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Volume 133 (2003)
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Volume 133, Issue 2
Displaying 1-16 of 16 articles from this issue
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Yasutomi Nishizuka2003 Volume 133 Issue 2 Pages 155-158
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSDownload PDF (608K) -
Akiko Nakatomi, Michio Yazawa2003 Volume 133 Issue 2 Pages 159-164
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCalcineurin has been inferred to function in meiosis and spermiogenesis in testis. Here, we identified a calcineurin-binding protein in scallop testis by Far-Western blot analysis using purified calcineurin as a probe. The molecular mass of the binding protein estimated on the blot was 75 kDa. The isolated cDNA clone encoded a novel 474-residue protein, named CaNBP75. The region between T6 and A210 of CaNBP75 was responsible for the interaction with calcineurin. CaNBP75 was predominantly expressed in testis and ovary of scallop. Thus, CaNBP75 may modulate the physiological function of calcineurin in the testis and ovary of scallop, such as in spermiogenesis or meiosis.View full abstractDownload PDF (3812K) -
Gyosuke Sakashita, Hiroshi Shima, Masakazu Komatsu, Takeshi Urano, Aki ...2003 Volume 133 Issue 2 Pages 165-171
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSInhibitor 2 (I-2) is a ubiquitous regulator of type 1 protein phosphatase (PP1). Previous in vitro studies suggested that its inhibitory activity towards PP1 is regulated by phosphorylation at Thr72 by glycogen synthase kinase-3β (GSK-3β), and at Ser86, Serl20, and Serl2l by casein kinase 2 (CK2). Here we report that GSK-3β expressed in COS-7 cells phosphorylates wild-type I-2 but not an I-2 mutant carrying a T to A substitution at residue 72, showing that GSK-3β phosphorylates I-2 at T72 in vivo as well. Co-immunoprecipitation study demonstrated that HA-GSK-3β and I-2-FLAG co-exist in a same complex in the intact cells, but they do not bind directly. It is noteworthy that co-expression of Myc-PP1C significantly increased co-precipitation of HA-GSK-3β with I-2-FLAG, showing a complex formation of HA-GSK-3β/Myc-PP1C/I-2-FLAG in vivo. Further studies using a GSK-3β kinase-dead mutant and LiCl, an inhibitor of GSK-3β, showed that the enzyme activity of GSK-3β is required for co-precipitation. IP-Western study using several I-2 mutants substituted at phosphorylation sites (T72, S86, S120, and S121) suggested that phosphorylation of I-2 by CK2 is also involved in enhancement of association between GSK-3β and I-2 in vivo. This study is the first demonstration that GSK-3β associates with PP1C/I-2 complex and phosphorylates I-2 at T72 in the intact cells.View full abstractDownload PDF (3810K) -
Shohei Akahane, Hideaki Kamata, Hitoshi Yagisawa, Hajime Hirata2003 Volume 133 Issue 2 Pages 173-180
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA novel gene encoding a small neutral amino acid transporter was cloned from the genome of the hyperthermophilic archaeon Thermococcus sp. KS-1 by functional cloning using Escherichia coli strain AK430, which is defective in transporting glycine and D-alanine. The cloned gene, snatA, encoded a protein of 216 amino acid residues, SnatA, and was predicted to be a membrane protein with six membrane-spanning segments. E. coli AK430 cells transformed with snatA transported glycine with an apparent Kt value of 24μM, which was one order of magnitude higher than that of other known glycine/alanine transporters, including cycA of E. coli and aep of thermophilic bacterium PS3. Competition studies revealed that SnatA transported various L-type neutral amino acids, but its substrate specificity was different from that of CycA or ACP. The glycine transport was inhibited by a protonophore, FCCP, or valinomycin plus nigericin, indicating that the process is dependent on an electrochemical potential of H+. Homology searches revealed no homology with any transporters known to date. However, several hypothetical genes in prokaryote cells enrolled in the gene bank showed significantly high homology scores, indicating that snatA and its homologues form a family of prokaryotes. To our knowledge, this is the first report on the cloning of a gene of an amino acid transporter from a hyperther-mophilic archaeon.View full abstractDownload PDF (1937K) -
Mikiko Takahashi, Yusuke Gotoh, Takayuki Isagawa, Tamako Nishimura, Em ...2003 Volume 133 Issue 2 Pages 181-187
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPKNαis a fatty acid- and Rho-activated serine/threonine protein kinase having a catalytic domain homologous to members of the protein kinase C family. Recently it was reported that PKNα is involved in the p38 mitogen-activated protein kinase (MAPK) signaling pathway. To date, however, how PKNα regulates the p38γ MAPK signaling pathway is unclear. Here we demonstrate that PKNα efficiently phosphorylates MLTKαa (MLK-like mitogen-activated protein triple kinase), which was recently identified as a MAPK kinase kinase (MAPKKK) for the p38 MAPK cascade. Phosphorylation of MLTKa by PKNα enhances its kinase activity in vitro. Expression of the kinasenegative mutant of PKNα inhibited the mobility shift of MLTKα caused by osmotic shock in SDS-PAGE. Furthermore, PKNα associates with each member of the p38γ MAPK signaling pathway (p38γ, MKK6, and MLTKα). These results suggest that PKNα functions as not only an upstream activator of MLTKα but also a putative scaffold protein for the p38γ MAPK signaling pathway.View full abstractDownload PDF (3819K) -
Yoshiya Ikawa, Tan Inoue2003 Volume 133 Issue 2 Pages 189-195
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe catalytic core of the Tetrahyemena group I ribozyme consists of two functionally different domains, P4-P6 and P3-P7, that are conjugated via multiple tertiary interactions. The sequence encoding the P3-P7 domain is divided into two fragments in its primary sequence although the two domains are physically separable in the three dimensional (3D-) structure of the ribozyme: The sequence encoding the P4-P6 domain is inserted into that of the P3-P7 domain. An artificial rearrangement was designed and attempted for the primary sequence of the P3-P7 domain on the basis of a 3D-structural model and the biochemical data on the ribozyme. The domain in the primary structure was relocated to form a contiguous region while retaining the 3D-structure of the ribozyme required for self-splicing. The topologically rearranged ribozyme exhibited self-splicing activity.View full abstractDownload PDF (4543K) -
Yoshiaki Hirako, Kohichiro Yoshino, Detlef Zillikens, Katsushi Owaribe2003 Volume 133 Issue 2 Pages 197-206
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSBullous pemphigoid antigen 180 (BP180)/type XVII collagen is a transmembrane hemidesmosomal protein. Previously, we demonstrated that the collagenous ectodomain of BP180 can be cleaved within the extracellular non-collagenous (NC) 16A domain adjacent to the cell membrane and released from the cell surface. Here, we report that the BP180 cleavage is mediated by a membrane-associated metalloprotease expressed in epithelial cells. A tissue inhibitor of metalloprotease 1 (TIMP-1), but not TIMP-2, like the synthetic metalloprotease inhibitor KB-R8301, significantly reduced the cleavage. Within epithelial cells cultured for more than 36h past confluency, antibodies to BP180 showed a reduced hemidesmosomal staining. Observed for the first time, addition of KB-R8301 to the cell culture preserved this staining. To examine the effect of the extracellular cleavage of BP180 on molecular interactions among hemidesmosomal components, we eliminated its collagenous extracellular portion, except for the NC16A domain, by collagenase digestion. Interestingly, this collagenase treatment caused partial disassembly of hemidesmosomal components in cultured human keratinocytes. Moreover, a monoclonal antibody specific for the cleaved extracellular fragment detected a unique tissue distribution of the fragment that might reflect an association of the cleavage process with the mitotic activity of epithelial tissues. Our observations demonstrate that the cleavage of BP180 occurring within the NC16A domain is mediated by a membrane-associated metalloprotease and suggest a possible involvement of the cleavage in hemidesmosomal disassembly.View full abstractDownload PDF (7218K) -
Keisuke Horiuti, Naoto Yagi, Shigeru Takemori, Maki Yamaguchi2003 Volume 133 Issue 2 Pages 207-210
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSEffects of ADP on the conformation of myosin cross-bridges were studied in x-ray diffraction experiments on single skinned fibers of frog skeletal muscle by photorelease of ADP from caged-ADP. The experiments were performed at the third-generation synchrotron radiation facility SPring-8 with a time resolution of 5ms. The intensity of the third-order meridional reflection from myosin filaments (at 1/14.4 nm-1) increased promptly after the ADP release with a time constant smaller than 5ms, which was similar to that of tension decline. The results show that ADP binding induces a conformational change of myosin in skeletal muscle fibers.View full abstractDownload PDF (510K) -
Akiko Shiratsuchi, Tomoe Mori, Yae Takahashi, Koichiro Sakai, Yoshinob ...2003 Volume 133 Issue 2 Pages 211-218
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe structure and subcellular localization of a number of molecules change during apoptosis. These molecules are recognized by the immune system, leading to the development of autoimmunity when apoptotic cells fail to be effectively cleared by phagocytosis. We searched for such molecules by analyzing sera from 12 individuals who suffered from autoimmune diseases and from 3 patients with amyotrophic lateral sclerosis. One serum sample, designated 681, detected an antigen that fulfilled the above criteria. In Western blotting of lysates of human Jurkat T cells, the 681 antigen appeared as a distinct signal with a molecular mass of 60 kDa in normal cells, and 2 additional signals with faster mobilities were detected in apoptotic cells. The results of subcellular fractionation and immunofluorescence experiments revealed this antigen to be strictly localized in the nucleus of normal cells, but to be translocated to a region near the plasma membrane, to membrane blebs in particular, after the induction of apoptosis. Under conditions in which membrane blebbing was inhibited in apoptotic cells, the antigen still moved away from the nucleus, but its accumulation at the periplasmic region was completely abolished. The apparent partial cleavage and intracellular redistribution of the 681 antigen in apoptotic cells mimics changes previously reported for the nuclear autoantigen La, but the 681 antigen was clearly distinct from La. These results suggest that cleavage-dependent exit from the nucleus during apoptosis is a phenomenon common to nuclear autoantigens.View full abstractDownload PDF (3553K) -
Haruhiko Sakuraba, Yuri Mitani, Shuichiro Goda, Yutaka Kawarabayasi, T ...2003 Volume 133 Issue 2 Pages 219-224
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe gene encoding the ATP-dependent glucokinase of hyperthermophilic archaeon Aeropyrum pernix was identified, cloned, and functionally expressed in Escherichia coli. The deduced amino acid sequence showed 40% identity to that of the putative glucokinase from hyperthermophilic archaeon Pyrobacurum aerophilum. The purified recombinant enzyme was a monomer with a molecular mass of 35 kDa. The enzyme retained its full activity on heating at 70°C for 10 min and retained 65% of the activity after 10-min incubation at 100°C. The enzyme exclusively catalyzed the phosphorylation of D-glucose using ATP as a phosphoryl donor. ITP was accepted in addition to ATP. The rate dependence with both glucose and ATP followed Michaelis-Menten kinetics, with apparent Km values of 0.054 and 0.50mM, respectively. The enzyme activity required divalent cations; Mg2+, which was most effective, could partially be replaced by Mn2+ or Ca2+. Phylogenetic analysis revealed that the glucokinase from A. pernix does not belong to the clusters of enzymes found in bacteria and eukarya. This is the first description of the characteristics of an ATP-dependent glucokinase from an archaeon.View full abstractDownload PDF (1665K) -
Hiroki Tsuruta, Yasuo Aizono2003 Volume 133 Issue 2 Pages 225-230
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA procedure was established for expression and purification of abundant recombinant cold-active protein-tyrosine-phosphatase (RCPTPase), which showed identical enzymatic characteristics to the native enzyme (NCPTPase). The purified RCPT-Pase showed high catalytic activity at low temperature and maximal activity at 30°C. RCPTPase has a thermodynamic characteristic in that its activation enthalpy was determined to be low, 4.3 kcal/mol, at temperatures below 19.3°C, where the Arrhenius relationship exhibited an inflection point, in comparison with 20.3 kcal/mol above 19.3°C. Also, the thermostability, ΔGwater, of the catalytic site in the RCPTPase molecule was increased with a decrease in temperature. It was considered that cold-active protein-tyrosine-phosphatase could maintain its catalytic site in a stable conformation for eliciting high catalytic activity with low activation enthalpy at low temperature.View full abstractDownload PDF (1748K) -
Fumitaka Kawakami, Yoshihito Shimoyama, Kenzo Ohtsuki2003 Volume 133 Issue 2 Pages 231-237
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe physiological interaction between glycyrrhizin (GL) and serum complement C3, and the inhibitory effects of GL, glycyrrhetinic acid (GA), and a GA derivative (oGA) on the phosphorylation of C3 by casein kinase 2 (CK-2), were investigated in vitro. C3 was found to be a GL-binding protein (gbP), because (i) of its high affinity for a GL-affinity HPLC column; and (ii) both GL and GA induce conformational changes in C3. At least four trypsin-resistant fragments (p30, p25, p18, and p15) were detected when the 32P-labeled C3α was digested with trypsin in the presence of 100 μM GA. Two of these (p25 and p15) were immuno-precipitated with anti-C3a serum. Furthermore, it was found that C3a contains GL-binding domains, because (i) C3a (anaphylatoxin) could be selectively purified from the synovial fluids of patients with rheumatoid arthritis by GL-affinity column chromatography (HPLC); and (ii) purified human C3a has a high affinity for a GL-affinity column. In addition, C3α (p115) of C3 was effectively phosphorylated by CK-2 in the presence of poly-Arg (a CK-2 activator) in vitro. This phosphorylation was completely inhibited by 10 μM oGA, 30 μM GA, or 100 μM GL. Taken together, these results suggest that the GL-induced inhibition of the physiological activities of C3a and C3a may be involved in the anti-inflammatory effect of GL in vivo.View full abstractDownload PDF (5713K) -
Kunihiko Itoh, Kazuyuki Inoue, Takehiko Tezuka, Hitoshi Tada, Yoshiyuk ...2003 Volume 133 Issue 2 Pages 239-245
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTo investigate the molecular structural and functional characteristics of tumor-suppressive anti-ErbB-2 monoclonal antibody (mAb) SER4, we performed mAb-gene cloning and epitope mapping by a phage display system. Structural analysis demonstrated that both the heavy chain (HC) and light chain variable regions are highly homologous with the derived germline sequences, while the HC complementarity determining region (HCDR) 3 has a relatively short length and biased amino acid usage. A cloned gene-derived recombinant Fab (rFab) fragment showed antigen binding activity and specificity comparable to the parent mAb. Cross-linking of the rFab fragment with the anti-Fab antibody elicited cell growth inhibition in vitro. These results imply that the cloned genes actually encode the Fab part of SER4. The epitope mimetic peptide (mimotope) isolated by panning a phage-displayed random peptide library against SER4 showed no cross-reactivity with mAbs other than SER4. The mimotope was found to be homologous with 87AHNQVRQVPLQR98 in the extracellular domain of ErbB-2 by means of a clustalw search. Since SER4 causes the growth inhibition of ErbB-2 positive cells, the predicted epitope sequence may constitute the putative functional domain of ErbB-2.View full abstractDownload PDF (1723K) -
Satoshi Tanaka, Jun-ya Kinoshita, Rieko Kurodat, Akio Ito2003 Volume 133 Issue 2 Pages 247-251
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSIntegration of cytochrome b5 (b5), a tail-anchored protein located in the endoplasmic reticulum (ER) membrane, into the membrane was studied. Mutation of three amino acids, -Leu-Met-Tyr, at the carboxy-terminal end of the transmembrane segment of b5 to alanines resulted in localization of the mutated protein, b5LMY/AAA, in the cytosol as well as in the ER membrane. When an N-glycosylation site was introduced at the carboxy-terminal end of b5LMY/AAA, a substantial amount of the glycosylated form of the mutant protein was recovered in the cytosol fraction. A portion of the mutant protein recovered in the ER was released from the membrane by incubation with the cytosol fraction, but no further release was observed in the second incubation, suggesting that b5 is present in two different states, loosely-bound and firmly-integrated forms, in the ER membrane. These results suggest that b5 is integrated into the ER membrane via the loosely bound state, in which the carboxy-terminal end of the molecule is inserted into the luminal side of the vesicle but is easily translocated back to the cytosol, and that the three amino acids are important for conversion of the loosely-bound state to the firmly-integrated state.View full abstractDownload PDF (2986K) -
Yuji Honda, Motomitsu Kitaoka, Ken Tokuyasut, Chiye Sasaki, Tamo Fukam ...2003 Volume 133 Issue 2 Pages 253-258
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSKinetic analyses of the hydrolysis reactions of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside [(G1cNAc)2-UMB (1), GlcN-G1cNAc-UMB (2), G1cNAc-GlcN-UMB (3), and (G1cN)2-UMB (4)] by ChiA and ChiB from Serratia marcescens were performed. Both enzymes released UMB from all compounds apart from 4. The S-v curves of the hydrolyses of 1 by ChiA and ChiB both exhibited atypical kinetic patterns, and the shapes of the two S-v curves were different from one another. However, both curve shapes were explained by assuming some of the enzyme present formed complexes with multiple molecules of the substrate. Conversely, the S-v curves generated in the cleavage of 2 and 3 by ChiA exhibited typical Michaelis-Menten profiles. Both enzymes hydrolysed 2 with an approximately 14-fold higher Km value relative to 1, indicating that the N-acetyl group was recognised at the -2 subsite. The keat value obtained with ChiA was identical to the heat value observed for 1. However, the keat value for ChiB was one-fourth that of 1, suggesting that the removal of the N-acetyl group caused an increase in the formation of a non-productive ES-complex. ChiA and ChiB hydrolysed 3 with 5- and 20-fold greater Km values relative to 1, respectively, and 60- and 30-fold smaller keat values relative to 1, respectively. The reaction mechanism of family 18 chitinases is discussed based upon the results obtained from the hydrolysis of these compounds.View full abstractDownload PDF (668K) -
Takuya Sugahara, T. Govind Pai, Masahito Suiko, Yoichi Sakakibara, Min ...2003 Volume 133 Issue 2 Pages 259-262
Published: 2003
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the M-form and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg2+ and Ca2+, but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.View full abstractDownload PDF (543K)
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