The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
121 巻, 2 号
選択された号の論文の32件中1~32を表示しています
  • Kazutomo Imahori, Tsuneko Uchida
    1997 年 121 巻 2 号 p. 179-188
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Alzheimer's disease (AD) is characterized by neuronal cell death and two kinds of deposits, neurofibrillary tangles (NFT) and senile plaques. The main component of NFT is paired helical filaments (PHF), which mainly consist of hyperphosphorylated tau protein. Tau protein kinases I and II were found as candidate enzymes responsible for hyperphos-phorylation of tau to induce the formation of PHF. Since prior phosphorylation of tau by TPKII strongly enhanced the action of TPKI, it was thought that TPKII was involved in the formation of PHF-tau in concert with TPKI. After cloning, TPKI was found to be identical with glycogen synthase kinase 3β (GSK3β), while TPKII consists of a novel 23 kDa protein activator and a catalytic subunit that is identical with cyclin-dependent kinase 5 (CDK5). The phosphorylation sites on tau by TPKI and TPKII could account for the most, but not all, of the major phosphorylation sites of fetal tau and PHF-tau. An antibody for a site specifically phosphorylated by TPKI (Ser413) could identify all three neurofibrillary lesions in the AD brain, and double staining for either TPKI or TPKII and NFT in the brain of Down's syndrome patients clearly demonstrated that TPKI and TPKII are both associat-ed with NFT in vivo, suggesting that the level of TPKI or TPKII is elevated in AD brain by some mechanism. On the other hand, the levels of both TPKs change developmentally, being high in the neonatal period when the phosphorylation of fetal tau proceeds actively, suggesting that the TPKI/TPKII cooperative system has an important physiological role in the formation of neural networks. In AD brain, aberrant accumulation of amyloid-β protein (Aβ) occurs ahead of the accumulation of PHF in NFT. When a primary culture of embryonic rat hippocampus was treated with 20 μM Aβ, induction of TPKI, extensive phosphorylation of tau and then programmed cell death were observed, indicating that TPKI induced by A$ phosphorylates tau, followed by disruption of axonal transportation and finally cell death. By using a yeast two hybrid system, TPKI was found to interact with pyruvate dehydrogenase (PDH), which is a key enzyme in the glycolytic pathway. PDH was phosphorylated in vitro by TPKI to reduce the activity converting pyruvate into acetyl-CoA, which is required for acetylcholine synthesis. In a primary culture of rat hippocampal cells treated with Aβ, PDH was inactivated in inverse relation to the activation of TPKI, resulting in accumulation of pyruvate or lactate, energy failure induced by the disturbance of glucose metabolism, and a shortage of acetylcholine owing to deficiency of acetyl-CoA, all of which are characteristic of AD brain. In cholinergic neurons such as those of the septum, non-aggregated Aβ, specifically Aβ (1-42), not Aβ (1-40), caused a shortage of acetylcholine by activation of TPKI and inactivation of PDH without cell death.
  • Fumio Fukai, Shinobu Hasebe, Masaaki Ueki, Mayuko Mutoh, Chizuru Ohgi, ...
    1997 年 121 巻 2 号 p. 189-192
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    We recently found that heparin-binding domain 2 (Hep 2) of fibronectin (FN) exhibits cryptic anti-adhesive activity. In order to locate the anti-adhesive site, a number of synthetic peptides which represent the primary structure of the Hep 2 domain were characterized as to their ability to decrease the adhesion of A375SM melanoma cells to FN substrate. Only one peptide (T-E-A-T-I-T-G-L-E-P-G-T-E-Y-T-I-Y-V-I-A-L, residues 1835-1855) (peptide 11114-2), which is situated between the previously identified adhesive sites, FN-C/H-I and II, decreased the cell adhesion to FN. Assaying of the anti-adhesive activities of sub-peptides showed that the hydrophobic moiety of peptide 11114-2 (under-lined sequence) seems to be indispensable for the anti-adhesive activity. These results suggest that anti-adhesive activity is closely associated with the sequence, Y-T-I-Y-V-I-A-L, that is usually buried within the Hep 2 domain structure because of its hydrophobic nature.
  • Terumasa Tsuchiva, Mitsuhiro Okada, Masatsugu Ueda, Yukio Yasukochi
    1997 年 121 巻 2 号 p. 193-196
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    We show here a role of the highly conserved GATA motif in the -30 region or me erythropoietin (Epo) promoter. Epo production is reduced in normoxia and activated in hypoxia to control the oxygen supply through erythropoiesis. Although the hypoxic inducibility has been analyzed, the molecular mechanism for the low basal activity in normoxia remained unclear. The GATA motif in the -30 region upstream of the transcrip-tion initiation site is highly conserved among the species. In many genes, the consensus motif in the -30 region is TATA. The GATA motif of the Epo promoter was mutated to TATA. The transcriptional activity of the mutant was enhanced even in normoxia. Binding assays showed that TATA-binding protein (TBP) could weakly bind to the wild-type GATA motif whereas TBP bound to the mutant TATA motif with high affinity. These results indicate that the highly conserved GATA motif in the -30 region of the Epo promoter can avoid binding and activation by TBP. This evidence is considered to be the molecular basis for the low physiological expression from the Epo promoter in normoxia.
  • Toru Nakanishi, Kenji Kadomatsu, Tomomitsu Okamoto, Keiko Ichihara-Tan ...
    1997 年 121 巻 2 号 p. 197-205
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Midkine (MK) is a 13 kDa heparin-binding growth/differentiation factor, and its interac-tion with heparan sulfate proteoglycan is important in promotion of neurite outgrowth. This study was performed to reveal the species of syndecans that interact with MK during embryogenesis of the central nervous system in the rat. Northern blot analysis and immunohistochemical staining using antibodies to syndecans showed that syndecan-1 was strongly expressed in the brain and spinal cord of the 10-day rat fetus, and the expression became weak as embryogenesis proceeded. On the other hand, syndecan-3 expression was stronger both in the brain and spinal cord in the later developmental period (day 14-16 of gestation). No significant expression of ryudocan (syndecan-4) was detected in fetal brains by Northern blot analysis. Syndecan-3 isolated from 16-day rat fetus bound to MK strongly, as did syndecan-1 isolated from 10-day rat fetus. Thus, both syndecan-1 and syndecan-3 were considered to be involved in interaction with MK during construction of the central nervous system; syndecan-1 is expected to play important roles in the earlier periods, and syndecan-3 in the later periods.
  • Hideaki Tanaka, Takahisa Akatsuka, Takashi Murakami, Yoshiro Ogoma, Ko ...
    1997 年 121 巻 2 号 p. 206-211
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    The morphology of protein-adsorbed stearic acid monolayers containing a fluorescent probe (rhodamine B octadecylester perchloride: RBO) was observed by using fluorescence spectroscopy, fluorescence microscopy, and Brewster angle microscopy (BAM). The quenching of fluorescence of RBO was observed at the limiting area of the stearic acid monolayer. Thus, the fluorescence of RBO could be a good marker for packing of the matrix monolayer. On the other hand, the BAM image showed the morphology of not the matrix monolayer, but the adsorbed protein monolayer. Thus, the packing processes of the two could be distinguished. The present method is available for a protein that does not have visible absorption, such as bovine serum albumin (BSA). It is suggested that electrostatic interaction between matrix and protein molecules greatly affects the change in the morphology of the protein-adsorbed monolayer.
  • Tsuyoshi Watanabe, Tohru Takemasa, Izuru Yonemura, Tamio Hirabayashi
    1997 年 121 巻 2 号 p. 212-218
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Troponin T (TnT), like other myofibrillar proteins, is expressed as various isoforms in different muscle fibers and/or at different development stages. A recent study suggested the expression pattern of chicken fast TnT isoforms is fixed in a given cell lineage. In the present study, we isolated genomic clones of the chicken fast TnT gene to carry out molecular analysis of its expression mechanism. One of the clones, pWETNTa, contained the 5' upstream region and approximately 20 kb downstream from exon 1. We constructed promoter/upstream segments of the chicken fast TnT gene linked to the bacterial chloram-phenicol acetyltransferase (CAT) gene and tested the regulatory function of the 5' upstream region by transient transfection of the gene constructs into muscle cells. We showed that a DNA segment between-264 and -44 by from the most 5' transcriptional initiation site, which has an MEF2, an M-CAT-like element, a CArG box and two E boxes, was essential for the expression of the fast TnT gene. Furthermore, mutation of the M-CAT-like element in the segment resulted in the most serious reduction in the fast TnT promoter activity. The results suggested that the M-CAT-like element plays an important role in transcriptional regulation of the fast TnT gene. The M-CAT-like element is very similar to the M-CAT element, but in electrophoretic mobility shift assay, the factor(s) that bound to this motif was found to be different from the M-CAT binding factor (MCBF).
  • Keigo Gohda, Noriyuki Matsuo, Yasushi Oda, Morio Ikehara, Seiichi Uesu ...
    1997 年 121 巻 2 号 p. 219-224
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    The effects of 2'-substituents of the first deoxyguanosine on EcoRI activity were examined using synthetic octadeoxynucleotides d(GG*AATTCC) containing 2'-substituted deriva-tives (G*), i. e., 2'-fluoro-2'-deoxyguanosine (dGfl), 2'-chloro-2'-deoxyguanosine (dGcl), and guanosine (rG). The overall structures of the octamers were very similar, as shown by CD and UV measurements, although their EcoRI reactivities were very different: 100% in 60 min for d(GGAATTCC) and d(GGflAATTCC), 5% in 24 h for d[G(rG)AATTCC], and no cleavage at all in 24 h for d(GGclAATTCC). However, the kinetics showed the octamers exhibit similar binding-affinity to the enzyme (10-6-10-7 M). 31P-NMR analysis suggested the modified octamers change the phosphate backbone conformation in a duplex, since an unusual downfield-shifted signal in the spectra was commonly observed for the modified octamers at low temperature (i.e., a duplex state), which was shifted upfield at high temperature (i.e., a single strand state). The order of the differences was dGcl>rG>dGfl-containing octamers, coinciding with that of the vdW volume of 2'-substituents (Cl>OH>F) and the cleavage reactivities. These findings suggest the steric hindrance by the 2'-substituents causes a conformational change of the phosphate backbone close to the scissile bond, and then interferes with the EcoRI reaction.
  • Takashi Masato, Takuya Numata, Tsuyoshi Katoh, Fumi Morita, Michio Yaz ...
    1997 年 121 巻 2 号 p. 225-230
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Telokin, which is a candidate for one of the factors stabilizing dephosphorylated myosin filaments in smooth muscle cells, was subjected to a chemical crosslinking study to determine its binding location on the myosin molecule. Telokin labeled with 5-iodoacet-amidofluorescein (Fl-telokin) retained the nature of unlabeled telokin: it bound to dephosphorylated gizzard myosin with a stoichiometry of 1 mol/mol myosin and induced filament assembly. The carboxyl groups of Fl-telokin were activated with 1-ethyl-3-(3-dimethyl-amino-propyl)carbodiimide and N-hydroxysuccinimide and then crosslinked to myosin. The production of three fluorescent peptides was observed on an SDS-gel, accompanying a decrease in the amount of regulatory light chain (LC20). The molecular weights of these products were estimated to be >200, 62, and 41 kDa. When unlabeled telokin was crosslink-ed to myosin of which LC20 was exchanged with 5- [ [2- [(iodoacetyl)amino] ethyl] amino] - naphthalene-1-sulfonic acid-labeled LC20, the 62- and 41-kDa bands were also fluorescent. These results suggest that the >200, 62, and 41-kDa species are telokin crosslinked with a heavy chain, with 2 LC20, and with 1 LC20, respectively. Myosin crosslinked with unlabeled telokin showed an extra structure with a small projection at the head-rod junction on electron microscopy and this structure was proved to be telokin by decorating it with anti-telokin antibodies. In addition, dephosphorylated myosin crosslinked with Fl-telokin was incapable of folding into the 10S conformation at 0.2 M NaCl in the presence of MgATP, and assembled into filaments at 0.15 M NaCl in the presence of MgATP. Thus, telokin may bind to LC20 and a heavy chain region at the head-rod junction and suppress folding into the 10S conformation, leading to the assembly of myosin into filaments.
  • Soo-Bok Lee, Kuniyo Inouye, Ben'ichiro Tonomura
    1997 年 121 巻 2 号 p. 231-237
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    The states of 28 tyrosyl residues of thermolysin have been characterized by means of pH-jump studies and nitration with tetranitromethane. The ionization states of phenolic groups of the tyrosyl residues have also been estimated by spectrophotometric titration of the absorption change at 295 nm. The ionization of 16 tyrosyl residues was completed within 15 s after a pH-jump, and these residues are considered to be located on the surface of thermolysin. On the other hand, the ionization of the other 12 residues required 15 s to 10 min, suggesting the occurrence of a conformational change which leads to the exposure of the buried tyrosyl residues to the solvent. Sixteen tyrosyl residues were nitrated and categorized into three classes according to reactivity. The second-order rate constants of the respective classes of tyrosyl residues for nitration were evaluated as 3.32, 0.52, and 0.18 M-1•min-1', and their apparent pKa values were estimated to be 10.2, 11.4, and 11.8. Tyrosyl residues in the first class were considered to be located almost freely on the surface, while those in the second and third classes might be in constrained states.
  • Kazuhiro Alba, Hui Fang, Nobuyuki Yamaguchi, Yoshimasa Tanaka, Hideko ...
    1997 年 121 巻 2 号 p. 238-243
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Sexual development of Dictyostelium discoideum is a unique and useful system for the study of sexual phenomena. We have been studying molecular mechanisms of sexual cell fusion in D. discoideum and have identified several relevant cell-surface proteins. One of the proteins, gp138, was identified as a target molecule for fusion-blocking antibodies, and two genes for gp138, GP138A and GP138B, were cloned. The participation of gp138 in the sexual cell fusion was confirmed by antisense RNA mutagenesis, but it is unclear which of the genes encodes gp138. Moreover, the presence of a third gene for gp138 was indicated by gene disruption. In the present study, we generated strains of D. discoideum overexpress-ing either GP138A or GP138B to investigate the products of these genes. The transformants overexpressing GP138A and GP138B overproduced glycoproteins with molecular masses of 135 and 130 kDa, respectively. Although their molecular masses were different from that of gp138, the results of peptide mapping and amino acid sequencing showed that they are related proteins, suggesting that the proteins encoded by GP138A and GP138B are isoforms of gp138 protein.
  • Teisuke Takita, Satoshi Hashimoto, Yuji Ohkubo, Takanori Muto, Naofumi ...
    1997 年 121 巻 2 号 p. 244-250
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    The formation of an enzyme. lysyladenylate complex was studied with a highly purified lysyl-tRNA synthetase [L-lysine:tRNALys ligase (AMP-forming); EC 6.1. 1.6] from Bacillus stearothermophilus. The apparent dissociation equilibrium constants of the enzyme with L-lysine and ATP in the process of the complex formation were estimated to be 50.9 and 15.5 μM, respectively, at pH 8.0, 30°C, by fluorometric measurement. The isolated enzyme•lysyladenylate complex was relatively stable with a rate constant of decomposition of 1.7×10-5 s-1 at pH 8.5 and 0°C The rate constant of transfer of L-lysine from the complex to Escherichia coli tRNA was 1.2×10-2 s-1 at pH 8.5 and 0°C. The effects of replacing L-lysine by several analogues on the complex formation were examined. L-Lysine hydroxamate, a strong inhibitor of the L-lysine dependent ATP-PP1 exchange reaction, produced a stable complex with the enzyme and ATP, enzyme•lysinehydroxamate-AMP probably being formed. The binding stoichiometry of the assumed L-lysinehydroxamate-AMP per mol of the dimer enzyme was 1:1.
  • Ko Onodera, Kentaro Yomogida, Naruyoshi Suwabe, Satoru Takahashi, Yasu ...
    1997 年 121 巻 2 号 p. 251-263
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Transcription factor GATA-1 was first identified in erythroid cells, but was later shown to also be expressed in Sertoli cells of the mouse testis. GATA-1 transcription in testis initiates from a different first exon (exon IT) than the erythroid mRNA (transcribed from exon IE). To begin to address the question of how expression of GATA-1 might be differentially regulated in Sertoli and erythroid cells, we have cloned and determined the structure of the IT promoters of both the rat and mouse GATA-1 genes. The transcription regulatory mechanism(s) controlling the synthesis of exon IT-derived mRNA was inves-tigated by transfection of wild-type and mutant reporter genes, with and without co-trans-fected GATA factor expression plasmids, into either fibroblasts or Sertoli cell lines. Two GATA binding sites in the IT promoter were found to be required for GATA factor-mediated activation in fibroblasts: GATA-IT-directed reporter gene expression was activated only after co-transfection with GATA-1, implying that transcriptional activation of GATA-1 in the testis might be at least partially mediated through these GATA regulatory elements. We also found that the endogenous GATA-1 gene was silent in primary culture and two different Sertoli cell lines, and that the repression of co-transfected GATA-IT reporter genes could not be relieved by forced expression of GATA-1 in Sertoli cells. Thus the GATA-IT promoter may be under the control of a regulatory network in Sertoli cells which involves both positive and negative regulation of transcription, and conserved GATA motifs found in the IT promoter may be required for transducing these effects.
  • Tamotsu Taketomi, Atsushi Hara, Kei-ichi Uemura, Hisashi Kurahashi, Ei ...
    1997 年 121 巻 2 号 p. 264-269
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Our rapid method of microwave-mediated saponification for preparing lysoglycosphingo-lipids from their parent glycosphingolipids was also able to prepare lysogangliosides or modified lysogangliosides, which were identified by delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric (DE MALDI-TOF MS) analy-sis. When GM3, GM2, and GM1 isolated from adult human brain gangliosides were subjected to the saponification, GM3 was found to give rise to only lyso-GM3 containing de-N-acetylneuraminic acid (de-N-acetyl lyso-GM3), whereas the GM2 produced both lyso-GM2 and the de-N-acetyl compound, and GM1 also gave both lyso-GM1 and the de-N-acetyl compound. In the saponification of GM1 and GD1a, isolated from rat brain gangliosides, GM1 similarly produced both lyso-GM1 and the de-N-acetyl compound, but GDIa was found to give rise to both dehydrated de-N-monoacetyl and dehydrated de-N-di-acetyl lyso-GD1a. However, the saponification of the GM1 fraction isolated from porcine brain gangliosides gave rise not only to both lyso-GM1 and the de-N-acetyl compound, but also unexpectedly to both lyso-fucosyl GM1 and its de-N-acetyl compound. The untreated GM1 fraction was examined by TLC and DE MALDI-TOF mass spectrometry, and proved to contain fucosyl-GM1. The DE MALDI-TOF MS analysis of the prepared lyso-gangliosides showed that their long chain bases consisted of d18:1 and d20:1 sphingosines in various ratios reflecting those of the different mammalian brain gangliosides.
  • Kiyotaka Hatsuzawa, Mitsuo Tagaya, Shoji Mizushima
    1997 年 121 巻 2 号 p. 270-277
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Newly synthesized mammalian secretory proteins such as preprolactin are translocated across the endoplasmic reticulum (ER) in a signal recognition particle (SRP)-dependent manner. Recent studies revealed that there are two recognition steps for signal peptides during this translocation. The first step is recognition by SRP, which results in elongation arrest, and the second step is interaction between signal peptides and the translocation channel embedded in the ER membrane. To determine the roles of the hydrophobic region of signal peptides in the recognition by SRP and the membrane-embedded translocation machinery, we constructed chimeric proteins consisting of the mature region of prepro-lactin and signal peptides containing different numbers of leucine residues. The transloca-tion of these chimeric proteins was completely dependent on SRP, and the efficiency increased as the number of leucine residues increased up to 10 and then decreased. Although the efficiency of elongation arrest also increased as the number of leucine residues increased up to 10, it only slightly decreased as the number increased up to 20. Similar results were obtained when the hydrophobic region was replaced by alternate leucine and alanine residues, except that the most efficient translocation occurred when the number was 14. Taken together, the present results suggest that the total hydrophobicity of the hydrophobic region of signal peptides is a determinant for recognition by both SRP and the membrane-embedded translocation machinery, although the specificities of the two signal recognition steps are slightly different from each other.
  • Seiji Madoiwa, Koichi Arai, Yasunobu Ueda, Masahiro Ishizuka, Jun Mimu ...
    1997 年 121 巻 2 号 p. 278-287
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Two groups of anti-plasminogen monoclonal antibodies, whose epitope was either in the kringle 1+2+3 domain (F3P2, F11P5, F11P6, and F12P18) or the kringle 5 domain (F1P6 and F12P16), were isolated and their effects on the conformation of plasminogen were explored. All antibodies except F1P6 had 3- to 10-fold higher affinity toward Lys-plasmino-gen than Glu-plasminogen. F1P6 exhibited a comparable affinity to Glu- and Lys-plasmino-gen. Among these, only F11P5 binding was inhibited by ε-amino-n-caproic acid (EACA) in a concentration-dependent manner, with half maximal inhibition at 3 mM. From a competi-tion assay, we concluded that the epitopes of F11P5, F11P6, and F12P18 should be very close, and located at or near the low affinity lysine binding site on the kringle 2+3. These three antibodies dramatically enhanced the binding of Glu-plasminogen to the other antibodies, except to F1P6. Interestingly, F3P2, whose non-overlapping epitope was in the kringle 2+3 domain, also augmented the binding of Glu-plasminogen to the other anti-bodies. In contrast, we did not observe enhanced binding of Lys-plasminogen to one antibody in the presence of the other antibodies, and the binding of Glu-plasminogen to these antibodies did not increase in the presence of 10 mM EACA. In the presence of these antibodies, including F1P6, Glu-plasminogen bound more efficiently to immobilized degraded fibrin, with a binding profile similar to Lys-plasminogen. All antibodies except F1P6 enhanced the conversion rate of plasminogen to plasmin remarkably. Taken together, we propose that these two groups of monoclonal antibodies can dissociate the intramole-cular interactions of Glu-plasminogen and induce the conformational transition of Glu-plasminogen to Lys-plasminogen. In addition, the kringle 2 +3 and kringle 5 structures of Glu-plasminogen liganded with EACA are distinct from the Lys-plasminogen structure.
  • Taiichi Sakamoto, Mi Hee Kim, Yasuyuki Kurihara, Nobuyuki Sasaki, Tomo ...
    1997 年 121 巻 2 号 p. 288-294
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    The properties of a mutant hammerhead ribozyme system, which consists of two RNA oligomer strands and in which stem II is deleted (replaced with a UUUU loop), are described. The effects of temperature, pH, and metal ions on the cleavage reaction were similar to those for the parent ribozyme with stem II. The mutant ribozyme showed a much lower cleavage rate (kcat=0.04min-1) in the presence of 10mM MgCl2, where the parent ribozyme showed full cleavage activity. However, increasing the concentration of MgCl2 from 10 to 100mM restored the cleavage activity of the mutant ribozyme to the original level (kcat=0.2min-1). CD titration experiments with MgCl2 using a noncleavable substrate were carried out. Deletion of stem II resulted in an about 20-fold reduction of the apparent Mg2+ binding affinity when the Mg2+ concentrations of half-saturation are compared. The results were analyzed by curve-fitting analysis and compared with those for the parent ribozyme. The analysis showed that the Mg2+ concentration dependence data in CD and cleavage experiments for the mutant enzyme can be explained by a two Mg2+ ion binding mechanism. These results suggest that stem II is important for maintaining the conformation of the catalytic core suitable for Mg2+ binding.
  • Toshitaro Ikeda, Bernhard Schmitt, Jacques Pouyssegur, Shigeo Wakabaya ...
    1997 年 121 巻 2 号 p. 295-303
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    To precisely identify the cytoplasmic subdomains that are responsible for the intracellular pH (pH_??_)-sensitivity, ATP depletion-induced inhibition and Ca2+ activation of the Na+/H+ exchanger (NHE1), we generated a set of deletion mutants of carboxyl-terminal cytoplasmic domain and expressed them in the exchanger-deficient cell line PS120. We evaluated pH_??_-sensitivity of these mutants by measuring the resting pH_??_, in cells placed in an acidic medium (pH 6.0) and pH_??_-dependence of 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Detailed analysis revealed that the cytoplasmic domain of NHE1 is consists of at least four subdomains in terms of pH_??_, -sensitivity of the unstimulated NHE1: I, aa 516-590/595; II, aa 596-635; III, aa 636-659; and IV, aa 660-815. Subdomains II and IV were silent for pH_??_, -sensitivity. Subdomain I had a pH_??_, -maintenance function, preserving pH_??_- sensitivity in a physiological range, whereas subdomain III, overlapping with the high affinity calmodulin (CaM)-binding site, exhibited an autoinhibitory function. Deletion of subdomain I abolished the decrease of pH_??_, -sensitivity induced by cell ATP depletion, indicating that domain I plays a crucial role in this phenomenon. Deletion of subdomain III rendered the inhibition by ATP depletion less efficient, suggesting the possible interaction between subdomains I and III. On the other hand, tandem elongation of subdomain II by insertion did not affect either the inhibitory function of domain III or the removal of this inhibition by ionomycin or thrombin. However, deletion of subdomain II partially abolished the inhibitory effect of subdomain III. Subdomain H thus seems to function as a mobile “flexible loop, ” permitting the CaM-binding subdomain III to exert its normal function. These findings, together with our previous data, support a concept that cell ATP, Ca2+, and growth factors regulate NHE1 via a mechanism involving direct or indirect interactions of specific cytoplasmic subdomains with the “H+-modifier site.”
  • Genji Kurisu, Takayoshi Kinoshita, Akiko Sugimoto, Akinobu Nagara, Yas ...
    1997 年 121 巻 2 号 p. 304-308
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    A zinc endoprotease produced by Streptomyces caespitosus (ScNP) specifically hydrolyzes the peptide bond at the imino side of aromatic residues and is the smallest protease found to date. Although ScNP carries the zinc-binding sequence HEXXH, its primary structure of 132 amino acid residues differs from those of other known zinc metalloendoproteases. X-ray structural analysis of ScNP at 1.6Å resolution revealed that despite a lack of sequence homology, the common topological feature of main-chain folding and a β-turn containing methionine, which is a feature of the zinc metalloendoprotease superfamily of metzincins, is conserved in ScNP. The zinc atom of ScNP is tetrahedrally ligated by the two histidines in the HEXXH sequence, an aspartate residue and a water molecule. Thus, ScNP represents a novel subfamily of metzincins with a HEXXHXXGXXD zinc-binding sequence. A plausible substrate recognition pocket to which aromatic residues bind is located near the catalytic zinc ion.
  • Yoshihito Beppu, Yasuhiro Imamura, Masato Tashiro, Takae Towatari, Hir ...
    1997 年 121 巻 2 号 p. 309-316
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Tryptase Clara, a trypsin-like protease localized exclusively in and secreted from Clara cells of the bronchial epithelium of rat, proteolytically activates the infectivity of influenza A virus [H. Kido, Y. Yokogoshi, K. Sakai, M. Tashiro, Y. Kishino, A. Fukutomi, and N. Katunuma (1992) J. Biol. Chem. 267, 13573-13579]. We report here that human mucus protease inhibitor (MPI), a major inhibitor of granulocyte elastase in the lining fluids of the human respiratory tract, significantly inhibited proteolytic activation of the infectivity of influenza A and Sendai viruses by tryptase Clara in vitroand multi-cycles of mouse-adapted influenza A virus replication in rat lungs in vivo. Recombinant MPI and the C- but not the N-terminal domain of MPI inhibited both the proteolytic activity of tryptase Clara and the activation of virus infection. The 50% inhibitory concentrations of recombinant MPI and the C-terminal domain for tryptase Clara with Sendai virus envelope glycoprotein as substrate were 7.4 and 61.6nM, respectively. These results indicate that MPI is a defensive compound against virus infection. Since there is evidence suggesting that concentrations of MPI in respiratory fluids are insufficient for prevention of virus infection, administration of MPI in the airway may be useful for treatment of these virus infections.
  • Eichi Tsuruga, Hiroko Takita, Hideaki Itoh, Yuichi Wakisaka, Yoshinori ...
    1997 年 121 巻 2 号 p. 317-324
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    To elucidate the biochemical mechanism of osteogenesis, the effect of matrix geometry upon the osteogenesis induced by bone morphogenetic protein (BMP) was studied. A series of five porous hydroxyapatites with different pore sizes, 106-212, 212-300, 300-400, 400-500, and 500-600μm, was prepared. A block (approximately 5×5×1mm, 40.0mg) of each hydroxy-apatite ceramics was combined with 4 μg of recombinant human BMP-2 and implanted subcutaneously into the back skin of rat. Osteoinductive ability of each implant was estimated by quantifying osteocalcin content and alkaline phosphatase activity in the implant up to 4wk after implantation. In the ceramics of 106-212μm, the highest alkaline phosphatase activity was found 2wk after implantation, and the highest osteocalcin content 4wk after implantation, consistent with the results observed with particulate porous hydroxyapatite [Kuboki, Y. et al. (1995) Connect. Tissue Res. 32: 219-226]. Comparison of the alkaline phosphatase activities at 2wk and the osteocalcin contents at 4 wk after implantation revealed that the highest amount of bone was produced in the ceramics implants with pore size of 300-400μm. In the ceramics with smaller or larger pore sizes, the amount of bone formation decreased as the pore size deviated from 300-400μm. The results indicated that the optimal pore size for attachment, differentiation and growth of osteoblasts and vascularization is approximately 300-400μm. This study using chemically identical but geometrically different cell substrata is the first demonstration that a matrix with a certain geometrical size is most favorable for cell differentiation.
  • Xiaoying Wang, Shigeru Yanagi, Cheng Yang, Ryoko Inatome, Hirohei Yama ...
    1997 年 121 巻 2 号 p. 325-330
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Thrombin and epinephrine in combination exert synergistic effects on platelet activation. On the other hand, tyrosine phosphorylation and activation of tyrosine kinases including Syk have been shown to play a critical role in the induction of platelet responses to thrombin stimulation. This study investigated the role of tyrosine phosphorylation and Syk activation in the synergistic mechanisms between thrombin and epinephrine. Although epinephrine alone (4μM) slightly induced protein-tyrosine phosphorylation and Syk activation, the presence of epinephrine caused a shift to the left in the dose-dependence of thrombin (0.01-0.5U/ml)- induced tyrosine phosphorylation and Syk activation, as well as platelet aggregation. Phenoxybenzamine, an a-adrenoceptor antagonist, canceled this potentiation by epinephrine. Since platelets dominantly express a2-adrenoceptor, this result indicates that epinephrine acts through the occupancy of a2-adrenoceptor. Furthermore, pretreatment with a tyrosine kinase inhibitor, genistein, or a cAMP-elevating agent, prostacyclin (PGI2), significantly reduced these synergistic effects of epinephrine. Taken together, our results suggested that the potentiation by epinephrine may be mediated via enhancement of tyrosine phosphorylation and Syk activation, in part through a decrease of intracellular cAMP levels.
  • Zheng Xu, Shengli Yang, Dexu Zhu
    1997 年 121 巻 2 号 p. 331-337
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    GroE, one of the molecular chaperones, facilitates correct protein folding both in vitro and in vivo. The refolding of recombinant human pro-urokinase, a protein with a high content of disulfide bonds, was used as a model system to illustrate the mechanism of action of GroE. Aggregation of this protein predominates during its in vitro refolding, as indicated by a strong, concentration-dependent increase in light scattering. The addition of GroE and Mg-ATP significantly increases the yield of the active protein. GroE specifically inhibits the aggregation reaction that competes with correct folding, as shown by a strong decrease in the intensity of light scattering. GroEL rapidly binds to unfolded or partially folded prourokinase molecules and thus protects them from the aggregation reaction. Interaction with GroES and ATP hydrolysis are required for the release of the polypeptide chain from GroEL and further acquisition of the completely folded, native conformation.
  • Norihiro Nakamura, Akira Matsuura, Yoh Wada, Yoshinori Ohsumi
    1997 年 121 巻 2 号 p. 338-344
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Acidification inside vacuoles has been shown to play a key role in a number of physiologi-cally important cellular events. We studied the role of vacuolar membrane H+-ATPase in the autophagic process of Saccharomyces cerevisiae. Mutants lacking VMA genes which encode their subunits of the vacuolarH+-ATPase accumulated autophagic bodies in vacuoles on starvation. vma mutants also had a defect in protein degradation induced by starvation. In vma mutants, the activities of vacuolar proteases were remarkably lower than those of the wild-type. Overexpression of vacuolar proteases did not overcome the defect in the disintegration of autophagic bodies in vma mutant, even the overexpressed proteinase A and proteinase B being substantially localized to the vacuolar compartment and undergoing proper proteolytic maturation. Our results showed that the acidification of vacuoles is not required for the formation and delivery of autophagosomes to vacuoles, but is essential for the disintegration of autophagic bodies.
  • Masayuki Ishihara, Yutaka Kariya, Hiroshi Kikuchi, Tosikazu Minamisawa ...
    1997 年 121 巻 2 号 p. 345-349
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    Complete drying of heparin with various concentrations of NaOH by lyophilization caused specific 2-O-desulfation to various degrees without detectable depolymerization or other chemical changes. In order to assess the importance of 2-O-sulfate groups in uronate residues to promote FGF-1 and FGF-2 activities, various 2-O-desulfated (2-O-DS-) heparins were quantitatively examined for their effects on FGF-1-and FGF-2-induced proliferation of BALB/c3T3 clone A31 (A31) cells and the chlorate-treated cells. Twenty-seven percent or less loss of the 2-O-sulfate groups had no effect on the ability to activate both FGF-1 and FGF-2, while 44% loss resulted in a significant loss of the ability. Complete loss of the ability was observed in 2- O-DS-heparins with 75% or more 2-O-desulfation. These results suggest that a high content of 2-O-sulfate groups in uronate residues of heparin is required for activation of both FGF-1 and FGF-2.
  • Takeshi Nakaguchi, Tsutomu Arakawa, John S. Philo, Jie Wen, Masao Ishi ...
    1997 年 121 巻 2 号 p. 350-354
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    The primary structures of two subunits of an α-amylase inhibitor (αAI-2) from a wild common bean (Phaseolus vulgaris) were revealed by a comparison of the amino acid sequence previusly deduced from the nucleotide sequence with the amino- and carboxyl-terminal amino acid sequences determined by conventional methods. The polypeptide molecular weight of αAI-2 obtained by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that αAI-2 has the subunit stoichiometry of an α2β2 complex. These structural features were closely similar to those recently elucidated for a white kidney bean (P. vulgaris) α-amylase inhibitor, which is quite different in the inhibitory specificity from αAI-2. The post-translational processing of the precursor glycoproteins to form the tetrameric structure appeared to require an Arg residue close to the processing site. Further, the proper associations of the subunits into the tetrameric structures seemed to be strictly controlled by a few amino acids on the subunit interfaces.
  • Shigehiro Osada, Shoko Daimon, Takayuki Ikeda, Tsutomu Nishihara, Keii ...
    1997 年 121 巻 2 号 p. 355-363
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    We have previously identified a silencer region in the glutathione transferase P (GST-P) gene, of which the expression is completely repressed in liver of the rat. At least three trans-acting factors bind to multiple cis-elements in this region. Since GST-P silencer 4 (GPS4) is a dominant element in this silencer, we purified the GPS4 binding protein, called Silencer Factor A (SF-A). Purified SF-A was separated into several proteins on an SDS-polyacrylamide gel, and the amino acid sequences of four major components of SF-A were determined. The amino acid sequences of three fragments were identical to those of rat NF1-L, and that of the other fragment was the same as that of hamster NF1/Red1. It is known that nuclear factor 1 (NF1) family proteins are encoded by at least four independent genes in vertebrates, and NF1-L and NF1/Red1 are derived from different genes, NFI-A and NFI-B, respectively. The microsequencing of SF-A revealed that at least two types of NF1 existed in rat liver. Functional analysis by using GAL4-fusion protein in HepG2 cells revealed that NFI-A repressed the transcription activitiy from human metallothionein HA promoter. Our findings indicate that multiple forms of the NF1 family bind to the silencer region and contribute to the negative regulation of the GST-P gene expression.
  • Ling Ling Jiang, Shoko Miyazawa, Masayoshi Souri, Takashi Hashimoto
    1997 年 121 巻 2 号 p. 364-369
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    When D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunc-tional protein was purified from human liver, two preparations were obtained. One contained a 77-kDa polypeptide as the main component and minor smaller polypeptides including a 46-kDa polypeptide, and this preparation showed both the dehydratase and dehydrogenase activities. The other preparation was a homodimer of the 46-kDa polypep-tide and showed only the dehydratase activity. Further analysis indicated that the native enzyme is a homodimer of 77-kDa polypeptide, which was proteolytically modified during purification. The cDNA for the human 77-kDa polypeptide was cloned. The amino acid sequences of the peptides derived from the components of the enzyme preparations were located in the deduced amino acid sequence of the cDNA. The preparation containing the 77-kDa polypeptide was treated with a protease, and two monofunctional fragments were separated. The dehydrogenase and dehydratase fragments were located on the amino- and carboxyl-terminal sides, respectively, of the deduced amino acid sequence of the cDNA. The protein expressed by the cDNA with the entire coding region exhibited both the dehydratase and dehydrogenase activities, and that expressed by a truncated version covering the carboxyl-terminal side exhibited only the dehydratase activity. The cloned cDNA was identical to the human 17β-hydroxysteroid dehydrogenase IV cDNA.
  • Thomas P. Horan, Frank Martin, Lizette Simonet, Tsutomu Arakawa, John ...
    1997 年 121 巻 2 号 p. 370-375
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    We have previously shown that the extracellular domain of granulocyte-colony stimulat-ing factor receptor (soluble G-CSFR), prepared from CHO cell conditioned media, dimerizes upon binding its ligand, G-CSF. The most stable ligand-receptor complex occurs at a 2:2 stoichiometry, unlike the growth hormone and erythropoietin ystems. In the latter cases, each ligand uses two binding sites to bring two receptors together. In this study, we have generated a truncated human G-CSF receptor, known to be sufficient for high affinity ligand binding, which consists of an Ig-like domain and a cytokine receptor homology module. With an affinity purified receptor, sedimentation equilibrium experiments clearly demon-strated that this truncated form of the receptor behaves very similarly to the entire extracellular domain. The sedimentation equilibrium data are consistent with the model that the truncated receptor has a weak tendency to self-associate into a dimer in the absence of a ligand, this receptor-receptor interaction is enhanced by ligand binding, and the most stable complex occurs at a 2:2 stoichiometry. These results are very different from those described by others for various murine G-CSF receptor constructs from either Escherichia coli or insect expression systems.
  • Taei Matsui, Shinji Kunishima, Jiharu Hamako, Masahiko Katayama, Tadas ...
    1997 年 121 巻 2 号 p. 376-381
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    The binding of human von Willebrand factor (vWF) to a variety of extracellular matrix components immobilized on plates and the binding of vWF to platelet glycoprotein Ib (GPIb) after interacting with these matrix components were examined by means of an enzyme-linked immunosorbent assay. vWF preferably bound to type III collagen, whereas it did not significantly bind to type I, IV, V, or VI collagen, fibronectin, laminin, elastin, or proteoglycans. Soluble type III collagen did not bind to vWF coated on plates and showed a little effect on the vWF binding to the immobilized collagen, suggesting that solid-phase collagen is important for the interaction with vWF. When glycocalicin, the N-terminal carbohydrate-rich extracellular domain of GPIb a exhibiting the vWF-binding activity, was added to vWF bound to collagen type III, no significant binding of glycocalicin was observed, but it bound to vWF in the presence of botrocetin, a vWF modulator protein isolated from Bothrops jararaca snake venom. These results indicate that vWF immobi-lized on collagen can interact with GPIb but that the binding of vWF to the collagen matrix alone is insufficient for modulating vWF so that it interacts with GPIb under static conditions. Another unknown physiological modulator functionally mimicking botrocetin or high-shear stress may be involved in the platelet adhesion to extracellular matrix in the early stage of hemostasis.
  • Beverly Smolich, Mynga Vo, Sharon Buckley, Greg Plowman, Jackie Papkof ...
    1997 年 121 巻 2 号 p. 382-388
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    We have isolated human and rat clones of the LIM motif-containing protein kinase, termed LIMK-2. LIMK-2 is related to the neuronally expressed LIM-kinase, whose hemizygous deletion appears to result in cognitive impairment in patients with Williams syndrome. The hallmark of this protein family is the presence of 1 or 2 N-terminal LIM motifs and an atypical C-terminal protein kinase domain. LIMK-2 mRNA was detected by Northern blot analysis in human tissues, most abundantly in placenta, lung, liver, and pancreas, and also in a variety of cell lines including neuronal, glioblastoma, and mammary carcinoma lines. The LIMK-2 transcript was also induced upon neuroectodermal differentiation of mouse P19 embryonal carcinoma cells. A 65 kDa recombinant LIMK-2 protein was identified in 293 cells stably transfected with a LIMK-2 expression vector. An in vitro kinase assay demonstrates LIMK-2 is autophosphorylated and exhibits serine/threonine kinase activity towards the exogenous substrate MBP. The endogenous 65 kDa LIMK-2 protein was detected in a variety of cell lines, and coprecipitates with a 140 kDa tyrosine phosphorylat-ed protein, but was not itself tyrosine phosphorylated. At the subcellular level, LIMK-2 is localized in both the nucleus and in a Triton X-100 soluble fraction.
  • Satoshi Kojima, Akira Kobayashi, Osamu Gotoh, Yoshiaki Ohkuma, Yoshiak ...
    1997 年 121 巻 2 号 p. 389-396
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
    BTEB2 is a GC box-binding transcription factor containing proline-rich and zinc finger domains as transactivation and DNA binding domains, respectively. We have identified a small region in the proline-rich domain indispensable for its transcription-enhancing activity by transfection experiments using various expression plasmids with point mutations or small deletions in the domain. This region comprising about 10 amino acids was relatively hydrophobic and rich in proline and alanine residues. BTEB2 purified from a baculovirus expression system could enhance transcription, depending on the presence of GC boxes in the promoter region of templates in an in vitro transcription assay. BTEB2 deleted of the hydrophobic region, however, lost the transcription-enhancing activity, in confirmation of the above results. Basic transcription factors which possibly interact with BTEB2 were examined. Initiation factors, TFIIB, TFIIERβ, and TFIIFRβ as well as the TATA box-binding protein (TBP) were found to interact with BTEB2 when analyzed by in vitro binding experiments, although these interactions could not be attributed to the proline-rich domain. We discussed factors which interact with and transmit the transcriptional activity of the BTEB2 activation domain to basic transcriptional machinery.
  • 1997 年 121 巻 2 号 p. 397
    発行日: 1997年
    公開日: 2008/11/18
    ジャーナル フリー
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