintained in the rat, but either one became extinct in the mouse, guinea pig, or human. The tree also shows that guinea pig murinoglobulin forms one Glade with mouse and rat murinoglobulins (a, -inhibitor, ) prior to joining the αM-2 lineage, and suggests that murinoglobulin is not a primitive form of tetrameric a-macroglobulin, but rather has evolved under selective pressure which is different from that of the tetrameric paralogues.View full abstractDownload PDF (1118K) -
Takashi Shimizu, Hisayuki Morii1996 Volume 120 Issue 6 Pages 1176-1181
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSncd is a microtubule motor protein from Drosophila, having a 40 kDa domain homologous to the kinesin motor domain. In the present study, we investigated the circular dichroism (CD) spectra of the ncd motor domain in comparison with those of the kinesin motor domain. Although the two are about 40% identical in amino acid sequence, and recent X-ray crystallographic studies [Sablin, Kull, Cooke, Vale, and Fletterick (1996) Nature 380, 555-559; Kull, Sablin, Lau, Fletterick, and Vale (1996) Nature 380, 550-555] indicate that their core structures are nearly identical, the far UV CD spectra of ncd and kinesin motor domains, both being monomeric, were considerably different from each other, suggesting a significant difference in the secondary, especially loop structures. The motor domain of ncd, like that of kinesin, contains tightly associating ADP even after purification. We removed ADP from the ncd motor domain by gel filtration in the presence of EDTA and high salt. The resultant protein, however, was likely to be in an inactive state, since it bound ATP slowly. The far UV CD spectrum of the ncd motor domain devoid of ADP was nearly identical to that of the ncd motor domain with bound ADP. This indicated that the removal of ADP did not affect the backbone structure in the presence of high salt. On the other hand, the near UV CD spectrum of the ADP-free ncd motor domain differed from that of the ned motor domain•ADP complex, one possibility being that the local conformation was changed upon removal of bound ADP. The near UV CD spectra of kinesin motor domain also showed a difference between the ADP-bound form and the nucleotide-free form, although the difference was much smaller.View full abstractDownload PDF (787K) -
Hironobu Kimura, Goshi Ishihara, Shoji Tajima1996 Volume 120 Issue 6 Pages 1182-1189
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Xenopus DNA methyltransferase cDNA was isolated from a Xenopus oocyte cDNA library by screening with the mouse DNA methyltransferase cDNA as a probe. The elucidated nucleotide sequence gave a 4, 470 nucleotide open reading frame, and the predicted protein was composed of 1, 490 amino acid residues, showing high homology to animal DNA methyltransferases, especially in the catalytic domain in the carboxylterminal region. The cysteine-rich region and the Lys-Gly repeat which were first found in the mouse sequence were conserved in Xenopus. However, 200 amino acid residues at the amino-terminus of Xenopus DNA methyltransferase were quite different from those of mouse and human, but showed 70% homology with those of chicken. The cloned Xenopus DNA methyltransferase cDNA expressed in COS1 cells showed a significant DNA methyltransferase activity. The size of the translation product of Xenopus DNA methyltransferase cDNA expressed in COS 1 cells was identical with that of the endogenous DNA methyltransferase in Xenopus A 6 cells and also with the size of newly synthesized DNA methyltransferase in Xenopus oocytes. However, a slightly larger immunoreactive band of about 205 kDa, and a small immunoreactive band of about 100 kDa, which were poorly labeled by short incubation with radiolabeled amino acids, were the main bands in stage I-III and stage IV-VI oocytes, respectively.View full abstractDownload PDF (2373K) -
Naoko Watanabe, Junko Suzuki, Yoshiro Kobayashi1996 Volume 120 Issue 6 Pages 1190-1195
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe role of calcium in the production of tumor necrosis factor-α (TNF-α) by the lipopolysaccharide (LPS)-stimulated macrophage cell line, J 774.1, was investigated. Flow cytometric measurement of intracellular calcium concentration ([Ca]1) using 2-(3, 6-bis (acetyloxy)-2, 7-dichloro-9H-xanthen-9-yl) benzoic acid (fluo-3)-loaded J 774.1 cells revealed that LPS at more than 0.1 μg/ml increased [Ca]1 transiently in the presence or absence of serum, and that a mixture of a calcium chelator, ethyleneglycol-bis(β-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), and a calcium release blocker from intracellular store sites, 8-(diethylamino)-octyl-3, 4, 5-trimethoxybenzoate hydrochloride (TMB-8), inhibited the [Ca]1 response induced by LPS. In concordance with this, production of TNF-α was inhibited by EGTA and/or TMB-8. These reagents also reduced the level of TNF-α mRNA significantly. These results indicate that the transient increase of [Ca]1 plays a role in LPS-induced expression of TNF-α by the macrophage cell line.View full abstractDownload PDF (1525K) -
Hiroto Mizushima, Yohei Miyagi, Yamato Kikkawa, Naoki Yamanaka, Hideta ...1996 Volume 120 Issue 6 Pages 1196-1202
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported a new laminin variant containing laminin γ2 (or B 2 t) chain, ladsin, which exerted prominent cell-scattering, cell-adhesion, and cell-migration activities. In the present study, this laminin was further characterized, and gene expression of its three subunits in various human tissues and cancer cell lines was examined by Northern blotting. cDNA cloning of the largest subunit of ladsin and partial amino acid sequencing of its β (or 131) subunit revealed that ladsin was identical to laminin-5 (kalinin/epiligrin/nicein). Among various human tissues, placenta, lung, and fetal kidney expressed high levels of mRNAs for the three subunits of laminin-5 (laminin α3EPA, β3, and γ2 chains). Most gastric and squamous carcinoma cell lines constitutively expressed all of the three subunit mRNAs, while other types of carcinoma cell lines expressed one or two of them. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) strongly enhanced the gene expression of the three subunits, increasing 2 to 8- fold the secretion of laminin-5 from carcinoma cells into culture medium. However, TPA treatment did not increase the secretion of laminin β1 chain, a subunit of laminins-1, -3, and -6. The unique properties and inducibility by TPA and EGF of laminin-5 suggest that it is associated with growth and migration of cancer cells.View full abstractDownload PDF (4626K) -
Akio Ohtani, Tamotsu Takagi, Ayako Hirano, Jun Murakami, Yasuhiko Sasa ...1996 Volume 120 Issue 6 Pages 1203-1208
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTissue-type plasminogen activator (t-PA) and its physiological inhibitor, plasminogen activator inhibitor-1 (PAI-1), are known to be synthesized by vascular endothelial cells and to play important roles in regulating the fibrinolytic activity of plasma. We found that a new butadiene derivative, (3E, 4E)-3-benzylidene-4-(3, 4, 5-trimethoxybenzylidene) pyrrolidine-2, 5-dione (T-686), inhibits PAI-1 production without affecting plasminogen activator (PA) synthesis in cultured bovine endothelial cells. T-686 (1-10 μM) dose-dependently decreased the accumulation of PAI-1 in conditioned medium from the treated cells and elevated PA activity in the conditioned medium. Analysis of the conditioned medium by the zymography technique indicated that T-686 decreased the activities of PAI-1 with an Mr of 55, 000 and t-PA/PAI-1 complex with an Mr of 99, 000. Furthermore, T-686 attenuated the augmentation of PAI-1 antigen induced by lipopolysaccharide in the conditioned medium. The decrease of PAI-1 antigen was in parallel with the reduction of the PAI-1 mRNA level (Northern blots). These results suggest that T-686 can promote net fibrinolytic activity through suppression of PAI-1 production without affecting PA elaboration in endothelial cells.View full abstractDownload PDF (1652K) -
Masaharu Terashima, Muhammad Badruzzaman, Mikako Tsuchiya, Makoto Shim ...1996 Volume 120 Issue 6 Pages 1209-1215
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExocytosis is a common phenomenon in neutrophil functions. We earlier reported the co-localization of arginine-specific ADP-ribosyltransferase [EC 2. 4. 2. 31] and its target protein p33 (mim-1 protein) in cytoplasmic granules in chicken polymorphonuclear leukocytes (so-called heterophils) [Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K., and Shimoyama, M. (1991) J. Biochem. 110, 388-394]. In the present study, we obtained evidence that the transferase and p33 were released into the extracellular space by the stimulus of calcium ionophore A23187 or serum-opsonized zymosan, but scarcely by phorbol myristate acetate (PMA) or N-formyl-Met-Leu-Phe (fMLP), thereby indicating the co-localization of the transferase and p33 in the azurophilic granules, and not in specific granules. [32P]ADP-ribosylation of p33 occurred in the extracellular space, induced by the stimulus of A23187 or opsonized zymosan in the presence of [32P]NAD. Our findings are interpreted to mean that heterophil transferase and p33 may be involved in neutrophil functions during processes of inflammation.View full abstractDownload PDF (3401K) -
Jun Funahashi, Kazufumi Takano, Kyoko Ogasahara, Yuriko Yamagata, Kats ...1996 Volume 120 Issue 6 Pages 1216-1223
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe physicochemical properties of an amyloidogenic mutant human lysozyme (Ile56Thr) were examined in order to elucidate the mechanism of amyloid formation. The crystal structure of the mutant protein was the same as the wild-type structure, except that the hydroxyl group of the introduced Thr56 formed a hydrogen bond with a water molecule in the interior of the protein. The other physicochemical properties of the mutant protein in the native state were not different from those of the wild-type protein. However, the equilibrium and kinetic stabilities of the mutant protein were remarkably decreased due to the introduction of a polar residue (Thr) in the interior of the molecule. It can be concluded that the amyloid formation of the mutant human lysozyme is due to a tendency to favor (partly or/and completely) denatured structures.View full abstractDownload PDF (946K) -
Tanweer Ahmed, Sharon M. Kelly, Anthony J. Lawrence, Mokdad Mezna, Nic ...1996 Volume 120 Issue 6 Pages 1224-1231
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBee venom PLA2 possesses a binding site for long-chain fatty acids that can be acylated by long-chain fatty acid imidazolides [Drainas, D. and Lawrence, A. J. (1978) Eur. J. Biochem. 91, 131-138]. Occupation of the site either by oleic acid or the oleoyl residue enhances the catalytic activity by 45.7-fold in the presence of 20% 1-propanol and occupation of the site by the oleoyl residue increases the lytic activity against rabbit erythrocytes by 60-fold. Treatment of the enzyme with oleic acid and glutaraldehyde is known to produce irreversible activation [Lawrence, A. J. and Moores, G. R. (1975) FEBS Lett. 49, 287-291]. Here we show that reduction of the glutaraldehyde-treated enzyme with borohydride stabilizes the activated state and enables the fatty acid to be removed, revealing that a large proportion of the induced activation does not require the presence of oleic acid and indicating that activation is due to a change in the conformation rather than the hydrophobicity of the protein. A kinetic study of enzyme activated by oleoyl imidazolide showed that this modification stabilizes the protein against reversible inactivation by 1-propanol. Comparison of the CD spectra of native and oleoyl imidazolide-activated enzyme shows a change in secondary structure with apparent increase in both α-helix and β-sheet content. During reaction of the enzyme with oleoyl imidazolide, the protein fluorescence shows a biphasic response with an initial fall attributed to occupation of the binding site followed by a progressive decrease with a shift of the emission maximum from 341 to 348 nm. The rate of the second phase closely matched the rate of increase in catalytic activity of the enzyme. Free oleic acid caused a rapid fall in fluorescence emission without the subsequent slow change. These results support the proposal that oleic acid or the oleoyl residue occupy a very similar site on the protein and that occupation of this site increases the exposure of one or both of the Trp residues to the aqueous environment. Binding studies show that activation by oleoyl imidazolide does not increase the affinity of the enzyme for the erythrocyte membrane. It is proposed that occupation of a long-chain fatty acid binding site on the enzyme enhances catalytic activity by changing the conformation of the protein rather than acting as a hydrophobic anchor to the substrate surface.View full abstractDownload PDF (1000K) -
Mariko Kato Hayashi, Tatsuya Haga1996 Volume 120 Issue 6 Pages 1232-1238
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have expressed human m2 muscarinic acetylcholine receptors tagged with six histidine residues at the carboxy-terminal region in insect cells (Sf9) and purified them using metal-immobilized Chelating Sepharose gels. Co2+-immobilized gels were found to be much more efficient for purification of m2 receptors than gels containing Ni2+ or other metal ions. Twenty-fold purification was attained by a simple, single-step procedure, and approximately 40% of solubilized receptors were recovered as a partially purified preparation with a specific activity of 1.6 nmol/mg of protein. Purified receptors were functionally active in that carbamylcholine stimulated binding of [35S] GTPγS to the G-protein G12 reconstituted in lipid vesicles with purified m2 receptors. The extent of stimulation of [35S] GTPγS binding to G12 by hexahistidine-tagged m2 receptors was essentially the same as that observed for m2 receptors that lack histidine tags. In addition, palmitoylation at the carboxy-terminal region was not impaired by the hexahistidine-tag fusion. The method described in this study should be applicable to the purification of other G-protein-coupled receptors in functionally active form.View full abstractDownload PDF (3441K) -
Nobuaki Adachi, Takeo Kubo, Shunji Natori1996 Volume 120 Issue 6 Pages 1239-1246
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we demonstrated that ABP-1 (arylphorin gene-specific binding protein-1), which is suggested to be the transcriptional activator of the arylphorin gene of Sarcophaga peregrina, is present in NIH-Sape-4 cells, which do not express arylphorin. As well as ABP-1, these cells were found to contain another protein (ABP-2) that probably binds to the same sequence as that to which ABP-1 binds [Adachi, N., Kubo, T., and Natori, S. (1993) J. Biochem. 114, 55-60]. We purified ABP-2 from a nuclear extract of NIH-Sape-4 cells and compared its DNA-binding activity with that of ABP-1. Both ABP-1 and ABP-2 were found to bind to the same sequence in the arylphorin gene with the same affinity and stability, but an ABP-2-specific hypersensitive site was detected by DNase I footprinting analysis. Analyses of proteolytic fragments suggested that both ABP-1 and ABP-2 have Zn fingers showing high similarity with that of AEF-1, a transcriptional repressor of the Drosophila melanogaster alcohol dehydrogenase gene that binds to a sequence very similar to that binding ABP-1 and ABP-2. We isolated a candidate cDNA for ABP-2, and the protein it encoded contained nine Zn fingers and regions rich in alanine, glutamine, serine/threonine, glycine, histidine, and asparagine.View full abstractDownload PDF (2999K) -
Tsuyoshi Ishimoto, Kyoichi Arisato, Satoshi Akiba, Takashi Sato1996 Volume 120 Issue 6 Pages 1247-1252
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism by which cytosolic phospholipase A2 (cPLA2) is not responsible for eicosanoid production in rat peritoneal mast cells upon antigen stimulation [Ishimoto et al. (1996) J. Biochem. 120, 616-623] was investigated in the mast cells stimulated by crosslinking of the IgE receptor or with thapsigargin. Stimulation with thapsigargin, but not with antigen, resulted in apparent lysophosphatidylcholine (lysoPC) formation. Antigen stimulation significantly increased the activities of mitogen-activated protein (MAP) kinase and cPLA2. These activities were further potentiated by phorbol ester. The antigen elicited a rapid and transient increase in intracellular Ca2+ concentration, while thapsigargin produced a slow and sustained increase. Furthermore, a combination of antigen and thapsigargin rapidly increased and prolonged the intracellular Call concentration. Under these conditions, lysoPC was apparently generated, whereas it was not in response to antigen alone. These results suggest that a prolonged increase in the intracellular Ca2+ concentration is required for cPLA2 to associate with membranes, thus leading to hydrolysis of membrane phospholipids by the enzyme.View full abstractDownload PDF (1668K) -
Shun-ichiro Kawabata, Ranko Nagayama, Michimasa Hirata, Takeshi Shigen ...1996 Volume 120 Issue 6 Pages 1253-1260
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSmall granules of horseshoe crab hemocytes contain two known major antimicrobial substances, taehyplesin and big defensin (S5), and at least five protein components (Sl to S6), with unknown functions. In the present study, we examined the biological properties and primary structure of a small granular component S2, named tachycitin. This component was purified from the acid extract of hemocyte debris by two steps of chromatography. The purified tachycitin was a single chain protein with an apparent Mr=8, 500 on Tricine-SDS-polyacrylamide gel electrophoresis. Ultracentrifugation analysis revealed tachycitin to be present in monomer form in solution. Tachycitin inhibited the growth of both Gram-negative and -positive bacteria, and fungi, with a bacterial agglutinating property. Moreover, tachycitin and big defensin acted synergistically in antimicrobial activities. The amino acid sequence and intrachain disulfide bonds of tachycitin were determined by amino acid and sequence analyses of peptides produced by enzymatic cleavages. The mature tachycitin consisted of 73 amino acid residues containing five disulfide bonds with no N-linked sugar. A cDNA coding for tachycitin was isolated from a hemocyte cDNA library. The open reading frame coded for an NH2-terminal signal sequence followed by the mature peptide and an extension sequence of -Gly-Arg-Lys at the COON-terminus, which is a putative amidating signal. The COON-terminal threonine amide released after digestion of tachycitin with lysylendopeptidase was identified. The NH2-terminal 28 residues of tachycitin shows sequence homology to a part of chitin-binding regions found in antifungal chitin-binding peptides, chitin-binding lectins, and chitinases, all of which have been isolated from plants. Tachycitin showed a specific binding to chitin but did not bind with the polysaccharides cellulose, mannan, xylan, and laminarin. Tachycitin may represent a new class of chitin-binding protein family in animals.View full abstractDownload PDF (2417K)
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