The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 122 , Issue 4
Showing 1-31 articles out of 31 articles from the selected issue
  • Tsuyoshi Shirai, Takashi Yamane, Teruo Hidaka, Kazuo Kuyama, Atsuo Suz ...
    1997 Volume 122 Issue 4 Pages 683-685
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The catalytic domain of an alkaline endo-1, 4-β-glucanase (family A) isolated from Bacillus sp. KSM-635 (Mr=40.2 kDa) was crystallized using the hanging drop vapor diffusion method. Two different crystal forms were obtained. Form 2 crystals (trigonal space group R3 with cell dimensions of a=b=111.9 and c=207.1 Å in a hexagonal lattice) were found to be more stable than form 1 ones upon X-ray irradiation. A full data set for form 2 crystals has been collected up to 3.3 Å resolution.
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  • Xiao-Bo Tang, Paul Dallaire, David W. Hoyt, Brian D. Sykes, Maureen O' ...
    1997 Volume 122 Issue 4 Pages 686-690
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    TGF-α, a 50 amino acid growth factor containing 3 disulfide bonds, was fused to the N-terminal domain of the pill protein of fusN, a derivative of phagemid fd-tet, to form a TGF-α phage. The fusion phage showed binding activity to epidermal growth factor receptor (EGFR). A library of approximately 4×107 variants of TGF-α was generated with substitutions of total of 10 amino acids located in the C-loop region. This C-loop subdomain of TGF-α consists of a small antiparallel double hairpin structure involving interactions between intra-polypeptide segments. Mutants isolated from the phage library with greatly increased binding affinity were selected through panning with A431 cells (a cell line expressing an elevated number of EGFRs). Following two rounds of stringent selection, variant phages with higher binding affinity than wild type TGF-α were identified and the phage DNAs were sequenced for the alignment analysis. Absolute selection at position 42 as Arg, preferential selection at position 38 and 45 as Tyr or Phe with aromatic side chain and selection at position 41 with acidic residues, were obtained. Although an amino acid residue with smaller side chain at position 35 and one with larger side chain at position 36 were preferred, the steric hindering of the structure in side chains was minimized between these adjacent amino acids.
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  • Shigemi Yoshioka, Ge-Hong Sun-Wada, Nobuhiro Ishida, Masao Kawakita
    1997 Volume 122 Issue 4 Pages 691-695
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    In our previous study, we demonstrated that UDP-galactose transporter cDNAs (hUGT1 and hUGT2) were able to complement the genetic defect of murine Had-1 cells that were deficient in the UDP-galactose transporter, and that the microsomal vesicles isolated from Had-1-transformants, which were obtained through transfection with these cDNAs, had recovered the ability to uptake UDP-galactose [Ishida, N. et al. (1996) J. Biochem. 120, 1074-1078]. In this report, we describe the preparation of peptide antibodies that recognize the hUGT isozymes, and the detection of hUGT proteins expressed in the transformants. The occurrence of the endogenous hUGT1 protein in HeLa cells was also detected. Using the hUGT1-specific antibodies, the subcellular localization of hUGT1 in the Golgi membrane was demonstrated by immunofluorescence microscopy and subcellular fractionation. These studies led us to develop a simple procedure, based on Percoll density gradient centrifugation, for preparing functional Golgi vesicles from the hUGT1-transformed Had-1 cells, that will facilitate future biochemical analyses of the UDP-galactose transporter for the elucidation of its structure-function relationship.
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  • Takashi Yamazaki, Takeshi Hasebe, Junko Shouguchi, Hitoshi Amano, Susu ...
    1997 Volume 122 Issue 4 Pages 696-702
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The Basidiomycete ras gene possesses a pyrimidine-rich stretch (CT-motif) with a short (7 bases) mirror repeat in which its major transcription start point is contained. To analyze the tertiary structure induced by the CT/AG-biased sequence and its effect on gene expression in supercoiled plasmids in Escherichia coli, the DNA fragment containing the ras CT/AG sequence was inserted into the EcoRI site on pBR322 in both orientations and the resulting pBR322 derivatives, named pBR-CT [ras] and pBR-invCT [ras] were introduced into E. coli strains DM800 (ΔtopA gyrB225) and JM109 (topA+ gyrA96). In pBR-CT [ras] the pyrimidine-rich sequence is on the pBR322 tetracycline-resistance gene (tet)-coding strand and in pBR-invCT [ras] the complementary purine-rich sequence is on this strand. DNAs of pBR-CT [ras] and pBR-invCT [ras] isolated from DM800 were frequently cleaved with single-strand-specific Sl nuclease within the CT/AG sequence, showing the formation of extended open structure. Compared with those carrying pBR322, DM800 and JM109 carrying pBR-CT [ras] showed much higher levels of tetracycline resistance (Tcr), while both strains carrying pBR-invCT [ras] showed clearly lower levels of Tcr. pBR-CT [ras] and pBR-invCT [ras], however, conferred reduced activity of β-lactamase on DM800 and JM109. pBR-CT [ras] derivatives lacking the counterpart of the mirror repeat did not form the Sl-cleavable open structure within the CT/AG sequence and conferred pBR322-like Tcr and β-lactamase activity. The tertiary structure formed in the CT/AG sequence via the mirror repeat was suggested to affect the expressions of pBR322-tet and -bla genes.
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  • Akira Kobayashi, Keiko Numayama-Tsuruta, Kazuhiro Sogawa, Yoshiaki Fuj ...
    1997 Volume 122 Issue 4 Pages 703-710
    Published: October 01, 1997
    Released: November 18, 2008
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    A heterodimer of AhR (aryl hydrocarbon receptor) and Arnt (AhR nuclear translocator) conveys a transactivation signal of aromatic hydrocarbons such as 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the genes for a group of drug-metabolizing enzymes. This inducible expression of the genes is inhibited by adenovirus E1A, suggesting that CBP/p300 is somehow involved in the transactivation of the genes by the AhR and Arnt heterodimer. Yeast and mammalian two hybrid systems revealed that CBP/p300 interacted with the transactivation domain of Arnt, but not with that of AhR, via the CREB-binding domain. The pull down assay using GST-Arnt hybrid protein confirmed the interaction between Arnt and CBP/p300. Considering these results and that Arnt or Arnt2 functions as a common partner in the formation of transcriptional regulators with other bHLH/PAS proteins such as AhR, HLF, and HIF-1α, the possibility arises that CBP/p300 is extensively involved as a coactivator in the transactivation process by bHLH/PAS (a conserved sequence motif among Per, Arnt, and Sim) heterodimer transcription factors through interaction with Arnt or Arnt2.
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  • Masataka Horiuchi, Yasuyuki Kurihara, Masato Katahira, Tadakazu Maeda, ...
    1997 Volume 122 Issue 4 Pages 711-716
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Max is a basic region/helix-loop-helix/leucine zipper (b/HLH/Z) protein that forms a hetero-complex with the Myc family proteins Myc, Mad, and Mxil, and a homo-complex with itself. These complexes specifically bind to double-stranded DNA containing CACGTG sequences. Here, we report on the structural properties in aqueous solution of a 109-aminoacid protein, Max110, corresponding to the N-terminal domain of Max containing the b/ HLH/Z motif (residues 2-110), as characterized by combined use of circular dichroism (CD) and sedimentation equilibrium experiments. The results showed that the α-helical content of Max 110 increases with increasing protein concentration. The sedimentation equilibrium data indicated that Max110 exists as a monomer at low protein concentration, and forms a dimer at high protein concentration. Further increases in the α-helical content of Max110 occur upon addition of DNA with the CACGTG recognition sequence. Thus, dimerization and binding to DNA of Max both favor an increase of the α-helical content.
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  • Shwu-Bin Lin, Sung-Hwa Hsieh, Hui-Lun Hsu, Ming-Yang Lai, Lou-Sing Kan ...
    1997 Volume 122 Issue 4 Pages 717-722
    Published: October 01, 1997
    Released: November 18, 2008
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    Insulin-like growth factor II (IGF-II) is expressed in many developing embryonic tissues and is involved in mammalian growth and development. After birth, serum IGF-II is mainly produced by liver cells. Many reports have indicated that IGF-II is overexpressed in some hepatocellular carcinoma (HCC) tissue. These findings imply the possible importance of this growth factor in carcinogenesis. We screened four human HCC cell lines and three rat HCC cell lines and found that HuH-7 and HepG2 cells produced fivefold more intracellular IGF-II than the other cell lines. Experimental data indicate that IGF-II functions through the intracrine mode for HuH-7 cells. To study whether the overexpression of IGF-II is significant for the growth of HCC or only a consequence of HCC development, we used antisense oligodeoxynucleotides (ATON) to arrest the translation of IGF-II mRNA, and then measured the effects on cell growth. We found that the production of IGF-II was suppressed by ATON, and the decrease of IGF-II resulted in growth inhibition of HuH-7 and HepG2. ATON had no effect on the other tested cell lines, which produced lower levels of IGF-II. The growth inhibition was mainly attributed to a decrease of cell proliferative activity. The results indicate that the IGF-II-overproducing cell lines do depend on IGF-II for growth, and ATON of IGF-II can selectively inhibit the growth of these cells. ATON may be a potential therapeutic agent for this type of HCC in vivo.
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  • Norio Takizawa, Masakazu Komatsu, Yusuke Mizuno, Takeshi Kawamura, Mas ...
    1997 Volume 122 Issue 4 Pages 723-729
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    To elucidate the roles of serine/threonine protein phosphatases PP1 and PP2A in the morphological changes of B-lymphocytes during development and in immune responses, we investigated alterations of protein levels of catalytic subunits of PP1 and PP2A and regulatory subunits of PP1 including M130/M133, inhibitor-1 (I-1) and inhibitor-2 (I-2) in B-cell lines at different maturational stages and during their aggregation induced by phorbol myristate acetate (PMA). The protein levels of PP1δ and/or M130/M133 were significantly lower in B-cell lines without pseudopods, WEHI-231, BAL-17, Daudi, and CESS, than in those with pseudopods, Bcl.1, A20, M12, and SKW6.4, whereas the amounts of PP1α and PP2A were similar among them. During aggregation of A20 and CESS cells induced by PMA, an activator of PKC, the amount of PP1δ was progressively decreased, and this decrease was blocked by H7, an inhibitor of PKC. The amount of PP1α was constant under these conditions. Okadaic acid, an inhibitor of PP1 and MA, also induced aggregation of A20 cells at concentrations sufficient to inhibit PP1, but not at lower concentrations that inhibit PP2A alone. These results suggest that myosin light chain phosphatase composed of PP1δ and M130/M133 is involved in the maintenance and regulation of cytoskeletal structures in B-lymphocytes.
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  • Norio Takizawa, Yusuke Mizuno, Masakazu Komatsu, Shu-Ichi Matsuzawa, T ...
    1997 Volume 122 Issue 4 Pages 730-737
    Published: October 01, 1997
    Released: November 18, 2008
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    In response to stimulation of B-cells through cell surface IgM, the activity of the serine/ threonine protein phosphatase PP1, but not PP2A, was transiently decreased and reached a minimum 10-20 min after the stimulation. The decrease was more profound in the immature B-cell line WEHI-231, than in the mature B-cell line BAL-17. Under these conditions, PP1α, an isoform of PP1, showed unique alterations in the patterns of several spots with distinct isoelectic points in the Western blot after two-dimensional electrophoresis, whereas another isoform, PP1δ, did not show any alteration. PP1γ1 and PP1γ2 were not detected in B-cells. Similar alterations in these spots were observed in B-cells stimulated by PMA. When partially purified PP1 consisting of PP1α and PP1β was incubated with [γ-32P] ATP and PKC, radioactive spots of PP1α could be detected, but no spot of PP1δ was detected. Because differences in sequence among PP1 isoforms are mostly restricted to their C-terminals, phosphorylation rates of the C-terminal peptides containing the PKC-phosphorylation motif were compared. The C-terminal peptide of PP1α is a better substrate for PKC than those of PP1γ1 and PP1γ2, and is phosphorylated at the serine residue corresponding to Ser-325 of PP1α. The corresponding C-terminal region of PP1δ does not contain the phosphorylation site. On the other hand, there was a large difference in subcellular distribution of PP1δ, but not PP1α, between immature and mature B-cells. From these results, it was strongly suggested that PP1α is involved, via phosphorylation by PKC, in the regulation of signal transduction in response to the stimulation of B-cells through cell surface IgM.
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  • Miwako K. Homma, Motoo Yamasaki, Shinobu Ohmi, Yoshimi Homma
    1997 Volume 122 Issue 4 Pages 738-742
    Published: October 01, 1997
    Released: November 18, 2008
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    It has been demonstrated that the phospholipase C-γ (PLC-γ) molecule contains within it a phospholipase C inhibitor (PCI) region and that synthetic peptides based on the sequence of this region (PCI peptides) suppress the enzymatic activity of PLC isoforms [Y. Homma and T. Takenawa (1992) J. Biol. Chem. 267, 21884-21889]. In order to improve the permeability of the plasma membrane to PCI peptides, we synthesized myristoylated PCI peptides, myr-GLYRKAMRLRYPV [myr-PCI(Y)] and myr-GLFRKAMRLRFPV [myrPCI(F)], which are identical except for the replacement of the two tyrosine residues in myr-PCI(Y) by phenylalanines in myr-PCI(F), and examined their inhibitory activity on PLC enzymes in vitro and in vivo. This fatty acid modification potentiated the inhibitory activity of the original PCI peptides and both myr-PCI(Y) and myr-PCI(F) suppressed the PIP2-hydrolyzing activity of purified PLC isoforms in vitro. The K1 values of myr-PCI(Y) and myr-PCI(F) for purified PLC-γ1 were 3.5 and 55μM, respectively. Myr-PCI(Y) at concentrations in the sub-micromolar range significantly suppressed IP3 formation induced by EGF, PDGF, bombesin, or serum in Swiss 3T3 cells. Furthermore, myr-PCI(Y) also strongly inhibited cell proliferation induced by these stimuli. The inhibitory effect on IP3 formation and proliferation of myr-PCI (F) was much less potent than that of myrPCI (Y). These results suggest that myristoylated PCI peptides could be applied to living cells as specific inhibitors of PLC signaling pathways and that PLC pathways are at least in part required for growth in Swiss 3T3 cells.
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  • Yoshihide Sorimachi, Kazuki Harada, Takaomi C. Saido, Tomio Ono, Sei-i ...
    1997 Volume 122 Issue 4 Pages 743-748
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The activities of calpain and its endogenous inhibitor, calpastatin, were measured in the soluble fraction of perfused rat heart after ischemia for 5-20min and reperfusion for up to 30 min. The method for m-calpain measurement was modified: washing of the DEAE-cellulose column with 0.18M NaCl instead of 0.15M NaCl increased the m-calpain activity 12.5-fold. Ischemia for 20min followed by reperfusion for 30min did not affect the m-calpain activity but decreased the calpastatin activity. m-Calpain was enriched in the nucleus-myofibril fraction but was not further translocated on ischemia-reperfusion. μ-Calpain was below the limit of detection on immunoblotting or casein zymography, but its mRNA was substantially expressed, as detected on Northern blotting. Casein zymography also revealed a novel Ca2+-dependent protease without the typical characteristics of μ- or m-calpain. The immunoblotting of myocardial fractions showed that calpastatin was proteolyzed on ischemia-reperfusion. The calpastatin proteolysis was suppressed by a calpain inhibitor, Ac-Leu-Leu-norleucinal. Calpastatin may sequester calpain from its substrates in the normal myocardium, but may be proteolyzed by calpain in the presence of an unidentified activator in the early phase of calpain activation during ischemia-reperfusion, resulting in the proteolysis of calpastatin and then other calpain substrates.
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  • Nobuko Kakiuchi, Yasumasa Komoda, Makoto Hijikata, Kunitada Shimotohno
    1997 Volume 122 Issue 4 Pages 749-755
    Published: October 01, 1997
    Released: November 18, 2008
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    To study the character of the hepatitis C virus (HCV) encoding serine proteinase and to search for inhibitors, a practical in vitro assay system using the purified enzyme and synthetic peptide substrates was established. The enzyme used was expressed in Escherichia coli as a fusion form with protein tags and purified to apparent homogeneity by single-step affinity chromatography. The purified enzyme exhibited proteolytic activity with pH optima of around eight, and the addition of NS4A fragments increased the activity as well as the thermal stability of the enzyme. The activity was inhibited by EDTA and some divalent ions, i.e., copper and zinc, though calcium, magnesium, and manganese were stimulative both in the presence and absence of the NS4A fragment. None of the common protease inhibitors, including serine protease inhibitors, effectively inhibited the activity. Based on the kinetic parameters of the cleavage reaction of the synthetic 20 mer peptides corresponding to the three cleavage sites, NS4A/4B, NS4B/5A, and NS5A/5B, the peptide with the NS5A/5B junction was found to be the most efficient substrate. Analysis of the minimal peptide substrate of NS5A/5B indicated that 5 to 7 amino acids on both sides of the junction were required for efficient cleavage. These findings are expected to contribute to the search for a proteinase inhibitor.
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  • Norihiko Tabuchi, Kenji Akasaki, Tomoko Sasaki, Naoko Kanda, Hiroshi T ...
    1997 Volume 122 Issue 4 Pages 756-763
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    We previously have purified and characterized a major lysosomal membrane glycoprotein termed LGP85 (LIMP II) in rat liver lysosomes. In this study, LGP85 in mouse liver lysosomes was identified and characterized by biochemical and molecular biological methods. Lysosomal membranes were isolated from murine liver by differential centrifugation. LGP85 was present in the lysosomal membrane fraction from mouse liver in a comparable amount to another lysosomal membrane glycoprotein, lamp-2. Mouse LGP85 (M-LGP85) from liver lysosomal membranes exhibited an Mr of 80, 000 on SDS-PAGE, which is smaller by 5, 000 than that of rat LGP85 (R-LGP85). M-LGP85 was immunochemically detected in the extracts of brain, heart, lung, liver, and kidney. A cDNA encoding M-LGP85 was cloned from mouse liver cDNA library. The primary protein structure deduced from a nucleotide sequence of M-LGP85 cDNA indicated that M-LGP85 consists of 478 amino acids with Mr of 54, 069. M-LGP85 showed 93.3 and 86.0% sequence similarities to its rat and human counterparts in amino acids, respectively. M-LGP85 contains 11 potential N-glycosylation sites which are heavily glycosylated, resulting in the increased Mr of M-LGP85 present in the mouse liver lysosomes. It is likely that M-LGP85 traverses the lysosomal membrane twice, with an NH2-terminal transmembrane domain, and another hydrophobic domain near the COOH-terminus. M-LGP85 has a protruding COOH-terminal cytoplasmic tail consisting of amino acid residues including the leucine-isoleucine sequence shown to be the lysosomal targeting signal of R-LGP85 and human LGP85 (H-LGP85). The high level of expression of M-LGP85 in the lysosomal membrane, the high structural similarities among M-, R-, and H-LGP85, and the occurrence of M-LGP85 in all the mouse tissues examined suggest the essential and constitutive function of LGP85 in lysosomes.
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  • Junshi Sakamoto, Yoshimi Handa, Nobuhito Sone
    1997 Volume 122 Issue 4 Pages 764-771
    Published: October 01, 1997
    Released: November 18, 2008
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    Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated several mutants from B. stearothermophilus defective in the caa3-type oxidase activity [Sakamoto, J. et al. (1996) FEMS Microbiol. Lett. 143, 151-158]. A novel oxidase was isolated from membrane preparations of one of the mutants, K17. The oxidase was composed of two subunits with molecular masses of 56 and 19 kDa, and contained protoheme IX, heme 0, heme A, and Cu in a ratio of 1:0.7:0.2:3. CO difference spectra indicate that the high-spin heme is mainly heme O. These results suggest that the enzyme belongs to the heme-copper oxidase family and is a cytochrome b(o/a)3-type oxidase, whose high-spin heme is mainly heme O and partly heme A. The enzyme oxidized cytochrome c-551, which is a membrane-bound lipoprotein of thermophilic Bacillus. The turnover rate of the activity (Vmax=190s-1) and its affinity for cytochrome c-551 (Km=0.15 μM) were much higher than those for yeast and equine heart cytochromes c. The oxidase activity was enhanced by the presence of salts and inhibited by sodium cyanide with a Ki value of 19μM. The enzyme kinetics suggests that cytochrome c-551 is the natural substrate to this oxidase. Furthermore, the oxidase had similarity to cytochrome ba3-type oxidase from Thermus thermophilus in the subunit composition, partial amino acid sequence, and prosthetic groups, and therefore is suggested to belong to a unique subgroup of the hemecopper oxidase family together with the Thermus enzyme and archaeal oxidases such as Sulfolobus SoxABCD.
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  • Shaoliang Li, Shigemi Norioka, Fumio Sakiyama
    1997 Volume 122 Issue 4 Pages 772-778
    Published: October 01, 1997
    Released: November 18, 2008
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    α-Lytic protease (alp) was purified from a bacteriolytic agent, Achromopeptidase from Achromobacter lyticus M497-1, and has been shown to possess staphylolytic activity. Cleavage sites of this enzyme on the peptidoglycan of Staphylococcus aureus were determined by N-terminal amino acid sequence and amino acid composition analyses. Alp cleaved the N-acetylmuramoyl-L-alanine amide bond, the junction between the polysaccharide and peptide moieties, in addition to the D-Ala-Gly and Gly-Gly peptide bonds, implying that this enzyme recognizes the amino acid of D-configuration at the P1 site and possesses N-acetylmuramoyl-L-alanine amidase activity. However, alp could not cleave the D-Ala-Gly peptide bond in a synthetic peptide, suggesting that this hydrolytic activity of alp is peptidoglycan-specific. The results obtained from different consecutive actions of alp and glycosidase on S. aureus peptidoglycan indicate that the presence of polysaccharide in the peptidoglycan is necessary for the bacteriolytic activity of alp.
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  • Kai Huang, Shuji Takahara, Toshiaki Kinouchi, Masami Takeyama, Tetsuo ...
    1997 Volume 122 Issue 4 Pages 779-787
    Published: October 01, 1997
    Released: November 18, 2008
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    Alanyl aminopeptidase (AAP) was purified to homogeneity from human seminal plasma. The calculated molecular weight of the purified enzyme was approximately 137, 000±5, 000 from light scattering, 140, 000 (main) and 137, 000 (minor) from non-denatured PAGE and 153, 000 from SDS-PAGE in the absence or presence of 2-mercaptoethanol (2-ME). These findings suggest that the enzyme is monomeric in form in human seminal plasma. The enzyme hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order of Kcat/Km values of AAP at optimal pH (pH 7.5) was Ala->Lys-Ala-≥Met->Leu->Phe->Arg-≥Arg-Arg->Lys->Gly-MCAs. AAP was potently inhibited by bestatin, leuhistin, actinonin, amastatin, and 1, 10-phenanthroline. These findings suggest that AAP is an aminopeptidase. We determined that the amino acid sequence of the first 22 residues of the enzyme was Ser1-Thr-Thr-Pro-Ser5-Ala-Ser-Ala-Thr-Thr10-Asn-Pro-Ala-Ser-Ala15-Thr-Thr-Leu-Asp-Gln20-Ser-Lys-. This sequence was completely coincident with that downstream of the transmembrane site of human intestinal alanyl aminopeptidase N (CD13). We also isolated cDNA encoding AAP from human prostate cDNA library, sequenced its structure, and confirmed human seminal plasma AAP to be identical with alanyl aminopeptidase N. We postulated that native human seminal plasma alanyl aminopeptidase is released into the seminal plasma after the specific site is cleaved by elastase or an elastase-like enzyme. The enzyme level in human seminal plasma determined by single radial immunodiffusion was 5.2±3.2 mg/100ml (mean±SD, n=20) in individuals 20-47 years of age. AAP was immunohistochemically stained in the luminal site-cell membrane of epithelial cells in the prostatic gland and ductuli efferentes of the testis.
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  • Sailaja S. Seeram, Kazumi Hiraga, Kohei Oda
    1997 Volume 122 Issue 4 Pages 788-794
    Published: October 01, 1997
    Released: November 18, 2008
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    Streptomyces metalloproteinase inhibitor (SMPI) is a small proteinaceous inhibitor which inhibits metalloproteinases such as thermolysin (K1=1.14×10-10M). When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis. To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action. The native inhibitor was resynthesized from the modified inhibitor (K1=2.18×10-8M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25°C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase inhibitors. Temporary inhibition was observed when the native inhibitor and thermolysin were incubated at a 1:100(mol/mol) enzyme-inhibitor ratio at 37°C. SMPI showed temporary inhibition towards all the enzymes it inhibited. The inhibitory spectrum of SMPI was analyzed with various metalloproteinases based on the K1 values and limited proteolysis patterns. Pseudomonas elastase and Streptomyces griseus metalloproteinase II formed more stable complexes and showed much lower K1 values (_??_2pM) than thermolysin. In the limited proteolysis experiments weak inhibitors were degraded by the enzymes. SMPI did not inhibit almelysin, Streptomyces caespitosus neutral proteinase or matrix metalloproteinases. SMPI specifically inhibits metalloproteinases which are sensitive to phosphoramidon.
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  • Mingxu Xu, Shigehiro Osada, Masayoshi Imagawa, Tsutomu Nishihara
    1997 Volume 122 Issue 4 Pages 795-801
    Published: October 01, 1997
    Released: November 18, 2008
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    The nuclear factor 1 (NF1) protein family functions as a cellular transcription factor as well as an adenovirus DNA replication factor. This family consists of four subtypes, NFI-A, NFI-B, NFI-C, and NFI-X, each encoded by a different gene. Each subtype possesses different isoforms generated by alternative splicing. To date, only a porcine NFI-C gene has been cloned, and the gene structures of the other NF1 proteins have not yet been identified. We recently isolated four kinds of NFI-A cDNA clones from the rat liver. To gain additional insight into the structure of NFI-A, we isolated the rat NFI-A gene. This gene is composed of 11 exons spanning over 70 kb. All of the exon/intron boundaries are consistent with the GT/AG rule, and consensus sequences surrounding the splice boundaries are also found. The 5'-flanking region lacks a canonical TATA box, but contains several GC-box and AP2 binding sites. A 5'-rapid amplification of cDNA end analysis indicated that the transcription of the NFI-A gene is initiated at multiple sites. We also found conservation in the genomic structure between the rat NFI-A and the porcine NFI-C, suggesting that duplication of an ancestral gene occurred rather recently to produce the NFI-A and NFI-C genes.
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  • Hayao Taguchi, Takahisa Ohta, Hiroshi Matsuzawa
    1997 Volume 122 Issue 4 Pages 802-809
    Published: October 01, 1997
    Released: November 18, 2008
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    For Lactobacillus pentosus D-lactate dehydrogenase, the binding of 2-ketoacids is markedly stabilized through interactions between the protonated imidazole of His-296, an acid/base catalyst of the enzyme, and the carbonyl oxygen of 2-ketoacids. The replacement of Arg-235 with Gln destabilized the inhibitory binding of oxamate much more than that of formate, acetate, or propionate, and the Arg to Lys substitution specifically diminished only oxamate binding. On the other hand, replacement of a conserved Glu, Glu-264, with Gin severely impaired the enzyme activity and markedly reduced affinity to 2-keto acids. The pH dependence of the oxamate inhibition revealed that the substitutions of Arg-235 and Glu-264 induced a great loss of the imidazole-carbonyl interaction. However, replacement of Glu-264 with Asp, another acidic amino acid, affected the enzyme function less than the Glu to Gln substitution. In addition, both the Arg-235 and Glu-264 substitutions induced marked increases in the primary isotope effect on the catalysis, suggesting that these amino acids stimulate the hydrogen transfer step in the catalysis. We concluded, therefore, that the guanidino and carboxyl groups of Arg-235 and Glu-264, respectively, cooperatively promote the essential imidazole-substrate interaction, enhancing the substrate binding and catalysis.
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  • Yong Ma, Hisatoshi Shida, Toshisuke Kawasaki
    1997 Volume 122 Issue 4 Pages 810-818
    Published: October 01, 1997
    Released: November 18, 2008
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    Human mannan-binding proteins (MBPs) occur in two forms, serum MBP (S-MBP) and liver MBP (L-MBP), both of which are synthesized in the liver from a single form of human MBP mRNA. To investigate further the mechanisms of post-translational modification, molecular assembly and differentiation of S-MBP and L-MBP in vitro, we expressed a full-length human MBP cDNA in three human hepatoma cell lines, using the vaccinia virus expression system. The expression of human MBP cDNA reproduced the native MBP differentiation of S-MBP and L-MBP in human hepatoma cells. The recombinant S-MBP was secreted into the medium, and the recombinant L-MBP retained in the cells. The former had the ability to activate the complement through the classical or lectin pathway but the latter did not. Furthermore, one notable difference between the two MBPs was the degree of oligomerization through interchain disulfide bonds between subunits. In addition, we showed that both S-MBP and L-MBP undergo hydroxylation of lysine and proline residues in collagen-like sequences, and that the hydroxylysine is glycosylated to form glucosylgalactosylhydroxylysine (GluGalHyl) and galactosylhydroxylysine (GalHyl). Hydroxylation was required for S-MBP to be assembled into large complexes, the apparent molecular sizes of which were estimated to be 200-1, 300 kDa by SDS-PAGE under non-reducing conditions and gel filtration under non-denaturing conditions. The hydroxylation and subsequent glycosylation and oligomerization were inhibited by α, α'-dipyridyl, an inhibitor of collagen lysyl and prolyl hydroxylases. These results suggested that newly synthesized lectins undergo post-translational modifications unique to the two forms of MBP, S-MBP, and L-MBP, in human hepatocytes and hepatoma cells, and that the collagen-like domains of the MBPs play an important role in promoting molecular assembly.
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  • Jeff C. F. Chen, Rong-Hwa Lin, Hsiou-Chen Huang, Jason T. C. Tzen
    1997 Volume 122 Issue 4 Pages 819-824
    Published: October 01, 1997
    Released: November 18, 2008
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    The oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosins are present in the oil bodies of diverse angiosperms, and classified as high and low Mr isoforms according to their relative molecular masses in each species. In sesame oil bodies, besides the two ubiquitous oleosin isoforms (17 and 15kDa), an additional minor oleosin (15.5kDa) was revealed on Tricine SDS-PAGE. A full-length cDNA fragment was cloned, sequenced and deduced to be a putative oleosin of 15, 446 Da. The gene was constructed in a fusion or non-fusion vector and then over-expressed with different efficiency in Escherichia coli. All three oleosins purified from sesame oil bodies were subjected to immunoassaying using antibodies raised against the over-expressed oleosin. The results confirmed that this gene encodes the sesame 15.5kDa oleosin. Sequence comparisons with other known oleosins revealed that sesame 15.5kDa oleosin does not represent a new oleosin isoform class but may have been derived through gene duplication and truncation of sesame 17kDa oleosin, and possesses the minimal structure of the high Mr oleosin isoform. A conserved amphipathic α-helix is predicted in sesame 15.5kDa oleosin, which may imply a potential biological function associated with this isoform.
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  • Retsu Miura, Chiaki Setoyama, Yasuzo Nishina, Kiyoshi Shiga, Hisashi M ...
    1997 Volume 122 Issue 4 Pages 825-833
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    As an extension of our recent X-ray crystallographic determination of the tertiary structure of D-amino acid oxidase (DAO) [Mizutani, H. et al. (1996) J. Biochem. 120, 14-17], we solved the crystal structure of the complex of DAO with a substrate analog, o-aminobenzoate (OAB). The alignment between flavin and OAB in the crystal structure of the complex is consistent with charge-transfer interaction through the overlap between the highest occupied molecular orbital of OAB and the lowest unoccupied molecular orbital of flavin. Starting with the atomic coordinates of this complex as the initial model, we carried out molecular mechanics simulation for the DAO-D-leucine complex and thus obtained a model for the enzyme-substrate complex. According to the enzyme-substrate complex model, the α-proton is pointed toward N (5) of flavin while the lone-pair of the substrate amino group can approach C (4α) of flavin within an interacting distance. This model as well as DAO-OAB complex enables the evaluation of the substrate-flavin interaction prior to electron transfer from the substrate to flavin and provides two possible mechanisms for the reductive-half reaction of DAO, i.e., the electron-proton-electron transfer mechanism and the ionic mechanism.
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  • Reiko Urade, Tsuneyuki Oda, Hiroshi Ito, Tatsuya Moriyama, Shigeru Uts ...
    1997 Volume 122 Issue 4 Pages 834-842
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The human ER-60 protease cDNA was expressed in Escherichia coli BL21 (DE3) cells using the pET-20b (+) T7 promoter. The recombinant ER-60 protease was obtained in a watersoluble form and purified through four sequential chromatographies. The ER-60 protease contains two CGHC motifs. When an alanine residue was substituted for the N-terminal cysteine residue in both motifs, the protease activity was not lost. However, when the C-terminal cysteine residue in both motifs was replaced by a serine residue, the cysteine protease activity, which was inhibited by p-chloromercuribenzoic acid (pCMB) but not by diisopropyl fluorophosphate (DFP), changed to serine protease activity, which was inhibited by DFP but not by pCMB. These results indicate that the C-terminal cysteine residue (s) of the CGHC motifs may constitute the active site (s) of ER-60 protease. The ER-60 protease has a C-terminal QEDL sequence, which was proved to serve as an ER-retention signal by deletion of the QEDL sequence. However, because QEDL could not serve as the ER-retention signal for protein disulfide isomerase or ERp72, it is suggested that amino acid residue (s) of ER-60 protease, other than the QEDL sequence itself, is complimentarily responsible for the ER retention of this protein.
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  • Susumu Tsunasawa, Yukiko Izu, Masaru Miyagi, Ikunoshin Kato
    1997 Volume 122 Issue 4 Pages 843-850
    Published: October 01, 1997
    Released: November 18, 2008
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    A gene for a methionine aminopeptidase (MAP; EC 3. 4. 11. 18), which catalyzes the removal of amino-terminal methionine from the growing peptide chain on the ribosome, has been cloned from the hyperthermophilic Archaeon, Pyrococcus furiosus, by a novel method effectively using its cosmid protein library, sequenced and expressed in Escherichia coli. The DNA sequence encodes a protein containing 295 amino acid residues with methionine at the N-terminus. From protein analyses of the recombinant protein expressed in E. coli, by using both amino acid sequence analysis from the N-terminus by automated Edman degradation and analyses of molecular masses of the peptides generated by two enzymatic cleavages performed independently, digestions with lysylendopeptidase and Endoproteinase Asp-N, with ionspray mass spectrometry, the primary structure of the protein has been elucidated to be completely identical with that deduced from its DNA sequence. Comparison of the amino acid sequence of P. furiosus MAP (P. f. MAP) with those of other MAPs from Eukarya and Bacteria showed that the protein has a high degree of sequence homology in the stretches surrounding the five cobalt-binding residues fully preserved in all of MAPs determined so far, but P. f. MAP belongs to Type II because it has an extra long insertion of about 60 amino acid residues between the fourth and fifth cobalt-binding ligands, similar to MAPs from human and rat, and to Met-AP2 from Saccharomyces cerevisiae, in comparison to Type I MAPs from Bacteria. Therefore, P. f. MAP seems to be rather close to those from Eukarya, although it is distinct in lacking the N-terminal extension of about 90-150 residues universally found in MAPs from Eukarya. These findings suggest that P. f. MAP is evolutionally located at the Eukarya-Bacteria boundary. The enzyme expressed in E. coli exhibits a considerable thermostability, with a half-life of approximately 4.5h at 90°C and an optimum temperature of around 90°C.
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  • Kiyoshi Nakazawa, Ikuko Takahashi, Yuko Ohno, Masami Sato
    1997 Volume 122 Issue 4 Pages 851-858
    Published: October 01, 1997
    Released: November 18, 2008
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    Corneal stromal cells, prepared from 2-day-old chicks and embedded in collagen gel on an insert dish, were co-cultured with corneal epithelial cells or endothelial cells on a fibronectin-coated companion plate. Cell growth and proteoglycan synthesis, as determined as the incorporation of [35S] sulfate and [3H]glucosamine, were examined for each cell type in both combinations. In comparison with single cultures, growth was affected little, while proteoglycan synthesis was appreciably altered in both combinations. Stromal and epithelial cells stimulated proteoglycan synthesis by each other, while endothelial cells stimulated the synthesis by stromal cells, but stromal cells reduced that by endothelial cells to some extent. Endothelial cells alone synthesized proteoglycans much more actively than did epithelial or stromal cells. Analysis of the radiolabeled proteoglycans revealed that endothelial and epithelial cells had different effects on proteoglycan synthesis by stromal cells: the former tended to increase keratan sulfate synthesis by stromal cells, while the latter tended to increase chondroitin sulfate/dermatan sulfate synthesis. Proteoglycan synthesis in the corneal stroma in vivo might thus be controlled by the balance between the antipodal actions of the epithelial and endothelial cell layers.
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  • Satoshi Akiba, Ryo Nagatomo, Misako Hayama, Takashi Sato
    1997 Volume 122 Issue 4 Pages 859-864
    Published: October 01, 1997
    Released: November 18, 2008
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    The present study investigated the effect of lipid peroxide on the ability of group IIA secretory phospholipase A2 (IIAsPLA2) to hydrolyze platelet membrane phospholipids. The treatment of rabbit platelets with tert-butyl hydroperoxide (BHP) and FeSO4, generated malondialdehyde, an index of lipid peroxidation, and slightly induced arachidonic acid liberation and lysophosphatidylcholine formation. Further addition of IIAsPLA2 purified from rabbit platelets synergistically enhanced the liberation and the formation induced by the oxidizing reagents, although the enzyme alone did not. When the IIAsPLA2 was pretreated with heparin, the enhancement was not observed. The combination of IIAsPLA2 with linoleic acid hydroperoxide and FeSO4 also caused synergistic arachidonic acid liberation. Furthermore, IIAsPLA2 enhanced thromboxane B2 generation and platelet aggregation induced by BHP and FeSO4. The synergistic aggregation was sensitive to indomethacin. With a membrane fraction as a substrate, IIAsPLA2 caused arachidonic acid liberation, which was enhanced in the presence of BHP and FeSO4. These results suggest that modification of membrane phospholipids by oxidizing reagents increases the accessibility of the membrane to platelet IIAsPLA2, and sequential enhancement of arachidonic acid liberation may contribute to the propagation of oxidative stress-induced cellular injury.
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  • Susumu Amano, Hiroshi Kawasaki, Shoichi Ishiura, Seiichi Kawashima, Ko ...
    1997 Volume 122 Issue 4 Pages 865-871
    Published: October 01, 1997
    Released: November 18, 2008
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    Drosophila calpain (Dm-calpain) produced in Escherichia coli has a distinct Ca2+-dependent activity. By using a recombinant Dm-calpain, we searched for its substrates occurring in Drosophila ovaries, where Dm-calpain is expressed. Among a number of major proteins, several proteins in a salt-extracted fraction were selectively degraded by Dm-calpain in a Ca2+-dependent manner. The major substrates were identified by microsequencing the lysylendopeptidase-digested proteins. Three ribosomal proteins, the L5, L7, and L8 subunits of the 60S ribosome, were found to be potential Dm-calpain substrates. In addition, the α subunit of elongation factor-1 (EF-1α), a multi-functional protein involved in both protein synthesis and cytoskeletal regulation, was shown to be cleaved by Dmcalpain into several distinct fragments when expressed as a GST-fusion protein. Endogenous EF-1α in ovary extracts was also shown by western blot analysis to be similarly degraded. These observations suggest that Dm-calpain may regulate protein synthesis and cytoskeletal structure through its degradative or processing activity.
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  • Akiko Nakamura, Masahira Hattori, Yoshiyuki Sakaki
    1997 Volume 122 Issue 4 Pages 872-877
    Published: October 01, 1997
    Released: November 18, 2008
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    Down syndrome is the most common birth defect, and is caused by trisomy 21. We identified a novel gene in the so-called Down syndrome critical region by means of computer-aided exon prediction and subsequent cDNA cloning. The gene, designated as DCRA (Down syndrome Critical Region gene A), consists of eight exons of 3, 252 by in total and encodes a large open reading frame of 297 amino acid residues. The open reading frame shows significant homology to Hβ58, a mouse gene essential for embryogenesis, PEPS, a yeast homologue of 11β58, and an expressed sequence tag of Arabidopsis thariana, suggesting that DCRA has some important function that has been conserved during the course of evolution. DCRA is expressed in most tissues examined, including fetal and adult brain, heart, lung, liver, and kidney. The cDNA of the DCRA mouse homologue, Dcra, was also cloned. It is 2, 157 by long and has an open reading frame of 297 amino acid residues, which shows 92% identity to human DCRA. Dcra is expressed in all the embryo and adult tissues examined.
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  • Yoshiya Ikawa, Ayako Okada, Hiroshi Imahori, Hideaki Shiraishi, Tan In ...
    1997 Volume 122 Issue 4 Pages 878-882
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    P5abc is a large extension of the P5 element characteristic of subclasses IC1 and IC2 of group I introns. It has a conserved region termed the A-rich bulge, that is responsible for activation of the Tetrahymena self-splicing intron. By employing a modified color-colony assay system, we identified four adenosines in the bulge that are responsible for an efficient splicing reaction. On comparison with the X-ray crystal structure of the P4-5-6 domains of the Tetrahymena intron, three adenosines at positions 183, 184, and 186 were found to be identical to those significantly contributing to the formation of its tertiary structure. However, our results show that an adenosine at 187 is involved in the formation of a Watson-Crick base pair with U135, although it forms a Hoogsteen base pair in the crystal structure.
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  • Ayako Takasu, Yoichi Nakanishi, Tokiko Yamauchi, Masayoshi Maeshima
    1997 Volume 122 Issue 4 Pages 883-889
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Vacuolar H+-translocating inorganic pyrophosphatase is a single-protein enzyme and uses a simple substance as an energy donor. Functional domains of the enzyme were investigated by using antibodies specific to peptides corresponding to the putative substrate-binding site (DVGADLVGKVE) in the hydrophilic loop and the carboxyl terminal part. The antibody to the former peptide clearly reacted with the pyrophosphatases of different plant species, and strongly inhibited the hydrolytic activity of the purified enzymes and the proton pumping activity of membrane vesicles. These results indicate that the sequence functions as an actual substrate-binding site and is a common motif. The antibody to the carboxyl terminal part reacted only to the mung bean enzyme, suppressing its hydrolytic and proton pumping activities. The results suggest that the carboxyl terminus is exposed to the cytosol and is close to the catalytic site. H+-Pyrophosphatase hydrolyzed triphosphate and tetraphosphate at low rates. Phytic acid, myo-inositol hexaphosphate, inhibited the enzyme even in the presence of Mg2+. The concentration for 50% inhibition was 0.15mM. The inhibition of H+-PPase by dicyclohexyldiimide was partly reversed by Mg2+. The catalytic site and the membrane topology of the enzyme are discussed.
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  • Takayuki Sugiura, Tomoko Kodaka, Sachiko Kondo, Shinji Nakane, Hironor ...
    1997 Volume 122 Issue 4 Pages 890-895
    Published: October 01, 1997
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The effects of Δ9-tetrahydrocannabinol and 2-arachidonoylglycerol on the intracellular free Ca2+ concentration ([Ca2+]1) in NG108-15 cells were examined in detail. We found that Δ9-tetrahydrocannabinol induces a rapid, modest increase in [Ca2+]i. The response was detectable with 3nM Δ9-tetrahydrocannabinol. We also found that very low concentrations of 2-arachidonoylglycerol elicit a rapid, more prominent increase in [Ca2+]1. Such a response was observed not only in NG108-15 cells but also in N18TG2 cells. The response induced by 2-arachidonoylglycerol in either NG108-15 cells or N18TG2 cells was abolished by pretreatment of the cells with a cannabinoid CB1 receptor-specific antagonist, SR141716A, suggesting that 2-arachidonoylglycerol interacts with the CB1 receptor to induce the response. The results of an experiment involving a phospholipase C inhibitor suggested that phospholipase C is involved in the rapid increase in [Ca2+]1 induced by 2-arachidonoylglycerol. We also found that 1 (3)-arachidonoylglycerol exhibits similar activity to that of 2-arachidonoylglycerol, although its activity at low concentrations was somewhat weak compared with that of 2-arachidonoylglycerol. We further confirmed that several structural analogues of 2-arachidonoylglycerol were less active compared with 2-arachidonoylglycerol. These results suggest that the structure of 2-arachidonoylglycerol is strictly recognized by the CB1 receptor, which raises the possibility that the CB1 receptor is originally a 2-arachidonoylglycerol receptor.
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