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Volume 119 (1996)
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Volume 119, Issue 5
Displaying 1-27 of 27 articles from this issue
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Crystallization and Preliminary X-Ray Analysis of Salicylate Hydroxylase from Pseudomonas putida S-1Tsuyoshi Yabuuchi, Kenzi Suzuki, Takao Sato, Kuniharu Ohnishi, Eiji It ...1996 Volume 119 Issue 5 Pages 829-831
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSApo-salicylate hydroxylase from Pseudomonas putida S-1 has been crystallized by the dialysis method, using ammonium sulfate as the precipitant. The crystals belong to hexagonal space group P62 or P64, with unit cell dimensions of a=b=142.8 Å and c=63.8 Å, and diffract X-rays at higher than 3.5 Å resolution. A heavy-atom derivative has been prepared by soaking a crystal in an ammonium sulfate solution containing p-chloromercuriphenylsulfonate.View full abstractDownload PDF (1201K) -
Mikio Shimizu1996 Volume 119 Issue 5 Pages 832-834
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was shown that a dipeptide, alanylhistidine, can act as a catalyst for the peptidyl transfer reaction in the absence of ribosomes between the amino acid moieties of phenylalanyl, lysyl, prolyl, and glycyl tRNAs depending on their model templates, poly U, poly A, poly C, and poly G, respectively. A template effect was observed: The peptidyl transfer reaction between tRNAGly y molecules (anticodon GCC) occurred in the presence of poly G but not in the presence of poly C. The reaction was most efficient for the best stacked poly A (tRNALys) and least efficient for the worst stacked poly U (tRNAPhe)View full abstractDownload PDF (1881K) -
Bum-Soo Hahn, Kyoung-Youl Yang, Eun-Mi Park, Il-Moo Chang, Yeong-Shik ...1996 Volume 119 Issue 5 Pages 835-843
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA thrombin-like enzyme, calobin, has been purified to homogeneity from the venom of Agkistrodon caliginosus by a procedure involving Bio-Gel P-100, Mono S, and Pro-RPC. The enzyme was identified as a monomer with a molecular weight of 34, 000 on SDS-PAGE, and its isoelectric point was 6.2. Calobin acted on fibrinogen to form fibrin with a specific activity of 226 NIH equivalent units, and also exhibited arginine esterase activity. The enzyme predominantly cleaved the α-chain of fibrinogen with little degradation of the β-chain. It contained abundant asparagine/aspartic acid residues, but very few tyrosine or methionine residues. The proteolytic activity of the enzyme with TAME as a substrate was higher than that of thrombin. However, it showed neither lysine esterase nor caseinolytic activity. The enzyme activity was strongly inhibited by PMSF, and moderately by benzamidine and soybean trypsin inhibitor, indicating it is a serine protease. On the other hand, the enzyme activity was not inhibited by hirudin or aprotinin. cDNA (1.6 kb) for calobin has been cloned from an A. caliginosus cDNA library. The cDNA sequence indicates that calobin is synthesized as a pre-zymogen of 262 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence encodes a 238-amino acid residue molecule exhibiting strong amino acid sequence homology to those of ancrod, batroxobin, and flavoxobin isolated from other snake venoms. Calobin contains 12 cysteine residues. As judged on alignment of the amino acid sequences of other thrombin-like enzymes (batroxobin, ancrod, and flavox-obin), calobin constitute the formation of six disulfide bridges. Amino acid residues, His43, Asp88, and Ser182, which are thought to be the catalytic active site are highly conserved. As calobin is a glycoprotein, its possible glycosylation site, Asn-X-Thr, is located at amino acid residues 81-83.View full abstractDownload PDF (1602K) -
Kensaku Hamada, Yasuo Hata, Yoshio Katsuya, Hajime Hiramatsu, Takaji F ...1996 Volume 119 Issue 5 Pages 844-851
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of Serratia protease from Serratia sp. E-15 was solved by the single isomorphous replacement method supplemented with anomalous scattering effects from both the Zn atom in the native crystal and the Sm atom in the derivative crystal, and refined at 2.0 Å resolution to a crystallographic R-factor of 0.194. The enzyme consists of N-terminal catalytic and C-terminal β-sandwich domains, as observed in alkaline protease from Pseudomonas aeruginosa IF03080. The catalytic domain with a five-stranded antiparallel β-sheet and five α-helices shares a basically common folding topology with those of other zinc metalloendoproteases. The catalytic zinc ion at the bottom of the active site cleft is ligated by His176, His180, His186, Tyr216, and a water molecule in a distorted trigonalbipyramidal manner. The C-terminal domain is a β-strand-rich domain containing eighteen β-strands and a short α-helix, and has seven Ca2+ ions bound to calcium binding loops. An unusual β-sheet coil motif is observed in this domain, where the β-strands and calcium binding loops are alternately incorporated into an elliptical right-handed spiral so as to form a two-layer untwisted β-sandwich structure. The Ca2+ ions in the C-terminal domain seem to be very important for the folding and stability of the β-sheet coil structure.View full abstractDownload PDF (1012K) -
Hiromichi Okuda, Chie Morimoto, Takahiro Tsujita1996 Volume 119 Issue 5 Pages 852-856
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNorepinephrine stimulated lipolysis in rat fat cells while (-)-alprenolol completely inhibited this lipolysis. (-)-Alprenolol competed for (-)-[3H]dihydroalprenolol(DHA) binding sites on rat fat cells. The specific (-)-[3H]DHA binding sites identified by competition with (-)-alprenolol were found to be transferred to the solubilized supernatant during preparation of endogenous lipid droplets from the fat cells. Although the lipid droplets did not exhibit specific (-)-[3H]DHA binding, norepinephrine induced lipolysis in a cell-free system consisting of the lipid droplets and hormone-sensitive lipase (HSL). Norepinephrine-induced lipolysis in the cell-free system was inhibited by propranolol and (-)-alprenolol, but not by phenoxybenzamine. The lipolytic action of norepinephrine and the anti-lipolytic actions of propranolol and (-)-alprenolol disappeared after sonication of the lipid droplets in the cell-free system. These results suggest that the adrenergic receptor concerned with lipolysis in fat cells may not be a specific (-)-[3H]DHA binding site, but may be closely related to the lipid droplets.View full abstractDownload PDF (616K) -
Yasuko Kawamura-Konishi, Naoki Hosomi, Saburo Neya, Sachiko Sugano, No ...1996 Volume 119 Issue 5 Pages 857-862
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe insertion of a copper (II) ion into mesoporphyrin by a monoclonal catalytic antibody has been investigated kinetically by measuring the increase in Soret absorbance due to the production of copper (II)-mesoporphyrin. The initial rate of the reaction showed saturation kinetics as a function of the mesoporphyrin concentration, while it increased linearly with an increase in the copper (II) concentration. Based on observations, a scheme for the reaction was proposed: mesoporphyrin binds to the antibody to form a complex, and copper (II) binds to the complex to yield copper (II)-mesoporphyrin. Kinetic parameters for the respective steps were estimated, and the thermodynamic parameters were calculated. The binding of mesoporphyrin to the antibody was endothermic and entropically driven. This implies that hydrophobic interactions are an important factor in the binding. Free energy profiles for the antibody-catalyzed and uncatalyzed reactions were drawn by use of the obtained thermodynamic parameter values. The results demonstrate that the rate acceleration by the antibody is ascribable to transition-state stabilization, and suggest that the structure of mesoporphyrin in the complex is more distorted than that of free mesoporphyrin.View full abstractDownload PDF (718K) -
Mitsuo Tagaya, Nobuhiro Henomatsu, Tamotsu Yoshimori, Akitsugu Yamamot ...1996 Volume 119 Issue 5 Pages 863-869
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNordihydroguaiaretic acid (NDGA) blocks intra-Golgi protein transport in a cell-free system and prolactin secretion from GH3 cells [Tagaya, M., Henomatsu, N., Yoshimori, T., Yamamoto, A., Tashiro, Y., and Fukui, T. (1993) FEBS Lett. 324, 201-204]. To determine which intracellular secretory pathway(s) is inhibited by NDGA, we investigated its effect on the transport of the vesicular stomatitis virus-encoded glycoprotein in BHK-21 cells. NDGA blocked protein transport from the endoplasmic reticulum to the Golgi apparatus, and from the trans-Golgi network to the plasma membrane. In addition, it retarded the brefeldin A-induced retrograde transport of mannosidase II to the endoplasmic reticulum. Although NDGA had an inhibitory effect on protein synthesis, it induced the expression of BiP, a chaperone located in the endoplasmic reticulum. The induction of BiP may be a consequence of the inhibition of protein transport by NDGA.View full abstractDownload PDF (6216K) -
Iori Maeda, Yasuyuki Shimohigashi, Koichi Ikesue, Takeru Nose, Yuzuru ...1996 Volume 119 Issue 5 Pages 870-877
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe dipeptide benzyl amide H-D-Thr-Phe-NH-CH2-C6H5 was found to inhibit chymotrypsin strongly (Ki=4.5×10-6M) in a competitive manner. When a series of phenyl amides H-D-Thr-Phe-NH-(CH2)n-C6H5 (n=0-4) were tested, inhibitory potency peaked at n=1 (benzyl amide). Incorporation of a methyl group into the benzyl methylene resulted in formation of stereoisomers, H-D-Thr-Phe-NH-(R or S)-CH(CH3)-C6H5, with considerably different inhibitory potencies. The R-isomer was as active as the benzyl amide, while the S-isomer was about 30-fold less active than the benzyl amide. Furthermore, when a fluorine atom was introduced into the para-position of the amide-benzyl group, the resulting H-D-Thr-Phe-NH-CH2-C6H4(p-F) showed considerably enhanced inhibitory activity (about 5-fold, Ki=9.1×10-7M). In conformational analysis by 400MHz 1H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upfield shifts of D-Thr-βOH (shifts in ppm, 0.09-0.17), D-Thr-βCH (0.23-0.32), and D-Thr-γCH3 (0.38-0.53), indicating the presence of shielding effects from the benzene ring. In addition, NOE enhancements between the D-Thr-γCH3 and Phe-phenyl groups were evidenced by measurements of two-dimen-sional NOESY spectra and NOE difference spectra. These observations demonstrated the spatial proximity of these side chains, which is due to side chain-side chain CH/π interaction. All these results support the idea that the amide-benzyl group binds at the chymotrypsin S1, site, while the hydrophobic core with CH/π interaction binds at the S2 or S1' site.View full abstractDownload PDF (976K) -
Kazuko Yamaoka, Arnd Ingendoh, Satoshi Tsubuki, Yoshitaka Nagai, Yutak ...1996 Volume 119 Issue 5 Pages 878-886
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously we demonstrated that overexpression of a, β-galactoside binding protein, galectin-1, caused the transformation of BALB3T3 fibroblast cells [Yamaoka, K., Ohno, S., Kawasaki, H., and Suzuki, K. (1991) Biochem. Biophys. Res. Commun. 179, 272-279]. We have now studied the structure-function relationships between the sugar-binding activity and the mitogenic activity of galectin-1 purified from an avian sarcoma virus-transformed rat NRK cell line, 77N1. The purified galectin-1 (t-galectin-1) had potent mitogenic activity in BALB3T3 cells, but no sugar-binding activity. Treatment of t-galectin-1 with 2-mercap-toethanol decreased its mitogenic activity, but resulted in the appearance of a sugar binding activity. Chemical modification of sulfhydryl groups in purified t-galectin-1 with [14C]-iodoacetamide suggested the presence of intramolecular disulfide bonds. MALDI-TOF mass spectrometric analysis of the native and reduced forms of the tryptic peptides from t-galectin-1 showed that t-galectin-1 has two intramolecular disulfide bonds (Cys2-Cys16 and Cys42-Cys60). These studies suggest that these intramolecular disulfide bonds of t-galectin-1 are essential for its mitogenic activity and that the different activities may be regulated by structural changes caused by intramolecular disulfide bond-breakage.View full abstractDownload PDF (3540K) -
Wortmannin and Li+ Specifically Inhibit Clathrin-Independent Endocytic Internalization of Bulk FluidSatoshi B. Sato, Takahisa Taguchi, Shohei Yamashina, Sakuji Toyama1996 Volume 119 Issue 5 Pages 887-897
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIncubation of a human fibrosarcoma cell line HT-1080 in Li+-containing medium inhibited internalization of a fluid marker, horseradish peroxidase (HRP), by more than 80%. The ion inhibited the activity enhanced by Ca2+ or phorbol 12-myristate 13-acetate. We also found that wortmannin (WT), a potent inhibitor of phosphoinositide (PI) 3-kinase (PI 3-k), inhibited the non-stimulated and the two stimulated types of endocytosis to the same extent as Li+. In contrast, neither WT nor Li+ influenced the early internalization of transferrin (Tfn), EGF or platelet-derived growth factor. Neither targeting to early endosomes nor recycling of the once-internalized Tfn was influenced. When the cytoplasmic pH was lowered by chasing cells that had been preincubated with 25mM NH4Cl in an amiloridecontaining Na+-free medium, more than 90% of internalization of Tfn in HT-1080 cells was inhibited, while that of HRP was reduced by only 35%. In contrast, WT reduced the uptake of HRP by KB cells by 34%, while 60% of the activity was inhibited by the treatment for cytoplasmic acidification. Comparison of other cells i. e., A-549 and a human diploid cell line Miyajima, indicated that cells showing higher sensitivity to WT were less sensitive to low cytoplasmic pH. These results suggest that, in all the cells studied, bulk fluid is internalized either via a clathrin-independent/PI 3-k-dependent route or via a clathrindependent/PI 3-k-independent one, though the ratio varied among them. We also found that internalization of a mAb directed toward the 116 (100)-kDa subunit of vacuolar ATPase [OSW2; Sato and Toyama (1994) J. Cell Biol. 127, 39-53] in the fluid phase was inhibited by WT, but the antibody was still internalized in a surface-bound form. Regardless of the treatment with WT, most of the antibody was transported to endosomes that were associated with Tfn receptor. These results suggest that both internalization routes are targeted to the same early endosomal compartments.View full abstractDownload PDF (7483K) -
Ken-ichi Saguchi, Takashi Tobe, Ken Hashimoto, Yuta Nagasaki, Eiichi O ...1996 Volume 119 Issue 5 Pages 898-905
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInter-α-trypsin inhibitor (ITI) family heavy chain-related protein (IHRP) is a novel human glycoprotein that shows significant homology in amino acid sequence to proteins of the ITI family heavy chains from human plasma. Three overlapping clones that encode the human inter-α-trypsin inhibitor family heavy chain-related protein (IHRP) gene (ITIHL1) were isolated and characterized. The IHRP gene spans 15 kb and is composed of 24 exons from 27 to 207 bp in size with consensus splice sites. The gene codes for the precursor of IHRP, which is similar to inter-α-trypsin inhibitor (ITI) family heavy chains. Two major transcription initiation sites were identified in the 5'-flanking region. They contain putative promoter elements, but no typical TATA box. Some exons of this gene showed significant similarities to those of the ITI-H1 gene in nucleotide length and in intron phasing. The tissue-specific transcription of this gene may be due to the presence of binding sites for the hepatocyte nuclear factors LF-A1, HNF-5, NF-IL6, and C/EBP. This gene was found to be localized very close to another unknown gene related to EST (GenBank accession #: R54643, R50663, R50563, H27139, and R54913).View full abstractDownload PDF (3587K) -
Masanori Mishima, Issei Mabuchi1996 Volume 119 Issue 5 Pages 906-913
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe studied enzymatic activities in sea urchin egg extracts that phosphorylate myosin regulatory light chain (MRLC) from chicken gizzard smooth muscle. The activity in the presence of EGTA showed cell cycle-dependent changes similar to that of histone Hl kinase, namely, it peaked shortly before cleavage, while that in the presence of Ca2+ ions did not show significant change during division cycle. Phosphopeptide mapping revealed that both the sites phosphorylatable by smooth muscle myosin light chain kinase (MLCK sites) and the sites phosphorylatable by protein kinase C (PKC sites) were phosphorylated in the presence or absence of Ca2+ ions. By analyses using an inhibitor of cdc2 kinase, butyro-lactone-I, and ion exchange column chromatography, at least three kinases were detected as kinases that phosphorylate MRLC in vitro. These kinases phosphorylated distinct sites on MRLC. The first one, which phosphorylated the PKC sites, was identified as cdc2 kinase. The second one phosphorylated the MLCK sites in the absence of Ca2+ ions. The third one phosphorylated unknown sites. Possible implication of these activities in regulation of cytokinesis is discussed.View full abstractDownload PDF (3334K) -
Kazuko Hanyu, Osamu Numata, Mihoko Takahashi, Yoshio Watanabe1996 Volume 119 Issue 5 Pages 914-919
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we succeeded in cloning a cDNA of the 23-kDa Tetrahymena Ca2+-binding protein, designated TCBP-23. Analysis of the deduced amino acid sequences showed that TCBP-23 is a member of the EF-hand family of Ca2+-binding proteins. However, its physiological function was not elucidated. In the studies reported here, recombinant TCBP-23 was expressed in Escherichia coli and purified. Since recombinant TCBP-23 binds Ca2+ in vitro, Ca2+-binding domains of the protein are likely to be functional in vivo. Rabbit antibodies against TCBP-23 were raised and used to determine the intracellular localization of the protein in Tetrahymena cells by indirect immunofluorescence. The antibodies strongly stained the whole cell cortex except for the oral apparatus and around the basal bodies. TCBP-23 remained in detergent-extracted cells, suggesting that it is associated with the epiplasm, the membrane skeleton of Tetrahymena. These results suggest that TCBP-23 may mediate Ca2+-regulated processes in the cell cortex.View full abstractDownload PDF (3077K) -
Kazuki Okamoto, Gang Liu, Wen-Gong Yu, Tetsuji Ochiai, Fumihide Isohas ...1996 Volume 119 Issue 5 Pages 920-925
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously purified macromolecular-translocation inhibitor-III (MTI-III), which inhibits the binding of the activated glucocorticoid-receptor complex (GR) to nuclei, to homogeneity from rat liver, and we found that the purified MTI-III bound to partially purified activated GR under low salt conditions at slightly acidic pH [Liu, G., Okamoto, K., and Isohashi, F. (1993) Eur. J. Biochem. 218, 679-687]. This was the first direct evidence that the inhibitor acts through a direct interaction with the activated GR. In this study, we examined whether the purified MTI-III could interfere with the binding of GR to a DNA fragment containing a specific glucocorticoid-response element (GRE). Under nearly isotonic salt conditions at neutral pH, the activated GR bound to the GRE but not to nonspecific DNA. Under similar conditions, the activated GR also bound to the purified MTI-III. The resulting GR/MTI-III complex did not bind to the GRE. We also found that addition of MTI-III to the GR/GRE complex resulted in time-dependent disruption of the GR/GRE complex and formation of the GR/MTI-III complex. The half-life of the GR/GRE complex in the presence of MTI-III was about 13min. These results suggest that MTI-III enhances the release of GR from the GR/GRE complex And immediately forms a stable GR/MTI-III complex.View full abstractDownload PDF (766K) -
Yuri Aoyama, Mitsuhide Noshiro, Osamu Gotoh, Susumu Imaoka, Yoshihiko ...1996 Volume 119 Issue 5 Pages 926-933
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the orthology of sterol 14-demethylase (P45014DM), the only known P450 enzyme distributed widely in eukaryotes with a conserved metabolic role, the full-length amino acid sequences of rat and human P45014DMs were determined from the cloned cDNA sequences, and compared with those of the corresponding fungal proteins (CYP51). The amino acid identity value between given pairs of P45014DMs ranged from 93% (human/rat) to 39% (human or rat/Saccharomyces cerevisiae). All the P45014DMs formed a single cluster in a phylogenetic tree constructed from representative P450 protein sequences currently available. The nearest neighbors to the P45014DM cluster in the phylogenetic tree were CYP7 (cholesterol 7α-hydroxylase) and CYP8 (prostacyclin synthase), and the divergence point of fungal and mammalian P45014DMs was clearly more recent than that of P45014DM and CYP7/CYP8. These lines of evidence show that fungal and mammalian P45014DMs are really orthologous. This is the first example of orthologous P450s occurring in distinct kingdoms. P45014DM may be an ancient P450 which arose before the divergence of major eukaryotic branches and has been conserved throughout evolution. The amino acid identity value (93%) between human and rat P45014DMs was comparable to those observed for some housekeeping enzymes. In addition, a processed pseudogene of P45014DM was found in a rat genomic DNA library, suggesting the expression of P45014DM in germ line cells. These facts suggest that P45014DM may be a housekeeping enzyme essential for the viability of mammals.View full abstractDownload PDF (1083K) -
Torn Matsukawa, Masatoshi Kitagawa, Kazuhiko Katayama, Yukiko Nagai, T ...1996 Volume 119 Issue 5 Pages 934-939
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA set of acute-phase proteins was found to have been induced in rat after triiodothyronine (T3) treatment. To study the mechanism of gene activation by T3, the rat gene of α1-acid glycoprotein (AGP), one of the major acute-phase proteins, was cloned and analyzed. CAT assay identified a thyroid hormone responsive element (TRE) in the first intron of the AGP gene and showed that mutations introduced into the TRE region destroyed the TRE activity. Gel shift assay and methylation interference assay showed that the thyroid hormone receptor bound to the TRE. The TRE identified here had a palindromic structure and was highly homologous to other known TREs.View full abstractDownload PDF (1471K) -
Qing Shao, Naokatu Arakaki, Tomokazu Ohnishi, Osamu Nakamura, Yasushi ...1996 Volume 119 Issue 5 Pages 940-946
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHepatocyte growth factor (HGF)/scatter factor is known to be the most potent mitogen for hepatocytes. In this paper, we report that lipogenesis in primary cultured rat hepatocytes treated with 10ng/ml of recombinant human HGF (rhHGF) for 24h was stimulated, as measured by the incorporation of 3H2O into long-chain fatty acids, to more than twice as much as the control. Insulin (0.1μM) was more effective than rhHGF but rhHGF did not show an additive or synergistic effect when added to insulin. We also showed that treatment with rhHGF increased the activities of glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme, key enzymes which supply NADPH for lipogenesis, and acetyl-CoA carbox-ylase, the rate-limiting enzyme of lipogenesis. The increase in G6PDH and acetyl-CoA carboxylase activities was accompanied by increases in the levels of mRNA for the enzymes. These results suggest that HGF is involved in liver regeneration not only by stimulation of cell proliferation but also by acceleration of differentiation of hepatocytes.View full abstractDownload PDF (2082K) -
Akio Shimizu, Masamichi Ikeguchi, Takuji Kobayashi, Shintaro Sugai1996 Volume 119 Issue 5 Pages 947-952
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe investigated the conformations of peptides that encompass the B helix or C helix region formed in the molten globule of bovine α-lactalbumin to get information on the molecular mechanism that stabilizes the molten globule. The CD spectra show that the isolated B and C helices are intrinsically unstable. The chemical shifts, NOE connectivities, and CD spectrum indicate that no helical structure is induced in the C helix region (86-99) by extending the peptide sequence to include the hydrophobic cluster region (101-107), although the hydrophobic cluster region can be regarded as a possible initiation site for folding of the protein. We also clarified that the isolated B helix (23-34) peptide does not directly interact with the C helix or hydrophobic cluster region. These results suggest that the B and C helices in the molten globule are stabilized by their interaction with other parts of the protein.View full abstractDownload PDF (1899K) -
Masayuki Funaba, Takuya Murata, Hisako Fujimura, Eri Murata, Matanobu ...1996 Volume 119 Issue 5 Pages 953-960
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInhibin-A is a glycoprotein composed of an α subunit containing a glycosylation site and a .αA subunit, whereas activin-A is a homodimer of two inhibin, βA subunits. We examined the recognition of activin-A and inhibin-A by several antisera to the α or βA subunit, and factors affecting the recognition. A total of six polyclonal antibodies to inhibin subunits, i. e., two antisera to a peptide fragment of the α subunit [α(1-19) and α(l-26)], and four antisera to the βA subunit [βA(1-10), βA(70-79), βA(87-99), and βA(94-105)], was generated. On Western blot analysis, the anti-βA(87-99) and βA(94-105) sera recognized recombinant human activin-A but not inhibin-A under non-reducing conditions. When inhibin-A was deglycosylated with N-glycosidase-F, inhibin-A could be recognized by the anti-βA(87-99) and βA(94-105) sera. In addition, when activin-A bound to a nitrocellulose membrane was pre-incubated with recombinant human follistatin, the recognition of activin-A by the anti-βA(87-99) and βA(94-105) sera was decreased. These results suggested that the lower affinity of follistatin to inhibin-A than to activin-A might be likely explained as reflecting a site associated with the glycosylation of inhibin-A. However, the exposure of amino acids 87-105 of the inhibin βA subunit on the molecular surface through deglycosylation did not increase the affinity of inhibin-A for follistatin but rather resulted in poor binding with follistatin. The present data suggest that (1) amino acids 87-105 of the inhibin/activin βA subunit are located on the molecular surface, although this region of inhibin-A is concealed by the carbohydrate chain of the α subunit, (2) the region responsible for follistatin binding within the activin, βA subunit is spanned by amino acids 87-105, and (3) the mode of binding of inhibin-A to follistatin is quite different from that of activin-A to follistatin, and the former may be influenced by glycosylation.View full abstractDownload PDF (5150K) -
Hiroshi Kaetsu, Teruto Hashiguchi, Don Foster, Akitada Ichinose1996 Volume 119 Issue 5 Pages 961-969
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe a subunit of coagulation factor XIII lacks a hydrophobic signal sequence for secretion from cells, while the b subunit has a typical signal sequence. To determine whether the a subunit can be synthesized and released, expression vectors containing the cDNA for either subunit were transfected into baby hamster kidney (BHK) cells. Western blotting analysis and gel filtration chromatography demonstrated that the recombinant a and b subunits (rXIIIa and rXIIIb) had the same molecular weights and subunit structures (a2, b2, and a2b2) as the native molecules. rXIIIa was enzymatically active when activated by thrombin. Most rXIIIb was secreted as measured by ELISA, while most rXIIIa was detected in the cytosol by subcellular fractionation. Co-expression with rXIIIb in the same cells did not promote the release of rXIIIa. Treatment of the cells with brefeldin A, a potent inhibitor of protein transportation, blocked the secretion of rXIIIb, although it had no effect on the release of rXIIIa. Several drugs and heat stress induced the release of rXIIIa, which correlated directly with that of cytoplasmic lactate dehydrogenase. These results suggest that the a subunit is released from cells as a consequence of cell injury, which is independent of the classical secretory pathway.View full abstractDownload PDF (3779K) -
Kuniko Ishikawa, Eizo Kaneko, Arata Ichiyama1996 Volume 119 Issue 5 Pages 970-978
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSerine: pyruvate/alanine: glyoxylate aminotransferase in the liver is a class IV aminotransferase. The present study was undertaken to characterize the pyridoxal 5'-phosphate (PLP) binding to a recombinant rat serine: pyruvate/alanine: glyoxylate aminotransferase (SPT10), which is a homodimer of 44.4 kDa subunits. Purified SPT10 exhibited absorption maxima at _??_330nm in addition to a 278nm protein peak and a _??_420nm peak of PLP bound via Schiff base, and contained 0.56-0.69 mol of PLP per mol of subunit. Apo-SPT10 without measurable bound PLP did not exhibit the absorbance at _??_420nm, but still showed the _??_330nm peak. Upon reconstitution, 0.73-0.79 mol of PLP per mol of subunit was bound to apo-SPT10 with an apparent Kd of _??_0.1μM, resulting in a holo-SPT10 preparation whose specific activity and A_??_420/A_??_330 absorbance ratio were higher than those of the original SPT10. On SDS/PAGE of BrCN-cleavage peptides of NaBH4-reduced SPT10 22-23 kDa fragments migrated as a pair of bands. On amino acid sequencing, the _??_22 and _??_23 kDa pair gave the same sequence, except that Lys was released only from the _??_22 kDa band material in the cycle corresponding to Lys209. NaB3H4-treated SPT10 also migrated as a pair of 44-45 kDa bands and 3H was incorporated only into the _??_45 kDa band. It appears that SPT10 has the capacity to bind 1 mol of PLP to Lys209 of every subunit, but usually binds less PLP in a Schiff base structure, probably due to the presence of a 330nm-absorbing chromophore.View full abstractDownload PDF (1234K) -
Akira Takagi, Tetsuhito Kojima, Shinobu Tsuzuki, Akira Katsumi, Tomio ...1996 Volume 119 Issue 5 Pages 979-984
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo better understand the regulation of ryudocan (syndecan-4) expression, we have determined the structural organization of the human ryudocan gene. The human ryudocan gene extends approximately 24 kilobases and is divided into five exons, which appear to be conserved in syndecan family members. Exon I encodes the signal peptide; exons II-IV, the extracellular domain; and exon V, the transmembrane and cytoplasmic domains, which are highly homologous among syndecan family members. Primer extension analysis showed that human ryudocan gene had a single transcription initiation site, located 3 bases upstream from the described cDNA [Kojima et al. (1993) BBRC 190, 814-822]. The 5'-flanking sequences of human ryudocan gene contain a TATA-like sequence as well as a variety of other potential binding sites for transcription factors, including Sp1, Ap-2, NF-kB, E-alpha H box, H4TF-2, and LBP-1, and were capable of functioning as a promoter. The determination of the human ryudocan gene structure will allow elucidation of constitutive, cell-specific, tissue-specific, and developmentally regulated expression.View full abstractDownload PDF (1756K) -
Yasuhiro Hashimoto, Akihiro Oshima, Hisashi Narimatsu, Akemi Suzuki1996 Volume 119 Issue 5 Pages 985-990
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe attempted to express the cholera toxin B subunit (CTXB) in the Golgi apparatus of cultured mammalian cells by means of gene transfection. Complementary DNA of CTXB was ligated with the Golgi-retention signal sequence of human β1, 4 galactosyltransferase cDNA, and the chimeric gene yielded was inserted into a mammalian expression vector. The resultant construct was transfected into COS-1 cells for transient expression and into Swiss 3T3 cells for stable expression. The expression of a fusion protein encoded by the chimeric gene was demonstrated according to the following criteria: first, detection of a protein exhibiting the expected molecular mass on Western blot analysis using an anti-CTXB antibody; second, detection of the protein located in the Golgi area by indirect immunofluoresence microscopy; and third, detection of GM1 binding activity in cell lysates. Stable transformants satisfying the above criteria were subjected to an assay for mitogen-induced DNA synthesis. These transformants exhibited significantly lower DNA synthesis than mock transfection cells on stimulation with basic fibroblast growth factor (bFGF), whereas the two types of cells exhibited similar responses to 10% fetal calf serum and other mitogens, such as epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, cal-cium ionophore A23187, and platelet-derived growth factor. Analysis of the binding of radio-iodinated bFGF to the cells revealed that the transformants did not exhibit a significant decrease in the binding affinity or the number of high affinity sites. These results suggest that the fusion protein specifically inhibits the bFGF signaling not at the binding step but rather at a later step(s) triggered by the binding.View full abstractDownload PDF (4001K) -
Masashi Kumano, Kaoru Omichi, Sumihiro Hase1996 Volume 119 Issue 5 Pages 991-997
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cytosolic neutral α-mannosidase was purified from bovine liver. Its molecular weight was found to be 500, 000 on gel filtration. The activity of the enzyme toward Manαl-6 (Manα1-3) Manα1-6 (Manα1-3) Manα1-4G1cNAc-PA was increased 26-fold by preincubation with 1mM Co2+. Manα1-6 (Manα1-3) Manα1-6 (Manα1-3) Manβ1-4GlcNAc was hydrolyzed by the enzyme to Manα1-3Manα1-6 (Manα1-3) Manβ1-4GlcNAc, whichwas further hydrolyzed to Manα1-6 (Manα1-3) Manβ1-4GlcNAe. The rate of hydrolysis was 15-fold greater than that of Manα1-6 (Manα1-3) Manα1-6 (Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc. This substrate specificity suggested that the enzyme could be involved in the degradation of oligomannose-type sugar chains with one GlcNAc residue released from glycoproteins by endo-β-N-acetylglucosaminidase, and supported a pathway for glycoprotein catabolism via oligomannosyl glycans with one GlcNAc residue proposed on the basis of an earlier study on a cytosolic neutral α-mannosidase from Japanese quail oviduct [Oku, H. and Hase, S. (1991) J. Biochem. 110, 982-989].View full abstractDownload PDF (1839K) -
Noriko Hamagashira, Hisashi Oku, Tomohiro Mega, Sumihiro Hase1996 Volume 119 Issue 5 Pages 998-1003
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn α-mannosidase capable of hydrolyzing three Man α1, 2-residues from pyridylamine (PA-) labeled Man9GlcNAc2 was purified from hen oviduct. The purity of the preparation was analyzed by PAGE; its molecular weight was 42, 000 by SDS-PAGE or 50, 000 by gel filtration. The pH optimum was 6.5. The enzyme was inactivated with EDTA; enzyme activity was restored by the addition of Ca2+. The enzyme acitivity was inhibited by 1-deoxymannojirimycin, but not by swainsonine. The substrate specificity of the purified enzyme was analyzed using PA-oligomannose-type sugar chains. When Man9GlcNAc2-PA was digested, Manα1-6 (Manαl-2Manαl-3) Manα1-6 (Manαl-3) Manβ1-4GlcNAcβ1-4Glc-NAc-PA was obtained as an end product, and the enzyme was incapable of hydrolyzing p-nitrophenyl α-D-mannoside and Man α1, 3- or Man α1, 6-residues. Judging from these characteristics, the enzyme was classified as a Man9-mannosidase or Golgi mannosidase I and speculated to participate in the processing or catabolism of glycoproteins.View full abstractDownload PDF (1366K) -
Ying Liu, Hiromi Kohara, Eikichi Hashimoto1996 Volume 119 Issue 5 Pages 1004-1013
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Mr 25, 000 protein, which was isolated from the cytosolic fraction of Xenopus laevis oocytes, is a newly identified substrate for casein kinase II and protein kinase C [Hashimoto et al. (1995) J. Biochem. 118, 453-460], and was recently shown to have the ability to modulate protein phosphatase 2A activity [Hashimoto et al. (1996) J. Biochem. 119, 626-632]. Acid phosphatase treatment of the protein shifted its electrophoretic mobility from 25 to 20 kDa on SDS-PAGE. The content of alkali-labile phosphate bound covalently to the protein was 53 mol per mol of Mr 25, 000 protein. Amino acid composition analysis revealed that there are 50 serine residues and 6 threonine residues per mol of this protein. Therefore, this Mr 25, 000 protein seems to be highly phosphorylated in vivo. The Mr 25, 000 protein, once partially dephosphorylated by acid phosphatase, served as an efficient substrate for casein kinase I and casein kinase II. When entirely dephosphorylated, the Mr 25, 000 protein was used as a substrate, the rate of phosphorylation with both casein kinases being decreased. This behavior of casein kinases toward the Mr 25, 000 protein reflects the possible mechanism of multisite phosphorylation in which the introduction of a phosphate group facilitates sequential phosphorylation.View full abstractDownload PDF (2398K) -
Takuro Yaoi, Kentaro Miyazaki, Tairo Oshima, Yutaka Komukai, Mitiko Go1996 Volume 119 Issue 5 Pages 1014-1018
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe coenzyme specificity of isocitrate dehydrogenase from an extreme thermophilic bacterium was converted from NADP-dependent to NAD-dependent by replacing a “module” involved in the coenzyme binding site. The conversion was not possible with the replacement of a few residues that interact with the coenzyme. In addition, the modulereplaced mutant dehydrogenase was as stable as the original, wild type enzyme. The results support a previous hypothesis that a module is a structural and functional unit of a protein.View full abstractDownload PDF (1765K)
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