The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
117 巻, 4 号
選択された号の論文の35件中1~35を表示しています
  • Ryohei Takeda, Mutsuhiko Mizobuchi, Koji Murao, Makoto Sato, Jiro Taka ...
    1995 年 117 巻 4 号 p. 683-685
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Two different sizes (approximately 1.0 and 1.6 kb) of transcripts of an isoaspartyl protein carboxyl methyltransferase (PIMT) were detected in eight cell lines derived from human hemopoietic cells on Northern blot analysis. We found three different sizes of cDNAs (907, 1, 553, and 1, 600 bp) in human erythroid leukemia cells (HEL) and a unique cDNA sequence corresponding to the 1.0 kb transcript was identified. These three cDNA sequences encoded two isozymes consisting of 226 (isozyme I) and 227 (isozyme II) amino acids. The 1.6 kb transcript was translated into two isozymes (isozyme I and II), while the 1.0 kb transcript was only translated into isozyme I. These results suggest that the two isozymes deduced from the cDNAs of the human erythroid leukemia cells may exist in normal human erythrocytes.
  • Sachiko Okuno, Takako Kitani, Hitoshi Fujisawa
    1995 年 117 巻 4 号 p. 686-690
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Rat brain calmodulin-dependent protein kinase IV (CaM-kinase IV) was activated approximately 20 to 30-fold by incubation with CaM-kinase IV kinase purified from rat brain under the Ca2+/calmodulin-dependent phosphorylation conditions. When CaM-kinase IV was incubated without CaM-kinase IV kinase, no significant activation was observed, indicating that the marked activation of CaM-kinase IV occurred as a result of the action of CaM-kinase IV kinase. More than 3 mol of phosphate were incorporated into 1 mol of the enzyme after incubation with CaM-kinase IV kinase at 30°C for 20min, but the activation occurred upon the initial incorporation of 1 mol of phosphate. The rate of the initial phosphorylation increased when the amount of CaM-kinase IV kinase added into the reaction mixture increased, but the rate of phosphorylation following the initial phosphorylation did not increase, suggesting that the initial phosphorylation was catalyzed by CaM-kinase IV kinase, and that subsequent phosphorylation was catalyzed by CaM-kinase IV itself activated by CaM-kinase IV kinase. The initial phosphorylation occurred in the amino-terminal serine-rich region of CaM-kinase IV. Kinetic analysis revealed that the increase in the activity upon phosphorylation was due mainly to an increase in the Vmax values.
  • Kazuhisa Kishimoto, Tohru Yoshimura, Nobuyoshi Esaki, Shigetoshi Sugio ...
    1995 年 117 巻 4 号 p. 691-696
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    We studied the catalytic role of leucine 201 residue of the thermostable D-amino acid aminotransferase: the residue was shown crystallographically to be in the vicinity of the active site to interact with the bound pyridoxal phosphate. We replaced the leucine 201 by alanyl or tryptophanyl residues by means of site-directed mutagenesis. The L201A and L201W mutant enzymes showed anomalous kinetic behavior in the overall reaction. The reaction rates of the L201A and L201W mutant enzymes gradually decreased with an increase in the reaction time to become practically zero at a high concentration of substrates. The mutant enzymes were also inactivated in the half reaction with D-alanine, although more slowly than in the overall reaction. The absorption spectra of the mutant enzymes in the presence of D-alanine and α-ketoglutarate suggest that the enzyme molecules were mostly in the pyridoxamine form under the conditions employed. These phenomena were explained by assuming two (or more) enzyme species showing kinetically different catalysis for pyridoxamine form of the mutant enzymes, and the rate of conversion from one of these pyridoxamine forms to the pyridoxal form should be very low. The leucine 201 residue probably regulates the function of cofactor during the reaction of D-amino acid aminotransferase.
  • Kiyoshi Nakazawa, Takako Isomura, Sadahiko Shimoeda
    1995 年 117 巻 4 号 p. 697-706
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Corneal explants with scleral rims were freshly prepared from 3-day-old hatched chicks, cultured in vitro for 5h in the presence of [35S]sulfate and [3H]glucosamine, then the constituent tissues (stroma, epithelium, endothelium, and corneo-scleral rim) were dissected from the explants. Incorporation of 35S activity into stromal tissue was the highest among the constituent tissues (54% of the total). Two predominant proteoglycans were identified in the stromal fraction: keratan sulfate proteoglycan and chondroitin sulfate/dermatan sulfate proteoglycan. Whereas heparan sulfate proteoglycan was found in epithelial tissue as the major proteoglycan therein, it was a minor component in the other tissues. In contrast, most of the 35S-labeled material in the culture medium was the sulfated glycoprotein, which was probably synthesized and secreted by epithelial cells. When each constituent tissue was separately cultured in the presence of radioactive precursors, incorporation of radioactivities into each tissue decreased markedly compared with those into the respective tissues obtained by dissection after culture of the whole cornea with scleral rim. This suggests that corneal constituent tissues interact with each other for proteoglycan synthesis. But the glycosaminoglycan compositions of stromal proteoglycans synthesized in stroma cultured alone and in stroma from the cultured cornea with scleral rim were similar to each other; the two major glycosaminoglycans were chondroitin sulfate/dermatan sulfate and keratan sulfate. In contrast, when stromal tissue was cultured alone, most of the 35S-labeled material in the culture medium was keratan sulfate proteoglycan and free keratan sulfate chain, suggesting that keratan sulfate species are catabolized and eliminated more readily from collagen lamina than chondroitin sulfate/dermatan sulfate species, unless the corneal stroma is surrounded with other tissues. Whereas keratan sulfate was not detected in epithelium cultured alone, a small amount of it was found in endothelium cultured alone.
  • Kiyoshi Nakazawa, Shigeo Suzuki, Kaoru Wada, Kaori Nakazawa
    1995 年 117 巻 4 号 p. 707-718
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Proteoglycan biosynthesis in developing chicken corneas was studied. Corneas free of scleral rims were prepared from 5- to 18-day-old chick embryos and labeled in vitro with [3H] glucosamine and [35S] sulfate. Then, the labeled medium and corneal proteoglycans were analyzed. Day 5 corneas synthesized mainly chondroitin sulfate/dermatan sulfate proteoglycan and a sudden increase in keratan sulfate proteoglycan synthesis was found on Day 7. Keratan sulfate proteoglycan in Day 7 cornea appeared to be synthesized by fibroblasts which invaded the stroma between Day 5 and Day 7. The proportion of keratan sulfate proteoglycan synthesis increased with advancing embryonic age until Day 14 and declined a little on Day 18. The relative proportion of chondroitin sulfate/dermatan sulfate proteoglycan synthesis changed in a reverse curve during Day 7 to Day 18. Because embryonic cornea begins to become transparent on Day 14, this change in proteoglycan synthesis may be correlated with the onset of transparency. The labeled glycosaminoglycans were isolated from cultured embryonic corneas by pronase digestion, and then digested with chondroitinase ABC or keratanase II. Disaccharides in the enzymatic digests were analyzed by HPLC, and the extents and positions of sulfation of proteoglycans were determined. Sulfation of keratan sulfate proteoglycan continued to increase with advancing age until Day 18. Moreover, it has been shown by HPLC of unsaturated disaccharides of chondroitinase ABC digests that, whereas a comparable amount of non-sulfated “chondroitin sulfate/dermatan sulfate” was synthesized during Day 7 to Day 9, its amount declined during late development (Day 12 to Day 18). Such increases in sulfation of proteoglycans with advancing age may be correlated with progress of corneal transparency, which begins on Day 14 and continues until hatching. Analysis of proteoglycans of the culture medium has shown that most of the medium proteoglycans were keratan sulfate proteoglycan and free keratan sulfate chain (or keratan sulfate chain with a short peptide) during all ages after Day 7, although the major proteoglycan of Day 5 medium was chondroitin sulfate/dermatan sulfate proteoglycan. Sulfation of medium keratan sulfate also increased with advancing age. In addition, the proportion of free keratan sulfate chain in the medium increased during late corneal development, suggesting that catabolism of keratan sulfate proteoglycan might be preferentially accelerated during those times.
  • Minoru Tanaka, Sachiko Fukada, Michiya Matsuyama, Yoshitaka Nagahama
    1995 年 117 巻 4 号 p. 719-725
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    We isolated the structural gene encoding cytochrome P-450 aromatase (P-450arom), for the first time from a nonmammalian vertebrate, the medaka (a teleost fish, Oryzias latipes), using the rainbow trout P-450arom cDNA as a probe. The structure of the entire P-450arom gene, the nucleotide sequence of its 5' flanking region, and the transcriptional initiation sites were determined. The medaka P-450arom gene consists of nine exons, but spans only 2.6 kb, being much smaller than the human P-450arom gene (at least 70 kb), as a result of extremely small introns (medaka, 73-213 by vs. human, 1.3-10 kbp). The splicing junctions are located at exactly the same positions as those found in the human P-450arom. The deduced amino acid sequence is 51-52% identical to those of mammals and chicken, and 75% identical to the rainbow trout amino acid sequence. Genomic Southern blots revealed the presence of a single medaka gene. Promoter analyses indicated two major transcription initiation sites 60 and 61 bp upstream from a putative initiation codon. The promoter region of medaka P-450arom gene also contains potential Ad4BP sites and estrogen responsive element (ERE) half-sites. These results suggest that the basic structural organization of P-450arom genes and the regulatory mechanisms of expression are well conserved through-out the vertebrates.
  • Akihiko Kimura, Kazuya Yamaguchi, Haruhiko Ikeda, Seiji Yasuda, Sueo M ...
    1995 年 117 巻 4 号 p. 726-729
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    The expressions of non-muscle-type (MIIA) and brain/embryonic-type (MIIB2) myosin heavy-chain isoforms in regenerating rat liver were examined. In regenerating liver after partial hepatectomy, the level of MIIA was nearly constant, while that of MIIB2 increased transiently. The level of MIIB2 was very low in normal livers, and increased gradually and then declined with a peak at the 4 th day, when the regeneration was almost completed. The level of proliferating cell nuclear antigen was highest at the 2 nd day. Serine dehydratase activity in the liver decreased on partial hepatectomy, began to increase at the 5 th day, and reached 71% of the control at the 7 th day. These results suggest that MIIB2 plays a role in reconstruction or differentiation of the regenerating tissues rather than in proliferation of hepatocytes.
  • Atsuo Suzuki, Eiichiro Matsueda, Takashi Yamane, Tamaichi Ashida, Hiro ...
    1995 年 117 巻 4 号 p. 730-740
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    The crystal structure of dimeric phospholipase A2 (PLA2) from the venom of Habu snake, Trimeresurus flavoviridis, has been determined by the molecular replacement method, and has been refined at 1.5 Å resolution to an R-factor of 0.175. In the crystal, T. flavoviridis PLA2 forms a dimer using two 14 kDa subunits related by a pseudo 2-fold axis. Along the axis, the dimer has a narrow channel passing through it. Although no calcium ion is present in the calcium binding site, the peptide-chain folding of the subunits, the conformation of the catalytic residues, and the hydrogen-bonding network around the active sites are almost identical to those of the group I/II monomeric or dimeric PLA2s. The catalytic residues in both subunits are buried in the interior of the dimer and are inaccessible to substrate from the bulk solvent. In addition, the subunits of the dimer interact with each other at the hydrophobic region of the molecular surface where the entrance to the active site opens and where PLA2 is presumed to interact with the phospholipid of the substrate. Therefore, it is inferred that dimerization of T. flavoviridis PLA2 is the result of free-energy minimization by excluding the hydrophobic molecular surface from the aqueous solvent, rather than being required for the enzymatic function.
  • Tomoko Hosoi, Masashi Uchiyama, Eiichi Okumura, Taro Saito, Koichi Ish ...
    1995 年 117 巻 4 号 p. 741-749
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    The major kinase capable of phosphorylating tau in a porcine brain extract was suggested to be a brain cdc2-like kinase, called cdk5. Tau protein components of microtubules assembled in the brain extract using ATP were phosphorylated to a higher level, and showed a slower electrophoretic mobility than those assembled with GTP. Most of this phosphorylation and electrophoretic mobility shift, that occurred in the brain extract incubated with ATP, were inhibited by butyrolactone I, a specific inhibitor of cdc2 kinase and cdk5. Further, butyrolactone I inhibited phosphorylation of tau exogenously added to the brain extract by approximately 70%. cdk5 purified from porcine brain decreased the electrophoretic mobility of dephosphorylated tau by in vitro phosphorylation of tau to the level present in microtubules polymerized with ATP. cdc2 kinase purified from starfish oocytes also phosphorylated tau and shifted its electrophoretic mobility to an extent greater than that obtained with cdk5. Western blot analysis showed that cdc2 kinase phosphorylated epitopes recognized by SMI31, 33, 34, and tau1 antibodies in tau proteins, while cdk5 phosphorylated the site recognized by SM133 (corresponding to phosphorylation at Ser235 in the longest human tau isoform) and partially phosphorylated the tau1 site. Phosphorylation experiments performed on tau in brain extracts, in the presence of okadaic acid, suggested the presence of both okadaic acid-sensitive and -insensitive phosphatases acting on phosphorylated Ser235. Rat tau that was prepared immediately after decapitation showed a similar phosphorylation state to tau in microtubules polymerized with ATP, suggesting that tau is relatively phosphorylated in vivo.
  • Kikuo Ogata, Ayumi Kurahashi, Rie Ohno, Kumi Takahashi, Kazuo Terao
    1995 年 117 巻 4 号 p. 750-757
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Rat liver cytosol was incubated with a trace amount of rat liver 5SrRNA which was highly labeled at the 3'-end with cytidine 3', 5'-[5'-32P] bisphosphate, and with [35S] methionine in the presence of ATP mixture, and then with an antibody against ribosomal protein L5. The mixture was analyzed by protein A-Sepharose chromatography. The following results were obtained. (i) The eluate with glycine-HCl buffer (pH 3.0) from the protein A-Sepharose column contained an overlapping peak of 32P- and 35S-radioactivities. In a control experiment using the same amount of 32P-labeled Escherichia coli 5SrRNA with the same specific activity, no fraction of the eluate contained 32P-radioactivity. (ii) The fractions containing both 32P- and 35S-radioactivities from the protein A-Sepharose column were crosslinked by UV irradiation. The products was subjected to PAGE, and RNA in each gel slice was eluted and purified. The fraction containing both 32P- and 35S-radioactivities was present in a region of somewhat higher molecular weight than that of 5SRNP, whereas very low 32P- and 35S-radioactivities were present in this region in the control experiment without UV irradiation. This finding suggested that [35S] methionyl-tRNA interacted with 32P-labeled 5SRNP. (iii) The fraction containing overlapping 32P- and 35S-radioactivities described above was subjected to Sephadex G-150 chromatography. The component containing both radioactivities was distributed in the region corresponding to molecular weights of 10, 000 to 250, 000 with a peak at about 200, 000, suggesting the presence of a complex containing Met-RS (Mr 108, 000), 5SRNP (Mr 74, 000), and methionyl-tRNA (Mr 25, 000). Furthermore, this fraction showed definite Met-RS activity. These results indicate that 32P-5SRNP, met-tRNA, and Met-RS form a complex which may be released from the macromolecular ARS complex during the preparation procedures described above. Formation of this complex may be the mechanism by which 5SrRNA(P) enhances the activity of Met-RS in the macromolecular ARS complex.
  • Sonoko Kobayashi, Shinobu Imajoh-Ohmi, Futoshi Kuribayashi, Hiroyuki N ...
    1995 年 117 巻 4 号 p. 758-765
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    In B lymphocytes, but not T lymphocytes, isolated from human peripheral blood, we detected the four protein components essential for “the respiratory burst” by immunoblot analyses using peptide-directed antibodies. These are two membrane proteins, namely, 91-and 22-kDa subunits of cytochrome b558, and two cytosolic proteins with molecular masses of 47 and 65 kDa. Like in neutrophils, cytochrome b558 was expressed on the cell surface of peripheral B lymphocytes. Mean amounts (n=8) of the 91-, 22-, 47-, and 65-kDa proteins, respectively, in peripheral B lymphocytes calculated from intensity of the blots were 0.011±0.003, 0.026±0.006, 0.179±0.022, and 0.039±0.013 relative to those in neutrophils on the basis of cell number. Epstein-Barr virus (EBV)-transformed cell lines derived from normal B lymphocytes and some B cell lines also possessed cytochrome b558 and two cytosolic proteins. Isolated human peripheral B lymphocytes generated the superoxide anion upon cross-linking of surface antigens such as IgM, IgD, IgG, HLA-DR, and CD19. EBV-transformants derived from normal peripheral B lymphocytes and B lymphoid cell lines also generated the superoxide anion when stimulated with various antibodies against surface antigens. These results indicate that peripheral B lymphocytes have substantial amounts of a superoxide-generating system identical to that in phagocytes and that the system is stimulated to generate the superoxide anion by the cross-linking of clonally expressed surface immunoglobulins or of certain surface antigens.
  • Jin-ichi Inokuchi, Seigou Usuki, Masayuki Jimbo
    1995 年 117 巻 4 号 p. 766-773
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Previous studies have demonstrated that the ceramide analog D-Threo-1-phenyl-2-decanoylamino-3-morpholino-l-propanol (D-Threo-PDMP) inhibits glucosylceramide (GlcCer) synthase and thus leads to extensive depletion of glycosphingolipids (GSLs) biosynthesized from GlcCer [reviewed by Radin, N. S., Shayman, J. A., and Inokuchi, J. (1993) Adv. Lipid Res. 26, 183-213). In the present study, stereospecificity of PDMP activity was demonstrated with an enantiomeric pair, D-threo-PDMP and L-threo-PDMP. Treatment of B 16 melanoma cells with the D-threo or L-threo isomer produced contrasting changes of GSL biosynthesis, as monitored by metabolic labeling with [3H]Gal. D-PDMP markedly inhibited incorporation of radioactivity into GlcCer, LacCer, and GM3 as expected, whereas the L-threo isomer significantly increased it. Homologs of L-PDMP having different N-acyl chains were synthesized and also tested for their effects. Among them, the compounds having C8-C14 acyl chains increased incorporation of the radioactivity into GSLs to different degrees, demonstrating that the stimulatory effect of the L-threo homologs depends on acyl chain length. In order to elucidate the biochemical mechanisms of these PDMP effects, the activities of GlcCer synthase, LacCer synthase, and GM3 synthase in B 16 cell lysates were measured in the presence of PDMP. D-Threo-PDMP but not the L-threo isomer inhibited both LacCer and GM3 synthases as well as GleCer synthase, suggesting that the ceramide-like structure of the D-PDMP molecule interacted stereospecifically with these GSL-synthesizing enzymes. On the other hand, L-PDMP had no effect in the in vitro assays. However, LacCer synthase activity was found to be significantly elevated when the intact B 16 melanoma cells were preincubated with L-PDMP before measuring enzyme activities. Furthermore, the increase of LacCer synthase activity by L-PDMP was observed even when protein synthesis of the cells was blocked by cycloheximide, suggesting that L-PDMP affects post-translational modification of the enzyme. Thus, we were able to demonstrate distinctive and contrasting stereospecific actions of the two enantiomers on GSL biosynthesis.
  • Masahiro Uritani, Megumi Takai, Koichi Yoshinaga
    1995 年 117 巻 4 号 p. 774-779
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Desiccation by vacuum-drying inactivates the restriction endonuclease HindIII completely. However, when dried in the presence of a disaccharide such as trehalose, maltose, or sucrose, the endonuclease retains its λ DNA-cleaving activity and produces the same digestive fragments as does the intact enzyme. Thus, the disaccharides are effective in protecting the restriction enzyme in terms of both recognition and accurate cleavage of the substrate. Among the disaccharides, trehalose protects the enzyme most effectively; and it also stabilizes the enzyme during dilution in aqueous solution. The restriction enzyme dried with trehalose maintains its activity without detectable loss for at least 4 days at 37°C, but it shows reduced activity after 30-day storage at either 4°C or room temperature. Trehalose also protects other restriction endonucleases, EcoRI and BamHI, from inactivation during vacuum-drying, whereas drying them alone leads to severe loss of their activity. The restriction endonucleases dried with trehalose retain their activities for at least 20 days at 4°C and for 7 days at room temperature.
  • Masayoshi Itoh, Masahiko Nakamura, Tohru Suzuki, Keiichi Kawai, Hiroyu ...
    1995 年 117 巻 4 号 p. 780-786
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    To investigate the role of hydrogen peroxide in Cr(VI) toxicity in vivo toward bacterial cells, we examined the effect of Cr(VI), hydrogen peroxide, sodium azide, and mannitol on the viability of Escherichia coli. Bacterial cells were incubated for 1h with shaking in the presence of Cr(VI), hydrogen peroxide, sodium azide as catalase inhibitor, and/or mannitol as radical scavenger. The colony-forming ability and double-strand DNA degradation were examined. The viability assays revealed that Cr(VI) toxicity depended on hydroxyl radicals generated in the reaction involving hydrogen peroxide and chromium. Moreover, incubation of E. coli cells with 10mM Cr(VI) and 3mM hydrogen peroxide caused the degradation of double-strand DNA in vivo, which was suppressed by the addition of mannitol. These results indicated that hydroxyl radicals generated in the incubation degraded DNA of E. coli cells, resulting in cell death. In the absence of added hydrogen peroxide, the intracellular concentration of hydrogen peroxide in E. coli was low (below 1 μM). A catalase-defective strain incubated in the absence of added hydrogen peroxide remained fully viable after 1h but showed decreased viability after prolonged incubation (4-8h). The addition of mannitol suppressed this decrease, suggesting that hydroxyl radicals may be involved in the expression of Cr(VI) toxicity even without added hydrogen peroxide.
  • Akira Nagayoshi, Norio Matsuki, Hiroshi Saito, Kazuhisa Tsukamoto, Kaz ...
    1995 年 117 巻 4 号 p. 787-793
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    We previously showed that fatty liver was easily induced in suncus by starvation and that the plasma level of apolipoprotein B (apo B) was very low. There are three possible explanations for the low level of apo B in the animals: low synthetic rate, low secretion rate, and rapid catabolism in the circulation of apo B. We measured post-heparin lipolytic activity (lipoprotein lipase activity), which plays a key role in the catabolism of apo B-containing lipoprotein, VLDL, and found no difference between rats and suncus. We also investigated the hepatic synthetic rate of apo B by liver perfusion studies. Newly synthesized apo B in the suncus liver was detected by immunoprecipitation and found to amount to 12.5% of that in rats. The secretion rate of VLDL in suncus, which was estimated by intravenous injection of Triton WR1339, was 13.8% of that in rats. These two results suggest that there is no major defect in the secretory process. We separated Golgi apparatus from rat and suncus livers, and found much fewer lipoprotein particles in suncus than in rat Golgi apparatus. This evidence suggests that there is no defect in the lipolytic process or hepatic secretory process of apo B-containing lipoprotein, VLDL, but there may be a defect in the assembly process of VLDL and/or in the synthetic process of apo B in suncus. Such a defect may be one of the reasons for starvation-induced fatty liver in suncus.
  • Shoji Yamada, Shigeko Araki, Sachiko Abe, Kazuo Kon, Susumu Ando, Mei ...
    1995 年 117 巻 4 号 p. 794-799
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    A novel phosphonoglycosphingolipid which contains three residues of 2-aminoethylphos-phonate (2-AEP) was isolated from eggs of a sea gastropod, Aplysia kurodai, and its structure was identified as follows.
    _??_
    The major aliphatic components of ceramide were palmitic acid, stearic acid, 4-sphingenine, and 16-methyl-4-sphingenine. Antibodies which recognize 3-O-methylgalactose linked β-glycosidically to phosphonoglycosphingolipids failed to react to the egg glycolipid. By comparing 1H-NMR spectra of native and HF-treated glycolipids, steric interactions of two residues of 2-AEP with ring protons of the glucose and the internal galactose were indicated.
  • Yasuzo Nishina, Kyosuke Sato, Iwaho Hazekawa, Kiyoshi Shiga
    1995 年 117 巻 4 号 p. 800-808
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    A catalytic intermediate, the so-called “purple complex, ” of acyl-CoA dehydrogenase is produced on its reaction with the substrate, acyl-CoA. The purple complex is a charge-transfer complex between the reduced enzyme and the product, enoyl-CoA. Resonance Raman spectra of the purple complexes of three acyl-CoA dehydrogenases [short-chain acyl-CoA (SCAD), medium-chain acyl-CoA (MCAD), and isovaleryl-CoA (IVD) dehydrogenases] were measured with excitation at 632.8 nm within charge-transfer absorption bands. The 1, 577cm-1 band of the SCAD purple complex formed in the reaction with butyryl-CoA is mainly associated with the C(1)=O stretching of crotonyl-CoA, judging from the isotopic frequency shifts upon 13C or 18O substitution of butyryl-CoA. The 1, 627cm-1 band of the C(1)=O moiety of crotonyl-CoA in solution shifted downward by 50cm-1 on complexation with reduced SCAD. This large frequency shift indicates a substantial interaction between C(1)=O and the enzyme, and is further evidence for an appreciable contribution of a polarized form of the C(1)=O moiety in the enzyme-bound enoyl-CoA. This frequency shift can be explained by the hydrogen bond of C(1)=O. The 1, 577cm-1 band of the MCAD purple complex remained constant, regardless of the acyl carbon-chain length (from C 4 to C 16 of the substrate, acyl-CoA); the alkyl chain scarcely affected the interaction of the C(1)=O moiety in the active site. The frequency of the 3-methylcrotonyl-CoA carbonyl C(1)=O moiety shifted from 1, 626cm-1 in solution downward by 45cm-1 when the CoA thioester bound to reduced IVD, but by 28cm-1 when the thioester bound to reduced SCAD or MCAD. This indicates that the hydrogen bond at C(1)=O of 3-methylcrotonyl-CoA in SCAD or MCAD is weaker than in the case of IVD; the steric repulsion of the 3-methyl group probably changes the orientation of the -C(3)H=C(2)H-C(1)=O moiety and thus affects the hydrogen bonding. Tyr-375, which is conserved in straight chain acyl-CoA dehydrogenases, may be responsible for the steric repulsion and thus play a role in the substrate specificity.
  • Kazufumi Kuroda, Ryu Ueda
    1995 年 117 巻 4 号 p. 809-818
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    An immunocytochemical method using specific antibodies was employed to detect DNA polymerase α-primase complex in Drosophila melanogaster embryos during the first 13 nuclear division cycles. A monoclonal antibody specific to the 72 kDa polypeptide stained interphase nuclei, but not metaphase chromosome, while at late anaphase and thereafter staining of the chromosome was regained. On the other hand, a polyclonal antibody specific to the 180 kDa polypeptide stained not only the interphase nuclei but also the cytoplasmic regions surrounding interphase nuclei. These results suggest that the distributions of the 180 kDa and the 72 kDa polypeptides of DNA polymerase α-primase complex are different. We detected the 180 kDa and the 72 kDa polypeptides in the extract prepared from a single Drosophila embryo by Western blotting, and a 130 kDa polypeptide immunologically related to the 180 kDa polypeptide was also detected in the extract. These polypeptides (180, 130, and 72 kDa) in the embryos were detected at similar levels at interphase and at the mitotic phase. These three polypeptides were also detected in unfertilized eggs, showing that they were maternally stored. The 130 kDa polypeptide was detected till cycle 10, then began to decrease, and finally disappeared at cycle 14, whereas the 180 kDa and the 72 kDa polypeptides were present without marked fluctuation in quantity throughout the developmental stages. Even in unfertilized eggs, the level of the 130 kDa polypeptide decreased gradually with a similar time course to that in fertilized ones, but the levels of the 180 kDa and the 72 kDa polypeptides remained unchanged. This is the first report suggesting the existence of the 130 kDa polypeptide in vivo in the early embryos of Drosophila. The significance of the 130 kDa polypeptide is discussed.
  • Moon-Kyu Kang, Chan-Ki Kim, Tae-Neung Johng, Young-Ki Paik
    1995 年 117 巻 4 号 p. 819-823
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    The membrane bound sterol-8-isomerase (isomerase) catalyzes the anaerobic conversion of sterol-8-ene to the sterol-7-ene isomer in eucaryotes. To examine the regulatory mechanism as well as molecular characteristics of the isomerase we investigated the consequences of alteration of the enzymic activity under various diet conditions. Feeding 5% cholesterol or 0.1% AY-9944 for a minimum of 2 days caused more than a 70% decrease in microsomal isomerase activity. Feeding 5% cholestyramine plus 0.1% lovastatin (CL-diet) for 7 days led to approximately 4.0-fold induction of the isomerase activity. In addition, diurnal variation in the enzymic activity was observed with this diet. Induction of the isomerase activity by the CL-diet was quantitatively reflected in an increase in the cholesterol synthetic rate in isolated rat hepatocytes. The isomerase was highly purified from liver of rats fed the CL-diet, and its molecular mass was determined to be 21, 000 Da by denaturing sodium dodecylsulfate gel electrophoresis.
  • Yong He, Tomomi Shimogori, Keiko Kashiwagi, Akira Shirahata, Kazuei Ig ...
    1995 年 117 巻 4 号 p. 824-829
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    The inhibitory effect on cell growth of a combination of α-difluoromethylornithine (DFMO) and an inhibitor of aminopropyl transferase was examined. N-(3-Aminopropyl)cyclohexylamine (APCHA) and trans-4-methylcyclohexylamine (4MCHA) were used as inhibitors of spermine and spermidine synthases, respectively. Combination of DFMO and APCHA showed strong inhibitory effects on the growth of FM3A cultured cells and P388 leukemia cells in mice, compared with DFMO alone. The prolongation of survival time of P388 leukemia-bearing mice by DFMO (1, 500mg/kg) was 1.12-fold, while that by DFMO (1, 500mg/kg) plus APCHA (25mg/kg) was 1.30-fold. The prolongation of survival time nearly paralleled the decrease of P388 leukemia cells in mice. However, the antiproliferative effect of DFMO was not strengthened by 4MCHA in the above two experimental systems. In the FM3A cell culture system, both putrescine and spermidine contents were decreased by DFMO, but spermine content did not decrease significantly. When APCHA was added to the medium with DFMO, spermine content was decreased greatly but a compensatory increase in spermidine was observed. Spermidine content in P388 leukemia cells was also decreased by DFMO, but spermine content was increased. When APCHA was administered together with DFMO, the increase in spermine was suppressed but a compensatory increase in spermidine was observed. Nevertheless, the spermidine content remained significantly low compared with the value in non-treated P388 leukemia cells. Thus, the results indicate that the antiproliferative effect of DFMO was strengthened by APCHA due to the decrease in spermine content, and that the decrease in total amount of spermidine and spermine, especially the decrease in spermine, is necessary for inhibition of cell growth.
  • Hiroshi Narita, Emi Morishita
    1995 年 117 巻 4 号 p. 830-835
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    We produced five monoclonal antibodies (mAbs 1, 2, 6, 7, and 9) that are specific to pyrroloquinoline quinone (PQQ). PQQ-conjugated hemocyanin was used for the immunization of mice and the hybridomas were selected using PQQ-conjugated BSA in an enzyme-linked immunosorbent assay. MAbs 2 and 9 were of the IgG1 isotype. Both could recognize free PQQ, the former probably at the o-quinone and the latter at the opposite side of the molecule. They did not bind with trihydroxyphenylalanine, dihydroxyphenylalanine, 1, 2, 4-trihydroxybenzene, ascorbic acid, riboflavin, or menadione. In contrast to the IgGs, mAbs 1, 6, and 7 (IgMs) did not bind with free PQQ. Using mAb 2, a competitive enzyme-linked immunosorbent assay was developed, which enabled us to determine 50 nM-1 μM free PQQ. Furthermore, we analyzed the covalently bound prosthetic groups of two quinoproteins (amine oxidase from Aspergillus niger and amine dehydrogenase from Pseudomonas putida) by Western analysis using these mAbs. However, the result was negative, indicating that the prosthetic groups are not PQQ.
  • Toshiyuki Miyata, Akinobu Funatsu, Hisao Kato
    1995 年 117 巻 4 号 p. 836-844
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    In the previous study involving a yeast expression system, a high molecular mass extracellular domain of human tissue factor (denoted as sTFα) with a high content of mannose residues was produced in abundance and 37 kDa sTFβ was obtained in a low yield [Shigematsu et al. (1992) J. Biol. Chem. 267, 21329-21337]. To obtain sTFβ in a high yield, we constructed four kinds of mutant sTF with partial or total replacement of the N-potential glycosylation Asn residues with Ala, and expressed them in yeast. We found that the yield of the β form of the Asn137-to-Ala mutant (designated as sTFβNNA) was threefold higher (3mg/liter) than that of the wild type, suggesting that the replacement of one of the three potential N-glycosylation Asn residues with Ala could be a good way to minimize the addition of mannose repeats. Since it has been reported that calcium ions are required for the effective hydrolysis of peptidyl substrates by the factor VIIa-sTF complex, it is believed to be essential for the expression of full protease activity. Here, we report the enzymatic characterization of a factor VIIa-sTFβNNA complex cross-linked with a homobifunctional reagent, bis(sulfosuccinimidyl) suberate. The factor VIIa-sTFβNNA complex cross-linked in the presence of 5mM calcium ions or 50mM EDTA was purified. The cross-linked complex did not show factor X activation in the presence of phospholipids. However, it showed essentially the same activity toward peptidyl substrates as before cross-linking, even in the presence of EDTA. The kinetic constants of the cross-linked complexes in the presence of 5mM calcium and 50mM EDTA were similar, indicating that once factor VIIa was cross-linked with sTF, calcium ions were required no more for its activity. Therefore, calcium ions are not required for full catalytic activity of the factor VIIa-sTF complex toward synthetic substrates once the bi-molecular complex has formed.
  • Claudio Pedraza R., Shyuichiro Matsubara, Takashi Muramatsu
    1995 年 117 巻 4 号 p. 845-849
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Midkine is a growth/differentiation factor which is found as a product of a reresponsive gene. The 2.3-kb upstream sequence of the human MK gene has cis acting elements which confer retinoic acid-induced expression of fused chloramphenicol acetyl-transferase (CAT) gene in F 9 embryonal carcinoma cells. In the 5'-region of the human gene, a sequence resembling the DR5-type retinoic acid-responsive element (AGGTCA-related direct repeats separated by 5 nucleotides) was present in a small block of highly homologous 5'-sequences shared by the human and mouse genes. Deletion of this direct repeat reduced retinoic acid-induced CAT gene expression. The core element in the human gene differs from the consensus sequence of retinoic acid-responsive element in two nucleotides and from the retinoic acid responsive element of the mouse MK gene in one nucleotide.
  • Akio Takenaka, Osamu Matsumoto, Yixin Chen, Sei-ichi Hasegawa, Toshiyu ...
    1995 年 117 巻 4 号 p. 850-855
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Eleven kinds of hammerhead ribozymes were designed and synthesized to investigate the structural composition of the complexes and to find suitable crystallization conditions, the substrate chains having been modified to prevent hydrolysis. Electrophoresis patterns indicated that the two strands except for the substrate chain form a binary complex and that they form a ternary complex when mixed with the substrate chain. Both complexes were crystallized. The crystal of the binary complex belongs to a Laue symmetry of 32 (space group of P321, P3121, or P3221) with cell dimensions of a=b=53.4 and c=59.4Å. The volume per one nucleotide allows the asymmetric unit to contain one binary complex. Our results suggest that the catalytic part forms a rigid ribozyme structure which induces a scissile reaction when the substrate is bound, in a similar manner to an enzyme protein.
  • In Sook Matsui Lee, Yasuteru Muragaki, Takashi Ideguchi, Toshiharu Has ...
    1995 年 117 巻 4 号 p. 856-862
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Alanine-glyoxylate aminotransferase (AGT) 2 is a pyridoxal 5'-phosphate dependent, mitochondrial enzyme which, in the rat, is expressed at a high level in the kidney. The amino acid sequences of nine tryptic and seven CNBr peptides of the rat kidney AGT2 were determined. Three overlapping cDNAs encoding the AGT2 were cloned on the basis of its partial amino acid sequences by means of a polymerase chain reaction-based approach involving rat kidney poly(A)+ RNA. The complete cDNA sequence comprised 1, 919 bases, and contained a 1, 536-base open reading frame which encodes a polypeptide of 512 amino acid residues with a putative presequence consisting of 39 amino acid residues at the amino terminus, giving a precursor protein with a molecular mass of 57, 150 Da. The sequence of AGT2 exhibits significant homology with neither peroxisomal AGT1 from human liver nor mitochondrial AGT1 from rat liver. However, the sequence of AGT2 exhibited 30.8, 29.2, and 27.1% identity with those of Escherichia coli 4-aminobutyrate aminotransferase, rat ornithine aminotransferase, and Pseudomonas cepacia 2, 2-dialkylglycine decarboxylase, respectively. The active site sequences were also well conserved among these aminotransferases. AGT2, thus, is more similar to the other aminotransferases than to AGT1. The results suggest that the rat kidney AGT2 may play a biological role in amino acid metabolism distinct from that of AGT1.
  • Long-Sen Chang, Kou-Wha Kuo, Jordge Lin, Shinne-Ren Lin, Chun-Chang Ch ...
    1995 年 117 巻 4 号 p. 863-868
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    The antibodies against cobrotoxin were separated into two antibody preparations by successive affinity chromatographies on reduced and S-carboxymethylated (RCM) -cobrotoxin-Sepharose, and cobrotoxin-Sepharose columns. The antibodies (abbreviated as Abcf•i) that bound with the RCM-cobrotoxin-Sepharose were verified to specifically recognize the continuous epitopes of cobrotoxin, which were insensitive to conformational changes. Whilst the antibodies (abbreviated as Abcf•d) that did not bind with the RCM-cobrotoxin-Sepharose column recognized the conformational epitopes in cobrotoxin. The two antibody preparations were employed to screen the antigenic peptides derived from the proteolytic hydrolysate of cobrotoxin and RCM-cobrotoxin. Five antigenic peptides (AP-4, AP-5, AP-10, AP-11, and AP-12) were obtained from the acid protease A-digested hydrolysate of cobrotoxin, and two antigenic peptides (V8-2 and V8-4) were found in the hydrolysate of RCM-cobrotoxin after hydrolysis with Saccharomyces aureus V8 protease. The segments at positions 1-21 and 22-38 encompassed the peptide fractions, AP-4, AP-5, V8-2, and V8-4, that reacted with Abcf•i, indicating that the two segments bore the continuous epitopes of cobrotoxin. Alternatively, AP-10, AP-11, and AP-12 reacted with both Abcf•i and Abcf•d. The structures of the three peptides had a common segment at positions 43-62, suggesting that this region comprised the conformation-independent epitopes as well as conformational epitopes in cobrotoxin. These results reflected that the conformation-independent and conformational epitopes in a protein can be separately identified.
  • Tetsuya Takeda, Yasuhiro Kurasawa, Yoshio Watanabe, Osamu Numata
    1995 年 117 巻 4 号 p. 869-874
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Tetrahymena 14-nm filament protein (49K protein) is a bifunctional protein with roles in the cytoskeleton and as citrate synthase. Previous studies in our laboratory showed that elongation factor 1α (EF-1α) copurifies with the 49K protein upon polymerization and depolymerization of the 49K protein. In this study, the 49K protein was isolated from partially purified 49K protein fraction containing EF-1α. Using the purified 49K protein and/or purified EF-1α, the interaction between 49K protein and EF-1α in filament formation was investigated electronmicroscopically and it was demonstrated that purified 49K protein was capable of forming 14-nm filaments without EF-1α. The 49K protein/citrate synthase has been suggested to form filaments in mitochondria. Here we show that the citrate synthase activity of 49K protein is decreased by polymerization and increased by depolymerization, suggesting a possible modulating mechanism of citrate synthase activity by monomer-polymer conversion in mitochondria in situ.
  • Shigeru Taketani, Hirao Kohno, Takako Furukawa, Rikio Tokunaga
    1995 年 117 巻 4 号 p. 875-880
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    To investigate the involvement of peripheral-type benzodiazepine receptors (PBR) in heme metabolism, we examined the interaction of [55Fe]heme with PBR. Transfection of the cloned mouse PBR-isoquinoline carboxamide-binding protein (PBR/IBP) cDNA into monkey kidney Cos-1 cells resulted in a 2.5-fold increase in [55Fe]hemin binding sites, concomitant with the increase in [3H]PK11195 binding sites, as compared with those seen in antisense PBR/IBP eDNA-transfected cells. The binding of hemin to the transfected receptors exhibited a relatively high affinity with a Kd of 12 nM, and was inhibited by several benzodiazepine ligands, including PK11195, Ro 5-4864, diazepam and protoporphyrin IX. When mouse liver mitochondria were incubated with [55Fe] hemin, the binding to PBR had a Kd of 15±1.8 nM. The Bmax of [55Fe] hemin binding to the mitochondria was 6.88±0.76 pmol/mg of protein, a value consistent with that of [3H] PK11195 binding, with a lower affinity. Coproporphyrinogen III, a precursor porphyrin produced in the cytosol, is translocated into mitochondria, then is converted to protoporphyrinogen IX; this conversion decreased in the presence of benzodiazepine ligands. To examine whether this decrease was related to a decrease in the binding of coproporphyrinogen to the mitochondria, the effects of benzodiazepines on the binding of coproporphyrinogen were examined. As the binding was dose-dependently inhibited by PK11195, Ro 5-4864, and diazepam, porphyrins are likely to be endogenous ligands for PBR. We propose that PBR play a role in the intracellular transport of porphyrins and heme.
  • Taizo Suzuki, Masao Kawakita
    1995 年 117 巻 4 号 p. 881-887
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    The characteristics of the interaction between N-(3-pyrene)maleimide (PMI) and the sarcoplasmic reticulum (SR) membranes were investigated, and the sites of labeling with PMI on Ca2+-transporting ATPase were identified. PMI was dissolved in the membrane lipids before reacting with the ATPase protein. The measurement of resonance energy transfer from PMI to 1-(dimethylaminophenyl) -6-phenyl-1, 3, 5-hexatriene revealed that the pyrene moiety of PMI stayed in the lipid layer after it had been covalently attached to the ATPase molecule. PMI-labeled SR membranes at an average labeling density of 1 mol PMI/mol ATPase were digested with trypsin, and the labeled peptides were purified through a series of reversed-phase HPLC procedures on C18 and C4 columns. The amino acid analysis of the purified peptides revealed multiple cysteine residues mainly distributed over the C-terminal half of the cytoplasmic domain of the ATPase molecule as the targets of PMI. This implied that PMI molecules mediated cross-linking between the cytoplasmic domain of the ATPase molecule and the membranes. The distortion of the structure of the former due to this cross-linking may explain the uncoupling of ATP-splitting from Ca2+-transport caused by PMI.
  • Hiroki Yamamoto, Atsushi Naruse, Takeshi Ohsaki, Junichi Sekiguchi
    1995 年 117 巻 4 号 p. 888-896
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    The nucleotide sequence of a large rRNA gene and its flanking regions in cloned fragments of mitochondrial DNA from a patulin producer, Penicillium urticae NRRL2159A, was determined by dideoxy sequencing, and the 5' end and intron-exon border of the 1-rRNA gene were determined by primer extension analysis and RNA sequencing, respectively. In addition to the extensive sequence homology of the 3' end of the P. urticae mt 1-rRNA gene with those of Aspergillus nidulans and Neurospora crassa, the P. urticae gene had a 1, 685 by intron which separates a 3, 307 bp 5' exon and a 583 bp 3' exon. In spite of being closely related Penicillium species, the size of the 5' exon of the P. urticae mt 1-rRNA is 472 bp larger than that of P. chrysogenum, whereas the sizes of the 3' exon and intron of P. urticae are very similar to those of P. chrysogenum (581 bp for the 3' exon and 1, 678 bp for the intron). The intron of P. urticae contains a structure similar to the consensus one of the self splicing group IA intron and a large open reading frame suggested to be a gene for ribosomal protein S 5. A sequence similar to the I-SceI recognition sequence was found at the exonintron border. Extensive sequence homology was observed between P. urticae and P. chrysogenum, exceptions being in four regions in the 5' exon. These non-homologous regions were located in the hairpin and variable regions outside of the core structures. Comparison of the mt 1-rRNA sequences of several filamentous fungi revealed that the above four non-homologous regions are greatly expanded, and two other non-homologous regions appear at the 3' ends of the 5' exon and 3' exon.
  • Kenji Akasaki, Hiroko Yoshimoto, Akihiro Nakamura, Hirohito Shiomi, Hi ...
    1995 年 117 巻 4 号 p. 897-902
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    We purified a major kyotorphin (L-Tyr-L-Arg) -hydrolyzing peptidase (KTPase) from the rat brain, to electrophoretic homogeneity using conventional chromatographic techniques. KTPase was purified 1, 660-fold with a specific activity of 161 μmol/min/mg protein and 6.8% recovery. The purified enzyme was composed of a single polypeptide with a molecular mass of 67 kDa and an isoelectric point (pI) of 5.5. KTPase has the ability to hydrolyze a variety of natural dipeptides. It also liberated NH2-terminal tyrosine from Tyr-Gly-Gly and Tyr-Tyr-Leu. Bestatin and arphamenine B were potent inhibitors of this enzyme, while amastatin and puromycin had little effect. An excess of anti-KTPase antibody raised in a white rabbit precipitated approximately 80% of the kyotorphin-hydrolyzing activity in the cytosol of rat brain. These data suggested that 67 kDa KTPase has a role in the degradation of kyotorphin within neuronal cells of the rat brain.
  • Kazuo Inaba
    1995 年 117 巻 4 号 p. 903-907
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Conformational changes of the dynein β heavy chain/intermediate chain 1 (IC1) complex from outer arm dynein of sea urchin sperm flagella were examined by means of cross-linking experiments using a bifunctional cross-linker, dimethylsuberimidate. Cross-linking of the β/IC1 complex in the absence of ATP and vanadate (Vi) produced five cross-linked products. Immunoblotting of the products with anti-β chain and anti-IC1 antibodies revealed that all of them were cross-linked between β chain and IC1. Cross-linking of the complex in the presence of ATP and Vi produced four cross-linked products, but their electrophoretic mobilities were different from those of the cross-linked products obtained in the absence of ATP and Vi. Immunoblotting showed that only one cross-linked product was formed by cross-linking between β and IC1 and others were formed by intramolecular cross-linking of the β chain. Quantitative analysis indicated that cross-linking between β and IC1 decreased in the presence of ATP and Vi. These results suggest that conformational changes of the β heavy chain occur and the interaction between β chain and IC1 changes during ATP hydrolysis.
  • Gilles Mithieux, Ahmed Ajzannay, Carol Minassian
    1995 年 117 巻 4 号 p. 908-914
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    Three recent reports have suggested that rat liver microsomal glucose-6 phosphatase (Glc6Pase) should be a 62-64 kDa polypeptide. In this work, we examine the possibility that the 62-64 kDa could represent a functional dimeric form of the 36.5 kDa glucose-6 phosphate (Glc6P)-phosphohydrolase, previously identified [Countaway et al. (1988) J. Biol. Chem. 263, 2672-2678]. From 32P-labeling experiments with 32P-Glc6P and analysis of 32P-labeled protein by SDS-PAGE and autoradiography, we show that three different rat liver microsomal polypeptides, the apparent molecular masses of which are 62, 54, and 37 kDa, may be specifically labeled with 32P-Glc6P. We demonstrate that the 62 kDa polypeptide is a microsome-bound form of cytosolic phosphoglucomutase, by combining labeling competition experiments and enzymatic assay. It should likely not account for a putative dimeric form of Glc6P-phosphohydrolase. The 37 kDa polypeptide fulfills the criteria of Glc6P-phosphohydrolase. We have not obtained any definitive evidence for its assembly as a dimer under functioning conditions. The 32P-Glc6P-labeling characteristics of the 54 kDa polypeptide are those expected for a protein displaying affinity in the millimolar range of concentration and a high binding capacity for Glc6P. They are consistent with those of a 54 kDa microsomal polypeptide, previously suggested to be involved in Glc6Pase activity.
  • Dae-Woong Jo, Trond P. Leren, Zung-Yoon Yang, Young-Ho Chung, John M. ...
    1995 年 117 巻 4 号 p. 915-922
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    We have previously demonstrated that expression of the human apolipoprotein (apo) E gene is controlled by multiple regulatory elements in the promoter [Paik et al. (1988) J. Biol. Chem. 263, 13340-13349; Chang et al. (1990) J. Biol. Chem. 265, 9496-9504]. To extend these studies, we have characterized an element in the apoE gene promoter that spans nucleotides -101 to -89, upstream regulatory element 3 (URE3). Transcription of promoter/marker gene constructs in vitro showed that URE3 modulates gene expression. Gel mobolity shift assays of URE3 using human placental nuclear extracts detected a specific binding protein whose activity can be modulated by micromolar amounts of divalent copper and zinc. Competitive binding and gel shift assays with mutant oligonucleotides revealed critical nucleotides within URE3 required for its specific nuclear protein-binding activity. Gel filtration and oligonucleotide affinity chromatography were employed to isolate a URE3-binding protein (URE3BP) from human placental nuclear extracts. Purified URE3BP appears to be a Mr=300, 000 protein that is composed of four equally-sized basic subunits of Mr=67, 000. These studies indicate that URE3 is an active regulatory component of the apoE gene.
  • Yumiko Komori, Jinjoo Hyun, Ken Chiang, Jon M. Fukuto
    1995 年 117 巻 4 号 p. 923-927
    発行日: 1995/04/01
    公開日: 2008/11/18
    ジャーナル フリー
    The role of thiols on the activation and/or stabilization of rat brain nitric oxide synthase (NOS) has been investigated. It was found that thiols are not necessary for stabilizing or protecting the protein during purification but are required during enzyme turnover for maximum activity. In the complete absence of thiols but with added tetrahydrobiopterin, the enzyme retained a low basal activity. Thiol addition to a thiol-deplete preparation of the enzyme resulted in a 4 to 7-fold increase in activity when measured after 15min. High concentrations of dihydropteridine reductase also caused an apparent activation of NOS and was capable of replacing thiols. The data presented is consistent with a cofactor role for thiols. The possibility that they serve as reducing agents for the regeneration of tetrahydrobiopterin from dihydrobiopterins is discussed.
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