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Volume 118 (1995)
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Volume 118, Issue 6
Displaying 1-30 of 30 articles from this issue
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Sen-itiroh Hakomori, Yasuyuki Igarashi1995 Volume 118 Issue 6 Pages 1091-1103
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycosphingolipids (GSLs), cell type-specific markers which change dramatically during ontogenesis and oncogenesis, have been implicated as playing major roles in cellular interactions and control of cell proliferation in multicellular organisms. These functional roles have been partially clarified through two types of studies: (i) Studies of cell recognition mediated by (a) GSL-GSL interaction, (b) GSL-lectin interaction, and (c) GSL-dependent modulation of integrin receptor function. (ii) Studies on control of transmembrane signaling by GSLs and/or sphingosine (Sph) derivatives, with emphasis on effects of these compounds on: (a) signaling pathways initiated by tyrosine kinase-linked receptors; (b) signaling systems mediated by protein kinase C, MAP kinase, other kinases, or cytosolic Ca2+ concentration, leading to changes in cellular phenotypes such as motility, proliferation, differentiation, and apoptosis.View full abstractDownload PDF (1796K) -
Masumi Eto, Toshihiro Ohmori, Masashi Suzuki, Kenji Furuya, Fumi Morit ...1995 Volume 118 Issue 6 Pages 1104-1107
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel phosphorylation-dependent inhibitory protein (IP) of porcine aorta myosin light chain phosphatase (PA-MLCP) was purified to homogeneity from porcine aorta media. The molecular mass of IP was 20 kDa. IP phosphorylated by endogenous potentiating kinase (IP-K) inhibited not only PA-MLCP activity, but also that of the catalytic subunit of protein phosphatase-1. The amino acid sequence of a peptide derived from IP phosphorylated with IP-K, RHARVT*VK, shared one of the consensus sequences phosphorylatable by protein kinase C (PKC), where T* was phosphorylated. IP was phosphorylated by PKC and the phosphorylated product inhibited PA-MLCP as strongly as IP phosphorylated with IP-K.View full abstractDownload PDF (2904K) -
Kazuki Sato, Akiko Wakamiya, Tadakazu Maeda, Kaori Noguchi, Akihiko Ta ...1995 Volume 118 Issue 6 Pages 1108-1111
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStructure-neurotoxicity relationships of amyloid β(25-35) peptide were studied by replacing each amino acid with Ala. In contrast to the general tendency in hydrophobicitytoxicity relationships, replacement of Asn27 yielded a more hydrophobic but less toxic analog and that of Met35 gave a less hydrophobic but more toxic one. Sedimentation profiles and CD spectra indicated that peptide aggregation via intermolecular β-sheet formation is essential for the neurotoxicity of amyloid β(25-35) peptide. The correlation between neurotoxicity and amyloid precursor protein accumulation suggested that the latter is one of the pathways of the neuronal death caused by amyloid β protein.View full abstractDownload PDF (497K) -
Kanenobu Kubo1995 Volume 118 Issue 6 Pages 1112-1117
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSα-Amylase from Aspergillus oryzae was investigated to better understand how disulfidebonded proteins behave during denaturation with dodecyl sulfate. It was previously reported that the α-amylase, when denatured with dodecyl sulfate, forms two species, D1 and D2. In D1, the disulfide bonds remain intact, while in D2, there is a restricted single sulfhydryl/disulfide (SH/SS) exchange reaction. This phenomenon was re-examined as follows: electrophoretic analysis of fragments created with and without modification of a free SH group or disulfide-reduced SH groups; N-terminal sequence analysis of the fragments; reactivity of a free SH group with Ellman reagent. Data from the former two analyses showed that the variability in the electrophoretic patterns results from cleavage at Asp163-Cys164 or Asp282-Cys283, provided that these Cys residues are reduced. Different reactivities of a SH group in D1 and D2 and the appearance of a polypeptide with a nonnative SS pairing in D2 fragments confirmed that the different electrophoretic patterns result from an intramolecular SH/SS exchange. This rearrangement accounts for the variety of electrophoretic patterns observed with D2 amylase and is a result of the creation of a specific free Cys283. Specifically, the variety appears to be due to the breaking of either Cys150-Cys164 or Cys240-Cys283, which in turn leads to the labilization of Asp163-Cys164 or Asp282-Cys283, respectively.View full abstractDownload PDF (3277K) -
María Orfa Rojas, Moisés Wasserman1995 Volume 118 Issue 6 Pages 1118-1123
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe studied the synthesis of calmodulin mRNA and of calmodulin during the asexual cell cycle of Plasmodium falciparum. The expression of the gene was low during the early stages of the erythrocytic asexual cycle. When the parasites were 28h old the level of mRNA increased steadily to reach a peak at the age of 40h. During the last 8h of the cycle a constant decrease in the amount of calmodulin mRNA was observed. The synthesis of the protein followed the expression pattern of the mRNA, but without the final decrease. The average amounts of calmodulin measured were 2.5±0.9ng/108 14-h-old rings, 4.38±0.35ng/108 36-h-old trophozoites, 11.8±1.45ng/108 44-h-old trophozoites, and 19.6±1.9ng/108 48-h-old schizonts. The in vivo stability of mRNA in different developmental stages was evaluated by pulse and chase experiments. The RNA of very mature trophozoites was quickly degraded, in contrast with the high stability shown by the RNA in other stages. The results here suggest that the calmodulin gene expression in Plasmodium does not follow a housekeeping pattern of expression such as in other eukaryotic cells, but that it is regulated at the level of transcription and RNA degradation.View full abstractDownload PDF (2019K) -
Keiichi Nomura, Ikuhiro Yoneda, Takashi Nanmori, Ryu Shinke, Yuhei Mor ...1995 Volume 118 Issue 6 Pages 1124-1130
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties of sulfhydryl (SH) and disulfide (S-S) groups in Bacillus cereus BQ10-S1 Spo III β-amylase have been investigated to clarify their roles in the enzyme action. Two out of three cysteine residues in B. cereus β-amylase were found to form an S-S bond, which was found to be located between Cys91 and Cys99 by the analysis of an S-S containing peptide. The replacement of the soybean β-amylase model around L3 loop 1 revealed that the S-S bond is located at the root of this flexible loop that moves between open and closed forms during catalysis. The analysis of fluorescence labeled peptides revealed that the remaining free SH group was Cys331. Modification of Cys331 with N-ethylmaleimide or p-chloromercuribenzoic acid (PCMB) caused inactivation of the enzyme. The rate constants for the reactions were consistent with those of Cys343 in soybean enzyme. The binding affinity of the PCMB-modified enzyme to maltose was also decreased. These results indicate that the modification of Cys331, which exists as a free SH group in B. cereus β-amylase caused inactivation by a similar mechanism to that in the case of Cys343 in soybean β-amylase as assumed from the sequence homology. This cysteine residue has a common role in β-amylases irrespective their origin.View full abstractDownload PDF (830K) -
Fumitomo Odawara, Masaaki Kurasaki, Mika Suzuki-Kurasaki, Shinji Oikaw ...1995 Volume 118 Issue 6 Pages 1131-1137
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA genetic approach was undertaken to investigate the physiological roles of human metallothionein-2. A constructed expression plasmid, pEXPMTII, in which human metallothionein-IIA cDNA was inserted downstream of a tryptophan-lactose promoter, was used to transform Escherichia coli JM105 strain. Cadmium-binding metallothionein was successfully expressed in E. coli in the medium containing cadmium, while copper and zinc-metallothioneins were scarcely observed in copper- or zinc-containing medium. The amino acid composition and sequence of the biosynthesized cadmium-metallothionein were analyzed. The selectivity of metals bound to metallothionein and the stability of metal-binding forms of metallothionein in E. coli were discussed. In addition, cadmium, zinc, or copper resistance of the cells expressing metallothionein was examined. Cells transformed with the plasmid pEXPMTII and cultured in a medium containing cadmium exhibited tolerance only to cadmium. It was demonstrated that human metallothionein-2 functioned for cadmium detoxification in E. coli.View full abstractDownload PDF (1736K) -
Hiroyuki Motoshima, Tadashi Ueda, Yoshio Hashimoto, Masako Tsutsumi, T ...1995 Volume 118 Issue 6 Pages 1138-1144
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe suggested for the introduction of a prolyl residue into a protein that if the N-terminus residue is glycine, an unfavorable interaction in the folded state caused by the introduction of the prolyl residue can be substantially avoided by use of mutant lysozymes in which Gly-Pro and Pro-Gly sequences are introduced to positions 101-102 in the loop region of the lysozymes [Ueda, T., Tamura, T., Maeda, Y., Hashimoto, Y., Miki, T., Yamada, H., and Imoto, T. (1993) Protein Eng. 6, 183-187]. In order to determine whether or not the information obtained is applicable to other regions, we prepared mutant lysozymes with Gly-Pro and Pro-Gly sequences at position 47, which is located in the β-sheet, positions 70-71, which are located in the loop, positions 117-118, which are located in the β-turn, and positions 121-122, which are located in the 310-helix. The free energy changes of the native and mutant lysozymes for unfolding were determined at pH 5.5 and 35°C. However, a mutant lysozyme with the Gly-Pro sequence was not always stabler than that with the Pro-Gly sequence at the same site. On the other hand, in order to determine whether or not strain caused by these sequences exists in the folded or unfolded state, the structures of these mutant lysozymes were determined by use of energy minimization. On comparison of the differences in the free energy change between the mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same site with those in their total local conformational energies, it was found there is a good correlation between them. Therefore, it was suggested that the difference in total local conformational energy caused by the introduction of a Gly-Pro or Pro-Gly sequence could be estimated by use of the energy minimized structure. Moreover, the correlation indicated that the differences in the free energy change between Gly-Pro and Pro-Gly lysozymes may be reflected by the differences in the total local conformational energies in their folded state. It was suggested that the energy levels in the unfolded states of mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same site in a Gdn-HC1 solution were almost identical.View full abstractDownload PDF (774K) -
Kenzo Ohtsuki, Sanae Nakamura, Yoshihito Shimoyama, Daisuke Shibata, H ...1995 Volume 118 Issue 6 Pages 1145-1150
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 96-kDa glycyrrhizin (GL)-binding protein (gp96) was purified to apparent homogeneity from an aqueous extract of soybeans by means of successive DEAE-cellulose column chromatography, gel filtration on Superdex 200pg, GL-affinity column chromatography, and ion-exchange chromatography on a Mono S column (HPLC). The protein was identified as a GL-binding protein since it specifically binds to [3H] GA. Moreover, it is a lipoxygenase (an enzyme that catalyzes the oxygenation of unsaturated fatty acids) since (i) it displays lipoxygenase (LOX) activity at pH 6.5; (ii) it is recognized on Western blot analysis by antibodies against LOX-1 and LOX-2; and (iii) the sequence of the N-terminal 21 amino acid residues (SNDVYLPRDEAFGHLKSSDFL) of a 42-kDa fragment (p42) proteolytically generated from gp96 is identical to a sequence of soybean LOX-3. In addition, GL, glycyrrhetinic acid (GA) and soyasaponin βg slightly inhibited LOX activity of the purified gp96 fraction, whereas oGA (a GA derivative) greatly inhibited its activity. Furthermore, CK-II catalyzed phosphorylation of gp96 was stimulated significantly by GL at doses between 1 and 10μM, but this phosphorylation was inhibited completely by 50μM GL. All these results taken together suggest that (i) gp96 purified from soybeans as a GL-binding protein belongs to the LOX family; and (ii) triterpenoid saponins, including GL, are involved in the regulation of the activities of CK-II and LOXs in plants, such as soybeans and roots of liquorice, which contain large quantities of saponins.View full abstractDownload PDF (1960K) -
Masayoshi Wakita, Goro Nishimura, Mamoru Tamura1995 Volume 118 Issue 6 Pages 1151-1160
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy extensively examining the experimental conditions for time-resolved spectrophotometry of non-transparent light scattering systems, we demonstrated the feasibility of quantitative analysis of both the fluorescence lifetime and intensity of reduced pyridine nucleotides in living tissues, suspensions of isolated liver mitochondria, and hepatocytes, as well as hemoglobin-free perfused rat liver being used systematically for measurements. The fluorescence decay was analyzed by the maximum likelihood method with a 4-compo-nent decay model. The lifetime of NADH observed in mitochondria (mean: 2.8±0.2ns) was much longer than that of the free form in an aqueous solution (mean: 0.43±0.01ns), and it was characterized as a protein-bound form. The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy state, though the intensity changed markedly.
The decay curves of isolated hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superimposed. Under the conditions of “unphysiological” acidosis, the mean lifetime became about 1.5 times longer than that under normal conditions.
With perfused liver, the relative contributions of cytosolic NADH and NADPH were determined by infusing lactate and tert-butylhydroperoxide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides. In contrast, about 70% of the total fluorescence intensity was due to cytosolic NADPH, but its decay parameters were essentially the same as those of mitochondrial NADH. No free form of either NADH or NADPH was detected in the cytosolic and mitochondrial spaces. We concluded that the changes in fluorescence intensity observed under the various conditions can be simply explained by a change in the amount of reduced pyridine nucleotides in tissues, rather than by changes in the microscopic environment. The wide applicability of time-resolved fluorescence photometry to in vivo studies is well documentedView full abstractDownload PDF (1211K) -
Sumio Maeda, Akemi Suzuki, Kong-Hua Lin, Hidetoshi Inagaki, Takao Sait ...1995 Volume 118 Issue 6 Pages 1161-1165
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that internucleosomal DNA fragmentation, a biochemical feature of apoptosis, was induced spontaneously in high-cell-density culture of adult rat hepatocytes. To understand better the intracellular mechanism of the DNA fragmentation in this system, we have examined the effects of several inhibitors of specific intracellular functions on the DNA fragmentation. We found that the DNA fragmentation could be suppressed by treatment of the cells with inhibitors of mitochondrial respiration, KCN and CCCP, or a protein synthesis inhibitor, cycloheximide. We also demonstrated that calmodulin inhibitors, chlorpromazine and W-7, could suppress the DNA fragmentation. Together, these results lead us to conclude that the DNA fragmentation in hepatocytes cultured at high cell density is an active process dependent on energy availability, gene expression, and calmodulin, rather than a passive event resulting from necrosis. However, by analyzing the incidence of apoptotic morphology during culture, we found that only 2-3% of cells exhibited apoptotic morphology, while the incidence of DNA fragmentation was estimated to be much higher. Based on these results, we estimated that the DNA fragmentation may result from the incomplete progression of apoptosis or from the occurrence of another type of active cell death.View full abstractDownload PDF (4155K) -
Hong Zeng, Takeshi Yoshida, Tomohiro Kurosaki, Hirohei Yamamura, Akihi ...1995 Volume 118 Issue 6 Pages 1166-1174
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman Fcγ receptor IIIA (hFcγRIIIA) cDNA was introduced into mouse macrophage/monocyte cell line P388D1, and several stable cell clones expressing hFcγRIIIA were isolated. This facilitated the study of the biological function of FcγRIIIA in monocytes/macrophages. The cloned cells showed the high phagocytic activity mediated by hFcγRIIIA, while the original P388D1 cells did not. In order to examine the phosphorylation of proteins involved in hFcγRIIIA signal transduction, these receptors were stimulated by cross-linking. The cross-linking of hFcγRIIIA induced a rapid increase in tyrosine phosphorylation of several proteins, including PLC- γ1, Syk, HS1, and p21ras ras GAP-associated p190 and p60 proteins. Immunoblotting with a polyclonal antibody specific for the GAP-associated p62 protein, which was originally found in fibroblasts and is homologous with an RNA-binding protein, revealed that the p60 phosphorylated after cross-linking of hFcγRIIIA seemed to represent a novel GAP-associated protein unrelated to the known GAP-associated p62 protein, which was also present in the P388D1 cells.View full abstractDownload PDF (5613K) -
Ryu-ichi Takezawa, Yoshifumi Watanabe, Toshihiro Akaike1995 Volume 118 Issue 6 Pages 1175-1183
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSControversy has surrounded origin and differentiation of tissue macrophages. We directly demonstrate the differentiation of bone marrow cells into macrophages in the liver in vivo using a cell-labeling fluorescence dye, PKH-26. Bone marrow cells labeled with PKH26 were intravenously injected into syngenic mice, and these cells were tracked by flow cytometric analysis. The majority of the labeled cells were detected only in the liver after 4 days. Interestingly, antigens specific for macrophage lineage cells (F4/80, FcγRII, and CD14) were detected on the liver-accumulated cells only 4h after the injection. The pattern of the antigen expression changed to that of Kupffer cells (F4/80+, FcγRII+, Mac-1-) after 4 days and remained so thereafter. These labeled cells in the liver were esterase stainingpositive and showed phagocytic activity at day 7. The number of labeled cells among the Kupffer cells in the liver increased with days after injection. This indicates that bone marrow cells accumulate in the liver and differentiate into liver macrophages on site. Roles of factors secreted from hepatocytes are also discussed.View full abstractDownload PDF (3769K) -
Hitoshi Sakashita, Takahiko Sakuma, Yoshiko Akitomo, Tadayasu Ohkubo, ...1995 Volume 118 Issue 6 Pages 1184-1191
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Escherichia coli Ada protein, a suicidal DNA methyltransferase, is converted into a transcriptional regulator for methylation-resistance genes by the transfer of a methyl group from a DNA methylphosphotriester to its own Cys69 residue. Here, we report the DNA recognition mode and the functional switch mechanism of the N-terminal 16 kDa fragment of the Ada protein. NMR analysis has revealed that the segment from residues 102 to 123 forms a helix-turn-helix structure. A site-directed mutagenesis study has shown that the second helix in the helix-turn-helix structure plays a crucial role in specific recognition of DNA. These results imply that the sequence-specific interaction of the Ada protein with DNA occurs through the helix-turn-helix motif. NMR experiments on the methylated protein-DNA complex showed line broadening for the amide proton signals from the helix-turn-helix motif and for the protons in the vicinity of Cys69. In the case of the nonmethylated protein-DNA complex, signal broadening was observed only for protons from the helix-turn-helix. These findings suggest that the residues in the vicinity of Cys69 come into direct contact with the cognate DNA after methylation. We propose that the direct contact of this region is a major factor for the “switch” that converts the Ada protein from a nonspecific DNA binding form to a transcription factor.View full abstractDownload PDF (2237K) -
Akira Mori, Kenshi Okubo, Dongchon Kang, Naotaka Hamasaki1995 Volume 118 Issue 6 Pages 1192-1198
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo peptides derived from the carboxyl terminal region of the human erythrocyte band 3 protein were identified as fragments releasable from cell membranes on trypsin digestion. These peptides, Asn-880-Lys-892 and Ala-893-Val-911-COOH, however, were resistant to trypsin, unless the cell membranes had been treated with high concentrations of NaOH. This suggests that the carboxyl terminal region is located in situ within the native band 3 molecule. Unlike in the cases of other portions of the band 3 protein, such as Gly-647-Arg-656, Ser-731-Lys-743, and Tyr-818-Lys-826, the release of the carboxyl terminal region was not inhibited by pretreatment of erythrocytes with 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), indicating that there is no major structural difference in the carboxyl terminal portion between the outward and inward facing forms. The carboxyl terminal region of the band 3 protein has a negative charge cluster. In the middle of the negative charge cluster, consensus sequences, Val-Asp-X-X-X-Leu-Asp-Ala-Asp-Asp and Thr-Phe-Asp-Glu (TFDE), were found in the carboxyl terminal regions of aquaporin CHIP and glucose transporter 1, respectively. The sequence, TFDE, exists in the highly amphipathic 11-residue sequence of glucose transporter 1, and this amphipathic sequence has been suggested to promote normal membrane insertion of polytopic membrane proteins such as glucose transporter 1 and serine chemoreceptor. The role of the carboxyl terminal region of the band 3 protein is discussed.View full abstractDownload PDF (2003K) -
Makoto Seki, Yoshikazu Honda1995 Volume 118 Issue 6 Pages 1199-1204
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphorothioate antisense oligodeoxynucleotides (S-ODNs) designed to hybridize to the 5' region of the hepatitis C virus (HCV) genome were evaluated as to their ability to inhibit HCV gene expression, using an in vitro translation system. Three effective regions were found to interfere with the translation of HCV RNAs. These regions were region A [nucleotides (nt) 124 to 153], region B (nt 100 to 123), and region C (nt 324 to 360). Further detailed evaluation of S-ODNs within each region allowed us to propose some HCV-specific antiviral agent candidates. Two of them, SMS16 (nt 328 to 347) and SMS17 (nt 326 to 345), caused over 90% inhibition of HCV gene expression when present in a less than fourfold molar excess; this effect was sequence-specific and dose-dependent.View full abstractDownload PDF (2702K) -
Takaaki Koike, Hidehiko Beppu, Hiroshi Kuzuya, Kazuhiro Maruta, Kan Sh ...1995 Volume 118 Issue 6 Pages 1205-1210
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel lectin was isolated from the leaf skin of “Kidachi Aloe” (Aloe arborescens Miller var. natalensis Berger) by sequential chromatographies on Sephadex G-25 gel filtration, DEAE ion exchange, and Superdex 75 gel filtration columns. The native lectin exhibited a molecular mass of about 35 kDa on both gel filtration on a Superdex 75 column and native-PAGE under nonreducing conditions. SDS-PAGE in the presence or absence of β-mercaptoethanol revealed two distinct peptides with molecular masses of about 5.5 and 2.3 kDa, respectively, in addition to a major 9.2 kDa subunit, indicating the presence of a partially processed subunit. The N-terminal amino acid sequence of the intact subunit showed homology with that of snowdrop lectin. The native lectin showed hemagglutinating activity toward rabbit but not human and sheep erythrocytes, and specifically bound to mannose like snowdrop lectin did, indicating that the Aloe and snowdrop lectins are structurally and functionally similar proteins. In addition, the native lectin showed strong mitogenic activity toward mouse lymphocytes.View full abstractDownload PDF (704K) -
Kunio Fujiwara, Hiroshi Kanetake, Koichi Furukawa, Yukinobu Masuyama, ...1995 Volume 118 Issue 6 Pages 1211-1215
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA monoclonal antibody (mAb), ASPM-2, produced against N-(γ-maleimidobutyryloxy)-succinimide (GMBS)-conjugated polyamine spermine [Spm; Fujiwara et al. (1994) Histochemistry 102, 397-404] was used for the development of an enzyme-linked immunosorbent assay (ELISA) for acetylpolyamines (Ac-PAs) in human urine. The ELISA is based on the principle of competition between an analyte and Spm-glutaraldehyde-bovine serum albumin conjugate-coated polystyrene microtiter wells for the mAb, followed by immunoreaction with biotinylated anti-mouse immunoglobulin and horseradish peroxidase-streptavidin. The ASPM-2 mAb showed strong immunoreaction with N1, N12-diacetylspermine (2Ac-Spm), N-monoacetylspermine (Ac-Spm), and N1-acetylspermidine (N1-Ac-Spd), the EC50 values being 29, 50, and 51μM, respectively, but no cross-reaction with other PA-related compounds or amino acids. The method was used to measure urinary Ac-PA levels in healthy subjects and cancer patients, without pretreatment of the specimens, mean concentrations of 3.25 and 2.80μmol per 24-h urine, respectively (as N1-Ac-Spd), being found. The ASPM-2 ELISA for N1-Ac-Spd, which is the PA most relevant to the analysis of human urine among the three Ac-PAs mentioned above, is specific and accurate, and can easily be used to analyze large numbers of specimens in parallel. It should thus have potential for studying the relationship between urinary N1-Ac-Spd levels and cancer.View full abstractDownload PDF (613K) -
Wataru Hashimoto, Hideyuki Suzuki, Satoko Nohara, Hiromi Tachi, Kenji ...1995 Volume 118 Issue 6 Pages 1216-1223
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSγ-Glutamyltranspeptidase [EC 2. 3. 2. 2] of Escherichia coli K-12 consists of one large subunit and one small Subunit, which can be separated from each other by high-performance liquid chromatography. Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39, 207 and 20, 015, respectively. The large subunit exhibited no γ-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity. The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity. The gene of γ-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order. Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit. A γ-glutamyltranspeptidase-less mutant of E. coli K-12 was transformed with each plasmid or with both of them. The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity. The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene. These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme. This suggests that the synthesis of γ-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct the intact structure of γ-glutamyltranspeptidase than the association of the separated large and small subunits.View full abstractDownload PDF (2893K) -
Hideyuki Yamamoto, Masato Hasegawa, Tsunehiko Ono, Kazuhiro Tashima, Y ...1995 Volume 118 Issue 6 Pages 1224-1231
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have reported that many sites of tau in fetal brain (fetal-tau) as well as in paired helical filaments (PHF-tau) are phosphorylated. In the present study, we used site-specific antibodies and peptide mapping to examine protein phosphatases involved in dephosphorylation of fetal-tau and PHF-tau. Immunoblot analysis and electrophoretic mobility showed that protein phosphatases 1 and 2A and calcineurin could dephosphorylate fetal-tau and PHF-tau. Phosphoserines 199, 202, 396, and 413 and phosphothreonine 231, numbered according to the longest human tau isoform, were dephosphorylated, as shown by the immunoblot analysis. Phosphoserine 422 was dephosphorylated by protein phosphatase 2A and calcineurin, but not by protein phosphatase 1. Peptide mapping with Achromobacter lyticus protease 1 showed that phosphoserines 199, 202, 235, and 396 and phosphothreonine 231 were dephosphorylated by protein phosphatases. Fetal-tau was more rapidly dephosphorylated by protein phosphatase 2A and calcineurin than PHF-tau. Interestingly, PHF-tau which had not been solubilized with guanidine HCl was little dephosphorylated by protein phosphatases. Thus, PHF-tau in neurofibrillary tangles of Alzheimer's disease brain is likely to be resistant to dephosphorylation by protein phosphatases.View full abstractDownload PDF (4530K) -
Kouichirou Hori, Fumi Morita, Fumiko Matsuzawa, Seiichi Aikawa1995 Volume 118 Issue 6 Pages 1232-1238
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we demonstrated that the 2.6-kDa peptide extending from Arg177 to Tyr198 in subdomain 4 of rabbit skeletal actin bound to actin itself, inhibited the elongation of actin filament, and severed F-actin. The corresponding segment in actin, therefore, is thought to contain the most critical actin-actin contact [Hori, K. and Morita, F. (1992) J. Biochem. 112, 401-408; Hori, K., Itoh, T., Takahashi, K., and Morita, F. (1994) Biochim. Biophys. Acta 1186, 35-42]. In this paper, we report on the binding site in actin for the 2.6-kDa peptide studied by using a zero-length cross-linker, 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC). We conducted limited digestion of actin cross-linked with the 125I-labeled 2.6-kDa peptide with various proteases, and developed peptide maps. The cross-linked region of the 2.6-kDa peptide was found to be within the region of Ala114 to Glu167 in actin by identifying the radioactive peptide fragments. The region was further restricted by isolation of radioactive peptide from α-chymotryptic digest of the cross-linked actin. The binding site of the 2.6-kDa peptide was finally assigned to be within the 24 amino acid segment from Ala144 to Glu167, which lies in subdomain 3 of actin. Using computer graphics, actin-actin contact provided by the two segments was suggested to be along the left-handed genetic helix of actin filament.View full abstractDownload PDF (2671K) -
Shunji Kunori, Tsuyoshi Katoh, Yuko Mogi, Fumi Morita1995 Volume 118 Issue 6 Pages 1239-1247
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe N-terminal 28-residue peptide of actin whose Cys10 was labeled with 5-iodoacetamidofluorescein (F3K peptide) was isolated from the fluorescently labeled thrombin digest of actin. The effect of myosin subfragment 1 (S1) on the fluorescence of F3K peptide was examined in the absence of ATP. With increasing concentration of S1 added, the fluorescence intensity of F3K peptide increased by maximally 7.3% with an apparent dissociation constant of 5.7μM, suggesting a role of this peptide region of actin in aeto-S1 binding in rigor. F3K peptide was crosslinked with S1 at 10mM NaCl using a zero-length crosslinker by the method of Grabarek and Gergely [Anal. Biochem. 185, 131-135 (1990)]. The crosslinking was greatly inhibited by the presence of either 0.2M NaCl or 5mM MgATP. The analyses of amino acid compositions and sequences of the fluorescent peptides isolated from a lysylendopeptidase digest of the crosslinked S1 indicated that F3K peptide was mainly crosslinked to residues 637-642 of the S1 heavy chain. The crosslinked S1 was isolated by selectively pelleting the uncrosslinked S1 with F-actin. ATPase activity of the isolated crosslinked S1 alone was twice as high as that of control S1. The actin-activated ATPase activity of the crosslinked S1 was much lower than that of uncrosslinked S1. The estimated Vm and Km values were 1.72s-1 and 125μM, respectively. The Vm decreased to less than 1/8, while Km increased only twofold. The results suggest that the N-terminal 28-residue segment of actin may be implicated in the rigor binding of actomyosin and in the actin-activation of myosin ATPase, but may not be the main determinant of actomyosin binding in the presence of ATP.View full abstractDownload PDF (3205K) -
Akira Ono, Masao Kawakita1995 Volume 118 Issue 6 Pages 1248-1254
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThapsigargin and ionomycin inhibited the intracellular transport of the Sendai virus F0 protein. Depletion of Ca2+ from intracellular Ca2+ store(s) is critical for the inhibition, since ionomycin was more effective in the absence of extracellular Ca2+ than in its presence. Transport of F0 was arrested between the trans-Golgi complex and the plasma membrane [Ono, A. and Kawakita, M. (1994) J. Biochem. 116, 649-656], but only when thapsigargin was added before the synthesis of F0. This implies that F0 was committed to later arrest in the endoplasmic reticulum. Non-reducing SDS-polyacrylamide gel electrophoresis revealed a conformational abnormality of F0 immediately after pulse-labeling in thapsigargintreated cells. The abnormality did not affect the exit of F0 from the endoplasmic reticulum, but paralleled its later arrest at the trans-Golgi stage. Pulse-labeled and 1-h-chased F0 was endoglycosidase H-resistant even in thapsigargin-treated cells, but was not recognized by mAb f-49, a monoclonal antibody that recognizes the corresponding F0 intermediate in uninhibited cells. The misfolded F0 may escape from recognition by means of the quality control system of the endoplasmic reticulum, but another system in the Golgi complex may complement the former. The arrested F0 was rapidly degraded.View full abstractDownload PDF (4431K) -
Kazuaki Takaku, Mayumi Futamura, Shio Saitoh, Yutaka Takeuchi1995 Volume 118 Issue 6 Pages 1268-1270
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe level of transglutaminase (TGase) expressed in various tumor cell lines was investigated. We found that each cell line could be categorized into three distinct groups, that is, (i) cell lines with low or negligible TGase activity, (ii) cell lines with significant or high TGase activity and immunoreactive to a monoclonal antibody against tissue type TGase, and (iii) cell lines with significant or high TGase activity but not immunoreactive to the antibody. Results reported here argue against the former proposal that tissue-type TGase is expressed at a lower level in malignant cells compared to the level in normal cells.View full abstractDownload PDF (1466K) -
Hiroshi Ohashi, Yasuo Itoh, Paul J. Birckbichler, Yutaka Takeuchi1995 Volume 118 Issue 6 Pages 1271-1278
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTransglutaminase (TGase) catalyzes an acyl-transfer reaction between peptidyl glutamine residues and primary amines including the ε-amino group of lysine residues in protein. TGase in the neuronal system has been suggested to be involved in neurotransmitter release, long-term potentiation, and so forth. In order to study the mammalian brain TGase at the molecular level, TGase was purified to apparent homogeneity from Sprague-Dawley rat brain, using DEAE ion exchange, and heparin and α-casein affinity column chromatographies. The brain TGase was concentrated 11, 400-fold and had a specific activity of 11, 000 nmol/h/mg protein. The purified protein migrated on SDS-PAGE to a position corresponding to a molecular size of approximately 75 kDa. The brain TGase activity was Ca2+-dependent (EC50_??_0.28mM), and its Km values for putrescine and N, N-dimethylcasein were 0.26 and 0.065mM, respectively. GTP inhibited the brain enzyme activity 100-fold more potently than ATP did, and the enzyme was photolabeled with [α-32P]8-azido-GTP, suggesting that the brain TGase is a member of the GTP-binding protein family. Monoiodoacetate and cystamine potently inhibited the enzyme activity, suggesting that cysteine residue(s) are essential for brain TGase. Zn2+ inhibited the enzyme, while Mg2+ was not inhibitory. The rat brain TGase reacted only weakly with monoclonal and polyclonal antibodies against tissue-type TGase such as guinea pig liver TGase and human red blood cell TGase, while it did not react with antibodies against non-tissue types of TGase, namely human epidermal TGase and human coagulation factor XIIIa. The results suggest that rat brain TGase shares major characteristics with tissue-type TGase, but is nevertheless distinct from tissue-type TGase.View full abstractDownload PDF (2590K) -
Yasuyuki Michigami, Keiko Abe, Hitoshi Obata, Soichi Arai1995 Volume 118 Issue 6 Pages 1279-1284
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIce nucleation-active (Ina) proteins of bacterial origin comprise three distinct domains, i.e., N-terminal (N-), central repeat (R-), and C-terminal (C-) domains, among which the R-domain is essential, and its length may be correlated with the ice nucleation activity. In addition, the short C-terminal domain of about 50 amino acid residues is indispensable for the activity. Using the Ina U protein of Erwinia uredovora, we carried out precise mutational analyses of its C-terminus. The ice nucleation activity (T50) assay showed that the C-terminal 12 amino acids were not necessary, and a deletion mutant (ΔC29) with a new C-terminal, Met29 (numbered from the first amino acid residue of the C-domain and corresponding to Met1022), exhibited almost the same activity as the wild-type Ina U protein did. However, deletion of the C-terminal 13 residues including Met29 resulted in almost complete loss of the activity. In the deletion mutant (ΔC29), amino acid replacement of the C-terminus, Met29, showed that the activity was retained when Met29 was replaced with a neutral, aromatic, or basic amino acid (Gly, Phe, or Lys), but was lost on the replacement with an acidic amino acid (Asp or Glu). In addition, two other residues in the C-terminal region commonly present in all Ina proteins were examined as to their importance, and it was shown that one of these residues, Tyr27, is important for the activity, although it is not exclusively required; the activity was lost to a great extent when this residue was replaced with Gly or Ala, but to a lesser extent when it was replaced with Leu. These results suggest the significance of the secondary and/or tertiary structure of the C-terminal region of the Ina U protein for the ice nucleation activity.View full abstractDownload PDF (3493K) -
C. Chandra Kumar, Jae-Hong Kim, Pierre Bushel, Lydia Armstrong, Joseph ...1995 Volume 118 Issue 6 Pages 1285-1292
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSmooth muscle α-actin promoter is repressed in ras-transformed fibroblast cells and derepressed in revertant cells. We have recently shown that serum response factor (SRF) which can bind to the serum response elements (SREs) present in the α-actin promoter, can activate α-actin promoter activity in ras-transformed cells and suppress transformation by ras. Agents that stimulate SRF expression and α-actin promoter activity in ras-transformed cells are expected to be potential candidates as antitumor agents. In this study, we show that treatment of ras-transformed cells with antitumor agents such as taxol, vincristine, vinblastine, colchicine, and nocodazole leads to 5- to 7-fold activation of α-actin promoter driven CAT activity, whereas there was very little effect on thymidine kinase promoter driven CAT activity. This activation occurred at subcytotoxic concentrations of these agents and correlated with inhibition of cell cycle progression. Furthermore, these agents stimulated SRF expression in ras-transformed cells, as measured by its SRE binding activity. The increase in α-actin expression is accompanied by the restoration of actin filaments into organized bundles. These results suggest a novel mechanism by which antimitotic agents suppress the ras-transformed phenotype.View full abstractDownload PDF (5377K) -
Yoshiyuki Matsuura, Yoshihide Yoshimoto, Kimiko Saeki, Takeyuki Wakaba ...1995 Volume 118 Issue 6 Pages 1293-1296
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have investigated the effect of methanol at concentrations below 15% (v/v) on actoheavy meromyosin (HMM)- and tropomyosin (Tm)-troponin (Tn)-acto-heavy meromyosin-ATPase activities. Methanol slightly enhanced ATPase activity of acto-HMM alone. It had no apparent effect on normalized Tm-Tn-acto-HMM-ATPase activity in the absence of Ca2+. In the presence of Ca2+, however, methanol markedly inhibited normalized Tm-Tn-acto-HMM ATPase activity. These results show that methanol affects the troponin-tropomyosin regulation of acto-HMM ATPase: methanol suppresses the Ca2+-sensitivity of the regulatory system and traps it in an “off-state.”View full abstractDownload PDF (530K) -
Mamoru Sato, Yoshiko Sato-Miyamoto, Keiichi Kameyama, Nobuyoshi Ishika ...1995 Volume 118 Issue 6 Pages 1297-1302
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe internal structure of the recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle was investigated by small-angle neutron scattering using the contrast variation method. Data were collected in aqueous buffer solutions containing 0, 40, 60, and 100% D2O in the q range of 0.005 to 0.2 Å-1 at 5°C. The radius of gyration at infinite contrast and the maximum dimension of the particle were estimated to be 107 and 290 Å, respectively. The contrast matching point of the particle was determined to correspond to about 30% D2O, indicating that a considerable portion of the vaccine particle is made up of lipids and carbohydrates from yeast. The distance distribution function of the particle at 40% D2O, at which the protein components are matched out to show only the distribution of the remaining lipids and carbohydrates, differed markedly from the functions at other D2Os, and showed the nearly symmetrical profile characteristic of a spherical particle with a diameter of 240 Å. The Stuhrmann plot and profile of the distance distribution function at 40% D2O showed that (i) the vaccine is a spherical particle with a diameter of 290 Å, in which two different regions in terms of scattering density are distributed radially, (ii) the lipids and carbohydrates form a spherical cluster with a diameter of 240Å in the core region of the particle, and (iii) the surface antigen protein is present in the peripheral region. This architecture of the vaccine particle is favorable for the induction of anti-virus antibodies.View full abstractDownload PDF (691K) -
Yoshio Shibagaki, Hideo Gotoh, Misako Kato, Kiyohisa Mizumoto1995 Volume 118 Issue 6 Pages 1303-1309
Published: 1995
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe yeast mRNA capping enzyme is composed of 52 (α) and 80 kDa (β) polypeptides, which are responsible for its mRNA guanylyltransferase and RNA 5'-triphosphatase activities, respectively. We isolated the gene encoding the α subunit (CEG1) and showed that CEG1 is essential for yeast cell growth [Shibagaki et al., (1992) J. Biol. Chem. 267, 9521-9528]. In this study, CEG1 was expressed in Escherichia coli and the α subunit protein was purified to near homogeneity. A [32P]GMP-bound tryptic peptide derived from the recombinant enzyme-[32P]GMP covalent reaction intermediate was converted to a [32P]phosphoryl-peptide through periodate oxidation followed by β-elimination. Hydrolysis of the [32P]phosphoryl-peptide with alkali resulted in [32P]Nε-phospholysine as the only phosphoamino acid, indicating that GMP in the enzyme-GMP complex is bound to a lysine residue via a phosphoamide linkage. Microsequencing of the [32P]GMP-peptide showed that the GMP binding site was located in the region between amino acids 60 and 75, which contained an internal trypsin-resistant lysine at position 70. CEG1 was subjected to site-directed mutagenesis and the mutant proteins were expressed in E. coli. Substitution of His or Ile for Lys70 entirely abolished the enzyme-GMP formation activity, and this mutation was lethal to yeast in vivo, supporting the notion that the active site in the α subunit is located at Lys70. Replacement of Lys70 with Arg reduced the ability to form the enzyme-GMP complex; however, yeast cells bearing this allele were not viable. A series of mutations, including 8 amino acid replacements and 3 insertions, near the active site (Lys70-Thr-Asp-Gly motif) were also introduced and the mutant polypeptides were examined for catalytic activity in vitro as well as yeast cell viability in vivo. There was a good correlation between the in vitro and in vivo functions of the mutant proteins, except when Asp72 was replaced with Glu, which allowed formation of the enzyme-GMP complex but failed to support cell growth. The results with Lys70 to Arg and Asp72 to Glu substitutions indicated that guanylyltransfer to RNA and/or additional roles besides cap formation per se are impaired in these mutant proteins.View full abstractDownload PDF (3745K)
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