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Volume 120 (1996)
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Volume 120, Issue 4
Displaying 1-26 of 26 articles from this issue
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Ichiro Niki, Hisayuki Yokokura, Toshiki Sudo, Masumi Kato, Hiroyoshi H ...1996 Volume 120 Issue 4 Pages 685-698
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChanges in cytosolic Ca2+ concentrations evoke a wide range of cellular responses and intracellular Ca2+-binding proteins are the key molecules to transduce Ca2+ signaling via enzymatic reactions or modulation of protein/protein interations (Fig. 1). The EF hand proteins, like calmodulin and S100 proteins, are considered to exert Ca2+-dependent actions in the nucleus or the cytoplasm. The Ca2+/phospholipid binding proteins are classified into two groups, the annexins and the C2 region proteins. These proteins, distributed mainly in the cytoplasm, translocate to the plasma membrane in response to an increase in cytosolic Ca2+ and function in the vicinity of the membrane. Ca2+ storage proteins in the endoplasmic or sarcoplasmic reticulum provide the high Ca2+ capacity of the Ca2+ store sites, which regulate intracellular Ca2+ distribution. The variety and complexity of Ca2+ signaling result from the cooperative actions of specific Ca2+-binding proteins. This review describes biochemical properties of intracellular Ca2+-binding proteins and their proposed roles in mediating Ca2+ signaling.View full abstractDownload PDF (3088K) -
Tatsuyuki Kobayashi, Ayako Shinozaki, Takashi Momoi, Kiichi Arahata, T ...1996 Volume 120 Issue 4 Pages 699-704
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined changes in proteinase activities in P19 embryonal carcinoma cells during retinoic acid-induced differentiation. The interleukin-1β converting enzyme (ICE)-like Ac-YVAD-MCA hydrolytic activity was increased about 6-fold by treatment with retinoic acid. This activity was inhibited by N-ethylmaleimide and Ac-YVAD-H but not by E-64, EDTA, PMSF, or amastatin. The ICE-like activity in P19 cells eluted as a single peak just after the void volume on gel filtration. No ICE-like activity was observed at a molecular mass of 30-50 kDa. Enzymatic purification, Western blot analysis, and an immunoabsorption study demonstrated that the ICE-like activity in P19 cells is caused by the proteasome, and is stimulated during retinoic acid-induced differentiation. The proteasome purified from mouse liver also cleaved Ac-YVAD-MCA. These results strongly suggest that the proteasome is a major ICE-like proteinase in P19 cells and may be involved in the neural differentiation and the apoptotic pathway.View full abstractDownload PDF (3175K) -
Jun-ichi Sasaki, Masafumi Yamaguchi, Satoshi Saeki, Hana Yamane, Naoki ...1996 Volume 120 Issue 4 Pages 705-709
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of sphingoid bases, sphingosine and dihydrosphingosine, which are protein kinase C (PKC) inhibitors, on NADPH oxidase were examined in a cell-free system. The bases inhibited cell-free activation of NADPH oxidase by arachidonic acid at lower concentration than N-acetylsphingosine. Thus, positive charge in the molecules may play a critical role in inhibition of the oxidase. Sphingosine did not change the Km value for NADPH, but shifted the optimum concentration of arachidonic acid for activation of the oxidase. Moreover, sphingosine suppressed the translocation of p47-phox, one of the cytosolic components of the oxidase, to the membrane fraction, suggesting that the base inhibits the assembly of the components.View full abstractDownload PDF (2136K) -
Yukitomo Arao, Naoto Miyatake, Etsuko Yamamoto, Hiroaki Usiku, Akiko M ...1996 Volume 120 Issue 4 Pages 710-715
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe stabilization of chicken ovalbumin (OVA) mRNA by different classes of steroid hormones (estrogen, progesterone, glucocorticoid, and androgen) was studied in the oviducts of chicks treated with combinations of four steroids. The combination of estrogen with progesterone, glucocorticoid, or androgen enhanced the induction of the OVA gene more than did estrogen alone. Run-on analysis of the isolated oviduct nuclei to measure the transcription rate of the OVA gene showed that the enhanced induction of the OVA gene by the combined hormone treatments was partly caused by an increased rate of transcription. The half-life of OVA mRNA as determined using a transcription inhibitor (actinomycin D) was estimated to be about 24h irrespective of the hormone treatment, though the half-life was about 6h in the absence of hormones. These results suggested that the prolongation of the half-life of OVA mRNA by steroid hormones is constant irrespective of differential transcription rates of the OVA gene.View full abstractDownload PDF (1839K) -
Shigehito Makifuchi, Masaki Takeuchi, Shigeru Kawahire, Shoji Odani, T ...1996 Volume 120 Issue 4 Pages 716-724
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 92k protein (p92) was purified from the wheat germ agglutinin-Sepharose (WGA-Sepharose) bound fraction of a rat liver nuclear envelope salt-extract by DEAE-5PW and hydroxyapatite HPLCs. Partial amino acid sequence analysis of p92 revealed that it is karyopherin β, which was found recently in the cytosolic fraction. It was shown using antip92 antiserum that the protein is present in the nuclear envelope and cytosolic fractions, in almost the same amounts, but not in other subcellular fractions of rat liver. p92 bound to N-acetylglucosamine-bearing nucleoporins (GNPs) on WGA-Sepharose, but not directly to WGA. The amount of p92 found in the rat liver nuclear envelope fraction corresponded to about 10% of the nuclear pore complex in mass, and to as much as 140 mol of p92 per mol of nuclear pore complex. Hydrodynamic analysis of the purified p92 suggested that the molecule is present as a monomer and that it is a rod-shaped molecule. The interaction of p92 and GNPs seemed to be hydrophobic and ionic. Based on these results, the participation of nuclear envelope p92 in protein nuclear transport is discussed.View full abstractDownload PDF (5743K) -
Tomomi Miyamoto, Masaru Hayashi, Akihiko Takeuchi, Takayuki Okamoto, S ...1996 Volume 120 Issue 4 Pages 725-730
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously reported the purification from human erythrocyte extracts of a novel growth-promoting factor with a wide target cell spectrum. The factor has been identified as catalase. As cell extracts from a variety of tumor cell types exhibited both growth-promoting and catalase activities, the relationship between the two activities was examined using cell extracts from three different cell types, human myeloid cells (U937), human melanoma cells (A375-C6), and human B cells (Daudi). The growth-promoting and catalase activities were well correlated in these cell extracts. The antibody against human catalase absorbed not only catalase activity, but also the growth-promoting activity of extracts from these cell types. Treatment of the cell extracts from these cells with an irreversible catalase inhibitor, aminotriazole, abolished both the catalase and growth-promoting activities. In contrast, glutathione peroxidase (GSH-Px) activity was neither absorbed with the anti-catalase antibody, nor inhibited by aminotriazole. In addition, GSH-Px exhibited growth-promoting activity only in the presence of glutathione (GSH). These results, in conjunction with the effect of aminotriazole on the growth-promoting activity of catalase, suggest that catalase is the major growth-promoting molecule in the cell extracts, and H2O2-decomposing activity is important. Northern blot analysis revealed that these cells contained authentic catalase mRNA, and the mRNA level was compatible with the catalase and growth-promoting activities in the cell extracts. These results suggest that the growth-promoting activity in the tumor cell extracts is due to catalase.View full abstractDownload PDF (2175K) -
Hiroyuki Kawachi, Haruyuki Atomi, Mitsuyoshi Ueda, Noriko Hashimoto, K ...1996 Volume 120 Issue 4 Pages 731-735
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an n-alkane-assimilating yeast, Candida tropicalis, carnitine acetyltransferase (CAT; EC 2. 3. 1. 7) was localized in both peroxisomes and mitochondria. Both CATs were encoded by one gene, CT-CAT, although the initiation sites of translation were suggested to be different. In the present study, the genes corresponding to the supposed C. tropicalis peroxisomal and mitochondrial CATs, which were truncated from the CT-CAT gene, were individually expressed in Saccharomyces cerevisiae, using the C. tropicalis isocitrate lyase promoter (UPR-ICL), which is inducible by oleic acid in concert with proliferation of peroxisomes in S. cerevisiae [Umemura, K., Atomi, H., Kanai, T., Teranishi, Y., Ueda, M., and Tanaka, A. (1995) Appl. Microbiol. Biotechnol. 43, 489-492]. The 71 kDa precursor of mitochondrial CAT, initiating at the first Met, was found to be processed to the mature size (66 kDa) in S. cerevisiae and immunoelectronmicroscopical observation revealed that this enzyme was localized in mitochondria. On the other hand, 68 kDa CAT, initiating at the second Met (residue No. 19), had no cleavable signal and was translocated into peroxisomes and cytosol, but not into mitochondria. The amino-terminal amino acid sequences of individually expressed CATs were identical to those of CATs isolated from alkane-grown C. tropicalis cells, respectively. These results demonstrated that only the 71 kDa protein yielded the 66 kDa protein and that peroxisomal and mitochondria) CATs arose from the difference in the initiation sites of translation.View full abstractDownload PDF (1876K) -
Shuhei Zenno, Hideaki Koike, Masaru Tanokura, Kaoru Saigo1996 Volume 120 Issue 4 Pages 736-744
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSnfsB, encoding a minor oxygen-insensitive nitroreductase, was isolated by PCR using primers corresponding to two amino acid sequences conserved among the major flavin reductase from Vibrio fischeri and classical nitroreductases from Salmonella typhimurium and Enterobacter cloacae. The gene product, NfsB, was purified to homogeneity from extracts of Escherichia coli cells overexpressing it. nfsB was found to be situated at 13min on the E. coli map. Biochemical analysis indicated NfsB to be a polypeptide having a calculated molecular weight of 23, 904, capable of forming a homodimer and associated tightly with FMN as a prosthetic group. Although it exhibited a lower affinity to the NfsB apoenzyme than FMN, FAD could serve as an effective substitute for FMN. It was also shown that NfsB has a broad electron acceptor specificity and is associated with a low level of the NAD(P)H-flavin oxidoreductase. The NfsB catalysis obeys the ping pong Bi-Bi mechanism. The Km value for NADH varied depending on the second substrate used.View full abstractDownload PDF (5963K) -
Satoru Harumiya, Kohsuke Takeda, Tsukasa Sugiura, Yuki Fukumoto, Hiroy ...1996 Volume 120 Issue 4 Pages 745-751
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel elastin-binding protein, EBP-37, was recently identified and purified from human plasma. Its partial amino acid sequences showed significant homology to porcine ficolins, which were originally purified from porcine uterus membranes as multimeric proteins with fibrinogen- and collagen-like domains. Here we report the presence of ficolins in an elastin-binding fraction of porcine plasma and the direct binding of recombinant porcine ficolin-α to elastin. In addition, a cDNA encoding a human counterpart of porcine ficolins that is composed of 319 amino acids and is different from EBP-37 was cloned and named human ficolin-1. Northern blotting of various human tissues revealed that human ficolin-1 mRNA is highly expressed in peripheral blood leukocytes. These data suggested that there are at least two kinds of ficolin-related proteins in both pig and human, and they may function as plasma proteins with elastin-binding activities.View full abstractDownload PDF (6013K) -
Munehiko Asayama, Hidechika Suzuki, Akio Sato, Tokujiro Aida, Kan Tana ...1996 Volume 120 Issue 4 Pages 752-758
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe performed molecular characterization of the RpoDl protein encoded by the rpoD1 gene isolated from a cyanobacterium, Microcystis aeruginosa K-81. The deduced amino acid sequence (416 aa, 48, 871 Da) of RpoDl exhibited extensive similarity to those of proteins of the eubacterial RpoD family (Escherichia coli σ70 homologs). We overproduced and purified RpoDl (54 kDa) from E. coli. Biological and biochemical analyses suggested that RpoD1 has a function homologous to that of E. coli σ70 as follows: (i) the RpoD1 protein complemented an rpoD mutant of E. coli strain YN543 (rpoD285) and (ii) the heterologous RNA polymerase holoenzyme reconstituted from the E. coli core enzyme and recombinant RpoD1 was specifically transcribed from E. coli promoters. Furthermore, Western blot analysis with antiserum against Synechococcus sp. strain PCC 7942 RpoD1 (a principal sigma factor of the σ70 type) indicated that M. aeruginosa K-81 RpoDl (σA1) is the principal σ factor, which is a major component of the σ subunit on exponential cell growth.View full abstractDownload PDF (6164K) -
Iwao Hiro, Yukio Tsugeno, Isamu Hirashiki, Fusahiro Ogata, Akio Ito1996 Volume 120 Issue 4 Pages 759-765
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe FAD-binding cysteine of rat liver monoamine oxidase A (MAO A), Cys406, was converted to an alanine by site-directed mutagenesis of the cDNA. The wild-type and mutated enzymes were expressed in yeast cells and catalytic activities were assayed, using as substrates serotonin, tyramine, and kynuramine. Specific activities of the Ala-mutant for these substrates, calculated as the activities per pargyline-sensitive molecule, were about half of those of the wild-type enzyme. The Km values of the mutant enzyme for the substrates were similar to those of the wild-type enzyme. An adduct between FAD and pargyline, a mechanism-based inhibitor, was attached to the apoprotein in the wild-type enzyme, while in the Ala-mutant it was detached from the apoprotein, thereby indicating the presence of noncovalently bound FAD in the mutant enzyme. The Ala-mutant rapidly lost activity during incubation, whereas the wild-type enzyme retained the initial activity. Partial protection from inactivation occurred in the presence of FAD, but not of FMN. Recovery of the enzyme activity was nil when FAD was added after the inactivation. Thus, while the covalent attachment of FAD in MAO A is not required for the catalytic activity, it may function as a structural core for the active conformation in the membrane.View full abstractDownload PDF (2061K) -
Motoshi Suzuki, Keiko Tamiya-Koizumi, Masaharu Takemura, Keigo Furuta, ...1996 Volume 120 Issue 4 Pages 766-772
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have shown that calf thymus DNA polymerase α-DNA primase complex (polα-primase) preferentially binds to pyrimidine-rich sequences and initiates RNA primer synthesis [Suzuki, M. et al. (1993) Biochemistry 32, 12782-12792]. Here we tested the association of polα-primase with a guanine-rich DNA fragment (SVG, 30-mer) containing in vivo initiation sites of simian virus 40 DNA replication. While pyrimidine-rich fragment (CTPPS 1, 30-mer), that is a preferred sequence for calf thymus DNA primase, was well co-precipitated with polα-primase using anti-polα antibody, SVG was hardly precipitated under the same conditions. Competition studies in either gel-retardation assay or during de novo DNA synthesis by polα-primase demonstrated that the interaction of polα-primase with SVG was much weaker than that with CTPPS 1. On the other hand, replication protein-A (RP-A) could bind SVG, although less efficiently than CTPPS 1. After preincubation with RP-A, SVG could bind polα-primase that was immobilized on Sepharose beads. The simian virus 40 large T antigen also enhanced association of SVG to polα-primase, while Escherichia coli single-stranded DNA-binding protein did not. However, polα-primase, bound to SVG in the presence of RP-A, failed to synthesize RNA primers. When SVG was extended 10 nucleotides at its 5'-end, polα-primase synthesized trace amounts of RNA primers, and this activity was stimulated more than 10-fold by adding RP-A. These results suggest a new role for RP-A, i.e., as a molecular tether that allows polα-primase to bind guanine-rich regions of DNA in order to initiate RNA primer synthesis.View full abstractDownload PDF (3645K) -
Rika Sugai, Masayuki Takeuchi, Sachiko Okuno, Hitoshi Fujisawa1996 Volume 120 Issue 4 Pages 773-779
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cDNA of a novel protein, which contains the association domain of α isoform of calmodulin-dependent protein kinase II (CaM-kinase Ilα), was cloned from rat skeletal muscle. This protein, called αKAP, consisted of 200 amino acid residues with a molecular weight of 22, 583. αKAP has a highly hydrophobic amino-terminal stretch of 25 amino acids which is absent from CaM-kinase IIα, suggesting that this protein is either a secretory protein or an integral membrane protein. Northern blot analysis with a probe specific for αKAP detected three distinct mRNA species of 4.0, 2.4, and 1.5 kb in rat skeletal muscle. The 4.0- and 2.4-kb RNAs were also detected in heart, and at much lower levels in lung, kidney, and testis. Western blot analysis, using antibody raised against a synthetic peptide corresponding to the carboxyl-terminal 15 amino acids, revealed a single band corresponding in mobility to a molecular weight of 21, 000 in crude extracts of both rat skeletal muscle and bacteria transformed with the cDNA, suggesting that no significant post-translational modification, such as excision of the amino-terminal hydrophobic segment, occurred. This, together with the fact that αKAP was recovered in the high-speed pellet in skeletal muscle, indicated that this protein may be an integral membrane protein.View full abstractDownload PDF (3076K) -
Sachiko Sugano, Retsu Miura, Naohiko Morishima1996 Volume 120 Issue 4 Pages 780-787
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDihydroxycholesterol and pregnenolone were clearly detected on HPLC when 22R-hydroxycholesterol was incubated with a reconstituted P450scc system containing equimolar amounts of P450scc and adrenodoxin. The dihydroxycholesterol, which has been accepted to be an intermediate in the conversion of 22R-hydroxycholesterol to pregnenolone, accumulated when adrenodoxin was at a subsaturating level with respect to P450scc. The formation of the intermediate increased with increasing pH in the range of 7.2 to 8.1, and the ratio of the intermediate to the product, pregnenolone, increased with increasing pH. When the binding of P450scc to adrenodoxin was weakened by elevation of the ionic strength, the formation of the intermediate relative to the product increased. The apparent Km for dihydroxycholesterol at a subsaturating level of adrenodoxin was about 7 μM, in contrast to 4 μM at a saturating level of adrenodoxin, implying that the affinity of dihydroxycholesterol is lower at a subsaturating level of adrenodoxin than at a saturating one. These results suggest that a subsaturating level of adrenodoxin weakened the binding of dihydroxycholesterol to P450scc and thus the intermediate, dihydroxycholesterol, was released. An intermediate other than dihydroxycholesterol, obtained when cholesterol was used as the substrate, was identified as 22R-hydroxycholesterol by HPLC and mass spectroscopic analysis. The intermediate obtained when 22R-hydroxycholesterol was used as the substrate was identified as 20R, 22R-dihydroxycholesterol by HPLC, mass, and 1H-NMR spectroscopic analyses. These results provide direct evidence that cholesterol is metabolized to pregnenolone by way of 22R-hydroxycholesterol and 20R, 22R-dihydroxycholesterol by P450scc.View full abstractDownload PDF (933K) -
Shigeru Chaen, Misako Nakaya, Xiao-Feng Guo, Shugo Watabet1996 Volume 120 Issue 4 Pages 788-791
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have examined the temperature-dependence of sliding velocity of fluorescent F-actin on myosins isolated from 10°C- and 30°C-acclimated carp. Activation energies for the sliding of F-actin were 63 and 111 kJ/mol for the 10°C- and 30°C-acclimated carp myosins, respectively. Arrhenius plots of the sliding velocity from 10°C- and 30°C-acclimated carp myosin were shown to intersect at high temperature (about 30°C). The thermostability estimated by measuring the Ca2+-ATPase activity was less for myosin from 10°C- than 30'C-acclimated carp. We suggest that a less thermostable structure in cold-acclimated carp myosin results in a reduced activation energy for the contractile process, which allows the F-actin to slide fast even at low temperatures.View full abstractDownload PDF (546K) -
Shigeo Takenaka, Wataru Masuda, Shingo Tsuyama, Yoshiyuki Tamura, Kazu ...1996 Volume 120 Issue 4 Pages 792-796
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSArginine:mono-ADP-ribosylhydrolase was purified from a protozoan, Euglena gracilis Z, using [32P] mono-ADP-ribosylated actin as a substrate. The enzyme showed molecular mass of 33 kDa both in SDS PAGE and gel filtration, indicating it to be a monomeric protein. It was strongly inhibited by ADP and ADP-ribose and activated by Mg2+, DTT, and 2-mercaptoethanol. These results suggest that it recognizes the ADP-ribose moiety of the modified protein. Since the enzyme activity increased in S phase and late G0 phase in a synchronous dividing culture, the enzyme may function in the regulation of the cell cycle.View full abstractDownload PDF (1713K) -
Abdul Motaleb, Yoshihiro Takihara, Midori Nomura, Yoichi Matsuda, Torn ...1996 Volume 120 Issue 4 Pages 797-802
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA putative murine homologue of the Drosophila polyhomeotic gene, named rae28, has been isolated from a genomic library of 129/SV mouse and its structural organization has been analyzed. rae28 is a single gene of approximately 22 kb long and consists of 15 exons. Its 5'-flanking region lacks typical transcriptional regulatory sequences, such as TATA and CCAAT boxes, but contains GC-rich sequences and seven putative binding sites for a transcription factor, Sp1. One major transcription start point has been determined. The overall exon-intron organization suggested that three different Rae28 mRNAs are generated through alternative splicing. Furthermore, the rae28 gene has been located on the R-positive F3 band of mouse chromosome 6 by the direct R-banding fluorescence in situ hybridization methods.View full abstractDownload PDF (2360K) -
Yasuko Nakano, Takashi Tobe, Nam-Ho Choi-Miura, Toshio Mazda, Motowo T ...1996 Volume 120 Issue 4 Pages 803-812
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy use of its affinity to gelatin-Cellulofine, a novel protein, GBP28 (gelatin-binding protein of 28 kDa), was obtained from human plasma. GBP28 bound to gelatin-Cellulofine could be eluted with 1M NaC1. By analysis of its amino-terminal amino acid sequences and the peptides obtained by protease digestion, GBP28 was identified as a novel protein. After repeated gel chromatography of the 1M NaCl eluate from gelatin-Cellulofine, about 50 μg of GBP28 was purified from 500 ml of human plasma. On gel chromatography, the protein migrated as a molecule of about 420 kDa. On SDS-PAGE, its molecular mass was 28 kDa under reducing conditions and 68 kDa under nonreducing conditions. Recently, human mRNA specific to adipose tissue, cDNA clone apM1, has been registered [Maeda, K., Okubo, K., Shimomura, I., Funahashi, T., Matsuzawa, Y., and Matsubara, K. (1996) Biochem. Biophys. Res. Commun. 221, 286-289]. The assumed amino acid sequence of cDNA clone apM1 contained all the sequences of GBP28 and its peptides. Therefore, it is evident that the cDNA clone apM1 encodes GBP28 and the protein is specific to adipose tissue. The clone encodes a polypeptide of 244 amino acids with a secretory signal sequence at the amino terminus, a small non-helical region, a stretch of 22 collagen repeats and a globular domain. Thus, GBP28 appears to belong to a family of proteins possessing a collagen-like domain through which they form homo-trimers, which further combine to make oligomeric complexes. Although its biological function is presently unclear, its adipocyte-specific expression suggests that GBP28 may function as an endogenous factor involved in lipid catabolism and storage or whole body metabolism.View full abstractDownload PDF (5683K) -
Masahiro Hosaka, Kyoko Toda, Hiroyuki Takatsu, Seiji Torii, Kazuo Mura ...1996 Volume 120 Issue 4 Pages 813-819
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSADP-ribosylation factors (ARFs) are a family of small GTP-binding proteins that are proposed to be involved in the formation of coated transport vesicles. Although six ARF sequences have been reported in mammals to date, it has been unclear how many ARF members are present in a single organism. In this study, we provide the first direct evidence by cDNA cloning for the presence of all six ARF members in mouse. These proteins are highly conserved across mammalian species and Northern blot analysis revealed that mRNAs for all the members were expressed ubiquitously. Transfection of cells with epitope-tagged ARFs revealed that ARFs 1-3 displayed a perinuclear Golgi localization, while ARFs 4-6 appeared to be widely dispersed throughout the cytoplasm. These results suggest that although all the ARF proteins play fundamental and critical roles in cellular function, they are involved in different vesicular transport processes.View full abstractDownload PDF (2578K) -
Fujiko Tsukahara, Masahira Hattori, Takamura Muraki, Yoshiyuki Sakaki1996 Volume 120 Issue 4 Pages 820-827
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe identified and cloned a novel 9, 078-bp cDNA, designated TPRDI, from the Down syndrome-critical region by exon trapping. The cDNA encodes a putative protein (TPRDI) of 2, 025 amino acid residues. Two isoforms, TPRDII (8, 992 bp) and TPRDIII (7, 416 bp), were also isolated. TPRDII, which is probably an alternative splicing product from the TPRD gene transcript, encodes two large open reading frames (ORFs) of 200 amino acid residues and 1, 792 amino acid residues, respectively. TPRDIII, which is probably generated by transcription from an alternative start site of the TPRD gene, encodes a putative protein of 1, 715 amino acid residues (TPRDIII). Northern blot analysis revealed that TPRDI and its isoforms are present in 7-17 day mouse embryo and in all the human adult and fetal tissues examined. TPRDI has three units of a 34-amino-acid repeat similar to the tetratricopeptide repeat (TPR) motif, which may mediate interaction with various proteins. A larger ORF encoded by TPRDII also has three units of TPR motif, but TPRDIII has only two-thirds of this motif unit. Thus, the TPRD gene may belong to the TPR gene family. Near-central and C terminal regions of TPRDs showed some homology to several matrix proteins such as trichohyalin and bullous pemphigoid antigen. It is possible that the TPRD gene is one of the genes whose overexpression causes several morphological anomalies observed in Down syndrome.View full abstractDownload PDF (3000K) -
Rieko Kuroda, Jun-ya Kinoshita, Masanori Honsho, Jun-ya Mitoma, Akio I ...1996 Volume 120 Issue 4 Pages 828-833
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome b5 is tail-anchored in the ER membrane and is composed of three functionally different portions; amino-terminal heme-containing catalytic, central hydrophobic membrane-anchoring, and carboxy-terminal ER-targeting portions [Mitoma, J. and Ito, A. (1992) EMBO J. 11, 4197-4203]. In situ topology of cytochrome b5 in the ER membrane was studied using immunofluorescence microscopy. Antibodies were raised against the hydrophilic portion (anti-b5) and the carboxy-terminal seven amino acid residues (anti-peptide) of cytochrome b5 and used for detection of the cytochrome in COS cells which expressed the rat cytochrome. Anti-b5 antibody detected the cytochrome in a reticular staining pattern characteristic of the ER, even when the cell plasma membrane was permeabilized with Streptolysin O. The anti-peptide displayed a fluorescence signal only with Triton-permeabilized cells in which the antibody was able to penetrate into the ER lumen. In a double immuno-staining of the cell using the antipeptide antibody and the antibody against protein disulfide isomerase, both antibodies showed the same staining pattern in the presence of either Triton X-100 or Streptolysin O. The results indicate that the carboxy-terminal hydrophilic stretch is exposed to the luminal side. Cytochrome b5 was tagged with c-myc peptide at the carboxy-terminal end and the topology of the c-myc peptide was analyzed by the same method. Anti c-myc monoclonal IgG detected the tagged cytochrome b5 only after Triton treatment of the fixed cells, suggesting that the addition of c-myc peptide to the carboxy-terminal end does not affect insertion or orientation of the cytochrome in the ER membrane.View full abstractDownload PDF (2703K) -
Akira Inoue, Takao Ojima, Kiyoyoshi Nishita1996 Volume 120 Issue 4 Pages 834-837
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA clone encoding troponin T of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle has been isolated and sequenced. The complete sequence deduced consists of 314 amino acid residues with a molecular weight of 37, 206. Akazara scallop troponin T contains 55 amino acid residues more and 82 residues fewer than rabbit skeletal muscle troponin T and Drosophila melanogaster troponin T, respectively, showing almost the lowest sequence homology with rabbit troponin T (26%) but the highest homology with Drosophila troponin T (33%). Further, high sequence homology was seen in the functional regions: residues 33-120 and 174-227, corresponding respectively to residues 71-158 and 197-250 of rabbit troponin T (tropomyosin-binding regions); and residues 200-204, corresponding to 223-227 of rabbit troponin T (troponin I-binding region). In residues 1-70 (tropomyosin-binding region), however, only six residues are identical with rabbit troponin T.View full abstractDownload PDF (1415K) -
Ken Karasawa, Osamu Kuge, Kiyoshi Kawasaki, Masahiro Nishijima, Yasuko ...1996 Volume 120 Issue 4 Pages 838-844
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn a previous study, we purified PAF-acetylhydrolase, which converts PAF to an inactive metabolite, lysoPAF, from peritoneal fluid of guinea pigs subjected to experimental endotoxin shock and found that this purified enzyme had similar biochemical properties to the plasma enzyme [Karasawa, K., Yato, M., Setaka, M., and Nojima, S. (1994) J. Biochem. 116, 374-379]. In this study, we isolated a homogeneous enzyme preparation from guinea pig plasma using a similar procedure. The molecular mass of this purified enzyme, as determined by SDS-PAGE was 58-63 kDa, larger than that (43 kDa) of the human enzyme. To elucidate the molecular structure of this enzyme and clarify its relationships with PAF-acetylhydrolases of other species, we isolated and sequenced a cDNA encoding this enzyme. Its cDNA contains an open reading frame encoding 436 amino acids and its predicted molecular mass (49 kDa) is lower than that of the native enzyme, suggesting that guinea pig plasma PAF-acetylhydrolase, unlike the human enzyme, is modified post-trans-lationally, perhaps by glycosylation.View full abstractDownload PDF (3476K) -
Masaaki Ito, Ben M. Dunn, Kohei Oda1996 Volume 120 Issue 4 Pages 845-850
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1, 2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3' (P5, P4, P3, P2, P2', P3': a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km=6.3 μM, kcat=51.4 s-1, and kcat/Km=8.16 μM-1•s-1. The kinetic parameters for XCP were Km=3.6 μM, kcat=52.2s-1, and kcat/Km=14.5 μM-1•s-1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general<0.5 μM-1•s-1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (>1.0 μM-1• s-1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a K1 value of 2.1 nM, as well as for PCP (K1=2.6 nM).View full abstractDownload PDF (782K) -
Takeomi Murata, Tomonori Itoh, Yumi Hayakawa, Taichi Usui1996 Volume 120 Issue 4 Pages 851-855
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-Galactosidase from porcine testes induced regioselective transglycosylation from lactose to the 3-position of 2-acetamido glycosides. When α-D-Ga1NAc-OC6H4NO2-p was used as an acceptor, the enzyme synthesized mainly β-D-Gal-(1-3)-α-D-GaINAc-OC6H4NO2-p with its (1→6) linked isomer. The use of an inclusion complex of the glycoside acceptor with β-CD increased the efficiency of transglycosylation by increasing the solubility of the acceptor. In the same way, the use of β-D-GalNAc-OC6H4NO2-p as acceptor led to the preferential synthesis of β-D-Gal-(1→3)-β-D-GalNAc-OC6H4NO2-p over that of its (1→6) linked isomer. α-D-Gal-(1→3)-β-D-GlcNAc-OC6H4NO2-p was also synthesized with β-D-GlcNAc-OC6H4NO2-p acceptor by the consecutive use of β-D-galactosidases from porcine testes and Bacillus circulans. These enzyme reactions are efficient enough to allow the one-pot preparation of the desired disaccharide glycosides.View full abstractDownload PDF (719K) -
Yuushi Okumura, Asako Kudoh, Miwa Takashima, Masahiro Inoue, Kentaro S ...1996 Volume 120 Issue 4 Pages 856-864
Published: October 01, 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat mast cell tryptase was purified to homogeneity from rat tongue by a series of standard chromatographic procedures. Since the enzyme gave band corresponding to molecular mass of 32-35 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and exhibited a molecular mass of 135 kDa on gel filtration, it was presumed to be a noncovalently associated tetramer. The N-terminal amino acid sequence of 50 residues of the enzyme showed the highest degree of homology with the same region in mouse mast cell protease 7 (92%), and less homology to those of tryptases from man and dog, and peritoneal cells of rats and Mongolian gerbils. The inhibitor specificity of rat tongue tryptase was similar to that of rat peritoneal mast cell tryptase free from trypstatin: it was inhibited by α1-antitrypsin, Kunitz-type soybean trypsin inhibitor and Bowman-Birk soybean trypsin inhibitor, but these inhibitors do not inhibit the tryptases from rat skin, human lung, and dog mast cells. Judging from these results, together with other enzymatic properties, the enzyme may be a novel isoform of tryptase in rat tongue. Analysis by differential staining with peroxidaselabeled lectins of the enzyme suggested that it has tri- and/or tetraantennary complex-type oligosaccharides containing a relatively high amount of sialic acid. The immunohisto-chemical distribution of this enzyme indicated that the reactive antigen was specific in connective tissue but not in mucosal mast cells.View full abstractDownload PDF (3312K)
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