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Volume 123 (1998)
- Issue 6 Pages 993-
- Issue 5 Pages 767-
- Issue 4 Pages 555-
- Issue 3 Pages 369-
- Issue 2 Pages 195-
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Volume 123, Issue 5
Displaying 1-32 of 32 articles from this issue
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Yoshikazu Sado, Megumi Kagawa, Ichiro Naito, Yasuyoshi Ueki, Tsugio Se ...1998 Volume 123 Issue 5 Pages 767-776
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSix distinct genes have been identified as belonging to the type IV collagen gene family. They can be organized into three sets, i.e., COL4A1/COL4A2, COL4A3/COL4A4, and COL4A5/COL4A6, which are localized on three different chromosomes in humans, 13, 2, and X, respectively. Within each set the genes are aligned head-to-head and their expression is regulated by bidirectional promoters between the genes. Transcriptional regulation of the COL4A1/COL4A2 set has been well characterized. The transcription of COL4A6 seems to be controlled by two alternative promoters. While collagen IV molecules composed of α1 and α2 chains are broadly distributed, molecules comprising combinations of the other four chains, α3-α6, are important components of specialized basement membranes. The precise chain composition of triple-helical molecules assembled from the α3-α6 chains is not entirely clear, but it is hypothesized that α3-α5 chains and α5 and α6 chains form heterotrimeric molecules. Several pieces of evidence indicate that α3/α4/α5 molecules and α5/α6 molecules are components of the basement membrane network. This helps explain the observation that the kidney and skin basement membranes from patients with Alport syndrome caused by mutations in the α5 coding gene, COL4A5, are defective in the α3, α4, and α6 chains together with the α5 chain. Large deletions involving the COL4A5 and COL4A6 genes have been found in rare cases of diffuse leiomyomatosis associated with Alport syndrome.View full abstractDownload PDF (2359K) -
Masao Kawakita, Nobuhiro Ishida, Nobuhiko Miura, Ge-Hong Sun-Wada, Shi ...1998 Volume 123 Issue 5 Pages 777-785
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNucleotide sugar transporters are mainly located in the Golgi membranes and carry nucleotide sugars, that are produced outside the Golgi apparatus, into the organelle, where they serve as substrates for the elongation of carbohydrate chains by glycosyltransferases. They are thus indispensable for cellular glycoconjugate synthesis and, moreover, may have regulatory roles in producing the structural variety of cellular glycoconjugates. Their occurrence has long been well recognized, but studies on the molecular bases of their strict substrate specificities and modes of action have been hampered by the lack of information on their precise molecular structures. Complementary DNAs encoding several of these transporters were cloned recently, which represented a substantial step forward as to the above mentioned issues. The products of these cDNAs are mutually related hydrophobic proteins consisting of 320-400 amino acid residues with multiple putative transmembrane helix domains, and are located in the Golgi apparatus. This review briefly summarizes the present status of the field of nucleotide sugar transporter research, and also presents an outlook of the study in this field.View full abstractDownload PDF (1354K) -
Masaki Akiyama, Junko Sugatani, Takashi Suzuki, Yasuo Suzuki, Masao Mi ...1998 Volume 123 Issue 5 Pages 786-789
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPlatelet-activating factor (PAF) acetylhydrolase from human serum/plasma was identified on a polyvinylidene difluoride (PVDF) membrane by electroblotting proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme activity was detected in the 43 kDa region on the membrane as a decrease in the β-radio-luminescence of [3H] acetyl-PAF or by the convenient method for determining PAF acetylhydrolase activity (the TCA precipitation method). The enzyme activity on treatment with N-glycosidase F shifted to the 34 kDa region on the PVDF membrane. On the other hand, only one band was observed, corresponding to a molecular mass of 53 kDa, on analysis by SDS-PAGE with silver staining. Treatment of the 53 kDa protein with N-glycosidase F changed its molecular mass to 43 kDa (protein A). The NH2-terminal 32 amino acid sequence of protein A completely corresponds to that of the heterogenous enzyme with 54 amino acids deleted from the NH2 terminus reported by Tjoelker et al. (Nature 374, 549-553, 1995). Even after trypsin treatment of the N-glycosidase F-digested enzyme, its PAF-AH activity remained in the 34 kDa region, but the contaminating protein A disappeared, on the PVDF membrane. In addition, the majority of serum PAF-AH was retained on a Sambucus sieboldiana agglutinin (SSA)-agarose column and was eluted with the hapten sugar, lactose. These results indicate that PAF acetylhydrolase consisting of a 34 kDa protein and about 9 kDa asparagine-conjugated sugar chain (s) is a major enzyme in human serum/plasma.View full abstractDownload PDF (1516K) -
Masood Jelokhani-Niaraki, Kenichi Nakashima, Hiroaki Kodama, Michio Ko ...1998 Volume 123 Issue 5 Pages 790-797
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction and orientation of a membrane protein ion channel model, an α-aminoiso-butyric acid analogue of gramicidin B (GBA), in egg yolk phosphatidylcholine vesicles was studied by means of fluorescence spectroscopic techniques. GSA helices form stable ionconducting pores in membranes [Jelokhani-Niaraki et al. (1995) J. Chem. Soc. Perkin Trans. 2, 801-808]. In an α-helical model for the peptide, all Trp residues (intrinsic fluorophores) are distributed near the C-terminus. Fluorescence quenching experiments revealed the exposure of the helical peptides' C-termini to aqueous environments. Dansyllabeled vesicles were used to investigate the GBA dynamism of the interaction with membranes. It was shown that considerable amounts of peptide reside on and in the vicinity of the outer surface of lipid bilayers. The transmembrane transfer to the inner layer is slow due to the high affinity of Trp residues for bilayer interfaces which anchor the peptide to the outer surface. A structural-functional interpretation of the GBA interaction with membranes is presented.View full abstractDownload PDF (1019K) -
Yukiko Saka, Tomoyoshi Mita1998 Volume 123 Issue 5 Pages 798-805
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of amphotericin B (AmB) with cholesterol was investigated in monolayers, aqueous solutions, and phospholipid vesicles. When AmB was mixed with cholesterol, it formed a stable monolayer, implying complex formation in which the stoichiometry was primarily 1:1 AmB:cholesterol. However, the interaction of AmB with cholesterol in aqueous solutions and lipid vesicles was more complex. In aqueous solutions, cholesterol at low concentrations increased the aggregation of AmB. But higher concentrations of cholesterol caused dissociation of the aggregates of AmB due to the formation of AmB-cholesterol complexes. In lipid vesicles, the effect of cholesterol was different from that in aqueous solutions. Both in aqueous solutions and lipid vesicles, the overall dissociation of AmB molecules occurred on interaction with cholesterol. In addition, the interaction of lipid membranes with AmB-cholesterol complexes was investigated by differential scanning calorimetry. The incorporation of AmB into lipid bilayers led to broadening of the lipid transition and a slight decrease in the transition enthalpy, showing that one lipid molecule per AmB molecule was immobilized. However, the number of immobilized lipid molecule per AmB molecule increased in the coexistence of cholesterol, due to the complex formation between AmB and cholesterol.View full abstractDownload PDF (991K) -
Shigeyuki Uno, Masaru Nakamura, Yasushi Ohomagari, Syutoku Matsuyama, ...1998 Volume 123 Issue 5 Pages 806-812
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have performed a comparative study on tPA and PAI-1 mRNA expression in primary cultures of rat hepatocytes and elucidated the possible regulation of these factors by certain hormonal stimulation. The tPA mRNA increased 2- to 4-fold in the presence of cholera toxin (CT), dibutyryl cyclic AMP (dbcAMP), or 3-isobutyl-1-methyl xanthine (IBMX), but slightly decreased in the presence of dexamethasone. The tPA activity was also changed by these agents in a similar fashion. On the contrary, PAI-1 mRNA decreased with CT, dbcAMP, or IBMX, but increased transiently with dexamethasone. From results obtained with cycloheximide, ongoing protein synthesis was judged to be required for both PAI-1 induction with dexamethasone and PAI-1 suppression with IBMX, but not for the tPA induction with IBMX. Dexamethasone exerted opposite regulatory effects on the tPA mRNA expression depending on its concentration: at 10-8 to 10-6 M, it suppressed the expression; whereas at 10-20 M, it elevated the expression.View full abstractDownload PDF (1959K) -
Toshiyuki Obata, Hiroshi Maegawa, Atsunori Kashiwagi, Tahir S. Pillay, ...1998 Volume 123 Issue 5 Pages 813-820
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have reported that high glucose conditions (27mM for 4 days) induces activation of protein tyrosine phosphatases (PTPases) which are associated with impaired insulin signaling in Rat 1 fibroblasts expressing human insulin receptors [Maegawa, H. et al. (1995) J. Biol. Chem. 270, 7724-7730]. In this study, we found increased mRNA-levels of a non-receptor type PTPase, protein tyrosine phosphatase 1B (PTP1B), and receptor type PTPases, leukocyte common antigen-related phosphatase (LAR), and LAR-related phosphatase (LRP), under high glucose conditions. In accordance with these results, LAR content was significantly increased, whereas LRP content was not increased. Cytosolic PTP1B content was increased, but membrane-associated PTP1B content showed no detectable change. Pioglitazone, a thiazolidinedione, normalized increased cytosolic PTPase activity through reduction of cytosolic PTP1B content, but it had no effect on mRNA levels of these PTPases. Under the high glucose condition, we also found that epidermal growth factor (EGF)-stimulated signaling, including tyrosine-phosphorylation of EGF receptor and phosphatidylinositol 3'-kinase activities, was attenuated. Nevertheless, pioglitazone failed to restore the attenuated EGF-signaling. These results indicate that the high glucose conditions cause dysfunction of EGF receptor. However, the increased cytosolic PTP1B content is not involved in the abnormal regulation of EGF-signaling, in contrast to insulin-signaling.View full abstractDownload PDF (4973K) -
Nobuyuki Tanaka, Hitoshi Saito, Hisashi Kimoto, Akira Taketot1998 Volume 123 Issue 5 Pages 821-826
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of cis-diamminedichloroplatinum(II) (cisplatin) on Escherichia coli cells and bacteriophages were investigated. The bacteriocidal effect of cisplatin was stronger on uvrA or recA mutants than on wild type cells. The drug, like UV, induced prophage development in lysogenic bacteria. Host cell reactivation of α3 replicative form (RF) I DNA treated with cisplatin in vitro was more efficient in wild type or recA cells than in uvrA host. When wild type cells were exposed to cisplatin, decay of the host's capacity to sustain the viral multiplication proceeded nearly in parallel with the loss of colony-forming ability, whereas the capacity of uvrA mutant was much more resistant to the drug, as compared with the viability. In the DNA preparation from cisplatin-treated α3-infected wild type cells, RF II was deficient, but the RF I molecules extracted from the cells were moderately infective. The microvirid gene A protein, required for RF I→RF II conversion, was hardly detectable in wild type cells exposed to cisplatin. The possible relationship between uvr+-dependent repair and synthesis of the viral protein is discussed.View full abstractDownload PDF (2984K) -
Hisashi Yasui, Wataru Ito, Yoshikazu Kurosawa1998 Volume 123 Issue 5 Pages 827-831
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined temperature dependency of thermodynamic parameters in the interactions between hen egg-white lysozyme (HEL) and anti-HEL antibody, D1.3, and two mutant antibodies. The ΔH° values appeared to decrease biphasically in the temperature range from 10 to 45°C with the apparent inflection point around 30°C. The ΔG° calculated from the KA values showed only small differences because of entropy and enthalpy compensation. It has been argued that large negative values of heat capacity change (ΔCp°), if observed, are mainly derived from hydrophobic interactions. However, the observed ΔCp° values were too high to be ascribed only to hydrophobic interactions. Moreover, addition of methanol did not cause a decrease in the absolute value of ΔCp°.View full abstractDownload PDF (514K) -
Wataru Ito, Yoshikazu Kurosawa1998 Volume 123 Issue 5 Pages 832-838
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe characterized a phage antibody in which an Fv fragment, namely, a free VH fragment noncovalently associated with a VL fragment that is fused with a truncated cpIII molecule (VL-ΔcpIII), is expressed on the phage surface. D1.3 antibody specific for hen egg-white lysozyme was used as a model system. Both VH and VL-ΔcpIII fragments were stably expressed and associated with each other to form a faithful antigen-binding site. The results of Western blotting indicated that more than 5% of phages expressed the Fv fragment on their surface. Analysis of the kinetics of binding of the phage antibody to the antigen suggested the possibility of presence of phages having multiple-binding sites on a single phage particle.View full abstractDownload PDF (1871K) -
Eiji Ohmae, Kenji Ishimura, Masahiro Iwakura, Kunihiko Gekko1998 Volume 123 Issue 5 Pages 839-846
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the role of a flexible loop (residues 142-149) in the stability and function of Escherichia coli dihydrofolate reductase, alanine-145 in this loop was substituted by site-directed mutagenesis with ten amino acids (Glu, Phe, Gly, His, Ile, Leu, Arg, Ser, Thr, and Val). The amount of three mutant proteins (A145E, A145I, and A145L) in cells was too small to allow the measurement of circular dichroism (CD) spectra and urea unfolding. The CD spectra of other seven mutants were identical with those of the wild-type DHFR, indicating that the native conformation of DHFR was not affected by the mutations. The free energy change of unfolding by urea decreased with an increase in the hydrophobicity of amino acid residues introduced, A145T>A145R>A145G_??_A145S_??_A145H>A145V>wildtype_??_A145F. The steady-state kinetic parameters for the enzyme reaction, Km and kcat, were only slightly influenced by the mutations. These results suggest that site 145 in the flexible loop plays an important role in the stability but has little or no effect on the native structure and function of this enzyme. The characteristics of the mutations are discussed in comparison with those of mutations at site 67 [Ohmae et al. (1996) J. Biochem. 119, 703-710] and at site 121 [Gekko et al. (1994) J. Biochem. 116, 34-41] in two other flexible loops.View full abstractDownload PDF (1012K) -
Kuniyo Inouye, Keiko Kuzuya, Ben'ichiro Tonomura1998 Volume 123 Issue 5 Pages 847-852
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThermolysin is remarkably activated in the presence of high concentrations (1-5M) of neutral salts [Inouye, K. (1992) J. Biochem. 112, 335-340]. The activity is enhanced 13-15 times with 4M NaCl at pH 7.0 and 25°C. In this study, the effect of neutral salts on the solubility of thermolysin has been examined. Although the solubility was only 1.0-1.2mg/ml in 40mM Tris-HCl buffer, pH 7.5, in the temperature range between 0 and 60°C, it was increased greatly by the addition of salts. With NaCl, the solubility showed a bell-shaped behavior with increasing NaCl concentration, and the maximum solubility (10mg/ml) was at 2.0-2.5M NaCl. With LiCl and NaI, it increased progressively to 20-50mg/ml with increasing salt concentration up to 5M. The solubility observed in the presence of salts decreased with increasing temperature from 0 to 60°C, and also with the order of chaotropic anion effect. The molecular weight of thermolysin was estimated to be 33.0(±2.5)×103 in the presence of 0-3M NaCl, suggesting that thermolysin exists as a monomer in the presence or absence of 3M NaCl. The possibility that aggregation and/or dispersion of thermolysin might be related to the remarkable activation by salt was ruled out.View full abstractDownload PDF (707K) -
Jitsuhiro Matsugi, Katsutoshi Murao, Hisayuki Ishikura1998 Volume 123 Issue 5 Pages 853-858
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus subtilis has been thought to have a high readthrough rate at the UGA stop codon because no opal suppressor tRNA has been isolated so far [Lovett et al. (1991) J. Bacteriol. 173, 1810-1812]. To examine whether a tRNATrP which we have characterized [Matsugi et al. (1992) Nucleic Acids Res. 20, 3514] has the ability to read the UGA codon, in vitro translation was performed with a synthetic mRNA containing a test codon, UGA, UAG, UAA, or UGG, in a reading frame. Addition of Trp-tRNATrP to the system significantly increased the readthrough rate only in the case of UGA. This suggests that this tRNATrP has a dual recognition pattern in B. subtilis, i.e., for the canonical tryptophan codon and for readthrough at the UGA stop codon.View full abstractDownload PDF (684K) -
Susumu Mitsutake, Katsuhiro Kita, Tetsuto Nakagawa, Makoto Ito1998 Volume 123 Issue 5 Pages 859-863
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis paper describes the synthesis of 14C-labeled glycosphingolipids using the reverse hydrolysis reaction (condensation) of sphingolipid ceramide N-deacylase. It was found that 50-70% of 14C-fatty acids were incorporated into various lyso-glycosphingolipids when a mixture of lyso-glycosphingolipids and fatty acids was incubated at 37°C with 1 mU of the enzyme for 20 h in 1 ml of 25mM phosphate buffer, pH 6.0-7.0, containing 0-0.1% Triton X-100. The optimum concentration of lyso-glycosphingolipids was 100-400 μM depending on the species of lyso-form when [14C] stearic acid was used at the concentration of 100 μM. Free 14C-fatty acids and lyso-glycosphingolipids were separated from the synthesized 14C-glycosphingolipids by using a Sep-Pak Plus Silica and a Sep-Pak CM or a QMA cartridge, respectively. After treatment of 14C-glycosphingolipids with endoglycocer-amidase or sphingolipid ceramide N-deacylase, digestion products were clearly separated from the parent glycosphingolipids on TLC and determined using an image analyzer with a sensitivity 100 times higher than that using non-radiolabeled substrates. Using this method, we found endoglycoceramidase activity in a seaflower, Condylactis sp., for the first time.View full abstractDownload PDF (1556K) -
Kazuhiro Fujisawa, Yoshihiro Matsumoto, Hisako Muramatsu, Torn Shinzat ...1998 Volume 123 Issue 5 Pages 864-869
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe heparin-binding growth factor midkine (MK) has been implicated in neuron growth, angiogenesis, and inflammation. In this study, to elucidate the involvement of MK in the development of pathologies associated with uremia, we examined the serum MK levels in patients receiving hemodialysis (HD) by a highly sensitive enzyme-linked immunoassay. Although no significant difference was found between control serum and serum before dialysis in HD patients, serum MK levels increased significantly at the early stage of HD sessions using heparin and gradually decreased after dialysis. In normal controls, intravenous administration of heparin induced a similar sudden increase of MK, but the subsequent decrease was also rapid. In an in vitro study, MK was released in time- and heparin-dose dependent manner from cultured vessels, but not from peripheral leukocytes. These results indicate that, in HD patients, MK is released mainly from endothelial cells immediately after administration of heparin during HD and disappears gradually from blood due to renal impairment. This phenomenon might affect some complications associated with HD.View full abstractDownload PDF (1427K) -
Koichi Kawabe, Tsutomu Kodaki, Kazuhisa Katayama, Shin-ichi Okamura, M ...1998 Volume 123 Issue 5 Pages 870-875
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhospholipase D (PLD) is implicated in important cellular processes, such as hormone action, inflammation, secretion, mitogenesis, and neural activity. Recent studies using cell-free systems have shown that the enzyme activity is modulated by both positive and negative regulators. During an attempt to purify PLD from pig colon mucosa, we noted the presence of a PLD inhibitor in the tissue extract. The inhibitor was purified and identified as comprising lysophosphatidylserine, phosphatidylinositol, and lysophosphatidylino-sitol, of which lysophosphatidylserine was the most potent. These lipids affected all of the PLD isoforms examined, oleate-dependent PLD, ARF-dependent PLD (PLD1a, PLD1b), and hosphatidylinositol 4, 5-bisphosphate-dependent PLD (PLD2), in the concentration range of the 1 or 10 μM order. In contrast to lysophosphatidylserine, the diacyl counterpart phosphatidylserine was without effect in the same concentration range. PLD inhibition by lysophosphatidylserine could not be reversed by an increase in the concentration of the substrate phosphatidylcholine or activator phosphatidylinositol 4, 5-bisphosphate.View full abstractDownload PDF (1581K) -
Yukiko Kimura, Megumi Nakazawa, Michiyuki Yamada1998 Volume 123 Issue 5 Pages 876-882
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOS-9 gene is frequently coamplified with CDK4 gene in human sarcomas. We isolated and characterized three isoforms of OS-9 cDNA found in a myeloid leukemia HL-60 cDNA library. Isoform 1 consisted of 2, 700 bp, from which a 667 amino acid sequence was deduced and found to be identical with that of OS-9 cDNA from osteosarcoma cells [Su et al. (1996) Mol. Carcinogen. 15, 270-275]. Isoform 2 cDNA lacked a 165 nucleotide sequence in the coding region. Isoform 3 cDNA had an additional 45 bp deletion in the coding region. Isoforms 2 and 3 encode 612 and 597 amino acid polypeptides, respectively. Comparison of their cDNA sequences with the genomic structure indicated that three isoforms are splice variants. Reverse transcription-polymerase chain reaction analysis showed predominant expression of isoform 2 mRNA in myeloid leukemia HL-60 cells, osteosarcoma OsA-CL cells and rhabdomyosarcoma Rh30 cells. Northern blotting revealed similar levels of expression of OS-9 gene in various tumor cell lines of sarcoma cells, carcinoma cells and myeloid leukemia cells, but 3-4 times higher expression in OsA-CL cells and Rh30 cells containing a homogeneously staining region of 12q13-15. OS-9 expression decreased in differentiation-induced HL-60 cells. Possible involvement of the OS-9 gene in cell growth is discussed.View full abstractDownload PDF (4076K) -
Tetsuo Shiomi, Kohtaro Fukushima, Nobuhiro Suzuki, Nobutaka Nakashima, ...1998 Volume 123 Issue 5 Pages 883-890
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSaccharomyces cerevisiae Dis3p, which interacts with Ran/Gsp1p, complements Schizo-saccharomyces pombe dis3-54. Consistent with the functional conservation of Dis3p in S. cerevisiae and S. pombe, the human ORF (accession number: R27667) was found to be highly homologous to yeast Dis3p. Based on its nucleotide sequence, we cloned a full-sized human DIS3 cDNA. The cloned human cDNA partly but significantly restored the temperature-sensitivity of S. cerevisiae dis3. Thus, Dis3p was found to be structurally and functionally conserved from yeast to mammals. Consistent with the report that S. cerevisiae Dis3p is identical to Rrp44p, which comprises the exosome involved in ribosomal RNA processing, S. cerevisiae Dis3p was found to be localized in the nucleolus. Similar to S. cerevisiae Dis3p, human Dis3p enhanced RCC1-stimulated nucleotide release from Ran, in a dose-dependent manner, and bound to GTP- or GDP-Ran.View full abstractDownload PDF (3586K) -
Masaya Kitamura, Takamasa Sagara, Masahiro Taniguchi, Masaki Ashida, K ...1998 Volume 123 Issue 5 Pages 891-898
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli. A 1.6-kbp DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SaiI and EcoRI, contained the flavodoxin gene and its regulatory region. An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E. coli. The purified protein was soluble and exhibited a characteristic visible absorption spectrum. HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D. vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38nM. In vitro H2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E1=-434 and E2=-151 mV using methyl viologen and 2-hydroxy-1, 4-naphthoquinone, respectively. Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode. The mutant, A16E, was also produced, which revealed the feature of a conserved Glu residue at the surface of the molecule.View full abstractDownload PDF (2885K) -
Keiko Hata, Tadashi Wada, Akira Hasegawa, Makoto Kiso, Taeko Miyagi1998 Volume 123 Issue 5 Pages 899-905
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA membrane-associated ganglioside-hydrolyzing sialidase was purified to apparent homogeneity from bovine brain. The enzyme was solubilized with Triton X-100 plus sodium cholate from the particulate fraction and purified over 100, 000-fold by sequential chromatography on DEAE-cellulose, octyl-Sepharose, heparin-Sepharose, Sephacryl S-200, MonoQ, RCA-agarose, thiol-activated Sepharose, and ganglioside-affinity Sepharose. The final enzyme preparation exhibited a specific activity of 4, 851.3 μmol/h/mg protein and an apparent molecular mass of 52 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme preferentially hydrolyzed gangliosides other than GM1 and GM2 but demonstrated hardly any activity against glycoproteins and oligosaccharides. Gangliosides GD3, GD1a, and GT1b were much better substrates than GM3 and GD1b in the presence of Triton X-100, but the latter became more sensitive to the sialidase with addition of sodium cholate. The enzyme was activated by dithiothreitol, strongly inhibited by 4-hydroxy-mercuribenzoate, and firmly adsorbed to thiol-activated Sepharose, indicating that free sulfhydryl groups are essential for its catalytic activity. Subcellular fractionation experiments revealed that the enzyme is mainly located in the synaptosomal fraction.View full abstractDownload PDF (1745K) -
Takeshi Mikami, Motoi Kashiwagi, Keiko Tsuchihashi, Toyoaki Akino, Shi ...1998 Volume 123 Issue 5 Pages 906-911
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDihydroceramide desaturase, which catalyzes the introduction of a double bond at the 4, 5-position of the sphingosine base in a dihydroceramide, was assayed in vitro using radiolabeled D-erythro-C18-dihydroceramide (N-stearoyl sphinganine) and homogenates of fetal rat skin, and some enzymatic properties, including substrate specificity, were determined. The ceramide structure, as the enzymatic product, was confirmed by (i) oxidation of the product with 2, 3-dicyano-5, 6-dichlorobenzoquinone, which revealed the conversion to 3-ketoceramide (3, 3'-didehydroceramide), indicating that a double bond was introduced at the adjacent to the C-3 hydroxyl residue of sphinganine, and (ii) mass spectrometry of a long chain base released from the enzymatic product, which revealed a spectrum identical to that of authentic sphingenine. A short chain dihydroceramide, which was radiolabeled at sphinganine through a newly established method, having a C2- or C6-fatty acid was not desaturated by the skin enzyme, whereas that having a C10-, C14-, or C18-acid was desaturated, maximal reactivity being observed for the C14-dihydroceramide. Other enzymatic properties were confirmed: NAD(H) or NADP(H) and a detergent were required for elevation of the activity; the optimum pH was approximately 6.7; and metal cations were not essential, but Zn2+, Cu2+, and Fe2+ were rather inhibitory. These properties of rat skin desaturase were partly similar to those of rat liver microsomes, as reported recently, however, their substrate specificities were different.View full abstractDownload PDF (1684K) -
Ge-Hong Sun-Wada, Shigemi Yoshioka, Nobuhiro Ishida, Masao Kawakita1998 Volume 123 Issue 5 Pages 912-917
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe describe the functional expression of the putative human Golgi UDP-galactose transporters (hUGT1 and hUGT2) in the yeast Saccharomyces cerevisiae. Both hUGT1 and hUGT2 were expressed under the control of the yeast constitutive GAPDH promoter. The expression level of hUGT1 seemed to be considerably lower than that of hUGT2, although hUGT1 has an amino acid sequence identical to that of hUGT2 except for 5 amino acid residues at the C-terminus. The hUGT product was expressed in the membranes of Golgi and other organellar compartments. The membrane vesicles prepared from the hUGT1- or the hUGT2-expressing yeast cells exhibited UDP-galactose specific transport activity. The apparent Km values of the yeast-expressed hUGT1 and hUGT2 for UDP-galactose were 1.2 and 2 μM, respectively, which were comparable with the Km obtained with mammalian Golgi vesicles. Transport was dependent on temperature and integrity of vesicles, and was inhibited by UMP, as observed with mammalian Golgi vesicles. Our results demonstrate that the previously described hUGT1 and hUGT2 encode the UDP-galactose transporters, rather than regulatory proteins. The development of a convenient yeast expression system should facilitate analysis of the structure-function relationships of the UDP-galactose transporters.View full abstractDownload PDF (3829K) -
S. Rauli, M. Del Puppo, F. Magni, M. Galli Kienle1998 Volume 123 Issue 5 Pages 918-923
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMeasurement of the concentrations of aldehydes in biological samples has become the object of much effort due to their relevance in relation to the toxic effects of lipid peroxidation, through which a number of aldehydes are derived. We have reconsidered a previously proposed method based on gas chromatographic mass spectrometric analysis of derivatives obtained by the treatment of aldehydes with O-pentafluorobenzyl hydroxylamine followed by a trimethylsilylating agent. In view of the possible use of the method for the simultaneous evaluation of the plasma levels of malondialdehyde and 4-hydroxy-2-trans-nonenal, we have studied the linearity of the analysis using various internal standards. Commercially available, inexpensive 2, 4-dihydroxybenzaldehyde gave optimal results, the correlation coefficient of the calibration curve for plasma being r>0.995 in the 0.1-5 μM range for both the tested aldehydes. The between-day imprecision (%CV) and accuracy (%bias) of the procedure determined using plasma samples spiked with the two aldehydes and with an internal standard reached maximum values of 3 and 8%, and 5 and 12% for HNE and MDA, respectively. The results obtained on analysis of plasma samples before and after oxidation with copper ions indicate the flexibility of the method for evaluation of the levels of MDA and HNE in plasma samples both under basal conditions and after an oxidative burst.View full abstractDownload PDF (677K) -
Masanori Mitta, Masaru Miyagi, Ikunoshin Kato, Susumu Tsunasawa1998 Volume 123 Issue 5 Pages 924-931
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcylamino acid-releasing enzyme (AARE) [EC 3. 4. 19. 1] is a tetrameric serine protease, which belongs to the oligopeptidase family and specifically removes acetyl amino acids from N-terminally acetylated peptides. By using diisopropyl fluorophosphate, we previously identified one of the residues comprising the catalytic triad of this enzyme as Ser587 [Miyagi, M. et al. (1995) J. Biochem. 118, 771-779]. To elucidate the other two residues forming the catalytic triad of this new serine-type protease, wild-type and four mutant AAREs, in which each candidate residue of the catalytic triad deduced from sequence alignment with other oligopeptidases was substituted by site-directed mutagenesis, were expressed in Escherichia coli as fusion proteins with short peptide chains at both N- and C-termini of a subunit of porcine liver enzyme. All of the recombinant AAREs were estimated to have similar conformational and quaternary structures to the native porcine liver enzyme from their CD spectra and behavior on gel-filtration, but the mutants in which Ala587, Asn675, or Tyr707 was substituted for Ser587, Asp675, or His707, respectively, did not show detectable hydrolytic activity toward acetyl-L-methionyl L-alanine. These facts suggest that Ser587, Asp675, and His707 are essential residues for the AARE activity and comprise the catalytic triad of the enzyme in this order. Thus, AARE has been shown to have a protease-like domain in its C-terminal region, as do other proteins classified as members of the oligopeptidase family.View full abstractDownload PDF (1933K) -
Kaoru Omichi, Sumihiro Hase1998 Volume 123 Issue 5 Pages 932-936
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn assay method for glycogen debranching enzyme involving fluorogenic dextrins as substrates was developed. Two dextrins were prepared from 6-O-α-D-glucosyl-α-cyclodextrin and glucose by taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase, and converted by pyridylamination to fluorogenic derivatives. Structural analysis of the fluorogenic dextrins by FAB-MS, partial acid hydrolysis, and glucoamylase digestion revealed that they were Glcα1-4(Glcα1-6)Glcα1-4Glcα1-4Gicα1-4Glc-PA (FD6) and Glcα1-4Glcα1-4(Glcα1-6)Glcα1-4Glcα1-4Glcα1-4Glc-PA (FD7). Using the glycogen debranching enzyme from rabbit muscle, FD6 and FD7 were, respectively, hydrolyzed to PA-maltopentaose and PA-maltohexaose, in addition to glucose, showing that these two fluorogenic dextrins are suitable substrates for assaying the glycogen debranching enzyme. An assay method involving the separation and quantification by HPLC of the characteristic fluorogenic products was successfully applied to determination of the distribution of the enzyme activity in mouse cerebrum.View full abstractDownload PDF (631K) -
Marc A. B. Kolkman, Bernard A. M. van der Zeijst, Piet J. M. Nuijten1998 Volume 123 Issue 5 Pages 937-945
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStreptococcus pneumoniae comprises 90 serotypes, each one having its own specific polysaccharide capsule. In order to explore the diversity of capsular polysaccharide synthesis (cps) gene clusters in S. pneumoniae, we performed cross-hybridizations between the 12 cps genes of S. pneumoniae serotype 14 and chromosomal DNA of 26 strains comprising 26 different capsule types. Large variations in the hybridization patterns were observed. The genes cps14A to cps14D are conserved in most serotypes. Sequences homologous to cps14I to cps14L were only observed in the four types of serogroup 15, which all have a capsule structure similar to that of type 14. By using a cps14E knock-out construct, cpsE mutants of the pneumococcal types 9N, 13, and 15B were obtained. These mutants were unencapsulated and showed reduced glycosyltransferase activity, indicating that the pneumococcal types 9N, 13, and 15B express a glucosyl-1-phosphate transferase which is homologous to Cps14E. Glycosyltransferase assays showed that among 21 pneumococcal types which contain glucose in the core of their capsule polysaccharide, 19 types express glucosyl-1-phosphate transferase activity. However, not all of these types hybridized strongly with Cps14E, the type 14 glucosyl-1-phosphate transferase gene. Thus, pneumococci possess glucosyltransferase genes distinct from cps14E, but encoding enzymes with identical activity. All serotypes which synthesized lipid-linked lactose intermediates in glycosyltransferase activity assays (type 11B, 13, 15F, 15A, 15B, 15C) hybridized with cps14G. This gene encodes a galactosyltransferase which catalyzes the addition of 1, 4-linked β-galactose to lipid-linked glucose. The cps14G homologues in type 11B, 13, 15F, 15A, 15B, and 15C may encode a similar β-galactosyltransferase activity as cps14G in type 14.View full abstractDownload PDF (2136K) -
Shun-ichi Wada, Shigeki Matsunaga, Shin-ya Saito, Nobuhiro Fusetani, S ...1998 Volume 123 Issue 5 Pages 946-952
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn actin-depolymerizing marine natural product, mycalolide B, and a related compound, kabiramide D, were labeled with biocytin, a biotin derivative, and used to specify target molecules in cultured rat 3Y1 fibroblasts. Mycalolide B exhibited the ability to bind to various intracellular proteins, probably through the Michael addition of a sulfhydryl group to C5 of mycalolide B. However, no intracellular proteins other than actin apparently reacted with biocytinylated kabiramide D, demonstrating that the binding of kabiramide D to actin was highly specific. Cells treated with biocytinylated kabiramide D followed by staining with fluorescein isothiocyanate-conjugated avidin showed that biocytinylated kabiramide D bound to stress fibers composed of F-actin, although the staining intensity was weaker than the fluorescent phalloidin staining. The assay for the binding of kabiramide D to actin, which had previously been treated with other actindepolymerizing agents, showed that the actin-binding site for kabiramide D was the same as that for bistheonellide A, but not those for latrunculin A and cytochalasin D.View full abstractDownload PDF (2884K) -
Takako Shimizu, Sei-ichi Ishiguro, Makoto Tamai1998 Volume 123 Issue 5 Pages 953-958
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt is known that exogenous 11-cis-retinol inhibits the recovery of photosensitivity of bleached rod outer segments (ROS) and 11-cis-retinol exists in the interphotorecepter matrix. We examined the conversion of 11-cis-retinol with bovine ROS. ROS was incubated with 11-cis-retinol under dim red light. Retinoids were extracted from the reaction mixture with hexane and analyzed by HPLC coupled with a fluorescence spectrophotometer. Isomerization of 11-cis-retinol to all-trans-retinol was observed in the presence of ROS. This isomerization was not suppressed by heat treatment and did not have stereospecificity. In addition, we incubated purified rhodopsin and phospholipids extracted from ROS with 11-cis-retinol. Rhodopsin was found to isomerize 11-cis-retinol to all-trans-retinol as well as ROS, but phospholipids did not. In contrast, the phospholipids inhibited the isomerization of 11-cis-retinol to all-trans-retinol by the purified rhodopsin. Commercially available phospholipids, especially phosphatidylserine, also inhibited the isomerization. Our results suggest that rhodopsin has activity for the isomerization of 11-cis-retinol to all-trans-retinol and may play an important role in the detoxification of 11-cis-retinol in the ROS.View full abstractDownload PDF (705K) -
Yasuhiro Isashi, Toshiyuki Yamashita, Shigeharu Nagasawa, Kumiko Tanak ...1998 Volume 123 Issue 5 Pages 959-967
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously shown that the ligand-binding activity of type II Fc receptor for IgG (FcγRIIB) on guinea pig peripheral blood polymorphonuclear leukocytes is very low and dramatically increases after treatment of the cells with proteolytic enzymes. In the present study, we analyzed the mechanism of this augmentation. We found that the protease treatment failed to enhance the binding of monomeric IgG to FcγRIIB, increased the binding of small immune complexes (IC) prepared under antigen-excess conditions only modestly, but markedly enhanced the binding of large IC prepared under antibody-excess conditions. These results suggest that proteolysis increases the ligand-binding avidity but not the intrinsic affinity of FcγRIIB. Confocal laser scanning microscopy revealed that the mobility of FcγRIIB on the cell surface was increased after protease treatment. In addition, transfection experiments indicated that the effect of proteolysis on IC binding to CHO cells expressing guinea pig FcγRIIB was strongly dependent on the receptor density. Finally, we demonstrated that the transmembrane and cytoplasmic domains of FcγRIIB were not involved in the proteolysis-induced augmentation of IC binding. Together our results suggest that the mobility of FcγRIIB, which may be restricted due to the association of the ectodomain of the receptor with unknown membrane proteins, is enhanced by proteolysis, allowing the receptors to bind multivalent ligands more readily and hence with higher avidity.View full abstractDownload PDF (2087K) -
Hisanobu Shibata, Mariko Fukushi, Atsuko Igarashi, Yoshio Misumi, Yuki ...1998 Volume 123 Issue 5 Pages 968-977
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have studied the biosynthesis and intracellular transport of tissue-nonspecific alkaline phosphatase (TNSALP) transiently expressed in COS-1 cells. Mutations were introduced into TNSALP to examine the effects of a single amino acid substitution on the activity and biosynthesis of TNSALP. The cells expressing wild-type TNSALP exhibited more than 200-fold higher alkaline phosphatase activity than untransfected ones. Pulse-chase experiments showed that TNSALP was synthesized as a 66-kDa endoglucosaminidase H (Endo H)-sensitive form and converted to EndoH-resistant forms with heterogenous molecular masses (_??_80 kDa), which finally appeared on the cell surface as judged by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC). In contrast, a TNSALP with a Glu218→Gly mutation exhibited no phosphatase activity at all and the 66-kDa Endo H-sensitive form was the only molecular species throughout the chase in the transfected cells. In accordance with this finding, digestion with PI-PLC and immunofluorescence observation confirmed that this mutant was never expressed on the cell surface. Another mutant with a Ala162→Thr substitution, which naturally occurs in association with a lethal hypophosphatasia, exhibited a low activity and only a small fraction of the 66-kDa form acquired Endo-H resistance and reached the cell surface. Since the wild-type and the mutant TNSALPs were labeled with [3H] ethanolamine, a component of glycosylphospha-tidylinositol (GPI), it is unlikely that the impaired intracellular transport of the two mutants is due to a failure in their modification by GPI. Interestingly, the 66-kDa Endo H-sensitive form of the TNSALP mutants but not that of the wild-type, was found to form an interchain disulfide-bonded high-molecular-mass aggregate within the cells. These results suggest that impaired intracellular transport of the TNSALP (Ala162→Thr) molecule caused by its aggregation is the molecular basis for the lethal hypophosphatasia carrying this mutation.View full abstractDownload PDF (5455K) -
David Oxley, Sharon L. A. Munro, David J. Craik, Antony Bacic1998 Volume 123 Issue 5 Pages 978-983
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSS-RNases are the stylar products of the self-incompatibility (S)-locus in solanaceous plants (including Nicotiana alata), and as such, are involved in the prevention of self-pollination. All cDNA sequences of S-RNase products of functional S-alleles contain potential N-glycosylation sites, with one site being conserved in all cases, suggesting that N-glycosylation is important in self-incompatibility. In this study, we report on the structure and localization of the N-glycans on the S7-allele RNase of N. alata. A total of nine N-glycans, belonging to the high-mannose- and xylosylated hybrid-classes, were identified and characterized by a combination of electrospray-ionization mass-spectrometry (ESI-MS), 1H-NMR spectroscopy, and methylation analyses. The glycosylation pattern of individual glycosylation sites was determined by ESI-MS of the glycans released from isolated chymotryptic glycopeptides. All three N-glycosylation sites showed microhetero-geneity and each had a unique complement of N-glycans. The N-glycosylation pattern of the S7-RNase is significantly different to those of the S1- and S2-RNases.View full abstractDownload PDF (682K) -
Naoya Hatano, Pamela Eversole-Cire, Anne C. Ferguson-Smith, Peter A. J ...1998 Volume 123 Issue 5 Pages 984-991
Published: 1998
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mouse insulin-like growth factor II (IGF-II) gene is subject to parental imprinting and is predominantly expressed from the paternal chromosome. This allele-specific expression is modified further by cell type, developmental stage, and growth conditions. We show that the ratio of the three major IGF-II mRNAs, each produced from a distinct promoter, is consistent in a variety of tissues and cells representing different modes and phases of the complex regulation. Nuclear run-on assays show that the major changes in total IGF-II mRNA level occur at the level of transcription. Moreover, a targeted disruption of the endoderm-specific enhancers, located 90 kb away from the gene, affects all promoters. The dependency of the promoters on distal enhancers is also shown by transgenesis experiments. Our findings suggest that enhancer-dependent, locus-wide mechanisms play a major role in the coordinate regulation of the multiple IGF-II promoters.View full abstractDownload PDF (5010K)
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