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Volume 126 (1999)
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- Issue 2 Pages 261-
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Volume 126, Issue 2
Displaying 1-30 of 30 articles from this issue
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Haruki Yamaguchi, Tomoko Nishiyama, Makoto Uchida1999 Volume 126 Issue 2 Pages 261-265
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis study advances direct evidence of the binding affinity of N-glycans for aromatic amino acid residues. The intrinsic fluorescence intensities of bovine pancreatic RNase A, bovine α-lactalbumin, and aromatic amino acids were markedly depressed in solutions (1mM or so) of free N-glycans of both the high-mannose and complex types. In addition, free N-glycans inhibited the chemical modifications of the solvent-exposed tyrosine and tryptophan residues of these proteins, confirming the affinity of N-glycans for aromatic amino acid residues. The results are discussed in connection with the roles of N-glycans in novel interactions between N-glycans and proteins.View full abstractDownload PDF (562K) -
Motoi Kanagawa, Shunji Kaya, Hideki Umezu, Shunsuke Watanabe, Katsuhik ...1999 Volume 126 Issue 2 Pages 266-270
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn vivo reversible phosphorylation of Tyr-7 and Tyr-10 of the pig stomach H/K-ATPase α-chain was initially demonstrated in mammals, rat, rabbit, and pig, in the presence of vanadate+H2O2. In vitro phosphorylation has also been unequivocally demonstrated via the use of protease inhibitors during membrane H/K-ATPase preparation. An amphoretic detergent permitted each intrinsic kinase to phosphorylate each fusion protein containing the requisite Tyr residues, along with a reduction in α-chain phosphorylation. These and other data suggest that some important enzyme systems are present in the apical membrane and that they are in sufficient proximity to participate in the reversible phosphorylation of the amino terminal soluble domain of the α-chain with an unknown physiological function in the membrane embedded H/K-ATPase.View full abstractDownload PDF (4953K) -
Shogo Ozawa, Makiko Shimizu, Takahiko Katoh, Atsuko Miyajima, Yasuo Oh ...1999 Volume 126 Issue 2 Pages 271-277
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe recently found single amino acid substitutions (213Arg/His and 223Met/Val) in polymorphic human phenol-sulfating phenol sulfotransferase (SULT: cDNAs encoding ST1A3, P PST or HAST1/2) among Caucasians and African-Americans. In a Japanese population (n=143), allele frequencies of 213Arg and 213His were 83.2 and 16.8%, respectively, but the 223Val allele was not found. 213His homozygosity was reportedly associated with both very low (>7-fold) sulfating activities of p-nitrophenol (at 4μM) and low thermostability in platelets. Sulfating-activity determinations using recombinant 213Arg- and 213His-forms (ST1A3*1 and ST1A3*2, respectively) did not, however, reveal appreciable deficiency in [35S] 3'-phosphoadenosine 5'-phosphosulfate (PADS)-dependent sulfation of p-nitrophenol (4μM) by ST1A3*2 (7.5vs. 10.2 nmol/min/nmol SULT for ST1A3). Kinetic parameters for p-nitrophenol for p-nitrophenol sulfation supported the slight decrease in sulfating activities at 4μM (Km, 0.82xs. 1.75μM; Vmax, 13.2vs. 13.1 nmol/min/nmol SULT, respec-tively, for ST1A3*1 and *2). p-Nitrophenyl sulfate-dependent 2-naphthol sulfation by ST1A3*2 was 69% of that by ST1A3*1 (p<0.05). However, ST1A3*2 was remarkably unstable at 45 and 37°C as compared to ST1A3*1. The lower p-nitrophenol sulfating activity of ST1A3*2 may explain the lower platelet p-nitrophenol sulfation in ST1A3*2 homozygotes. Protein instability and ST1A3 gene regulation may be both involved in the polymorphism of p-nitrophenol sulfation in human tissues.View full abstractDownload PDF (3860K) -
Nobuko Kawazoe, Toshihiro Aiuchi, Yutaka Masuda, Shigeo Nakajo, Kazuya ...1999 Volume 126 Issue 2 Pages 278-286
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an attempt to characterize the mechanisms that are operative at the early stages of the induction of apoptosis by bufalin, a component of the traditional Chinese medicine chan'su, we examined the effects of bufalin on plasma membrane potential, as determined by monitoring the uptake by cells of rhodamine 123. Bufalin induced apoptosis in human monocytic leukemia THP-1 cells, in human lymphoblastic leukemia MOLT-3 cells, and in human colon adenocarcinoma COLO320DM cells but not in normal human leukocytes, for example, polymorphonuclear cells and lymphocytes, and not in murine leukemia P388D1 and M1 cells. Treatment for 3h with bufalin at 10-6M caused a decrease in the plasma membrane potential in several lines of human tumor cells but not in murine leukemia cells. No changes in mitochondrial membrane potential, as monitored with the fluorescent dye JC-1, and no release of cytochrome c were observed within at least 6h after the start of treatment with bufalin. Moreover, overexpression of bel-2 in human leukemia HL60 cells that had been transfected with cDNA for bcl-2 prevented bufalin-induced apoptosis but had no significant effect on the change in plasma membrane potential induced by bufalin. Since bufalin specifically inhibits the Na+, K+-ATPase of human but not murine tumor cells, and since this inhibition leads to a change in intracellular concentration of Na+ ions, our findings suggest that bufalin induces apoptosis in human tumor cells selectively via inhibition of the Na+, K+-ATPase, which acts upstream of the bcl-2 protein.View full abstractDownload PDF (4471K) -
Norifumi Konishi, Yasuhiro Torii, Tatsuo Yamamoto, Atsushi Miyagi, Hir ...1999 Volume 126 Issue 2 Pages 287-295
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlucosyltransferase-I (GTF-I: 175kDa) of a cariogenic bacterium, Streptococcus sobrinus 6715, mediates the conversion of water-soluble dextran (α-1, 6-glucan) into a water-insoluble form by making numerous α-1, 3-glucan branches along the dextran chains with sucrose as the glucosyl donor. The structures and catalytic properties were compared for two GTF-I fragments, GTF-I' (138kDa) and GS (110kDa). Both lack the N-terminal 84 residues of GTF-I. While GTF-I' still contains four of the six C-terminal repeats characteristic of streptococcal glucosyltransferases, GS lacks all of them. Electron microscopy of negatively stained samples indicated a double-domain structure for GTF-I', consisting of a spherical head with a smaller spherical tail, which was occasionally seen as a long extension. GS was seen just as the head portion of GTF-I'. In the absence of dextran, both fragments simply hydrolyzed sucrose with similar Km and kcat values at low concentrations (<5mM). At higher sucrose concentrations (>10mM), however, GTF-I' exhibited glucosyl transfer activity to form insoluble a-1, 3-glucans. So did GS, but less efficiently. Dextran increased the rate and efficiency of the glucosyl transfer by GTF-I'. On removal of the C-terminal repeats of GTF-I' by mild trypsin treatment, this dextran-stimulated transfer was completely lost and the dextran-independent transfer became less efficient. These results indicate that the N-terminal two-thirds of the GTF-I sequence are organized as a structurally and functionally independent domain to catalyze not only sucrose hydrolysis but also glucosyl transfer to form α-1, 3-glucan chains, although not efficiently; the C-terminal repeat increases the efficiency of the intrinsic glucosyl transfer by the N-terminal domain as well as rendering the whole molecule primer-dependent for far more efficient insoluble glucan synthesis.View full abstractDownload PDF (3413K) -
Minao Asano, Shunichi Suzuki, Misako Kawai, Tetsuya Miwa, Hiroshiro Sh ...1999 Volume 126 Issue 2 Pages 296-301
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe enzymatic properties of novel cysteine proteases D 3-α and β which were purified from germinating soybean cotyledons were investigated. The enzyme activities were exhibited in the presence of a thiol reagent, such as 2-mercaptoethanol, and apparently inhibited by E-64, a cysteine protease inhibitor. Hydrolytic activities toward carbobenzoxy-Phe-Arg-MCA were detected at a pH above 4.0. The optimum temperature for activities was about 40°C. The isoelectric point of D 3-α and β was 4.4 and 4.7, respectively. The molecular mass of D 3-α and β, measured by MALDI/TOF mass spectrometry, was 26, 178 and 26, 429 Da, respectively. The substrate specificities of the enzymes were examined using peptide-MCAs and peptides, and cathepsin L-like broad specificity was observed at pH 4.0. These results demonstrated that these enzymes are cysteine endopeptidases [EC 3. 4. 22.-] like papain [EC 3. 4. 22. 2].View full abstractDownload PDF (1733K) -
Jing-Ze Lu, Fen Huang, Jian-Wen Chen1999 Volume 126 Issue 2 Pages 302-306
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe investigated the behavior of a membrane protein, Ca2+-ATPase, in interdigitated phospholipid bilayers. The results showed that Ca2+-ATPase does not cause significant alterations in the interdigitation of 16:0 LPC/DPPC (27.0 mol% LPC) vesicles when it is reconstituted with lipids. Intrinsic fluorescence, acrylodan fluorescent adducts, and CD spectra indicated that Ca2+-ATPase, when embedded in interdigitated bilayer structures, is more exposed to the hydrophilic environment and has a looser structure than when embedded in non-interdigitated bilayers. The interdigitation of acyl chains induces a rapid loss of enzyme activity. It is suggested that interdigitated bilayer structures may play an important role as negative regulatory factors in physiological functions.View full abstractDownload PDF (622K) -
Sabu Kasai1999 Volume 126 Issue 2 Pages 307-312
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn previous studies involving Photobacterium species we proposed that (i) P-flavin is the product of luciferase, (ii) the physiological function of the lux operon is not to produce light but to produce FP390 (luxF protein), including its prosthetic group, P-flavin, and (iii) FP390 reactivates oxidatively inactivated cobalamin-dependent methionine synthase similar to flavodoxin but at relatively high ionic strength. It seems difficult to extend this idea to all luminous bacteria because the luxF gene is not present in the lux operon in Vibrio or Xenorhabdus. But we predicted that a luciferase fragment which binds P-flavin should function like FP390 in these species. In this study, we isolated P-flavin binding protein from Vibrio fischeri ATCC 7744. The obtained protein was a modified luciferase as expected, in which the β-subunit was intact but about 25 amino acid residues at the C-terminus of the α-subunit were deleted and the prosthetic group was the fully reduced P-flavin. These results strongly support that the physiological function of the lux operon is as described above even in luminous bacteria other than Photobacterium species. We propose that chromophore B reported by Tu and Hastings [Tu, S. -C. and Hastings, J. W. (1975) Biochemistry 14, 1975-1980] is the reduced P-flavin.View full abstractDownload PDF (1658K) -
Ken Kitano, Youichi Niimura, Yoshitaka Nishiyama, Kunio Miki1999 Volume 126 Issue 2 Pages 313-319
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAhpC protein, purified from Amphibacillus xylanus with a molecular mass of 20.8kDa, protects cells against oxidation damage. The enzyme catalyses the reduction of hydroperoxides in cooperation with the 55kDa flavoprotein, A. xylanus NADH oxidase (NADH oxidase-AhpC system). A. xylanus AhpC has two disulfide linkages between monomers and can act in the homodimer form. Gel-filtration column chromatography and dynamic light scattering (DLS) suggest that A. xylanus AhpC also forms a large oligomeric assembly (10-12 mers). A. xylanus AhpC was crystallized and X-ray diffraction data were collected to 3.0 Å. The self-rotation function revealed fivefold and twofold axes located perpendicu-larly to each other, suggesting that the molecular assembly of A. xylanus AhpC is composed of ten monomers. The oligomerization of A. xylanus AhpC is affected by ionic strength in the DLS measurements. The H2O2 reductase activity of the A. xylanus NADH oxidase-AhpC system is also affected by ionic strength, and it was found that the decamerization of AhpC might be required for the activation of the NADH oxidase-AhpC system.View full abstractDownload PDF (1835K) -
Sun Jung Kim, Bo Hwa Sohn, Sangkyun Jeone, Kyung-Won Pak, Jung-Sun Par ...1999 Volume 126 Issue 2 Pages 320-325
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn our previous study, transgenic mice were generated that expressed human lactoferrin (hLF) in milk using cDNA under control of the 2 kb bovine β-casein promoter. The expression level of the protein in milk of 7 mice ranged from 1 to 200μg/ml; 1 to 34μg/ml in 6 mice and 200μg/ml in 1 mouse. With the aim of inducing higher expression of the protein, we constructed an expression cassette comprised of 10 kb of the bovine β-casein gene promoter and the hLF genomic sequence in place of the cDNA. The hLF genomic sequence of about 27 kb, spanning 23 kb of the entire coding region and 4 kb of the 3'-flanking sequence, was placed downstream the bovine β-casein promoter. In total, 8 transgenic mice were generated from 31 mice (transgenic rate of 25.8%) born from the embryos microinjected with the 40-kb hLF expression cassette. Mammary-specific expression of the transgene was addressed by performing Northern hybridization of the total RNAs from various tissues of transgenic mice. Immunoblot analysis showed that the recombinant protein expressed in milk has the same molecular weight as the native protein. The amount of the protein in milk of 5 mice ranged from 60 to 6, 600μg/ml when judged by ELISA analysis. Three mice expressed the protein at the level higher than 500μg/ml. These data suggest that the genomic lactoferrin sequence represents a valuable element for the efficient expression of the protein in milk of transgenic animals.View full abstractDownload PDF (2179K) -
Eliza Wyszko, Jens-Peter Fuerste, M. Barciszewska, M. Szymanski, R. Ad ...1999 Volume 126 Issue 2 Pages 326-332
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo hammerhead ribozymes derived from plant pathogenic RNAs were used to cut off the HIV TAR RNA from the T 7 RNA transcript through a cis cleavage reaction. Stem I of the (+) vLTSV ribozyme comprises 8 nucleotides of the 5' terminus of TAR RNA, but stem III of the (+) sTRSV ribozyme consists of 8 nucleotides of the 3' end of TAR RNA. The construct containing two GUC hammerhead ribozyme target sequences identified the cleavage sites to cut off a required RNA molecule. This method was applied for preparation of 35 nt long TAR RNA. Its activity was proved by the complex formation with the Tat protein. It seems that this approach based on RNA scissors can also be used for the generation of required RNA molecules, RNA decoys or RNA aptamers in vivo.View full abstractDownload PDF (1966K) -
Byung-Sik Shin, Soo-Keun Choi, Seung-Hwan Park1999 Volume 126 Issue 2 Pages 333-339
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe enzyme, phosphotransacetylase (Pta), catalyzes the conversion of acetyl coenzyme A to acetyl phosphate. The putative pta gene of Bacillus subtilis, which had been sequenced as part of the Genome Project, was cloned and overexpressed in Escherichia coli. We confirmed that the gene encodes Pta by measuring the enzymatic activity of the purified protein. Insertional mutagenesis of the pta gene resulted in complete loss of the Pta activity, indicating that B. subtilis contains only one kind of pta gene. Expression of a pta-lacZ fusion was induced in the presence of excess glucose in the growth medium, and the intact ccpA gene was required for this activation. The transcriptional start site of the pta gene was located at 37 nucleotides upstream of the pta start codon, and a cre (catabolite responsive element) sequence, a cis-acting element that is responsible for the catabolite repression of a number of carbon utilization genes in B. subtilis, was identified upstream of the tentative promoter site. Experiments involving oligonucleotide-directed mutagenesis showed that the cre sequence is involved in glucose-mediated transcriptional activation.View full abstractDownload PDF (2227K) -
Hirokazu Kimura, Isao Sekiguchi, Akira Shoji, Hisanori Minakami, Hisan ...1999 Volume 126 Issue 2 Pages 340-346
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlthough phorbol 12-myristate 13-acetate (PMA) inhibits apoptosis and promotes the growth of some types of cells, it induces apoptosis in other cells. We evaluated the apoptotic effects of PMA on murine fibroblasts (L-929) that had been exposed to ultraviolet-B (UV-B) radiation at 312 nm, which promotes tumor cell growth. Exposure to PMA alone did not induce Fas, Fas-L, or apoptosis. Cells exposed to mild UV-B irradiation (80 J/m2) alone exhibited a slight expression of Fas and Fas-L 36 to 48h after the exposure, and exhibited apoptosis as evidenced by DNA fragmentation 72h after exposure. The addition of PMA (0.8×10-5 to 3.2×10-5M) to the medium 24h after the UV-B exposure markedly and dose-dependently enhanced these cell responses. Confluent untreated cells, cells cocultured with PMA, and cells cocultured with PMA for 24h after the UV-B exposure consistently expressed mRNAs for wild-type p53, bcl-2, and ICE. Expression of c-myc mRNA was initially observed, but became undetectable in the cells cocultured for 24h with a high concentration of PMA (3.2×10-5M) following UV-B exposure. Such cells subsequently exhibited the maximal apoptotic response. We conclude that mild exposure to UV-B altered murine fibroblast cells in such a way as to facilitate their death by apoptosis upon addition of PMA.View full abstractDownload PDF (3394K) -
Yukihito Mizutani, Tatsuya Narikawa, Takanori Satoh, Nobuhiko Sakurai, ...1999 Volume 126 Issue 2 Pages 347-353
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS4-Aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline. For use as a clinical reagent, the gene encoding 4 ABH from Agaricus bisporus was cloned by the RACE method. Also, the cDNA encoding 4 ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST). The expressed GST-4 ABH fusion protein (recombinant 4 ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities. We investigated a new ultraviolet spectrometric method for determining serum γ-glutamyltransferase (γ-GT) using recombinant 4 ABH as a coupling enzyme. The principle of the method is as follows. Using γ-glutamyl-3-choloro-4-aminobenzoate (L-γ-glu-PACIBA) and glycylglycine as the donor and acceptor sub-strates, 3-choloro-4-aminobenzoate (PACIBA) is formed by the catalysis of serum γ-GT. PACIBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD+ by 4 ABH and NADH. However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent. Using recombinant 4 ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity. The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum γ-GT activity using L-γ-glu-PACIBA as the donor substrate and recombinant 4 ABH as a coupling enzyme.View full abstractDownload PDF (981K) -
Shin-ichiro Takeda, Akinori Matsushika, Takeshi Mizuno1999 Volume 126 Issue 2 Pages 354-360
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Escherichia coli sdhCDAB operon encodes suecinate dehydrogenase, an enzyme complex involved in the tricarboxylic acid (TCA) cycle. Expression of this operon is under complex transcriptional regulation in response to growth conditions, such as anaerobiosis and carbon sources. Typically, the expression of sdhCDAB is known to be subjected to “an aerobic repression” and “a glucose repression.” The molecular mechanism underlying the anaerobic repression has been well documented, involving both the ArcB-ArcA two-com-ponent system and the Fnr global anaerobic regulator. However, the mechanism underlying the glucose repression is not yet clear, because the involvment of the general catabolite regulators such as CRP and CRA has been dismissed. In this study, we conducted a series of genetic analyses to identify the regulator gene (s) involved in the glucose repression of sdh. The results demonstrate that the EIICBG1c protein (the ptsG gene product), a component of the major glucose transporter, acts as a crucial mediator in glucose repression. These results support the view that the EIICBG1c protein functions not only as a glucose transporter, but also as a glucose-sensing signal transducer that modulates the glucose repression of the sdhCDAB operon.View full abstractDownload PDF (2161K) -
Katsura Nozawa, Yasuhiro Kurumiya, Akiko Yamamoto, Yuka Isobe, Motoshi ...1999 Volume 126 Issue 2 Pages 361-367
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTelomerase is a unique reverse transcriptase involved in the maintenance of telomeric DNA, which is generally undetectable in normal human somatic cells. However, it has been found in organs of normal adult rodents including the liver. In order to elucidate relevant control mechanisms operating in normal somatic cells, we examined telomerase activity in primary cultured rat hepatocytes. During culture under serum-free conditions, rat hepato-cytes rapidly lose the ability of organ-specific expression of serum albumin, apolipoprotein A-I, and hepatocyte nuclear factor 4, and the capacity for cytochrome P-450 induction by xenobiotics. The telomerase activity was found to be concomitantly increased about 2.5-fold at 48h and 3-fold at 72h. Northern blot and RT-PCR analyses with primary cultured hepatocytes revealed the associated accumulation of rat telomerase RNA subunits (TR), and the mRNAs for a telomerase reverse transcriptase (TERT) and a telomerase-associated protein (TEP 1). The activity of hepatocyte telomerase, which was elevated during the primary culture, increased further when the cells were stimulated with hepatocyte growth factor. In this case, however, the levels of TR, TERT, and TEP 1 mRNA did not show any detectable changes.View full abstractDownload PDF (4544K) -
Hiroyu Hiraishi, Fumiaki Yokoi, Akira Kumon1999 Volume 126 Issue 2 Pages 368-374
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 13-kDa phosphoamidase was isolated as a single band on SDS-PAGE from bovine liver. Its Stokes' radius, sedimentation coefficient, molecular mass, and optimal pH were estimated to be 1.6 nm, 1.8s, 13kDa, and 6.5, respectively. The enzyme released Pi from 3-phosphohistidine, 6-phospholysine, and amidophosphate at rates of 0.9, 0.6, and 2.6μmol/min/mg protein, respectively. However, it did not dephosphorylate phosphocreatine, Nψ-phosphoarginine, imidodiphosphate, or O-phosphorylated compounds including inorganic pyrophosphate. It also dephosphorylated succinic thiokinase and nucleoside diphosphate kinase autophosphorylated at His residues, indicating that it works as a protein histidine phosphatase. A thiol reagent, 30μM N-ethylmaleimide, depressed the activity by half, while a thiol compound, 2-mercaptoethanol, protected the enzyme from heat-inactivation. Five millimolar divalent cations, such as Mg2+ and Mn2+, and 5mM EDTA, had no effect on the activity.View full abstractDownload PDF (4738K) -
Yukinobu Goso, Kazuhiko Ishihara, Makoto Kurihara, Tsukiko Sugaya, Kyo ...1999 Volume 126 Issue 2 Pages 375-381
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhole mucins and reduced subunits were extracted from the corpus of the rat stomach. After purification by Sepharose CL-4 B chromatography followed by cesium trifluoroace-tate equilibrium centrifugation, they were analyzed by Sepharose CL-2 B chromatography, rate-zonal sedimentation centrifugation, and Q-Sepharose chromatography. Monoclonal antibodies RGM 21 and HIK 1083, which histochemically stained mucins in the surface and glandular mucosa of the rat stomach, respectively, were used to detect the site-specific mucins. Although RGM 21- and HIK 1083-reactive mucins both had a multimerized structure, the density and size of both the whole mucins and reduced subunits differed, thus indicating the presence of distinct mucin species in the surface and glandular mucosa. The mucin subunits were separated into four fractions, UB, B 1, B2a, and B2b, by Q-Sepharose chromatography. HIK 1083 reacted mainly with UB, while RGM 21 reacted with B 1, B2a, and B2b. These results, combined with dot-blot, amino acid, and carbohydrate composition analyses, showed that the surface mucins may consist of three kinds of subunits. In contrast, the glandular mucins may consist of one kind of subunit which differs from that of surface mucins.View full abstractDownload PDF (834K) -
Akiyoshi Tanaka, Kozue Okuda, Keishi Senoo, Hitoshi Obata, Kuniyo Inou ...1999 Volume 126 Issue 2 Pages 382-386
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe guanidine hydrochloride-induced denaturation of Pseudomonas cepacia lipase (PCL) was studied at pH 7 by monitoring the changes in the fluorescence and circular dichroism of the enzyme. The denaturation was irreversible as a whole, and the addition of Ca2+ ions decreased the velocity of the denaturation. The denaturation process was well explained consistently by a two-step mechanism, as follows:
where N is the native state of PCL, D1 an intermediate denatured-state which can be refolded into the native state, and DF the final denatured-state that can not be renatured. Ethanol (10%) increased the denaturation velocity by decreasing the refolding step, DI+Ca2+→N•Ca2+, which would be caused by the stabilization of DI by ethanol.View full abstractDownload PDF (542K) -
Kazunori Miura, Satoru Ohgiya, Tamotsu Hoshino, Nobuaki Nemoto, Masato ...1999 Volume 126 Issue 2 Pages 387-394
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRD3, a new antifreeze protein (AFP) extracted from antarctic eel pout is a single polypeptide divided into homologous N-terminal (residues Asn1-Glu64) and C-terminal (residues Ser74-Glu134) domains, each of which has a high sequence identity with Type III AFP. A 9-residue linker (-D65GTTSPGLK73-) connects these two domains in tandem and is thought to play a significant role in defining the nature of the intact molecule. The present paper shows for the first time the solution structure and preliminary 15N-NMR backbone dynamics data of the N-domain plus the linker of recombinant RD3 protein (RD3-Nl: residues 1-73) by employing homo- and heteronuclear multidimensional NMR spectroscopy. Forty converged structures of RD3-N1 were successfully calculated by using a total of 958 NMR-derived structural restraints. It was found that the N-domain of RD3-N1 has a globular form comprising six β-strands, three type III turns, and several loops, which stabilize a flat, ice-binding site formed on one side of this domain. Further, the linker portion appears to have a definitive structure, which is independent of the globular N-domain. This definitive linker is roughly divided into two short strands, -D65GTTSP70-and-G71LK73-, which are bent around -T67TSPG71-at an angle of approximately 60°. This bending motif of the linker may function to orient the two ice-binding sites of the N-and C-domains of RD3 in the same direction, leading to their simultaneous interactions with the ice crystal surface.View full abstractDownload PDF (1856K) -
Shuichi Akaba, Mitsunori Seo, Naoshi Dohmae, Koji Takio, Hiroyuki Seki ...1999 Volume 126 Issue 2 Pages 395-401
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPolyclonal antibodies were raised against synthetic peptides or recombinant polypeptides encoded by Arabidopsis atA0-1 and atAO-2 cDNAs, which have sequences similar to maize and animal aldehyde oxidase (AO) cDNAs. Anti-atAO-1 antibodies recognized AOα and AOβ among the three isoforms, AOα, AOβ, and AOγ, detected in Arabidopsis seedlings after native PAGE, while anti-atAO-2 antibodies reacted with AOβ and AOγ. The polypeptide specifically recognized by each antibody was collected as the Protein-A/IgG/antigen complex. The 150- and 145-kDa polypeptides were purified by SDS-PAGE and digested with Achromobacter Protease I. From the amino acid sequences and molecular masses of the derivative peptides, it was revealed that the 150- and 145-kDa polypeptides were the products of atAO-1 and atAO-2, respectively. Molecular masses of the native forms of AOα, AOβ, and AOγ were estimated as approximately 290-300 kDa. These results suggest that AOα and AOγ are homodimers consisting of atAO-1 and atAO-2 products, respectively, and that AOβ is a heterodirner of the atAO-1 and atAO-2 products.View full abstractDownload PDF (3453K) -
Ken-ichi Nunoya, Tsuyoshi Yokoi, Yuki Takahashi, Kanzo Kimura, Moritos ...1999 Volume 126 Issue 2 Pages 402-407
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo clarify the molecular mechanisms involved in the generation of the CYP2A6 gene deletion (E-type variant), we analyzed the CYP2A7 gene, which is located in the 5'-flanking region of the CYP2A6 gene, from individuals with the E-type variant and compared it with the sequences of wild type CYP2A7 and CYP2A6 genes. The 3'-downstream sequence (up to 324 bp from the SacI site in exon 9) of the CYP2A7 gene of the E-type variant is identical to that of the wild CYP2A7 gene. However, the 3'-downstream sequence (starting from 325 bp from the SacI site in exon 9) of the CYP2A7 gene of the E-type variant is identical to that of the wild CYP2A6 gene, indicating that the 3'-downstream region of CYP2A7 and the 3'-downstream region of CYP2A6 linked directly eliminating the whole CYP2A6 gene. PCR analysis using primers specific to the CYP2A7 gene and the CYP2A6 and CYP2A7 genes confirmed that all DNA samples obtained from 7 individuals carrying the E-type variant possessed the same break points. These results indicate that the breakpoint of the CYP2A6 gene deletion lies in the 3'-downstream region of the CYP2A7 and CYP2A6 genes.View full abstractDownload PDF (4000K) -
Kazuo Kobayashi, Seiichi Tagawa, Deligeer, Shinnichiro Suzuki1999 Volume 126 Issue 2 Pages 408-412
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSElectron transfer over 12.6 Å from the type 1 copper (T1Cu) to the type 2 copper (T2Cu) was investigated in the copper-containing nitrite reductases from two denitrifying bacteria (Alcaligenes xylosoxidans GIFU 1051 and Achromobacter cycloclastes IAN 1013), following pulse radiolytical reduction of T1Cu. In the presence of nitrite, the rate constant for the intramolecular electron transfer of the enzyme from A. xylosoxidans decreased 1/2 fold to 9×102 s-1 (20°C, pH 7.0) as compared to that for the same process in the absence of nitrite. However, the rate constant increased with decreasing pH to become the same (2×103 s-1) as that in the absence of nitrite at pH 6.0. A similar result was obtained for the enzyme from A. cycloclastes. The pH profiles of the two enzymes in the presence of nitrite are almost the same as that of the enzyme activity of nitrite reduction. This suggests that the intramolecular electron transfer process is closely linked to the following process of catalytic reduction of nitrite. The difference in redox potential (ΔE) of T2Cu minus T1Cu was calculated from equilibrium data for the electron transfer. The pH-dependence of dE was in accord with the equation: ΔE=ΔE°+0.058 log (Kr[H+]+[H+]2)/(K0+[H+]), where Kr and Ko are the proton dissociation constants for the oxidized and reduced states of T2Cu, respectively. These results raise the possibility that amino acid residues linked by the redox of T2Cu play important roles in the enzyme reaction, being located near T2Cu.View full abstractDownload PDF (627K) -
Rumi Ishisaka, Noriyuki Sato, Kenji Tanaka, Tomonori Takeshige, Hiroyu ...1999 Volume 126 Issue 2 Pages 413-420
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the minimum requirement in the 76-residue leader sequence of pro-tumor necrosis factor (TNF) for membrane translocation across the endoplasmic reticulum (ER) and for the maturation of pro-TNF, we constructed pro-TNF mutants in which a part of the transmembrane domain of pro-TNF was directly linked to the N-terminus of the mature domain, and evaluated their translocational behavior across the ER-membrane and their secretion from the transfected cells. The in vitro translation/translocation assay involving a canine pancreatic microsomal membrane system including a mutant, 4-75-47, -32-1, revealed that the N-terminal half of the transmembrane domain of pro-TNF consisting of 14 residues functioned as a cleavable signal sequence; it generated a cleaved form of TNF having a molecular mass similar to that of mature TNF. Analysis of the cleavage site by site-directed mutagenesis indicated that the site was inside the leader sequence of this mutant. When the mutant, Δ-75-47, -32-1, was expressed in COS-1 cells, efficient secretion of a biologically active soluble TNF was observed. Further deletion of the hydrophobic domain from this mutant inhibited the translocation, indicating that some extent of hydrophobicity is indispensable for the membrane translocation of the mature domain of TNF. Thus, the N-terminal half of the transmembrane domain of pro-TNF could function as a cleavable signal sequence when linked to the mature domain of TNF, and secretion of a biologically active secretory form of TNF could be achieved with this 14-residue hydrophobic segment. In intact pro-TNF, however, this 14-residue sequence could not function as a cleavable signal sequence during intracellular processing, indicating that the remainder of the 76-residue leader sequence of pro-TNF inhibits the signal peptide cleavage and thus enables the leader sequence to function as a type II signal-anchor sequence that generates a transmembrane form of TNF.View full abstractDownload PDF (5538K) -
Ryo Taguchi, Noriaki Hamakawa, Naoya Maekawa, Hiroh Ikezawa1999 Volume 126 Issue 2 Pages 421-429
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe applied the improved sensitivity and soft ionization characteristics of electrospray Ionization (ESI)-MS/MS and matrix-assisted laser desorption/ionization(MALDI)-time of flight (TOF) mass spectrometry (MS) to analysis of the GPI-anchored C-terminal peptide derived from 5'-nucleotidase. ESI-MS/MS analysis was applied to the core structure (MW, 2, 743). In the collision-induced dissociation (CID) spectrum, single-charged ions such as m/z 162 (glucosamine), 286 (mannose-phosphate-ethanolamine), and 447 ([mannose-phosphate-ethanolamine]-glucosamine) were clearly detected as characteristic fragment ions of the GPI-anchored peptide. On MALDI-TOF-MS analysis, heterogeneous peaks of GPI-anchored peptides were detected as single-charged ions in the positive mode. Product ions were obtained by post-source decay (PSD) of m/z 2, 905 using curved field reflectron of TOF-MS. Most of the expected product ions derived from the GPI-anchored peptide, containing the core structure and an additional mannose side chain, were successively obtained. Thus, ESI-MS/MS and MALDI-TOF-PSD-MS proved to be effective and sensitive methods for analyzing the GPI-anchored peptide structure with less than 10 pmol of sample. These characteristic fragments or fragmentation patterns seem to be very useful for identification of GPI-anchored C-terminal peptides derived from any kind of GPI-anchored protein.View full abstractDownload PDF (803K) -
Yoshiyuki Mizushina, Naoko Takahashi, Akitsu Ogawa, Kyoko Tsurugaya, H ...1999 Volume 126 Issue 2 Pages 430-436
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA DNA polymerase β (pol. β) inhibitor has been isolated independently from two organisms; a red perilla, Perilla frutescens, and a mugwort, Artemisia vulgaris. These molecules were determined by spectroscopic analyses to be the cyanogenic glucoside, D-mandelonitrile-β-D-glucoside, prunasin. The compound inhibited the activity of rat pol. β at 150 μM, but did not influence the activities of calf DNA polymerase α and plant DNA polymerases, human immunodefficiency virus type 1 reverse transcriptase, calf terminal deoxynucleotidyl transferase, or any prokaryotic DNA polymerases, or DNA and RNA metabolic enzymes examined. The compound dose-dependently inhibited pol. β activity, the IC50, value being 98 μM with poly dA/oligo dT12-18 and dTTP as the DNA template and substrate, respectively. Inhibition of pol. β by the compound was competitive with the substrate, dTTP. The inhibition was enhanced in the presence of fatty acid, and the IC50 value decreased to approximately 40 μM. In the presenceof C10-decanoic acid, the K1 value for substrate dTTP decreased by 28-fold, suggesting that the fatty acid allowed easier access of the compound to the substrate-binding site.View full abstractDownload PDF (891K) -
Hitomi Fukuda, Nobuko Iritani1999 Volume 126 Issue 2 Pages 437-444
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTranscriptional regulation of ATP citrate-lyase (ACL, one of the lipogenic enzymes) gene by glucose/insulin, polyunsaturated fatty acid (PUFA), and leptin has been investigated in hepatocytes and adipocytes of obese Wistar fatty rats and their lean littermates. The sequence spanning nucleotides -64 to -41 of the ACL gene, which is responsive to glucose/insulin stimulation [Eur. J. Biochem. 247, 497-502, 1997], was linked to a reporter gene and transfected into rat hepatocytes or adipocytes. The chloramphenicol acetyltransferase (CAT) activities in the presence of glucose alone were similar in primary cultured cells from both obese and lean rats. In the presence of glucose/insulin, however, the CAT activities were markedly increased in the hepatocytes of lean rats, but were not significantly increased in those of obese rats. The stimulation by glucose/insulin was reduced in PUFA-treated cells of lean rats. The stimulation was also reduced in leptin-treated cells or ob gene expression vector-containing cells. However, PUFA-or leptin-treated cells from obese rats did not show a significant reduction in insulin stimulation. The same effects were observed at the endogenous mRNA and enzyme levels. Similar results were seen in adipocytes, although the stimulation and suppression levels were much smaller than in hepatocytes. The expression of endogenous insulin receptor in hepatocytes and adipocytes was reduced in the presence of leptin or PUFA. We previously found that insulin-binding capacities are also reduced in the presence of leptin or PUFA and are very low in obese rats in comparison with lean. Moreover, gel mobility shift assays using end-labeled ACL-(-64/-41) revealed that nuclear factor (s) including Spl bind specifically to the sequence, and DNA-protein complex formation is reduced in the obese rats. Thus, the reductions in the insulin-stimulated ACL transcription may be ascribed in part to reductions in insulin binding to receptors and DNA-protein complex formation.View full abstractDownload PDF (4021K) -
Ken Karasawa, Kazuaki Yokoyama, Morio Setaka, Shoshichi Nojima1999 Volume 126 Issue 2 Pages 445-448
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe deduced the amino acid sequence of Escherichia coli lysophospholipase L1 by determining the nucleotide sequence of the pldC gene encoding this enzyme. The translated protein was found to contain 208 amino acid residues with a hydrophobic leader sequence of 26 amino acid residues. The molecular weight of the purified enzyme (20, 500) was in good agreement with the predicted size (20, 399) of the processed protein. A search involving a data bank showed that the nucleotide sequence of the pldC gene was identical to those of the apeA and tesA genes encoding protease I and thioesterase I, respectively. Consistent with the identity of the pldC gene with these two genes, the enzyme purified from E. coli overexpressing the pldC gene showed both protease I and thioesterase I activities.View full abstractDownload PDF (470K) -
Kotaro Sugimoto, Masaaki Takahashi, Yumiko Yamamoto, Kohei Shimada, Ka ...1999 Volume 126 Issue 2 Pages 449-455
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSErosion of cartilage is a major feature of joint diseases, i.e., osteoarthritis and rheumatoid arthritis, which leads with time to a loss of joint function. Proteolytic cleavage of the aggrecan core protein is a key event in the progress of these joint diseases. Aggrecan degradation has been believed to be mediated by a putative proteinase, aggrecanase. We identified aggrecanase activity in conditioned medium from explant culture of bovine nasal cartilage stimulated by retinoic acid. The activity was partially purified more than 10, 000-fold. The enzyme cleaves at the aggrecanase site (Glu373-Ala374) but not at the MMP site (Asn341-Phe342) in the interglobular domain of the aggrecan. It also cleaves at Glu1971-Len1972, which is located in the gap region in the chondroitin sulfate attachment region prior to the aggrecanase site. The enzyme is a typical Ca2+-dependent metalloproteinase with a unique salt-dependency and is inhibited by several hydroxamate-based inhibitors for matrix metalloproteinases. Heparin and chondroitin sulfate inhibited the enzyme in a dose-dependent manner, suggesting that the large carbohydorate in aggrecan is important for substrate recognition by aggrecanase.View full abstractDownload PDF (3195K) -
1999 Volume 126 Issue 2 Pages 456
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (20K)
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