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Volume 118, Issue 1
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Manabu Sato, Li-Hong Ye, Kazuhiro Kohama1995 Volume 118 Issue 1 Pages 1-3
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin light chain kinase (MLCK) was prepared from smooth muscle of bovine aorta. MLCK inhibited the ATP-dependent movement of actin filaments on a glass surface coated with smooth muscle myosin that had been phosphorylated. The inhibitory effect was abolished by calmodulin in the presence of Ca2+ (Ca-CaM). The abolition was also observed when the concentration of actin filaments was increased. The inhibitory effect and its abolition were related to the actin-binding activity of MLCK, that is antagonized by Ca-CaM.View full abstractDownload PDF (378K) -
Keiko Hirota, Kanako Momoeda, Kenichi Ono, Kazuo Hanaoka, Kentarou Hor ...1995 Volume 118 Issue 1 Pages 4-8
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe antigens for a monoclonal antibody, VJ-41, established by alloimmunization of B10. A (3R) mice with lymphocytes from B10. A (5R) mice and screening of its reactivity toward Con A-stimulated human T lymphocytes, were found to be phosphorylcholine-containing ceramides (sphingomyelin) and disaturated fatty acyl glycerol (phosphatidyl-choline, PC), but neither deacylated sphingomyelin nor unsaturated fatty acid-containing PC reacted with the antibody. Although the reactivity of disaturated fatty acyl PC increased with increasing chain length, that of sphingomyelin was stronger than that of di-20:0-PC. The binding of the antibody to Con A-stimulated lymphocytes was inhibited by sphingomyelin-containing liposomes, but not by di-18:0-PC containing ones, and the concentration of sphingomyelin in Con A-stimulated human T-lymphocytes was the same as that in non-stimulated ones, indicating that the reactivity of sphingomyelin in lymphocytes is altered by Con A-stimulation.View full abstractDownload PDF (1828K) -
Takumi Noguchi, Yorinao Inoue1995 Volume 118 Issue 1 Pages 9-12
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFourier transfer infrared (FTIR) signals originating from a high-potential electron accep-tor in PS II were studied by flash-induced FTIR difference spectroscopy. Redox titration of these signals using ferri-ferrocyanide mixtures showed midpoint potentials (Em) of 489±12 and 426±9mV at pH 5.5 and 6.5, respectively, revealing a pH dependence of about -60mV/pH unit. These Em, values and pH dependence were in good agreement with those of the non-heme iron, so-called Q400, located between QA and QB. This indicates that the observed FTIR signals are due to changes in ligands of the non-heme iron and surrounding protein moieties induced on photoreduction from Fe3+ to F2+.View full abstractDownload PDF (421K) -
Chun Ren Lim, Yukio Kimata, Masahiro Oka, Koji Nomaguchi, Kenji Kohno1995 Volume 118 Issue 1 Pages 13-17
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTagging proteins with the green fluorescent protein (GFP) from Aequorea victoria is a good means of analyzing protein localization in living cells. Nevertheless, GFP and a chimeric protein, GFP-nucleoplasmin, expressed in Saccharomyces cerevisiae were less fluorescent at high culture temperatures. Proteins synthesized at a low temperature retained their fluorescence despite a shift to a higher temperature. Hence, when a temperature-sensitive nspl mutant expressing GFP-nucleoplasmin was cultured at 23°C and then shifted to 35°C, we were able to exclusively monitor the localization of the protein synthesized prior to the temperature shift. This protein accumulated in novel nuclear-like compartments devoid of DNA.View full abstractDownload PDF (2588K) -
Chi-Fang Lee, Jung-Yaw Lin1995 Volume 118 Issue 1 Pages 18-22
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo squash family trypsin inhibitors, CMeTI-A and CMeTI-B, were isolated from the melon (Cucumis melo) seeds, by ion exchange chromatography, gel filtration, affinity chromatography, and high-performance liquid chromatography, and their amino acid sequences were determined. All inhibitors contain 29 amino acid residues including 6 half-cystine residues. They differ by twelve amino acid residues. These polypeptides are strong inhibitors of bovine trypsin, with K1 values of 1.6×10-10M (CMeTI-A) and 4.7×10-10M (CMeTI-B). The products of CMeTI-A and CMeTI-B cleaved at their reactive sites by tryptic digestion during the purification by trypsin-Sepharose 4B affinity column chromatography are active against trypsin activity, but a molar ratio of inhibitor to trypsin of 2:1 for trypsin-treated CMeTI-B or 1:1 for trypsin-treated CMeTI-A is required.View full abstractDownload PDF (456K) -
Chikashi Nakamura, Tomoko Kikuchi, J. Grant Burgess, Tadashi Matsunaga1995 Volume 118 Issue 1 Pages 23-27
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe magA gene from Magnetospirillum sp. strain AMB-1 is required for the synthesis of bacterial magnetic particles (BMPs). This gene has been cloned, sequenced and found to encode a protein which is homologous to the Escherichia coli potassium efflux membranebinding protein, KefC. By using the firefly luciferase gene (luc) cloned downstream of the magA promoter, the effect of iron on regulation of magA expression was investigated, and transcription of magA was found to be enhanced by low concentrations of iron. Intracellular localization of the MagA protein was studied using magA-luc fusion proteins. The luc gene was cloned downstream of the magA hydrophilic C-terminal domain. Detection of luciferase activity in the cytoplasm, cell membrane, and magnetic particle membrane subcellular fractions confirmed that the MagA fusion protein was localized in the cell membrane. The fusion protein was also detected on the surface of the lipid bilayer covering the magnetic particles. These results suggest that MagA is a membrane-bound protein, the expression of which is enhanced at low iron concentrations.View full abstractDownload PDF (565K) -
Shinji Saijo, Naoko Watanabe, Yoshiro Kobayashi1995 Volume 118 Issue 1 Pages 28-32
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBiologically active TNF-α is a trimer. Our newly developed monoclonal antibody against recombinant human TNF-α (3F2) bound to TNF-α immobilized on a solid phase precoated with the same monoclonal antibody, 3F2. We therefore examined the possibility that 3F2 preferentially recognized trimeric TNF-α, and obtained the following results. (i) Treatment of TNF-α with 1% NP40 enhanced the dissociation from the trimer to the monomer. The treatment also reduced the reactivity of 3F2 to TNF-α immobilized on a solid phase precoated with either 3F2 or a rabbit anti-TNF- α antibody (PT50). (ii) When trimeric and monomeric TNF-α, obtained by gel filtration of NP40-treated TNF-α, were assayed with either PT50 (solid phase)/3F2 (fluid phase) or PT50/PT50, PT50/3F2 showed a higher response to trimeric TNF-α than to monomeric TNF-α, while PT50/PT50 showed the opposite result. (iii) The values for two fractions containing only trimeric TNF-α obtained on ELISA with PT50/3F2 fitted the standard curve for ELISA with PT50/3F2 which was made by using unseparated TNF-α. Taken together, these findings suggested that 3F2 preferentially recognizes trimeric TNF-α.View full abstractDownload PDF (1294K) -
Cheng Yang, Shingo Maruyama, Shigeru Yanagi, Xiaoying Wang, Minoru Tak ...1995 Volume 118 Issue 1 Pages 33-38
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRadiation-induced biochemical events that mediate the intracellular signal transduction leading to cell apoptosis are largely unknown. Limited evidence suggests the possible involvement of one or more protein-tyrosine kinases (PTKs) in radiation-induced cellular responses, including apoptosis. However, so far, a PTK(s) responsible for the radiationinduced tyrosine phosphorylation of cellular substrates has not been identified and the role of the PTK(s) in the radiation-induced apoptosis remains unclear. To examine the roles of Syk and Lyn in radiation-induced signal transduction and radiation-induced apoptosis, we analyzed Syk-deficient or Lyn-deficient DT40 B cells along with wild-type cells following radiation. When DT40 B cells were exposed to radiation, the activity of Syk kinase dramatically increased and reached a maximum with 0.25 Grays (Gy) (15s), and then decreased, whereas Lyn kinase activity increased and reached a maximum with a dose of 1.00 Gy (1min). However, an apparent difference was not observed in radiation-induced apoptosis among wild-type, Syk-deficient, and Lyn-deficient DT40 B cells. These results indicate that Syk and Lyn kinases are involved in radiation-induced signal transduction, with different kinetics. In addition, our results revealed that functional inactivation of Syk or Lyn alone is not sufficient to prevent radiation-induced apoptosis. Thus, it is suggested that the activation of Syk or Lyn kinase alone may be sufficient to mediate the radiationinduced apoptosis in DT40 B cells, or both kinases may not be required for this biological process.View full abstractDownload PDF (2509K) -
Jun-ichi Nikawa, Akiko Murakami, Etsuko Esumi, Kohei Hosaka1995 Volume 118 Issue 1 Pages 39-45
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSaccharomyces cerevisiae ire15 mutant has a defect in the expression of INO1, showing the inositol auxotrophic phenotype [Nikawa, J. (1994) Gene 149, 367-372]. We have isolated five yeast genes which suppress the ire15 mutation in multiple copies by genetic complementation. Among them, one gene, designated as SCS2, also suppressed the cholinesensitive dominant mutation, CSE1 [Hosaka, K. et al. (1992) J. Biochem. 111, 352-358]. The CSE1 mutation is not allelic to ire15. Sequencing analysis revealed that the SCS2 gene encodes 244 amino acid residues with a calculated molecular mass of 26, 866. INO2/SCS1, which is another suppressor gene for CSE1 and is known to be a positive factor for INO1 expression, also suppressed the growth defect of the ire15 mutant. These results clearly indicate that the ire15 and CSEI mutations genetically interact and the SCS2 and INO2/SCS1 genes are involved in the regulation of INO1 expression.View full abstractDownload PDF (3253K) -
Yukio Nishimura, Keitaro Kato, Kimimitsu Oda, Toyoko Ishikawa, Yukio I ...1995 Volume 118 Issue 1 Pages 46-55
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOrganophosphate compounds are known to cause a selective increase of β-glucuronidase activity in rat serum. Previous data suggested that increase of serum β-glucuronidase activity was well correlated with decrease of that activity in rat liver microsomal fraction, thereby, suggesting a role of the microsomal enzyme in mediating the organophosphate effect. To investigate further the intracellular sorting pathway of β-glucuronidase in dibutyl phosphate-treated rats, liver subcellular fractions were prepared at 12 or 48h after in vivo administration of [3H]leucine and it was established that microsomal β-glucuronidase was the origin of the increased serum enzyme. To characterize the intracellular secretory pathway of β-glucuronidase in dibutyl phosphate-treated rats, Golgi subfractions were isolated and a time course study was carried out. At 30 min after administration of dibutyl phosphate, specific activity of β-glucuronidase in GF-2 (Golgi intermediate fraction) and GF-3 (Golgi heavy fraction) was significantly increased to the maximum. Furthermore, colchicine pretreatment of rats caused a delay of the peak of specific activity for 30min in GF-2 and GF-3, and accumulation of enzyme activity in Golgi subfractions was observed. Colchicine pretreatment also had an inhibitory effect on release of β-glucuronidase into serum until 30 min after dibutyl phosphate injection. The electrophoretic pattern of microsomal β-glucuronidase on polyacrylamide gel was found to show two major bands of microsomal enzyme type and lysosomal enzyme type in dibutyl phosphatetreated rats. Taken together, these findings indicate that microsomal β-glucuronidase follows the intracellular secretory pathway and is secreted into serum via Golgi complex in response to dibutyl phosphate.View full abstractDownload PDF (4598K) -
Yukio Nishimura, Keitaro Kato, Masaru Himeno1995 Volume 118 Issue 1 Pages 56-66
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOrganophosphate compounds are known to cause the selective release of rat liver micro-somal β-glucuronidase into plasma. To investigate the alterations of molecular forms and oligosaccharide moieties of liver β-glucuronidase in organophosphate compound-admin-istered rats, β-glucuronidase was isolated from microsomal, Golgi, lysosomal, and serum fractions. In SDS-polyacrylamide gel electrophoresis, a single polypeptide band was observed on gels in Golgi and serum β-glucuronidases. This result indicated that Golgi and serum β-glucuronidases of treated rats did not undergo post-translational proteolytic processing, in contrast to those in control rat livers. Biochemical characterization of the isolated β-glucuronidases by employing lectin affinity chromatography revealed that interaction of serum and Golgi enzymes with Ricinus communis agglutinin- and wheat germ agglutinin-Sepharose was fairly strong, and that microsomal and lysosomal enzymes were poorly retained on those columns. These results suggested that the serum and Golgi β-glucuronidases are sialoglycoproteins. A clearance study also showed that infused serum β-glucuronidase was slowly cleared from plasma with a half-life of about 60min, but the asialo-serum enzyme was rapidly cleared with a half-life of about 5min. These results imply that microsomal β-glueuronidase undergoes extensive modification of the oligosaccharide moieties by terminal glycosyltransferases at the trans Golgi when it is destined for secretion into serum in response to treatment with an organophosphate compound.View full abstractDownload PDF (5612K) -
Masahiro Iwakura, Tomokuni Kokubu1995 Volume 118 Issue 1 Pages 67-74
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs a result of the expression of a single open reading frame composed of the coding sequence for a cysteine-free mutant (Cys85→Ala, Cysl52→Ser) of Escherichia coli dihydrofolate reductase (DHFR; 18K monomeric protein), that for the E. coli thymidylate synthase (TS; dimeric protein with a 30K protomer), and a spacer sequence (coding 7 amino acids) with a Shine-Dargarno sequence, an active hetero-dimeric bifunctional enzyme with 50K DHFR-TS and 30K TS polypeptides was stably produced in the transformed E. coli cell in addition to an overproduction of the TS dimeric enzyme. The highly purified hetero-dimeric enzyme has similar Vmax and Km values in both DHFR and TS activities to those of the natural counterparts, monomeric DHFR and dimeric TS. Although the hetero-dimeric enzyme did not show an apparent channeling transfer of dihydrofolate (the intermediate substrate) between the spatially discrete DHFR and TS active sites, the coupling efficiency of the TS and DHFR reactions in the artificial enzyme was better than that in the separated enzymes, as shown by a decrease in the intermediate concentration at the steady state in the coupled reaction.View full abstractDownload PDF (2521K) -
Wataru Hashimoto, Hideyuki Suzuki, Kenji Yamamoto, Hidehiko Kumagai1995 Volume 118 Issue 1 Pages 75-80
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSγ-Glutamyltranspeptidase [EC 2. 3. 2. 2] of Escherichia coli K-12 is thought to be synthe-sized from a single precursor polypeptide into a heterodimeric form through post-translational processing. Cells of a γ-glutamyltranspeptidase-overproducing transformant of E. coli K-12 were fractionated and the localization of the enzyme was examined by Western blot analysis. The periplasmic fraction only contained the mature form of γ-glutamyltranspeptidase, membrane fraction only contained the precursor of γ-glutamyltranspeptidase, and no precursor of γ-glutamyltranspeptidase was detected in the cytoplasmic fraction. Amino acid residues at the cleavage site for processing into the large and small subunits were substituted by site-directed mutagenesis. The processing phenotypes of six mutants were examined by Western blot analysis, and their γ-glutamyltranspeptidase activities were measured. Mutations at the N-terminal amino acid residues of the small subunit (Thr-391, Thr-392, and His-393) prevented the maturation of the enzyme and the immature mutants exhibited no enzymatic activity. A mutation at the C-terminal residue of the large subunit (Gln-390) had less effect on the processing and enzymatic activity. These results suggest that the sequence of threonyl-threonyl-histidinyl residues at the N-terminal of the small subunit is very important for the processing of E. coli K-12 γ-glutamyltranspeptidase and this processing is essential to the expression of γ-glutamyltranspeptidase activity of E. coli K-12.View full abstractDownload PDF (3104K) -
Timothy K. Lee, William N. Drohan, Henryk Lubon1995 Volume 118 Issue 1 Pages 81-87
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMaturation of human Protein C (HPC) precursor to a zymogen in the liver requires endoproteolytic cleavages after a basic dipeptide, Lys-2-Arg-1in the propeptide and Lys156-Arg157 connecting the light and heavy chain. Recombinant human Protein C (rHPC) was expressed in the mammary gland of transgenic swine and its proteolytic processing was monitored. We found that about 10-20% of rHPC purified from the milk still retained the propeptide and 30-40% was in the single-chain form, indicating inefficient proteolytic cleavage. This demonstrates that endoprotease(s) of the swine mammary epithelial cells do not process fully the precursor of heterologous protein. rHPC was fractionated by anion exchange chromatography and polypeptides with novel N-termini at positions -1, 152, and 157 were detected in addition to the known N-termini at residues -24, +1, and 158. Since rHPC was found to be stable both in milk and after purification, it is possible that these new cleavages on the amino side of arginine at dipeptide sites Lys-2-Arg-1, Lys151-Arg152, and Lys156-Arg157 could have occurred in the mammary gland. Thus, our results suggest that a portion of HPC precursor was proteolytically processed in swine mammary gland differently than those in other expression systems.View full abstractDownload PDF (2868K) -
Hiroya Obama, Jun-ichiro Tsutsui, Masayuki Ozawa, Hiroki Yoshida, Yosh ...1995 Volume 118 Issue 1 Pages 88-93
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMidkine (MK) and pleiotrophin (PTN) constitute a new family of heparin-binding growth factors. Extraembryonic membranes and the placenta of the mouse expressed MK mRNA at 11.5 days gestation. While the MK mRNA level in extraembryonic membranes decreased during embryogenesis, that in the placenta remained unchanged. Immunohistochemical studies showed that MK was located in the yolk sac and in the amnion at 11.5 days gestation. PTN mRNA expression was weak in extraembryonic membranes and was scarcely detectable in the placenta. Western blot analysis revealed the presence of MK in amniotic fluid and cerebrospinal fluid, in amounts of more than 1μg/ml, raising the possibility that MK delivered by these fluids participates in the regulation of organogenesis. Transport of MK from the site of its synthesis appears to also occur in the adult kidney, since MK mRNA and the MK protein are localized in different regions of the kidney.View full abstractDownload PDF (3221K) -
Kaneo Sekiguchi, Chika Yokota, Makoto Asashima, Tadashi Kaname, Qi-Wen ...1995 Volume 118 Issue 1 Pages 94-100
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMidkine (MK) is a heparin-binding growth factor and forms a novel protein family together with another member, pleiotrophin (PTN)/heparin-binding growth-associated molecule (HB-GAM). A cDNA clone isolated from Xenopus laevis specifies for the Xenopus counterpart of MK (XMK), and the mode of XMK expression was studied by in situ hybridization and Northern blot analysis. XMK was first expressed at stage 11 (middle gastrula) and was located in the neural anlage at stage 12 (late gastrula). Through stage 13 to 15 (early neurula), XMK expression was restricted to the neural folds. At stage 23 (tailbud stage), XMK was predominantly localized in the brain and neural tube. At the larva stage, XMK expression was again restrictedly observed in the brain, the optic vesicles, the otic vesicle, and the spinal cord, all of which are derivatives of the neural tube, as well as in the branchial arches, derivatives of the cranial neural crest. Comparing the mode of MK expression between Xenopus and the mouse, we propose that MK plays evolutionally conserved roles in neurogenesis and development of the craniofacial architecture of ectomesenchymal origin. We also found that XMK was expressed in various adult organs; strong expression was observed in the brain, the eye and the spinal cord, all of which showed intense MK expression at the larva stage.View full abstractDownload PDF (4067K) -
Kwang-Hwan Jhee, Tohru Yoshimura, Nobuyoshi Esaki, Kazuo Yonaha, Kenji ...1995 Volume 118 Issue 1 Pages 101-108
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThermostable L-ornithine: α-ketoglutarate δ-aminotransferase (L-ornithine:2-oxo-acid 5-aminotransferase) [EC 2. 6. 1. 13] was purified to homogeneity from Bacillus sp. YM-2. The enzyme has a molecular weight of about 82, 000 and consists of two subunits with identical molecular weights. The enzyme catalyzes transamination from L-ornithine to α-ketoglutarate, producing L-glutamate and L-glutamate γ-semialdehyde, which is spontaneously dehydrated to L-Δ1-pyrroline-5-carboxylate, and the enzyme is most active at 70°C. In addition to L-ornithine, the enzyme unexpectedly acts on D-ornithine, the reaction rate being 6% of that for L-ornithine. The enzyme contains 1 mol each of pyridoxal 5'-phosphate and another vitamin B6 compound per mol. The enzyme released the bound pyridoxal 5'-phosphate, as judged from the absorption at 425 nm on incubation with 2.0M guanidine hydrochloride. The resultant inactive enzyme still gave a 340-nm peak and contained 1 mot of the vitamin B6, compound. The partial amino acid sequence shows high homology with those of mammalian and yeast ornithine δ-aminotransferases.View full abstractDownload PDF (1968K) -
Chiaki W. Nakagawa, Norihiro Mutoh, Yukimasa Hayashi1995 Volume 118 Issue 1 Pages 109-116
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExposure of Schizosaccharomyces pombe cells to various stresses including 0.2mM hydrogen peroxide, 50μM menadione, 10J/m2 of UV irradiation at 255 nm, and high osmolarity (0.5M sorbitol or 0.3M NaCl) induces catalase [EC 1. 11. 1. 6] activity. A part of the catalase gene of S. pombe was amplified by PCR with oligonucleotide primers designed from amino acid sequences conserved in several species of catalases. The catalase gene including its flanking sequence of S. pombe was cloned from a genomic DNA library of S. pombe, which was constructed on the EMBL3 vector, using the PCR-amplified DNA as a radioactive probe. A 3.5 kb HindIII fragment, which hybridized with the PCR-amplified probe, was subcloned into pUC19 and sequenced. The fragment contains one long open reading frame without any intron. The polypeptide deduced from the nucleotide sequence consists of 512 amino acid residues and is homologous to several other catalases. Amino acid sequences of the proteolytic peptides obtained from the purified catalase of S. pombe coincided with the amino acid sequence predicted from the DNA sequence. Transcription of this gene starts at 370 bases upstream of the initiation methionine codon. Northern blot analyses of the catalase mRNA revealed that the stresses which induce the catalase activity also induce the transcription of the catalase gene. The induction of the catalase mRNA by hydrogen peroxide is not inhibited by cycloheximide or staurosporine.View full abstractDownload PDF (6805K) -
Masakazu Oyama, Kou Kubota1995 Volume 118 Issue 1 Pages 117-121
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding of an intrinsic agonist (cyclic AMP) to specific receptors on the cell surface induces transmembrane signals for the activation of adenylate cyclase in the cellular slime mold Dictyostelium discoideum. We found that stimulation by CaCl2, MgSO4 or polyamines having two to four positive charges induced the activation of adenylate cyclase in cells treated with saponin. The activation was roughly identical to the stimulation of the intrinsic agonist both in amount and in time course. The intact cells (saponin-untreated cells) responded to neither divalent cations nor polyamines. While saponin is known to have a detergent-like effect and to make the plasma membrane permeable, low molecular weight dyes did not penetrate the plasma membrane under our conditions for the saponintreatment. Caffeine is known to inhibit the cAMP-induced activation of adenylate cyclase by blocking signal transduction, but not by acting directly on the enzyme [Brenner, M. and Thorns, S. D. (1984) Dev. Biol. 101, 136-146]. We found that caffeine inhibited the cationinduced activation. These results suggest that these divalent and polyvalent cations do not act directly on adenylate cyclase but that they mimic or induce the transmembrane activation signal for adenylate cyclase in the saponin-treated cells.View full abstractDownload PDF (655K) -
Erika Suzuki, Nobuhiko Kojima, Kazunori Yoshimura, Keiichi Uyemura, Ku ...1995 Volume 118 Issue 1 Pages 122-128
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMonoclonal antibody 5E5 recognized an intranuclear antigen of neurons in the rat. We isolated 5E5cDNA and determined its nucleotide and deduced amino acid sequences. The 5E5cDNA had an open reading frame of 825 amino acids and its amino acid sequence showed no significant homology to any protein or to any DNA binding motif so far known. 5E5 protein had an abundance of basic amino acids, especially arginine, and included a glycine-rich region and a proline cluster. Monoclonal antibody 12H raised against 5E5cDNA fusion protein recognized an intranuclear substance in rat brain sections and a single protein band of about 98 kDa in the brain nuclear extract fraction on immunoblotting. DNA-cellulose column chromatography indicated that 5E5 protein might have DNA-binding ability. Transfection studies indicated that 5E5 protein expressed in COS-1 is localized in cell nuclei. These results suggest that 5E5 protein is a possible DNA-binding protein which is expressed especially in neurons.View full abstractDownload PDF (4084K) -
Yoshihisa Hagihara, Yuji Goto, Hisao Kato, Tetsuro Yoshimura1995 Volume 118 Issue 1 Pages 129-136
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ2-Glycoprotein I (β2-GPI) is a cofactor in the recognition of the phospholipid antigen cardiolipin by anti-cardiolipin antibodies in autoimmune diseases such as systemic lupus erythematosus. We examined the interactions of various forms of bovine β2-GPI, such as its intact form, desialylated form (Asialo-β2-GPI), N-terminal domain (Domain I), and modified forms of β2-GPI and Asialo-β2-GPI with nicks in their C-terminal domains, with phospholipid liposomes under different conditions of pH and ionic strength. We found that at neutral pH and low ionic strength, β2-GPI became bound to liposome membranes containing cardiolipin, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, or phosphatidylinositol, but not phosphatidylcholine alone. The number of phospholipids involved in the binding seemed to depend on the head group structure of the negatively charged phospholipids, but the dissociation constant did not, being about 10-8M, except that for the interaction with phosphatidylinositol, which was one order of magnitude lower. We also found that Domain I and Asialo-β2-GPI bound to liposome membranes containing negatively charged phospholipids, and that in the interaction with cardiolipin, their dissociation constants were about 10-6 and 10-8M, respectively. At neutral pH and both low and high ionic strengths, the affinities of the nicked forms of β2-GPI and Asialoβ2-GPI for cardiolipin were both lower than those of their intact forms but similar to that of Domain I. The pH dependencies at low and high ionic strengths and the ionic strength dependency at neutral pH of the binding of β2-GPI to liposome membranes containing cardiolipin were similar to those of Asialo-β2-GPI, but the pH and ionic strength dependencies of the two species were different from those of their nicked forms, the latter being similar to those of Domain I. These results suggest that the N-terminal as well as C-terminal domains have an important role in the interaction of β2-GPI with cardiolipin, and that the three residual domains containing sialic acid have no significant effect on the interaction.View full abstractDownload PDF (1793K) -
Yo Matsuo, Haruki Nakamura, Ken Nishikawa1995 Volume 118 Issue 1 Pages 137-148
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new approach to evaluate protein structure (3D)-sequence (1D) compatibility has been developed. This approach uses four functions: side-chain packing, solvation, hydrogenbonding, and local structure functions. The side-chain packing function takes into account not only inter-residue distance but also inter-residue contact and angle. Parameters defining the functions were statistically derived from 101 proteins with known 3D structures. The functions are combined to give a score quantifying the 3D-1D compatibility. The ability to identify the native structure of a protein among a large number of incorrect structures was tested. For 69 out of the 70 proteins examined, the native structures were successfully identified. Particularly, the side-chain packing function showed clear improvement over our previous function. The only unsuccessful case was observed for cytochrome c3 containing four haems; such prosthetic groups were ignored in the calculation. In addition to the above functions, two procedures for removing membrane-spanning regions and estimating electrostatic interactions were also adopted. A sequence for which membrane-spanning regions were predicted was considered incompatible with a structure irrespective of the compatibility score. A method to calculate the electrostatic interaction energy was developed and used for the detection of electrostatistically undesirable interactions between residues of a sequence threaded onto a structure. These procedures greatly help to remove false positives in the 3D-1D compatibility search.View full abstractDownload PDF (1413K) -
Kanefusa Kato, Hidenori Ito, Kaori Hasegawa, Yutaka Inaguma, Atsushi S ...1995 Volume 118 Issue 1 Pages 149-153
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe levels of two small stress proteins, hsp27 and αB crystallin, were low in C6 glioma cells confluency. However, the levels of the two proteins increased after exposure of cells to heat (42°C for 30 min) or arsenite (50-100μM for 1 h) stress. When cells were exposed to arsenite or heat in the presence of mastoparan, a peptide toxin from wasp venom, the induction of hsp27 and αB crystallin was markedly stimulated, as detected by means of specific immunoassays, Western blot analysis, and Northern blot analysis. The response of hsp70 to each stress was also enhanced in the presence of mastoparan. Treatment of cells with 40μM mastoparan alone barely induced the accumulation of hsp27 and αB crystallin. The stimulatory effect of mastoparan was little affected in cells that had been treated with pertussis toxin, but it was strongly suppressed in the presence of quinacrine, an inhibitor of phospholipase A2. These results suggest that mastoparan, which is an activator of phospholipase A2, enhances the responses to stress of hsp27, αB crystallin and hsp70 by increasing the metabolic activity of the arachidonic acid cascade.View full abstractDownload PDF (2008K) -
Masayuki Ozawa1995 Volume 118 Issue 1 Pages 154-160
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSReticulocalbin, an endoplasmic reticulum (ER)-resident Ca2+-binding protein, is a member of the EF-hand Ca2+-binding protein superfamily [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Reticulocalbin has six repeats of a domain containing the EF-hand motif. In addition, the protein has an amino-terminal leader sequence which serves as a signal for transfer of the protein into the lumen of the ER, and a His-Asp-Glu-Leu sequence at its carboxy terminus which functions as a signal to retain the protein in the ER. In this paper, we describe the genomic structure of this unique Ca2+-binding protein. Southern blot analysis of mouse genomic DNA revealed that there is a single copy of the reticulocalbin gene per haploid genome. The gene spans over 13 kilobase pairs and encodes six separate exons. Thus, reticulocalbin differs from the cytosolic Ca2+-binding protein calbindin D28 which also has six EF-hand motif domains, but the gene for which is divided into 11 exons. While there is some correlation between exon division and protein domain structure, these relationships are not as clear as they are in other genes. Comparison of the gene organization of reticulocalbin with that of other EF-hand proteins revealed that reticulocalbin diverged very early from other members of the EF-hand protein superfamily.View full abstractDownload PDF (2007K) -
Y. P. Tu, F. Y. Yang1995 Volume 118 Issue 1 Pages 161-167
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSZn2+ could inhibit the anion transport activity of spectrin-stripped inside-out human erythrocyte membrane vesicles (IOVs). Removal of the cytoplasmic domain from Band 3 by trypsin could eliminate Zn2+ inhibition. The location of a Zn2+-binding site was confirmed by atomic absorbance spectrometry. The results of time-resolved fluorescence and intrinsic fluorescence quenching by KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus growing in Yunnan, China) showed that the cytoplasmic domain is necessary for the Zn2+-induced conformational changes of the whole molecule as well as the membrane domain of Band 3. It is suggested that Zn2+ induced a conformational change in the cytoplasmic domain of Band 3, which in turn was transmitted to the membrane domain, resulting in an inhibition of activity of Band 3. Such long-range conformational changes may imply that the cytoplasmic domain is poised to function as a cytosolic arm in order to modulate the structure of the membrane domain of Band 3.View full abstractDownload PDF (1742K) -
Yukio Nishimura, Hiroaki Takeshima, Masao Sakaguchi, Katsuyoshi Mihara ...1995 Volume 118 Issue 1 Pages 168-177
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the intracellular transport mechanisms of lysosomal cathepsin D in yeast cells, we produced cathepsin D in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D. Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein. To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells. Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction. Immunofluorescence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles. Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.View full abstractDownload PDF (7058K) -
Takeo Abumiya, Kei-ichi Enjyoji, Toshinori Kokawa, Yu-ichi Kamikubo, H ...1995 Volume 118 Issue 1 Pages 178-182
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor with three tandem inhibitory domains, which inhibits the initial reactions of the extrinsic blood coagulation pathway through the first and second Kunitz domains. We prepared a monoclonal antibody against recombinant human TFPI (rTFPI) and determined the epitope as the third Kunitz domain, using fragments derived from rTFPI (K1-K2 fragment and K3 fragment) and synthetic peptides. We then developed an enzyme immunoassay (EIA) method using a combination of the monoclonal antibody and a polyclonal antibody. Although TFPI activity is distributed among LDL/VLDL-associated, HDL-associated, and free forms of TFPI after gel-filtration of human plasma, only the free form was detected by the EIA method. After incubation with LDL, the antigenicity of rTFPI was reduced, but that of K3 fragment was not. Gel-filtration analysis of the mixture of radiolabeled rTFPI or K3 with LDL demonstrated that rTFPI, but not K3, bound LDL. From these results, we concluded that the monoclonal antibody against TFPI recognized only a free form of TFPI in plasma, since the epitope of lipoprotein-associated TFPI had been masked by the interaction with lipoproteins.View full abstractDownload PDF (2141K) -
Rong Zhang, Tatsuo Kai, Yasushi Sugimoto, Takahiro Kusakabe, Yozo Taka ...1995 Volume 118 Issue 1 Pages 183-188
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree isozymic forms, α, β, and γ, of Drosophila melanogaster aldolase are produced from a single gene by alternative usage of the triple exons 4 (4α, 4β, and 4γ) [Shaw-Lee et al. (1992) J. Biol. Chem. 267, 3959-3967; Kim et al. (1992) Mol. Cell. Biol. 12, 773-783; Kai et al. (1992) J. Biochem. 112, 677-688]. The expression plasmids for the respective isozymes were transfected into Eseheriehia coli cells, and the isozymes α and β were purified to homogeneity by a simple procedure, though isozyme γ was only partially purified. These isozymes are active towards two substrates, fructose-l, 6-bisphosphate (Fru-1, 6-P2) and fructose-l-phosphate (Fru-1-P), with a preference for Fru-1, 6-P2 over Fru-1-P, but they have different kcat/Km values towards these two substrates; isozyme α shows the highest kcat/Km value for Fru-1-P among the three isozymes, whereas isozyme β has the highest value for Fru-1, 6-P2. These isozymes show similarity in optimal pHs, thermal stability, and Km values for both Fru-1, 6-P2 and Fru-1-P. They are composed of four identical subunits of 40 kDa, forming a tetramer with a molecular weight of approximately 160 kDa. The three isozymes are different in primary structure only at the carboxyl-terminal region encoded by the respective exon 4. Therefore, this region should be primarily responsible for the distinct characteristics of these isozymes.View full abstractDownload PDF (3011K) -
Hiroko Kojima, Joe Chiba, Yoshio Watanabe, Osamu Numata1995 Volume 118 Issue 1 Pages 189-195
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 14-nm filament protein (designated as 49K protein) was purified from a ciliated protozoan, Tetrahymena, using the polymerization and depolymerization procedure. Previous studies in our laboratory showed that its primary structure shared a high sequence identity with citrate synthases known so far and that the 49K protein possessed citrate synthase activity. To ascertain whether or not Tetrahymena's mitochondrial citrate synthase is identical to the 49K protein, citrate synthase was purified from Tetrahymena mitochondria using ammonium sulfate fractionation, Butyl-Toyopearl and SP-Toyopearl column chromatographies, based on monitoring of the enzymatic activity. The molecular weight of the purified citrate synthase was estimated to be 49 kDa, as was that of the 49K protein and the enzyme cross-reacted with an anti-49K protein antiserum. The purified citrate synthase showed much the same optimum pH, optimum KCl concentration, effects of substrate concentrations (acetyl-CoA and oxaloacetate), and inhibitory effect by ATP as those of purified 49K protein. Furthermore, an anti-49K protein monoclonal antibody strongly suppressed the enzymatic activity of the purified citrate synthase. Thus, we suggest that mitochondrial citrate synthase and the 49K protein are identical and that the 49K protein has dual functions in the cytoskeleton in cytoplasm and as a TCA cycle enzyme, citrate synthase, in mitochondria.View full abstractDownload PDF (3304K) -
Tadayoshi Imai1995 Volume 118 Issue 1 Pages 196-203
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeven pyridine nucleotide analogs were synthesized, including nicotinic acid mononu-cleotide, nicotinic acid-adenine dinucleotide, and nicotinic acid-adenine dinucleotidephos-phate, by means of the transglycosidase activity of rabbit spleen pyridine nucleotide glycohydrolase. The velocity ratio of the transglycosidation to the hydrolysis in the pres-ence of 16mM nicotinamide mononucleotide and 0.3M nicotinic acid at pH 5.2 was greater than 10 and the transglycosidation/hydrolysis partition ratio was estimated to be (12±2)×103. The partition ratio values obtained with 3-acetylpyridine and nicotinylglycine were smaller than that with nicotinic acid. Configurational analysis of pyridinium C-N glycosidic linkages of the transglycosidation products by means of 1H-NMR, UV, CD, and paper chromatography indicated that the configuration was completely retained and absolutely β. Retention of the configuration, together with the substrate specificity of the enzyme for the β-pyridinium linkage and formation of the intermediary enzyme-phosphoribosyl (or ADP-ribosyl) complex during the enzyme catalysis, indicated that the transglycosidation reaction catalyzed by the enzyme proceeds through the double replacement mechanism.View full abstractDownload PDF (978K) -
Mamoru Wakayama, Toshiyuki Ashika, Yoshiro Miyamoto, Tomoya Yoshikawa, ...1995 Volume 118 Issue 1 Pages 204-209
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene coding the N-aryl-D-glutamate amidohydrolase of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was cloned and its complete DNA sequence was determined. The N-acyl-D-glutamate amidohydrolase structural gene consists of 1, 464 nucleotides and encodes 488 amino acid residues. The molecular weight of the enzyme was calculated to be 51, 490. This value is close to the apparent molecular weight of 59, 000 determined for the purified enzyme from Alcaligenes A-6 by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The N-terminal amino acid sequence of the recombinant protein exactly matches the amino acid sequence derived from the DNA sequence and that determined from the Alcaligenes A-6 enzyme (NH2-MQEKLDLVIEGGWVIDGLGG). The deduced amino acid sequence of the cloned N-aryl-D-glutamate amidohydrolase showed high sequence homology with those of N-acyl-D-aspartate amidohydrolase (46%) and D-aminoacylase (47%) from Alcaligenes A-6. This fact strongly suggests that these three enzymes have evolved from a common ancestral gene.View full abstractDownload PDF (709K) -
Hisamitsu Ide, Hiroshi Itoh, Masaki Tomita, Yuko Murakumo, Takahiko Ko ...1995 Volume 118 Issue 1 Pages 210-215
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA encoding rat mast cell tryptase (rMCT) was successfully cloned, and sequenced, from peritoneal cells of Lewis rats infected with Nippostrongylus brasiliensis by the reverse transcription-polymerase chain reaction and rapid amplification of eDNA ends methods. The cDNA was 1, 097 base-pairs long, and included 822 base-pairs of an open reading frame. As judged from the deduced amino acid sequence, rMCT is highly homol-ogous to mouse mast cell protease-6, and is considered to be translated as a prepro-enzyme with a 19-amino acid signal peptide, a 10-amino acid activation peptide, and a 245-amino acid mature enzyme. The rMCT mRNA was not detected in peritoneal cells of mast cell-deficient Ws/Ws rats, though it was strongly detected in ones of littermate +/+ and Lewis rats. In addition to in peritoneal mast cells, the rMCT mRNA was detected in the tongue. However, mRNA signals were not detected in the small intestine regardless of N. brasilien-sis infection. Nor were mRNA signals detected in RBL2H3 rat basophilic leukemia cells. In the lung, the rMCT mRNA was strongly detected after infection with N. brasiliensis, though it was only faintly detected before infection. These results suggest that the rMCT is basically specific for connective tissue mast cells, but not for mucosal mast cells and that it is up-regulated in the lung during the inflammatory process of a parasitic infection.View full abstractDownload PDF (1729K) -
Yoshinao Azuma, Kiyoshi Yasui, Masahiro Yamagishi, Akira Ishihama1995 Volume 118 Issue 1 Pages 216-220
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubunit α of prokaryotic RNA polymerases plays key roles in protein-protein contacts for both Subunit assembly and transcription activation. To gain an insight into the roles of subunit 3, the eukaryotic homologue of α, temperature-sensitive mutants of the fission yeast, Schizosaccharomyces pombe, have been isolated after transformation of the mutagenized rpb3 gene encoding mutant Subunit 3 of RNA polymerase II. A total of 68 is mutants were classified into two groups: mutants comprising one group ceased growing immediately after a temperature up-shift, while mutants comprising the other group exhibited delayed growth arrest at high temperatures. RNA polymerase II partially purified from Ts54, one of the group 2 mutants, was thermolabile in vitro, as measured by a non-specific transcription assay. This mutant carries double mutations in domain A of subunit 3, and thus can be used as a reference mutant of RNA polymerase II.View full abstractDownload PDF (571K) -
Ho Jeong Kwon, Minoru Yoshida, Kenkoh Muroya, Seisuke Hattori, Eisuke ...1995 Volume 118 Issue 1 Pages 221-228
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRadicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or, its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene (s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.View full abstractDownload PDF (6925K) -
Takashi Kato, Kinya Ogami, Yoshihiro Shimada, Akihiro Iwamatsu, Yoshia ...1995 Volume 118 Issue 1 Pages 229-236
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA thrombopoietic factor, termed thrombopoietin (TPO), was highly purified directly from the plasma of sublethally irradiated 1, 100 rats by measuring the production of megakaryocytes from a highly enriched population of rat megakaryocyte progenitor cells (CFU-MK). The rat plasma TPO is a glycoprotein and strongly hydrophobic. The total activity and purification yields obtained were about 29% and 1.49×108, respectively. The amino acid sequences of the two peptide fragments prepared from the purified 19 kDa TPO were analyzed, and used for the cloning of rat and human TPO eDNAs. It was found that the 19 kDa TPO was truncated but comprised at least 163 amino acids. The sequence of human TPO eDNA revealed that the TPO was identical to the c-Mpl ligand. Both rat and human TPOs expressed in COS-1 cells exhibited significant activity toward the CFU-MK in vitro, and were active in stimulating platelet production in mice. These results indicate that a thrombopoietic factor originally found in the irradiated rat plasma is a ligand for the rat c-Mpl.View full abstractDownload PDF (3057K) -
Naoki Matsumoto, Kiyoshi Kita, Somei Kojima, Kazuo Yamamoto, Tatsuro I ...1995 Volume 118 Issue 1 Pages 237-243
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSp50 is phosphorylated in mitogen-stimulated T cells, and translocated from the membrane to the cytosol after activation of protein kinase C. Sequence analysis of p50 revealed that it is identical with LSP1, a putative calcium-binding and actin-binding protein. The lymphocyte form of p50 exhibits heterogeneity in the apparent molecular mass on SDS-PAGE, 50 and 52 kDa (pp50 and pp52), and each isoform exhibits heterogeneity in the isoelectric point, when examined by two-dimensional PAGE. When the two molecular mass variants of p50 were dephosphorylated with alkaline phosphatase, both isoforms showed the same apparent molecular mass of 50 kDa on SDS-PAGE, but could be distinguished by their distinct isoelectric points. Dephosphorylated pp50 (p50a) has an acidic pI compared with dephosphorylated pp52 (p50b). Comparison of the peptide maps of purified p50a and p50b on HPLC revealed that the difference was limited to one peptide peak. NH2-terminal sequence and mass spectrometric analyses of these peptides showed that the peptides derived from p50a and p50b had the same NH2-terminal amino acid sequence up to eight residues, but had distinct molecular masses, 5, 533.4 and 6, 318.6 Da, respectively. These data suggested that pp52 (p50b) is the product of the previously cloned cDNA and the reduction in the molecular mass of the p50a-derived peptide could be explained by deletion of six amino acid residues, EHLIRH or HLIRHQ. To verify this prediction, we examined the mRNA encoding p50 by means of the reverse transcriptase-polymerase chain reaction and found the presence of an mRNA that had a deletion of 18 bp, that results in a deletion of six amino acid residues, HLIRHQ (158-163), after translation. These data, and the results of transfection and expression of the two types of p50 cDNA in BALB/3T3 fibroblasts indicate that alternative splicing and phosphorylation generate the multiple isoforms of p50 in lymphocytes.View full abstractDownload PDF (6113K) -
Isao Suetake, Shoji Tajima, Akira Asano1995 Volume 118 Issue 1 Pages 244-249
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe detected a novel nuclear protein, MMBP-3, that bound to the c-Myc binding motif (CACGTG) in which deoxycytidine in the CpG sequence was methylated. MMBP-3 was partially purified by chromatography on heparin-agarose and hydroxyapatite, followed by affinity adsorption to a matrix coupled to the methylated binding motif. Its binding to the methylated c-Myc binding motif was specific, although it also recognized the unmethylated motif weakly. MMBP-3 was further found to recognize only one of two differently hemimethylated forms of the double-stranded c-Myc binding motif. MMBP-3 activity was detected in proliferating C2C12 and C3H/10T1/2 cells, and down regulated when the growth of these cells was inhibited. We propose that MMBP-3 plays a role in regulating the c-Myc function by recognizing the methylation state of the c-Myc binding motif in a growth-dependent manner.View full abstractDownload PDF (4683K)
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