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Volume 116 (1994)
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Volume 116, Issue 2
Displaying 1-38 of 38 articles from this issue
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Kenji Yamamoto1994 Volume 116 Issue 2 Pages 229-235
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMicrobial endoglycosidases are useful for elucidating the structure and function of the oligosaccharide chains of glycoconjugates. Most of the microbial endo-β-N-acetylglucosaminidases including Endo-H can preferentially act on high-mannose type chains of asparagine-linked oligosaccharides of various glycoproteins. Among them, Flavobacterium sp. enzyme is produced in large amounts by the inducing cultivation. Using this enzyme, the role of oligosaccharide chains in various microbial glycoenzymes such as Rhizopus glucoamylase, and yeast invertase was examined. The findings suggested that the oligosaccharide chains of them are essential participants in the stabilization of the enzyme and in the protection from proteolytic inactivation. Novel endo-β-N-acetylglucosaminidases were also found in the culture broths of microorganisms. Unlike most microbial endo- β-N-acetylglucosaminidases, Endo-M of Mucor hiemalis could act on a complex type oligosaccharide chains, which is similar to Endo-F2 in multiple form of Endo-F from Flavobacterium meningosepticum. The complete amino acid sequences of Endo-F1, -F2, -F3, -H, and Flavobacterium sp. enzyme were determined. All of them had two highly conserved regions common to a number of chitinases. Endo-α-N-acetylgalactosaminidase which hydrolyzes the O-glycosidic linkages in glycoproteins was found in the culture broth of only a few microorganisms. The production of Alcaligenes sp. enzyme was highly induced by the addition of porcine gastric mucin in the culture medium. There is some evidence that endo-α-N-acetylgalactosaminidases may recognize not only the glycon but also the aglycon amino acids. Alcaligenes sp. enzyme was used for developing a sensitive method for detection and determination of T-antigen which is proposed as a unique carcinoma marker, by combination of HPLC.View full abstractDownload PDF (909K) -
Yo Sasaki, Fumiaki Tsunomori, Takashi Yamashita, Kazuyuki Horie, Hideh ...1994 Volume 116 Issue 2 Pages 236-238
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe fluorescence intensity has been reported to increase 10 to 25 times when N-(1-pyrene)-iodoacetamide (PIAA)-conjugated actin polymerizes from the G- to the F-form. To elucidate the molecular mechanism underlying this process, we measured the time-averaged anisotropy of PIAA-actin in both the G- and F-forms. The anisotropy ratio of PIAA-G-actin (0.137±0.008) was smaller than that of PIAA-F-actin (0.221±0.008). Similar results were obtained when N-(1-pyrene)-3-iodopropionamide (PIPA), a PIAA analogue with an extra carbon-chain in its reactive group, was conjugated with actin. The anisotropy ratio increased from 0.096±0.042 to 0.199±0.035 when PIPA-actin was transformed from the G-to the F-form. Further more, we measured the anisotropy decay of PIAA-actin in both the G- and F-forms. Least-square fitting revealed that the decay pattern was well fitted the wobbling-in-cone model. In the G-form, the pyrene of PIAA actin diffused in a cone region with a vertical half angle of 33.4°. This value decreased to 25.9° when the actin was transformed to the F-form. Because PIAA and PIPA were conjugated at Cys-374 of actin, our results suggest that a small cleft exists in the actin molecule in the vicinity of Cys-374, and this cleft becomes narrow upon polymerization, resulting in an increase in fluorescence intensity.View full abstractDownload PDF (283K) -
Nobuya Inagaki, Masanari Mizuta, Susumu Seino1994 Volume 116 Issue 2 Pages 239-242
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRab3A, a ras p 21-related small GTP-binding protein, is implicated in the exocytosis of neurotransmitters. Recently, Rabphilin-3 A, a putative target protein for Rab3A, was identified and its cDNA has been cloned from bovine brain. In this study, we isolated a cDNA encoding a mouse Rabphilin-3 A homolog from the insulin-secreting cell line, MIN 6. Mouse Rabphilin-3 A is a protein of 681 amino acids exhibiting overall 88.5% identity with bovine Rabphilin-3 A. The amino acid identity between mouse and bovine Rabphilin-3 A is highest in their carboxyl-terminal halves (97.8% identity) and amino-termini (93.0% identity), which contain the region of the two internal repeats homologous to the regulatory domain (C2 domain) of protein kinase C and the putative Rab3A-binding region, respectively. RNA blot analysis revealed that Rabphilin-3 A mRNA is expressed in endocrine and hormone-secreting clonal cells, including rat adrenal glands, MIN 6, the hamster insulinsecreting cell line, HIT-T 15, and the rat catecholamine-secreting cell line, PC 12, as well as rat brain. These results suggest that Rabphilin-3 A might be involved in the exocytosis of secretory vesicles in hormone-secreting cells as well as in neurons.View full abstractDownload PDF (1430K) -
Akihiko Nakano, Hideshi Otsuka, Mami Yamagishi, Eiichi Yamamoto, Keita ...1994 Volume 116 Issue 2 Pages 243-247
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSAR1 encodes a 21-kDa GTPase, which is required for vesicle formation from the endoplasmic reticulum in yeast. Although it belongs to the expanding small GTPase superfamily, there are interesting structural features that are unique to the Sari protein. We performed a site-directed mutational study to identify the amino acid residues that are essential for the Sarlp function. Among seven mutants we constructed, four are functionless by themselves, while two confer temperature sensitivity to cells. When the mutant proteins are overproduced in wild-type cells, all of these six show a dominant negative effect on cell growth. The replacement by serine of the only cysteine residue present in Sarlp caused no significant change in the growth phenotype. These findings are not only important for analyzing the mechanism of the Sarlp action in yeast, but will also be very useful for studying the function of Sarlp counterparts in higher eukaryotes.View full abstractDownload PDF (1326K) -
Ji-Quan Liu, Tatsuo Kurihara, Nobuyoshi Esaki, Kenji Soda1994 Volume 116 Issue 2 Pages 248-249
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHis20 of L-2-halo acid dehalogenase from Pseudomonas cepacia MBA4 was suggested to serve as a catalytic base [Biochem. J. (1993) 292, 69-74]. In this study, we substituted Asn or Leu for His19 of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which corresponds to His20 of the P. cepacia enzyme. Although the substrate specificity was affected by the substitution, the susceptibilities of substrate halo acids were not substantially diminished, and the Km, and kcat values of the mutant enzymes for L-2-chloropropionate were not significantly different from those of the wild-type enzyme. In addition, the wild-type and mutant enzymes showed the same pH optimum. Accordingly, His19 is not essential for catalysis of L-2-halo acid dehalogenase.View full abstractDownload PDF (224K) -
Jian Du, Sheng-Guang Li, Zhi-Huan Lin1994 Volume 116 Issue 2 Pages 250-256
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIndomethacin showed a dose-, time-, and pH-dependent, noncompetitive inhibitory effect on hog gastric H+/K+-ATPase. Four percent of total indomethacin in the buffer (0.20 mmol/ liter) bound to the H+/K+-ATPase vesicles (15 μg/ml). It markedly quenched the intrinsic fluorescence of the enzyme, and decreased the membrane fluidity. Thus, the inhibitor effect of indomethacin may arise from both a direct effect on the hydrolytic and H+ transport functions of the enzyme and a disturbing effect on the lipid bilayer of the vesicle.View full abstractDownload PDF (887K) -
Masato Ishigami, Shizuya Yamashita, Naohiko Sakai, Takeshi Arai, Ken-i ...1994 Volume 116 Issue 2 Pages 257-262
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHigh-density lipoprotein (HDL) has been speculated to have an anti-atherogenic function. Many in vitro studies have demonstrated that HDL has the ability to remove cholesteryl ester (CE) from lipid-laden macrophages. However, the effect of alteration in chemical composition and particle diameter on the in vivo function of HDL is unknown. In the study described here, we have isolated the HDL from patients homozygous for cholesteryl ester transfer protein (CETP) deficiency and examined its function in vitro, in order to clarify the anti-atherogenic property of HDL in CETP-deficient subjects. Apolipoprotein (apo) E-free HDL2 from the patients, separated by heparin-Sepharose column chromatography, was rich in CE, poor in triglycerides (TG), and enlarged in size on 4-30% nondenaturing polyacrylamide gradient gel electrophoresis. In contrast, HDL3 from the patients was normal in size and in its chemical composition. First, we examined the effect of HDL on CE accumulation in macrophages. After mouse peritoneal macrophages had been incubated with both acetylated low-density lipoproteins (Ac-LDL) and HDL, cellular CE content was determined by an enzymatic, fluorometric method. Ac-LDL alone induced a 9-fold accumulation of CE. The addition of apo E-free HDL2 and HDL3 from controls and patients' HDL3 prevented CE accumulation in macrophages, while patients' HDL2 had no preventive effect. We next investigated the in vitro ability of HDL to remove cellular CE from lipid-laden macrophages after incubation with Ac-LDL. After loading of macrophages with cholesterol by Ac-LDL, HDL was added to the culture medium and the cellular CE content was measured. The apo E-free HDL2 and HDL3 from controls and patients' HDL3 reduced the CE content in macrophages, while the patients' HDL2 had no significant effect. These results suggest that the large and CE-rich HDL2 from CETP-deficient patients is functionally abnormal in vitro and that particle diameter and chemical composition of HDL may be important for its anti-atherogenic function in vivo. CETP may play a crucial role in preventing CE accumulation in HDL, thereby making HDL more active in its antiatherogenic function.View full abstractDownload PDF (1618K) -
M. Isabel Mínguez-Mosquera, Beatriz Gandul-Rojas, Lourdes Galla ...1994 Volume 116 Issue 2 Pages 263-268
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChlorophyllase activity in olive fruit (Olea europaea) was measured using the enzyme solubilized from the protein precipitate. The reaction was stopped by freezing the mixture at -20°C, to avoid dilution of the sample and consequent reduction of the substrate levels to below the detection limits of the analytical system. Separation of the substrates and products of the enzymatic reaction was performed by reverse-phase HPLC using a gradient solvent system of water and ion suppressor/methanol/acetone. These conditions allowed direct resolution of the reaction mixture prior to centrifugation, without the need for the transfer of any of the components to other solvents. Olive chlorophyllase in the crude enzymatic extract showed maximum activity at 50°C and the optimum pH was 8.5 in acetate-phosphate-borate buffer for all substrates used, chlorophylls (a and b) and pheophytins (a and b). The Km and Vmax values obtained for hydrolysis of these substrates showed that chlorophyllase had a greater affinity for chlorophyll b, while the highest maximum rate of reaction occurred with pheophytin a. Substrate inhibition was observed with pheophytin b.View full abstractDownload PDF (676K) -
Masamichi Fujita, Yasuhito Nakagawa, Ryoichi Osawa, Isao Kijima-Suda, ...1994 Volume 116 Issue 2 Pages 269-273
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of collagen and thrombin on the liberation of free arachidonic acid were investigated in human platelets by fluorometric high-performance liquid chromatography. Collagen induced a concentration-dependent increase in the extent of platelet aggregation, as well as an accumulation of arachidonic acid in human platelets. By contrast, thrombin effectively provoked a potent aggregation at relatively low concentration without any accumulation of free arachidonic acid, although the accumulation of arachidonic acid was detected at a high concentration of thrombin (>0.1 U/ml) that induced full aggregation. The selective liberation of arachidonic acid was found in thrombin-stimulated platelets. Non-selective liberation of fatty acids occurred in platelets that had been stimulated with a high concentration of collagen (10 μg/ml), as well as in platelets stimulated with A 23187. The net amount of free arachidonic acid in collagen-stimulated platelets was estimated by use of eicosatetraenoic acid (ETYA), which is an inhibitor of both cyclooxygenase and lipoxygenase. ETYA markedly potentiated the accumulation of free arachidonic acid in collagen-stimulated platelets without changing the amounts of other fatty acids in the cell.View full abstractDownload PDF (649K) -
Nobuhiro Ohshima, Takahiro Iwamoto, Munekazu Shigekawa1994 Volume 116 Issue 2 Pages 274-281
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe characterized Ca2+ entry in rat aortic smooth muscle cells (SMCs) maintained in primary culture by measuring uptake of 45Ca2+ or Mn2+ from a normal balanced salt solution and the extracellular Ca2+-induced increase in the intracellular Ca2+ concentration ([Ca2+]1) in a medium [high pH (pH 8.8)/high Mg2+ (20mM) medium containing a sarcoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin] that inhibits removal of Ca2+ from the cytoplasm. Such measurements in the presence or absence of a dihydropyridine (DHP) calcium channel antagonist (PN200-110) or hyperpolarizing agent (valinomycin) revealed that DHP-sensitive voltage-gated Ca2+ channels (VGCCs) are activated in these SMCs under resting conditions and that DHP-sensitive Ca2+ entry occurs mostly via these VGCCs. We found that receptor stimulation by endothelin-1 in these SMCs resulted in activation of neither DHP-sensitive nor -insensitive Ca2+ entry, but rather resulted in marked suppression of the former. Utilizing the DHP-sensitive extracellular Ca2+-induced increase in [Ca2+]1 as a monitor of activity of the DHP-sensitive VGCCs, we investigated the effects of protein kinase activators and phosphatase inhibitors on the regulation of these VGCCs. We found that the DHP-sensitive VGCCs were inhibited by endothelin-1 through the activation of protein kinase C. We also found that they were inhibited by 8Br-cGMP, okadaic acid, and calyculin A.View full abstractDownload PDF (1914K) -
Tomoko Inubushi, Hisao Kakegawa, Yasuo Kishino, Nobuhiko Katunuma1994 Volume 116 Issue 2 Pages 282-284
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have established a new differential assay method for cathepsin L-type proteinases using specific inhibitors, E-64 for all cysteine proteinases, CA-074 for cathepsin B, and PLCPI for cathepsin L-type proteinases with Z-Phe-Arg-MCA as the substrate. The value of cathepsin B calculated by this method did not coincide with value assayed directly in terms of the hydrolysis of Z-Arg-Arg-MCA, a specific substrate for cathepsin B. The activities of cathepsin L-type proteinases, cathepsins B and J in rat liver and kidney were assayed at the same time using this new assay method as a representative example.View full abstractDownload PDF (411K) -
Toshi Shimamoto, Kei Inaba, Paul Thelen, Tetsuya Ishikawa, Edward B. G ...1994 Volume 116 Issue 2 Pages 285-290
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe isolated a mutant of Escherichia coli which was defective in an Na+/H+ antiporter and grew poorly under alkaline conditions [Ishikawa, T., Hama, H., Tsuda, T., and Tsuchiya, T. (1987) J. Biol. Chem. 262, 7443-7446]. Later, it was concluded that the defective Na+/H+ antiporter in the mutant was the NhaB system, and the nhaB gene was mapped to 25.6min on the E. coli chromosome [Thelen, P., Tsuchiya, T., and Goldberg, E. B. (1991) J. Bacteriol. 173, 6553-6557]. We found that the NhaB-defective cells cannot grow in a high pH medium. Furthermore, intracellular pH in the mutant cells was almost the same as extracellular pH between 7.9 and 9.1, that is, intracellular pH was not regulated at this pH range. On the other hand, intracellular pH of the wild-type cells was maintained at about 7.6 when the extracellular pH was between 7.6 and 8.5. Thus, the NhaB Na+/H+ antiporter is essential for the regulation of intracellular pH under alkaline conditions in E. coli. Introduction of nhaA gene into the mutant cells increased Na+/H+ antiporter activity, but did not restore the defective growth and defective intracellular pH regulation under alkaline conditions.View full abstractDownload PDF (711K) -
Yoshiko Aoki, Toshimitsu Uenaka, Junken Aoki, Masato Umeda, Keizo Inou ...1994 Volume 116 Issue 2 Pages 291-297
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRo09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum which recognizes strictly the structure of phosphatidylethanolamine (PE) and forms an equimolar complex with the phospholipid on biological membranes. To use the peptide as a probe for analyzing the transbilayer movement of PE, we labeled the amino-terminal amino acid of the peptide with biotin without changing either the reactivity or specificity of the peptide. The amount of the peptide bound to the membrane was measured by enzyme linked immunosorbent assay (ELISA) after extraction of the peptide from the membrane. The peptide showed a strict temperature-dependent binding to human erythrocytes and the binding increased with increasing temperature. Since the peptide bound to PE in a temperature-independent manner and the binding to membrane PE is not affected by membrane proteins, the present temperature-dependent binding of the peptide to the cell membranes was likely to reflect temperature-dependent translocation of PE. The binding of the peptide to erythrocytes differed greatly among animal species. The peptide also showed temperature-dependent binding to a human histocytic lymphoma cell line, U 937, suggesting that the peptide will provide a novel and convenient probe for analyzing the transbilayer movement of PE in eukaryotic cells.View full abstractDownload PDF (821K) -
Tokio Yamaguchi, Izuru Shioji, Akiko Sugimoto, Yasuo Komoda, Hiroshi N ...1994 Volume 116 Issue 2 Pages 298-303
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeven bilirubin metabolites negative to the diazo reaction were identified in the urine of healthy persons by enzyme-linked immunosorbent assay (ELISA) using the anti-bilirubin monoclonal antibody 24G7. Two of the seven metabolites were isolated and their chemical structures were determined using fast-atom bombardment-mass spectroscopy (FAB-MS) and 1H-NMR. The two metabolites are 1, 14, 15, 17-tetrahydro-2, 7, 13-trimethyl-1, 14-dioxo-3-vinyl-16H-tripyrrin-8, 12-dipropionic acid and 1, 14, 15, 17-tetrahydro-3, 7, 13-trimethyl-1, 14-dioxo-2-vinyl-16H-tripyrrin-8, 12-dipropionic acid. They are regioisomers of each other. The two bilirubin metabolites are novel tripyrrole biocompounds and belong to a third group of bile pigments following biliverdin and bilirubin. We gave these compounds the generic names biotripyrrin-a and biotripyrrin-b, respectively.View full abstractDownload PDF (2924K) -
Sam J. Mansour, Katheryn A. Resing, Julian M. Candi, April S. Hermann, ...1994 Volume 116 Issue 2 Pages 304-314
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitogen-activated protein kinase kinase (MKK) phosphorylates and activates mitogenactivated protein kinase (MAPK) in response to stimulation of various eukaryotic signaling pathways. Conversely, a recent report showed that MAPK phosphorylates MKK in vitro [Matsuda, S., Gotoh, Y., and Nishida, E. (1993) J. Biol. Chem. 268, 3277-3281]. To gain insight into the function of this feedback phosphorylation, we identified the major sites targeted for phosphorylation by MAPK and examined whether such a modification plays a role in regulating the basal and stimulated MKK activities. Two phosphopeptides generated by tryptic digestion of MAPK-phosphorylated MKK were identified by electrospray ionization mass spectrometry. Cyanogen bromide cleavage also yielded two phosphopeptides whose sequence overlapped with the tryptic phosphopeptides. Both sets of phosphopeptides contained candidate MAPK target sites at Thr292 and Thr386 that fit the consensus sequence ProXThr*Pro. Replacement of either Thr292 or Thr386 with alanine by site-directed mutagenesis reduced the phosphate incorporation respectively to 32 or 75% that of wild type MKK. Replacement of both threonine residues with alanine reduced phosphate incorporation to 2.5% that of wild type enzyme. Comparison of MAPK-phosphorylated vs. unphosphorylated MKK showed no significant differences in basal or Raf-l-stimulated MKK activity. We conclude that the phosphorylation of MKK at Thr292 and Thr386 does not interfere with catalysis in vitro.View full abstractDownload PDF (5358K) -
Norihisa Mera, Shigeru Ohmori, Koshiro Itahashi, Masahiro Kiuchi, Taka ...1994 Volume 116 Issue 2 Pages 315-320
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSImmunoblot analysis showed that alpha-class glutathione S-transferase (GST), which is one of the major forms in adult human liver, was expressed in human fetal liver. Mu-class GST was also expressed in fetal liver. The majority of mu-class GST expressed in adult liver consisted of a subunit with a molecular weight of 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two subunits of 27 and 26 kDa were detected in fetal liver as proteins immunochemically related to mu-class GST. On reverse-phase HPLC, these two subunits cross-reactive with antibodies to rat GST 3-3 in fetal liver were indistinguishable from each other in their retention time; though, they could be separated by chromatofocusing analysis. The molecular weights of GSTs immunochemically related to rat GST 3-3, eluted at pH 7.1, 6.4, and 5.7, were 27, 27 and 26, and 26 kDa, respectively. In addition, the N-terminal amino acid sequence of these subunits suggested that GSTs related to rat GST 3-3 expressed in fetal liver may be homodimeric and heterodimeric proteins. As expected, pi-class GST was found to be a major form of GST in fetal liver but not in adult liver. In contrast, the GST immunochemically related to rat GST Yrs-Yrs, which is classified as theta-class GST, was detected in adult liver but not in fetal liver. These results indicate that several isoenzymes of GST are expressed in human fetal liver, but they are not the same as those in adult liver.View full abstractDownload PDF (2848K) -
Junko Watanabe, Shin-ichi Hayashi, Kaname Kawajiri1994 Volume 116 Issue 2 Pages 321-326
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of two genetically associated polymorphisms [c 1 allele: PstI -, RsaI +; c 2 allele: Psti +, RsaI -] in the 5'-flanking region on the regulation and expression of the human CYP2E1 gene were investigated. DNase I footprinting and CAT activity analyses using various deletion mutants constructed with DNA segments from genotype A (c1/c1) or C (c2/c2) and SV40 or an endogenous promoter showed that the RsaI polymorphism affects the binding of a transcription factor and the transcriptional activation of CYP2E1, while the PstI polymorphism has little effect. The correlation between the genotypes and expression levels of CYP2E1 mRNA were examined in peripheral lymphocytes of 86 individuals by RT-PCR, the alcohol consumption by the subjects being taken into account. In non-drinkers, the mean ratio of the expression of CYP2E1 mRNA to that of GAPDH mRNA in genotype B (c1/c2) was 1.7-fold higher than that in genotype A (0.05<p<0.10). As compared to non-drinkers with genotype A, subjects with genotype B who drank alcohol on a daily basis expressed about 2.0-fold higher levels of CYP2E1 mRNA (p<0.01). These results indicate that a RsaI polymorphism in the 5'-flanking region of CYP2E1 may lead to inter-individual differences in CYP2El-mediated microsomal drug oxidation activities, including oxidation of N-nitrosamines.View full abstractDownload PDF (2502K) -
Kohei Shimada, Yasuo Ohnishi, Sueharu Horinouchi, Teruhiko Beppu1994 Volume 116 Issue 2 Pages 327-334
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA large COOH-terminal domain of Serratia marcescens serine protease (SSP) is essentially required for the excretion of the mature protease region through the outer membrane in Escherichia coli. For investigation of the possibility of transporting foreign periplasmic proteins by utilizing the function of the COOH-terminal domain of SSP, the pseudoazurin (PA) gene of Alcaligenes faecalis was fused to three different lengths of the COOH-terminal domain via a BglII linker sequence (AGATCT) which would be translated into Arg-Ser, one of the cleavage sequences of the OmpT protease. The outer membrane fractions of E. coli JM105 harboring each of the fused genes on a multicopy plasmid was found to contain the PA-SSP hybrid protein without the signal sequence as well as the COOH-terminal domain of SSP processed at the Arg-Ser sequence. Trypsin-treatment of intact and cold osmotically shocked cells suggested that the COOH-terminal domain of SSP in the hybrid protein was rigidly integrated in the outer membrane, while the PA part of the hybrid protein was exposed on the cell surface. The localization of the PA part was confirmed by immunolabeling of intact cells with the anti-PA antibody. Furthermore, exogenous addition of purified SSP to the intact E. coli cells containing the hybrid protein liberated the PA part from the cells into the medium, probably by cleavage at or very near the site processed during the normal secretion of intact SSP. All these data suggest that the PA part of the hybrid protein exported to the cell surface with the aid of the COOH-terminal domain of SSP is liberated by the cleavage between the Arg-Ser sequence derived from the linker sequence by the OmpT protease, although the liberated PA was not detected, probably because of rapid degradation by E. coli proteases.View full abstractDownload PDF (5039K) -
Masaki Iino, Hiroyuki Takeya, Junji Nishioka, Tomohiro Nakagaki, Koji ...1994 Volume 116 Issue 2 Pages 335-340
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe studied the interaction of factor X activation peptide (XAP) with factor IXa and factor Xa and the effect of XAP on factor IXa-catalyzed activation of factor X. XAP associated with factor Xa in the presence of 5mM Ca2+ was dissociated from factor Xa by gel chromatography using Ultrogel AcA54 in 5mM EDTA, or in 8M urea-0.1% SDS. An exogenous isolated XAP inhibited the factor IXa-catalyzed factor X activation both in the presence and absence of factor VIIIa. 4-Amidinophenylmethylsulfonyl (aPMS)-factor Xa independent of XAP also inhibited the factor X activation more effectively than XAP alone in the presence of factor VIIIa. However, aPMS-factor Xa independent of XAP hardly inhibited the factor X activation in the absence of factor VIIIa. The binding of 125I-labeled factor X to the aPMS-factor IXa fixed to a microwell plate was inhibited by unlabeled factor X or XAP, but not by aPMS-factor Xa with or without XAP. Factor IXa directly bound to XAP and aPMS-factor Xa with XAP, but did not bind to aPMS-factor Xa without XAP. These findings suggest that the region of XAP in factor X directly interacts with factor IXa, and factor Xa region other than XAP interacts with factor VIIIa.
Desialation or deletion of N-linked carbohydrates of XAP reduced the inhibitory activity of XAP for the factor X activation by factor IXa to approximately 50% of that of the intact XAP. This suggests that the sialic acids in the carbohydrate chains of the XAP region partly contribute to the interaction with factor IXa during its activation.View full abstractDownload PDF (2995K) -
Nobuaki Okumura, Masato Okada, Katsuya Nagai, Hachiro Nakagawa1994 Volume 116 Issue 2 Pages 341-345
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVasoactive intestinal peptide (VIP) is a neuropeptide that induces neuronal differentiation through a cAMP-dependent mechanism. We have previously shown that VIP induces tyrosine phosphorylation and activation of MAP kinases in PC12h cells [J. Biochem. 115, 304-308 (1994)]. In the present study, we showed by Western blotting with anti-phosphotyrosine antibodies that in PC12h cells VIP induced tyrosine phosphorylation of proteins of 140, 120, 110, and 70 kDa in addition to MAP kinases. The immunoprecipitates with anti-phosphotyrosine antibody from VIP-treated cells contained high activity of protein kinase phosphorylating poly(glu-tyr) and enolase; the activity from VIP-stimulated cells was 1.5-2 times higher than that from unstimulated cells. In vitro kinase reaction without extrinsic substrates resulted in tyrosine phosphorylation of doublet proteins which migrated slower than pp125FAK on SDS-PAGE. An increase in kinase activity of the immunecomplex was detected when the cells were stimulated with forskolin. These results suggest that protein tyrosine phosphorylation is involved in differentiation of neuronal cells stimulated by VIP and that it is regulated by a cAMP-dependent mechanism.View full abstractDownload PDF (3954K) -
Nobuaki Okumura, Masato Okada, Hachiro Nakagawa1994 Volume 116 Issue 2 Pages 346-350
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDepolarization induced by KCl was found to induce tyrosine phosphorylation of cellular proteins in PC12h cells. By Western blotting with anti-phosphotyrosine antibody, we detected tyrosine phosphorylation of proteins with molecular weights of 120, 110, 105, 95, 75, 70, 66, 44, and 42 kDa in response to KCl. The immunoprecipitates from KCl-treated cells with the antibody contained large amounts of tyrosine-phosphorylated proteins and increased activity of tyrosine kinase. Incubation of the immunoprecipitates with [γ-32P]ATP resulted in tyrosine phosphorylation of two proteins with the molecular weights of 120 and 140 kDa. These effects were completely abolished by the addition of EGTA before KCl treatment, suggesting that the depolarization-induced tyrosine phosphorylation may require calcium entry into the cells from the medium. Increased activity of tyrosine kinase phosphorylating the 120 and 140 kDa proteins was also recovered from cells stimulated with nerve growth factor, basic fibroblast growth factor, epidermal growth factor, and vasoactive intestinal peptide. Among them, depolarization by KCl elicited the strongest effect. These results indicate that a protein tyrosine kinase that phosphorylate the 120 and 140 kDa proteins is phosphorylated or activated in response to calcium ion, cAMP, and growth factors acting through tyrosine kinase receptors.View full abstractDownload PDF (4022K) -
Takao Ohyashiki, Tomomi Takino, Katsuhiko Matsui1994 Volume 116 Issue 2 Pages 351-356
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of lipid peroxidation on Ca2+-induced aggregation of porcine intestinal brush-border membranes were examined using a system consisting of ascorbic acid/Fe2+/tert-butyl hydroperoxide (t-BuOOH). Incubation of the membranes with ascorbic acid/Fe2+/t-BuOOH resulted in inhibition of Ca2+-induced aggregation of the membranes with the formation of thiobarbituric acid-reactive substances, depending on the hydroperoxide concentration and the incubation time. The inhibition of the membrane aggregation associated with ascorbic acid/Fe2+/t-BuOOH treatment was effectively prevented by the addition of an antioxidant, 3(2)-tert-butyl-4-hydroxyanisole, to the reaction mixture. Studies with 8-anilino-1-naphthalenesulfonate (ANS) revealed that there is a linear relationship between the apparent dissociation constants (Kd) of ANS-membrane complexes and the aggregating efficiencies of the membranes with different levels of lipid peroxidation. This suggests that inhibition of the membrane aggregation by lipid peroxidation involves a change in the membrane surface charge density. Modification of the membranes with malondialdehyde also resulted in a decrease in the aggregating efficiency of the membranes with a decrease in the Kd, value of ANS-membrane complex. In addition, the contribution of the lipid organization to membrane aggregation was examined by measuring the fluorescence anisotropy of diphenylhexatriene-labeled membranes in the presence of a lipid fluidizer, benzyl alcohol. The results are discussed in terms of peroxidationinduced inhibition of intramembrane interactions of the membranes.View full abstractDownload PDF (757K) -
Fumihiko Saito, Kazuko Hori, Masayuki Kanda, Toshitsugu Kurotsu, Yoshi ...1994 Volume 116 Issue 2 Pages 357-367
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus brevis Nagano grs2 gene, which encodes gramicidin S synthetase 2 (GS2) catalyzing activation and combination of four constituent amino acids of gramicidin S, namely, proline, valine, ornithine, and leucine, has been sequenced. The open reading frame of grs2 gene specifies a 4, 450-amino acid protein with a calculated molecular weight of 508, 658. There are four domains with a mean of 1, 042 amino acid residues containing a repeated sequence of about 600 amino acids, which is highly homologous to the amino-terminal half of gramicidin S synthetase 1 (GS1) (about 40-50% identity). Three domains of grs2 protein, excluding the first one, show homology over the entire sequences of 1, 042 amino acids, but the first domain only shows homology in the conserved 600-amino acid sequence. The last 300-amino acid sequence of grs2 protein following the fourth domain has no homology with any of the above sequences. Translation products of subcloned fragments containing the third or the fourth domain catalyzed ornithine- or leucine-dependent ATP-32PP1 exchange, respectively. These results, together with a previous report on a proline-activation domain indicated that the repeated and conserved domains are the individual activation sites of the constituent amino acids; the activation sites are arranged in the order of peptide elongation on GS2. Several motifs of grs2 protein are conserved among the multiple domains of peptide synthetases and aminoacyl or acyl adenylate-forming enzymes.View full abstractDownload PDF (3568K) -
Ken Karasawa, Hideyuki Kato, Morio Setaka, Shoshichi Nojima1994 Volume 116 Issue 2 Pages 368-373
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the production of PAF, a mediator of shock, and LysoPAF, an inactive metabolite of PAF, in the guinea pig peritoneal cavity after i. p. administration of Escherichia coli LPS. Within 1h of LPS administration, the level of PAF in the peritoneal fluid increased from 4.9 to 37.2 pmol/animal and decreased to the control value thereafter. In contrast, the level of lysoPAF gradually rose from 63.5 to 268 pmol/animal for upto 6h. The activity of acetylhydrolase, which converts PAF to lysoPAF, in the peritoneal cavity increased in parallel with the increase in the lysoPAF level. The enzyme was distinguishable from phospholipase A2, because p-bromophenacylbromide (p-BPB), Ca2+, and ethylenediaminetetraacetic acid (EDTA) did not affect its enzymatic activity. In addition, this acetylhydrolase revealed similar biochemical properties to that detected in plasma. Both acetylhydrolases were resistant to trypsin treatment and had the same apparent molecular weight, as shown by gel-filtration column chromatography. These results suggest that the acetylhydrolase, which accumulates in the peritoneal cavity, infiltrates from the plasma in response to LPS, and then participates in the exclusion of PAF during endotoxin shock.View full abstractDownload PDF (759K) -
Ken Karasawa, Masayuki Yato, Morio Setaka, Shoshichi Nojima1994 Volume 116 Issue 2 Pages 374-379
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPlatelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) acetylhydrolase is an enzyme that hydrolyzes the acetyl ester of PAF. We purified this enzyme, which accumulated in the peritoneal cavity during endotoxin shock, by ammonium sulfate precipitation, and sequential use of butyl-Toyopearl, heparin-Sepharose, Con A-Sepharose, chelating-Sepharose, and MonoQ column chromatographies. We identified a monomeric polypeptide with a molecular weight of approximately 63 kDa on SDS-PAGE. This molecular weight differs from those of previously described acetylhydrolases. The purified enzyme did not degrade phospholipids with a long chain fatty acyl group at the sn-2 position. In addition, the enzyme activity was not inhibited by either pBPB or quinacrine. Accordingly, this enzyme is distinct from phospholipase A2. In addition, this enzyme also hydrolyzed some oxidatively fragmented phospholipids with PAF-like biological activities such as 1-O-hexadecyl-2-glutaroyl-sn-glycero-3-phosphocholine and 1-O-hexadecyl-2-succinoyl-sn-glycero-3-phosphocholine.View full abstractDownload PDF (2460K) -
Hitoshi Ohmori, Masako Kuba, Akira Kumon1994 Volume 116 Issue 2 Pages 380-385
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA phosphatase hydrolyzing 3-phosphohistidine and 6-phospholysine was purified from rat brain cytosol to 90% homogeneity on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. One milligram of protein of the purified phosphatase released inorganic phosphate from 3-phosphohistidine, 6-phospholysine, AMP, GMP, and p-nitrophenyl phosphate at velocities of 6.5, 15.6, 15.0, 6.9, and 8.3 μmol/min, respectively. However, the purified enzyme could not hydrolyze Nω-phosphoarginine and phosphocreatine, which are substrates for phosphoamidase [EC 3. 9. 1. 1]. The molecular masses of the holoenzyme and the subunit were 94 and 50 kDa, respectively, and the sedimentation coefficient of the native enzyme was 6.3 s, indicating that it was a dimeric enzyme of identical subunits. The enzyme functioned well under acidic conditions, and 50% of the activity was inhibited by 30 μM tartrate, 4 μM vanadate, 20 μM molybdate, 4 μM VCl3, or 13 μM MoCl5. These results indicate that the present hydrolase belongs to the acid phosphatase group [EC 3. 1. 3. 2].View full abstractDownload PDF (2780K) -
Mika Inomata, Yoshiharu Takayama, Hiroshi Kiyama, Shigeyuki Nada, Masa ...1994 Volume 116 Issue 2 Pages 386-392
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have so far suggested that the functions of Src family protein-tyrosine kinases are under the control of their regulator kinase, Csk. To evaluate the role of Csk-mediated regulation in neural tissues, we examined the correlation between the activities of Src family kinases and the expression level of Csk during development of the rat brain. Csk was expressed at high levels in the developing embryonic brain and then rapidly decreased as the brain matured. Consistent with the decrease in the Csk level, the kinase activity of a member of the Src family, Fyn, was greatly enhanced, but that of Src was not correlated inversely with the level of Csk expression. Src exhibited elevated activity in the developing brain, in which a neuronal form of Src (N-Src) is expressed as the dominant form of Src. Although N-Src was readily down-regulated by Csk when coexpressed in yeast, it showed much higher specific activity than c-Src, even in the repressed form. These findings suggest that neural tissues acquire high activities of Src family kinases, which might be important for differentiation and development of the nervous system, through induction of the active form of Src (N-Src) and down-regulation of their suppresser, Csk.View full abstractDownload PDF (6151K) -
Shamima Nasrin, Hiroshi Ichinose, Hiroyoshi Hidaka, Toshiharu Nagatsut1994 Volume 116 Issue 2 Pages 393-398
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman tyrosine hydroxylase (hTH) exists in four isoforms. The four recombinant hTH isoenzymes types 1-4 (hTH1-4) produced in and purified from Escherichia coli showed regulatory kinetic properties for the natural (6R)-L-erythro-tetrahydrobiopterin (RBPH4) as a cofactor. In contrast, the unnatural cofactor (6S)-L-erythro-tetrahydrobiopterin (SBPH4) and a synthetic cofactor (6RS)-methyl-tetrahydropterin (6MPH4) showed usual kinetic characteristics with each of hTH1-4. Substrate inhibition by tyrosine was observed for each of hTH1-4 with natural RBPH4. Two different Km values for pterin cofactor were observed at a high concentration (200 μM) of L-tyrosine only with natural RBPH4, in contrast to a single Km value for unnatural SBPH4 or synthetic 6MPH4. The present results suggest that in the presence of relatively high concentrations (approximately 100 μM) of tyrosine in vivo, RBPH4 cofactor may have a regulatory role for the activity of all four human isoenzymes in vivo. We also found that recombinant hTH1 and 3 were more unstable than hTH2 and 4, suggesting that the 4-amino-acid insertion in hTH2 and 4 may be responsible for the relative stability of hTH2 and 4 isoenzymes.View full abstractDownload PDF (1686K) -
Eiichi Saitoh, Satoko Isemura1994 Volume 116 Issue 2 Pages 399-405
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cDNAs encoding the precursors of cystatin SN, cystatin S, and two mutants of cystatin S (-18R→W; 117R→W) were expressed in Escherichia coli JM109 with isopropyl-β-D-thiogalactoside (IPTG) induction. Premature cystatin S with the original signal [-20MARPLCTLLLLMATLAGALA] was processed and a large amount of the mature form was produced. A mutation (-18R→W) in the signal reduced its accumulation in periplasmic space remarkably. The amount of cystatin SN accumulated in the periplasm was slightly smaller than that of cystatin S. The periplasmic fraction was prepared by cold osmotic-shock treatment and the expressed cystatins were detected using anti-cystatin S antibody. Recombinant cystatin S and its mutant (117R→W) were purified from the periplasmic fractions with an ion exchange column of DEAE-cellulose. The amino (N-) terminal 10 residues of recombinant cystatin S was sequenced to be SSSKEENRII-, which is exactly identical to that of the authentic mature cystatin S. Recombinant cystatin S and the mutant showed virtually the same inhibitory properties for ficin, papain and cathepsin B as the native cystatin S and its monophosphorylated form. The inhibitory activity of the both recombinant cystatins for cathepsin C was weaker than those of the native cystatin S and phosphorylated cystatin S.View full abstractDownload PDF (3046K) -
Jun Suzuki, Sumiko Kimura, Koscak Maruyama1994 Volume 116 Issue 2 Pages 406-410
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConnectin (titin) is an extraordinarily long filamentous protein of striated muscle. The particle lengths of α-connectin (titin 1) and its proteolytic products, β-connectin (titin 2) and 1, 200 kDa fragment, were measured with rotary-shadowed images of the filaments after orientation by centrifugation. It was observed that the 1, 200 kDa fragment was frequently folded into a double strand, β-connectin was partly folded, and α-connectin was easily split into β-connectin and 1, 200 kDa fragment. Taking these features into consideration, the average lengths of α- and β-connectin and 1, 200 kDa fragment were estimated to be approximately 1, 250, 920, and 360 nm, respectively.View full abstractDownload PDF (3791K) -
Norio Takizawa, Yusuke Mizuno, Yasushi Ito, Kunimi Kikuchi1994 Volume 116 Issue 2 Pages 411-415
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amounts of four isoforms of the catalytic subunit of type-1 protein phosphatase, PP1α, PP1γl, PP1γ2, and PP1δ have been determined in extracts of various mouse tissues including brain, liver, skeletal muscle, kidney, small intestine, heart, lung, spleen, thymus, and testis by Western blot analysis. Immunoreactive bands for PP1 isoforms were detected at 39.5, 38.5, and 40 kDa for PPlα, PP1γ1, and PP1γ2, respectively, and at 39 and 37 kDa for PP1δ. The amount of PP1α was at comparable levels in all tissues examined except skeletal and heart muscles, in which it was detected slightly or not detectable, respectively. The amount of PP1γ1 was at higher levels in brain, small intestine, and lung, being 2 to 3 times those in other tissues except heart and spleen, in which PP1γ1 was not detectable. The amount of PP1γ2 was extremely large in testis, small in brain, lung, spleen, and thymus, but it was not detectable in the other tissues. The amount of PP1δ was at comparable levels in all the tissues except skeletal muscle, in which it was at a low but detectable level. Then, the amounts of the four PP1 isoforms were determined in non-obese diabetic (NOD) mice. The amounts of PP1α were progressively decreased in livers of NOD mice as a function of increasing concentrations of blood glucose, whereas the amounts of PP1γ1 and PP1δ were unchanged. These tissue-characteristic expression patterns and the diabetes-induced alteration strongly suggest different roles and significance of each isoform of type-1 protein phosphatase in these mouse tissues.View full abstractDownload PDF (1966K) -
Rashidul Alam, Naomi Hachiya, Masao Sakaguchi, Shun-ichiro Kawabata, S ...1994 Volume 116 Issue 2 Pages 416-425
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe identified a liver cytosolic protein factor that stimulated the import of wheat germ lysate-synthesized precursor proteins into mitochondria. It was termed mitochondrial import stimulation factor or MSF [Hachiya, N. et al. (1993) EMBO J. 12, 1579-1586]. It consisted of 32-kDa (MSFL) and 30-kDa (MSFS) polypeptides as assessed by SDS-PAGE. MSF recognized the presequence portion of mitochondrial precursor proteins and catalyzed the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP-dependent manner. We report here the cDNA cloning and characterization of MSF. Microsequencing of MSFL and MSFS showed that they belonged to a highly conserved, widely distributed eukaryotic protein family, collectively designated as 14-3-3 proteins. We cloned the cDNA of MSFL and that of one component of MSFS (MSFS1) from a rat liver cDNA library. The cloned cDNAs were separately expressed in Escherichia coli and the expressed proteins were purified to homogeneity. The purified recombinant MSFL and MSFS1 stimulated mitochondrial import of adrenodoxin precursor (pAd) synthesized in vitro with wheat germ lysate translation system. Recombinant MSFL or MSFS1 had the ability to bind with denatured pAd and they kept the precursor in an import-competent state. Rabbit polyclonal antibodies raised against the recombinant proteins inhibited the import-stimulation activity of rat liver cytosol as well as that of authentic purified MSF. Identification of MSF as 14-3-3 proteins establishes a novel function for this family of proteins and indicates their role as cytosolic chaperones to aid many important cellular events.View full abstractDownload PDF (2469K) -
Kunio Tsujimura, Jin Tanaka, Shoji Ando, Yoichiro Matsuoka, Masashi Ku ...1994 Volume 116 Issue 2 Pages 426-434
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe identified the phosphorylation sites of glial fibrillary acidic protein (GFAP) for cdc2 kinase and Ca2+-calmodulin (CaM)-dependent protein kinase II. GFAP was phosphorylated to _??_0.2 mol of phosphate/mol of GFAP by cdc2 kinase, and this phosphorylation did not induce disassembly of the filament structure. On the other hand, GFAP was phosphorylated to _??_1.9 mol of phosphate/mol of GFAP by Ca2+-CaM-dependent protein kinase II, and this phosphorylation did induce disassembly of the filament. Sequential analysis of the purified phosphopeptides revealed that only Ser8 on GFAP was phosphorylated by cdc2 kinase, whereas Ser13, Ser17, Ser34, and Ser389 on GFAP were phosphorylated by Ca2+-CaM-dependent protein kinase II.View full abstractDownload PDF (3904K) -
Naoki Noda, Ryuichiro Tanaka, Kazushige Tsujino, Yoko Takasaki, Masami ...1994 Volume 116 Issue 2 Pages 435-442
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeven glycosphingolipids were obtained in the pure state from the clam worm, Marphysa sanguinea, by preparative HPLC with a reversed-phase column in a recycling mode. Their structures were elucidated based on detailed analyses of 1H- and 13C-NMR spectra of the intact compounds. The position and the geometry of double bonds in the long-chain base (LCB) were determined by the two-dimensional NMR (COSY, NOESY, and HMBC) analyses. All compounds are phosphocholine-bonded monogalactosylceramides and two of them have tri-unsaturated long-chain bases.View full abstractDownload PDF (686K) -
Takuo Kosaka, Nobuhiro Suzuki, Yoshihiro Ishibashi, Hirokazu Matsumoto ...1994 Volume 116 Issue 2 Pages 443-449
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo understand the biosynthesis of the human ET family, three kinds of cloned cDNA for human prepro-endothelin-1 (prepro-ET-1), prepro-ET-2, and prepro-ET-3 were stably expressed in Chinese hamster ovary cells (CHO-K1). Immunoreactive (ir-) ET polypeptides in the culture media of the transformants were purified by reverse-phase high-performance liquid chromatography (HPLC) coupled with sandwich-type enzyme immunoassay (EIA). Amino acid sequencing and FAB mass spectrometry of the purified ir-ET polypeptides revealed the presence of human big ET-1 (1-38), big ET-2 (1-38), and big ET-3 (1-41)NH2 as intermediate forms. These results directly revealed the biosynthetic pathways of three human ETs at the peptide level.View full abstractDownload PDF (759K) -
Richard Jennemann, Matthias Schulze, Bernhard L. Bauer, Christian Kurt ...1994 Volume 116 Issue 2 Pages 450-456
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nephropathic effects of Heymann's experimental nephrites involve autoallergic serum antibodies directed against rat kidney membrane constituents. In assessing the action of glycolipids as possible autoallergens in these conditions, it was found that heterologous and autologous Heymann's nephritis sera antibodies recognize the rat kidney sulphatides, II3SO3(-)-Gg3Cer (Strl1), and III3SO3(-)-, II3SO3(-)-Gg3Cer (Strl2). Two antibody populations in Heymann's sera, each reacting with only one of the two sulphatides, could be observed. It was further shown that human factor-H and properdin, pivotal regulators of the alternative pathway of complement activation, both bound to Strl2 in vitro. This binding of factor-H and properdin was differentially affected by affinity-purified anti-Strl2 antibodies of Heymann's nephritis sera. Whereas the interaction between factor-H and Strl2 was inhibited by the antibody, that of properdin was enhanced.View full abstractDownload PDF (2919K) -
Olin D. Liang, Jan-Ingmar Flock, Torkel Wadström1994 Volume 116 Issue 2 Pages 457-463
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding of heparin-binding form of vitronectin to Staphylococcus aureus was inhibited completely by heparin or by the same form of vitronectin. The binding was inhibited only to about 50% by the non-heparin-binding form of vitronectin, indicating an apparent involvement of the heparin-binding properties in the interaction between vitronectin and S. aureus. This was supported by experiments in which a synthetic peptide (Ala347-Arg361, comprising heparin-binding consensus sequences) was found to partly inhibit bacterial adherence to immobilized vitronectin. A bacterial cell surface protein could bind to the quinquedecapeptide, but not to the highly charged peptides consisting entirely of arginine or lysine, immobilized on microtiter plates and the binding could be competitively inhibited by an excess of soluble peptide. Direct binding of radiolabeled peptide to bacterial cells was also demonstrated, which was rapid, saturable, and pH-dependent. Furtherly a bacterial surface protein having molecular mass of 60 kDa was isolated by affinity chromatography on a quinquedecapeptide-HiTrap-NHS column. Our data suggest that the heparin-binding properties of vitronectin play a role in bacterial recognition.View full abstractDownload PDF (1519K) -
Kuniko Akama, Kimio Terao, Yukiharu Tanaka, Akihiko Noguchi, Naoto Yon ...1994 Volume 116 Issue 2 Pages 464-470
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA trypsin-like protease was extracted with 1% cetyltrimethylammonium bromide (CTAB) at pH 7.0 from boar cauda epididymal sperm nuclei whose acrosin had previously been removed by acid extraction. The CTAB-extracted sperm protease (CSP) was purified by ion-exchange chromatography on CM-23, gel filtration on Sephadex G-100, affinity chromatography on benzamidine-CH-Sepharose 4 B, and HPLC on CM-5PW. CSP is a two chain protein composed of Mr 2.6 K and Mr 37 K chains, which are covalently cross-linked by disulfide bonds. CSP exhibited a pH optimum between pH 8.0 and 9.0, and was inhibited by diisopropyl phosphorofluoridate, antipain, leupeptin, and 1-chloro-3-tosylamide-7-amino-L-2-heptanone. The activity of CSP was enhanced about 1.2-fold with 50mM CaCl2, with which acrosin is enhanced 2.0-fold. The catalytic efficiency (kcat/Km) of CSP toward Bz-L-Arg-OEt, Tos-L-Arg-OMe, and Tos-L-Lys-OMe in the presence of 50mM CaCl2 differed from that of acrosin by factors of 0.53, 1.2, and 0.80, respectively. Amino acid sequencing of V8-digested peptides of CSP, and its L- and H-chains showed that the amino acid sequence of CSP was closely related to, but different from, that of acrosin. These results suggest that CSP is a novel acrosin-like enzyme that differs from acrosin in its location in the sperm head, the effect of calcium ions on its activity, and its substrate specificity.View full abstractDownload PDF (901K)
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