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Volume 122 (1997)
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Volume 122, Issue 2
Displaying 1-31 of 31 articles from this issue
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Taroh Kinoshita, Kazuhito Ohish, Junji Takeda1997 Volume 122 Issue 2 Pages 251-257
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtein GPI anchors are ubiquitous in eukaryotic cells. More than 50 mammalian proteins are anchored to the membrane via GPI. GPI anchoring is a posttranslational modification occurring in the endoplasmic reticulum where preassembled GPI anchor precursors are transferred to proteins bearing a C-terminal GPI signal sequence. The GPI anchor precursors are synthesized in the endoplasmic reticulum by sequential addition of sugar and other components to phosphatidylinositol. More than ten genes participate in this biosynthetic pathway, eleven of the mammalian genes having been cloned by means of complementation of mutant cells that are defective in this pathway or based on sequence homology to previously cloned yeast counterparts. A somatic mutation in one of those genes, PIG-A, involved in the first reaction step, is responsible for the hemolytic disease, paroxysmal nocturnal hemoglobinuria.View full abstractDownload PDF (874K) -
Masashi Honjo, Tetsuo Ishida, Kihachiro Horiike1997 Volume 122 Issue 2 Pages 258-263
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrontal gel chromatography is a convenient and accurate method to obtain the free ligand concentration of a protein-ligand mixture. Because a large amount of sample (more than 6ml) is required for the method, it has been rarely used for binding experiments. We have developed a system to carry out frontal gel chromatography on a semi-micro scale using short gel filtration columns (4.6mm×50-100mm); frontal chromatograms could be obtained with small amounts of samples (1-2.5ml) within 20min. We used this technique to examine the binding of warfarin, L-tryptophan, or FMN to human serum albumin, the binding of warfarin to bovine serum albumin, and the interaction of catechol 2, 3-dioxygenase with o-nitrophenol. The data fitted to a binding model in which a protein has one or several independent binding sites. Both human and bovine serum albumin showed the high-affinity bindings of two warfarin molecules. The binding number for L-tryptophan on human serum albumin was confirmed to be one, whereas maximal binding of FMN was 0.6 molecule per albumin molecule. o-Nitrophenol showed high-affinity binding only to holocatechol 2, 3-dioxygenase. The absorption spectrum of the bound o-nitrophenol resembled that of anionic o-nitrophenol. These results demonstrated that frontal gel chromatography on a semi-micro scale is useful for the study of binding systems; the method is rapid and would be easy to automate fully.View full abstractDownload PDF (800K) -
Kazuhiro Tsujita, Takuo Shiraishi, Katsuko Kakinuma1997 Volume 122 Issue 2 Pages 264-270
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA highly sensitive microspectrophotometer was developed to measure spectral changes of oxyhemoglobin (oxy Hb) in single red blood cells (RBC) incubated with stimulated macrophages as a model of nitric oxide (NO)-dependent cytotoxicity. Our microspectrophotometer, using a modified acousto-optic tunable filter (AOTF) and a 2-dimensional CCD array, allows fast spectrophotometric data acquisition. Human RBC treated with various concentrations of NO showed spectral changes due to the conversion of oxy Hb to methemoglobin (met Hb), in which the change in absorption differences at α (557-590nm) and β (542-525nm) bands showed a linear relationship with the concentration of NO up to 100μM. In contrast to highly diffusible NO, nitrite ions (NO2-) seem to enter RBC very slowly, resulting in negligible formation of met Hb in the presence of 5mM glucose even during a prolonged incubation period. RBC were incubated with murine macrophages with and without lipopolysaccharide (LPS) in the presence of glucose for 24 and 40h and subjected to the microspectrophotometric assay. The RBC incubated with LPS-stimulated macrophages showed significant changes in the spectrum due to NO-dependent conversion of oxy Hb to met Hb, which corresponded to the spectral changes of RBC treated with a several times higher concentration of NO than that in the culture medium. The trapping efficiency was calculated from the amounts of the NO released from macrophages and of the met Hb formed in the RBC, which gave a high efficiency (43%). The results suggest that RBC trap NO directly by cell-cell interaction with macrophages. This spectrophotometric system is available for use with just a few drops of samples to study NO-specific cytotoxicity as a model of RBC without the use of any chemical reagent, in parallel with microscopic observations on changes of the cellular morphology under physiological conditions, such as membrane damage leading to hemolysis, adherence, and phagocytosis.View full abstractDownload PDF (1912K) -
Toshihiko Aki, Shuichi Yanagisawa, Hiroshi Akanuma1997 Volume 122 Issue 2 Pages 271-278
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is expressed at high levels in almost all tissues. However, the molecular mechanism which sustains high-level expression of this house-keeping enzyme is still unknown. Here we show that transcriptional activity is reduced by deletion of the nucleotides from -181 to -144 (relative to the transcriptional start site) in the promoter of human GAPDH gene, both in CHO (derived from Chinese hamster ovary) and HepG2 (derived from human hepatoma) cells. Gel retardation assays revealed that at least two nuclear factors, termed GAPBF1 and GAPBF2, bind to this region. Mutations in the GAPBF1 binding site (-178 to -169) or the GAPBF2 binding site (-168 to -163) reduced this promoter activity in vivo, showing that these two sites contribute to the activity of the GAPDH gene promoter. Since mutations in the region from -162 to -146 also reduced the promoter activity, this region seemed to function as an added cis-element, although we failed to find a factor that interacted specifically with this region in vitro. Accordingly, we propose that there are multiple cis-elements in the region from -181 to -144, each of which contributes to the promoter activity of GAPDH gene; the GAPBF1 binding site has the unique feature of having a stretch of repeated A nucleotides.View full abstractDownload PDF (3578K) -
Ken-ichi Yoshida, Kazuki Harada1997 Volume 122 Issue 2 Pages 279-285
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnkyrin links cytoskeleton and integral membrane proteins and is proteolyzed in vitro by calpain, a Ca2+-dependent protease. In the present study, we examined the localization of two ankyrin isoforms, erythrocyte (red blood cell)-type (ankyrinR) and brain-type (ankyrinB), and their proteolysis after ischemia-reperfusion in the subcellular fractions of perfused rat heart by immunoblotting and by immunohistochemistry using specific antibodies. Both isoforms were observed to be distributed chiefly in the myofibril-nucleus (1, 000×g pellet: P1) fraction, while ankyrinR was located substantially in the membrane (100, 000×g pellet: P2) fraction. Reperfusion after 10min or more of global ischemia induced preferential proteolysis of ankyrinR in the P2 fraction and ankyrinB in the P1 fraction. The proteolysis of ankyrinR, but not ankyrinB, was effectively inhibited by the synthetic calpain inhibitor acethyl-leucyl-leucyl-norleucinal. The immunohistochemical examination showed that anti-ankyrinR delineated striations, sarcolemma and nuclei, and the staining was decreased after ischemia-reperfusion, while anti-ankyrinB showed diffuse staining. The proteolysis of ankyrinR may interfere with force conduction through disruption of the linkage between integral membrane proteins and the myofibril-cytoskeleton.View full abstractDownload PDF (3630K) -
Kazuyoshi Chiba, Eisuke Sato, Motonori Hoshi1997 Volume 122 Issue 2 Pages 286-293
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method was investigated for monitoring the activity of protease (s) in cytosol of a single starfish oocyte using succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid as the substrate, which was injected into the cell. After preincubation of immature oocytes with a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norvalinal, the initial hydrolysis of the substrate was remarkably inhibited. The inhibitor blocked 1-methyladenine-triggered cyclin degradation, which is known to be mediated by proteasome. However, calpain inhibitor E-64 did not inhibit the hydrolysis of the substrate. These results suggested that the protease activity measured by this method is mainly attributable to cytoplasmic proteasome. The hydrolysis of the substrate was partially inhibited by bestatin, suggesting that the substrate was cleaved by aminopeptidase. Thus, the initial velocity of hydrolysis of the substrate (V0) by proteasome was assayed in a living oocyte after preinjection of bestatin. The values of V0 increased gradually after 1-methyladenine addition and reached the maximum level at the time corresponding to cyclin degradation. The calculated maximum velocity of hydrolysis by a mature oocyte was approximately three times higher than that by an immature oocyte. The Michaelis-Menten constant value was also higher in mature than immature oocytes. These results suggest that proteasomedependent proteolysis is regulated not only by ubiquitination of substrates, as is generally believed, but also by the proteasome activity itself.View full abstractDownload PDF (4633K) -
Hiroo Yonezawa, Tetsuya Uchikoba, Makoto Kaneda1997 Volume 122 Issue 2 Pages 294-299
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN-Pepstatinyl-N'-dansyldiaminopropane (dansyl-pepstatin) was prepared by the coupling of pepstatin A and N-dansyl-diaminopropane. The dansyl-pepstatin obtained strongly inhibited pepsin activity by forming a 1:1 complex. The fluorescence of the dansyl group (excitation at 320nm, and emission near 520nm) increased with the formation of the complex. The increase in fluorescence of dansyl-pepstatin solution was proportional to the amount of added pepsin, chymosin and cathepsin D until dansyl-pepstatin was saturated by these enzymes and at higher protease concentrations the fluorescence did not increase further. Therefore, the net amounts of active pepstatin-sensitive carboxyl proteases could be determined by detecting the inflection point of increased fluorescence upon addition of the protease to a dansyl-pepstatin solution of known concentration. Moreover, the protease concentrations of many samples were obtained easily by measurements of increased fluorescence compared with that caused by authentic protease solution. The minimum detectable amount of pepsin was about 20 pmol. On the other hand, the fluorescence did not increase upon mixing with inactivated pepsin, chymotrypsin, or trypsin. The Ki value of dansyl-pepstatin for pepsin was similar to that of pepstatin A. It was possible to determine the amount of chymosin contained in rennet by this method. The inactivation curve of pepsin in pH 6.5 buffer was also determined quickly and easily by the use of this method. This assay method for pepstatin-sensitive carboxyl proteases is very simple and easy, and it is possible to determine the net amounts of active pepstatin-sensitive carboxyl proteases even in crude mixtures.View full abstractDownload PDF (1658K) -
Eizo Tatsumi, Masaaki Hirose1997 Volume 122 Issue 2 Pages 300-308
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStructural characteristics of ovalbumin at acidic pH were investigated by a variety of analytical approaches. At pH 2.2, the protein appeared to assume a partially denatured, molten globule-like conformation as evaluated by the binding of a hydrophobic probe, anilino-1-naphthalene-8-sulfonate. The protein was, however, resistant to proteolysis with pepsin under conditions in which the urea-denatured form was extensively hydrolyzed. Furthermore, under more drastic proteolytic conditions, the acid ovalbumin was specifically proteolyzed at the N-terminal site of Ala351, which is located in close proximity to the canonical serpin cleavage site A1a352-Ser353 that is known to be the cleavage site at neutral pH with subtilisin and elastase in native ovalbumin. Among the four cysteine residues (Cys11, Cys30, Cys367, and Cys382), which are all known to be buried in the native ovalbumin molecule, only Cys367 was specifically modified with 2, 2'-dithiodipyridine, generating a mixed-disulfide protein derivative. Upon incubation of the derivative with a high concentration of L-cysteine, the thiopyridine mixed disulfide did not undergo any bimolecular exchange reaction with the thiol in the absence of an added denaturant, indicating that the mixed disulfide group is inaccessible. The far-UV CD spectra indicated that the native secondary structure is retained in either the modified or non-modified protein; but as evaluated by the near-UV CD spectra, the asymmetric nature of aromatic side chains in the non-modified ovalbumin and of the mixed-disulfide group in the modified protein was almost lost at pH 2.2. These results are consistent with a highly ordered molten globule-like state for OVA at pH 2.2, in which side chains, but not the backbone chain, significantly fluctuate.View full abstractDownload PDF (2314K) -
Takahisa Sugita, Hideki Aya, Makoto Ueno, Tohru Ishizuka, Keisuke Kawa ...1997 Volume 122 Issue 2 Pages 309-313
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cDNA encoding human 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase has been cloned from a placenta cDNA library, utilizing a PCR-derived probe. It encodes a peptide of 592 amino acids. The amino (N)-terminal sequence of this enzyme, purified from HeLa cells and CCRF-CEM cells, was found to be APGQLALF-. Both sequencing results revealed a difference of six N-terminal residues when compared to the reported sequence of cloned cDNA from a hepatoma cDNA library. Northern-blot analysis of human AICAR transformylase mRNA showed the expression of a single 2.0kb mRNA in all tissues examined. With the cloned cDNA fragment, we constructed expression vectors for mature and GST-fused AICAR transformylase. Both recombinant molecules possessing AICAR transformylase activity were overproduced in Escherichia coli. GST-AICAR transformylase can be purified to homogeneity by a single-step affinity procedure with glutathione Sepharose. Mutational analysis, utilizing this expression system, showed that His213 and His267 were essential for AICAR transformylase activity.View full abstractDownload PDF (3271K) -
Masaji Tachikawa, Hiroyuki Nakagawa, Asako G. Terasaki, Hajime Mori, K ...1997 Volume 122 Issue 2 Pages 314-321
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 260-kDa protein, termed cgABP260, which localized in the dense plaques and dense bodies of smooth muscle cells, was found in a low-salt alkaline extract of chicken gizzard smooth muscle. An antibody against cgABP260 was used to screen a chicken gizzard cDNA library, and the nucleotide sequence of the partial cDNA encoding this protein was determined. Comparison of predicted amino acid sequences revealed that the protein had significant homology with human ABP-280 and chicken retina filamin [Barry, C. P. et al. (1993) J. Biol. Chem. 268, 25577-25586], but despite the high homology, cgABP260 was immunologically distinguishable from filamin. Immunoblot analysis showed that an anticgABP260 antibody reacted exclusively with the cgABP260 band of smooth, skeletal, and cardiac muscle tissues. By indirect immunofluorescence, the membrane-affinity-purified antibody against cgABP260 intensely stained the dense plaques of the isolated smooth muscle cells. Immunoelectron microscopy showed that immunogold particles representing cgABP260 were found abundantly on the dense plaques and less abundantly on the dense bodies. Its amino acid sequence, molecular size, immunological reactivity, and localization in smooth muscle thus indicated that cgABP260 is a new component of the dense plaques and dense bodies of smooth muscle cells.View full abstractDownload PDF (7023K) -
Chikako Suzuki, Hiroshi Ueda, Eiji Suzuki, Teruyuki Nagamune1997 Volume 122 Issue 2 Pages 322-329
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have designed and constructed a bacterial expression vector to produce a fusion protein of hapten-specific single-chain Fv (ScFv) and alkaline phosphatase (PhoA) in Escherichia coli. The ScFv gene was assembled using genes encoding the heavy and light chain variable domains of anti-NP (4-hydroxy-3-nitrophenyl acetyl) mouse monoclonal antibody. The ScFv gene was then fused to the 5'terminus of the E. coli PhoA coding region. The expressed fusion protein ScFv (NP)-PhoA was purified using an NP affinity column, and gel-filtration. Characterization of the fusion protein was then performed. The estimated molecular weight by gel filtration was approximately 151 kDa, suggesting the dimerization of the protein. Kinetic constants of ScFv (NP)-PhoA were calculated and compared with those of wild-type PhoA. The kcat values of ScFv (NP)-PhoA and wild-type PhoA were 103 (s-1) and 96.1 (s-1), respectively, showing that PhoA activity was somewhat increased by tethering the molecules. The equilibrium binding constant of ScFv(NP)-PhoA was determined using two different haptens, NP-capronate and NIP(3-iodo-4-hydroxy-5-nitrophenyl acetyl) by means of fluorescence quenching measurements. The obtained binding constants were 2.2×105(M-1) for NP-capronate and 1.0×105(M-1) for NIP, respectively. No apparent difference in binding constants was seen between ScFv(NP) and ScFv (NP)-PhoA, showing that sufficient specificity and binding affinity were retained when ScFv(NP) was tethered to alkaline phosphatase. ScFv (NP)-PhoA can be used to detect nanogram concentrations of NP-BSA in ELISA without the use of chemically conjugated secondary antibodies.View full abstractDownload PDF (2557K) -
Atsushi Tanaka, Sho Ozaki1997 Volume 122 Issue 2 Pages 330-336
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-N-Acetylgalactosaminidase [EC 3. 2. 1. 53] was purified to homogeneity from the culture media of Bacillus sp. AT173-1. The enzyme has a molecular weight of 48, 000 as estimated by SDS-PAGE under reducing conditions and an isoelectric point of 4.3. The enzyme requires dithiothreitol as an activator and is most active at pH 6.0. Analysis of its substrate specificity using 2-aminopyridine-labeled oligosaccharides as substrates revealed the enzyme specifically hydrolyzes β-N-acetylgalactosaminyl linkages of GalNAcβ1-4Galβ1-4Glc, GalNAcβ1-3Galα1-4Galβ1-4Glc, and N-glycans terminating with β-N-acetylgaactosamine residues but not those with β-N-acetylglucosamine residues. The enzyme is thus a novel β-N-acetylgalactosaminidase with practically no β-N-acetylglucosaminidase activity.View full abstractDownload PDF (1886K) -
Sachiko Okuno, Takako Kitani, Hitoshi Fujisawa1997 Volume 122 Issue 2 Pages 337-343
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+/calmodulin-dependent protein kinase (CaM-kinase) kinase α, which is known to activate CaM-kinases IV and I by phosphorylation of Thr196 and Thr177, respectively, can only phosphorylate Thr196 among many phosphorylation sites of CaM-kinase IV [Kitani, T., Okuno, S., and Fujisawa, H. (1997) J. Biochem. 121, 804-810], indicating its high degree of substrate specificity. In the present study, the substrate specificity of CaM-kinase kinase α was examined using various proteins and synthetic peptides as substrates as a means to address its physiological function. Among a number of proteins and synthetic peptides, including several known as good substrates for various protein kinases, only CaM-kinases IV and I and peptides containing the sequence surrounding Thr196 of CaM-kinase IV or Thr177 of CaM-kinase I were significantly phosphorylated by CaM-kinase kinase α, while the heat-denatured (at 60°C for 5min) CaM-kinases IV and I were not phosphorylated. Peptides containing the phosphorylation site of CaM-kinase IV or I were far less active as substrates for CaM-kinase kinase α than were native CaM-kinase IV or I. Thus, CaM-kinase kinase α showed a high degree of substrate specificity, recognizing not only specific amino acid sequences but also the native conformation of CaM-kinases IV and I.View full abstractDownload PDF (2321K) -
Toshihiro Fujii, Tsuneyoshi Hiromori, Masateru Hamamoto, Tatsuo Suzuki1997 Volume 122 Issue 2 Pages 344-351
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalponin, a major actin-, tropomyosin-, and calmodulin-binding protein in smooth muscle, interacted with tubulin, a main constituent of microtubules, in a concentration-dependent fashion in vitro. The apparent Kd value of calponin to tubulin was calculated to be 5.2μM with 2mol of calponin maximally bound per 1mol of tubulin. At low ionic strength, tubulin bound to calponin immobilized on Sepharose 4B, and the bound protein was released at about 270mM NaCl. Chemical cross-linking experiments showed that a 1:1 molar covalent complex of calponin and tubulin was produced. The amount of calponin bound to microtubules decreased with increasing ionic strength or Ca2+ concentration. The addition of calmodulin or S100 to the mixture of calponin and microtubule proteins caused the removal of calponin from microtubules in the presence of Ca2+, but not in the presence of EGTA. Calponin-related proteins including tropomyosin, SM22, and caldesmon had little effect on the calponin binding to microtubules, whereas MAP2 inhibited the binding. Interestingly, there was little, if any, effect of mycalolide B-treated actin on the binding of calponin to microtubules. Furthermore, only about 20% of calponin-F-actin interaction was inhibited in the presence of an excess amount of tubulin (4mol per mol of calponin), indicating that tubulin binds to calponin at a different site from that of actin. Compared with MAP2, calponin had little effect on microtubule polymerization.View full abstractDownload PDF (2946K) -
Shin Hisamatsu, Yuki Morikawa, Rie Tomita, Terumichi Tanaka, Shigenori ...1997 Volume 122 Issue 2 Pages 352-357
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe negative strand of the satellite RNA of tobacco ringspot virus [(-) sTRSV] is a selfcleaving RNA, of which self-cleaving domain is called the hairpin ribozyme. The negative strand of the satellite RNA of arabis mosaic virus [(-) sArMV] has been suggested to have a hairpin ribozyme-like secondary structure, and we have previously shown that this hairpin domain of (-) sArMV has ribozyme activity. Here we report characterization of the cleavage reaction of the (-) sArMV hairpin ribozyme. Mutagenesis analyses in a trans-acting system revealed, surprisingly, that the wild-type ribozyme was less active than almost all the other mutant ribozymes tested. In a cis-acting system (self-cleaving reaction), however, the reaction of the RNA containing the wild-type sequence proceeds highly efficiently. This result suggests that the inefficient cleavage of the wild-type substrate in trans-acting system may be due to low efficiency at the substrate-binding step but not at the chemical cleavage step in the reaction. We also constructed a chimeric ribozyme between the catalytic hairpin domain from (-) sArMV and the substrate-binding site from (-) sTRSV. This chimeric ribozyme had the highest activity among the trans-acting hairpin ribozymes tested.View full abstractDownload PDF (690K) -
Kuniyo Inouye, Soo-Bok Lee, Koji Nambu, Ben'ichiro Tonomura1997 Volume 122 Issue 2 Pages 358-364
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activity of thermolysin in the hydrolysis of N-[3-(2-furyl) acryloyl] (FA)-dipeptide amides and N-carbobenzoxyl-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced by high concentrations (1-5M) of neutral salts. The activation is due to an increase in the molecular activity, kcat, while the Michaelis constant, Km, is not affected by the addition of NaCl. In the present study, the effect of NaCl on the thermolysin-catalyzed hydrolysis of FA-glycyl-L-leucine amide (FAGLA) has been examined by changing the pH and temperature, and by adding alcohols to the reaction mixture. The enzyme activity, expressed by kcat/Km, is pH-dependent, being controlled by two functional residues with pKa values of 5.4 and 7.8 in the absence of NaCl. The acidic pKa is shifted from 5.4 to 6.7 by the addition of 4M NaCl, while the basic one is not changed. The degree of activation at a given concentration of NaCl is pH dependent in a bell-shaped manner with the optimum pH around 7. Although the activity increases in both the presence and absence of NaCl with increasing temperature from 5 to 35°C, the degree of activation decreases. Alcohols inhibit thermolysin, and the degree of activation decreases with increasing alcohol concentration. The degree of activation tends to increase with increasing dielectric constant of the medium, although it varies considerably depending on the species of alcohol. Electrostatic interactions on the surface and at the active site of thermolysin are suggested to play a significant role in the remarkable activation by salts.View full abstractDownload PDF (3826K) -
Gabriel A. Rabinovich, Nidia M. Modesti, Leonardo F. Castagna, Carlos ...1997 Volume 122 Issue 2 Pages 365-373
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-Galactoside-binding lectins or galectins are a family of closely related carbohydrate-binding proteins which functions still remain to be elucidated. Several evidence suggest they could play a role in different biological processes, such as cell growth regulation and immunomodulation. In the present study we report that affinity-purified CLL-I (chicken lactose lectin-I), an acidic 16-kDa galectin exhibits specific growth regulatory properties. Con A-stimulated rat spleen mononuclear cells showed a marked dose-dependent growth inhibition upon incubation with the galectin protein. Cell growth arrest was highly prevented by galectin-specific sugars. In addition, biochemical, cytofluorometrical, and morphological evidence are also provided to show that these inhibitory properties are related to a positive control in the apoptotic threshold of spleen mononuclear cells. Flow cytometric analysis showed a dose- and time-dependent increase of cells with hypodiploid DNA content upon exposure to CLL-I. Moreover, cells treated with CLL-I displayed the typical ultrastructural changes compatible with apoptosis, mainly chromatin condensation and margination along the inner surface of the nuclear envelope. Finally, the highly characteristic “ladder” pattern of DNA fragmentation into oligonucleosome-length fragments of_??_180-200 bp could be found within 6h of cell culture with CLL-I, mainly in the T cell-enriched population. Induction of apoptosis by a β-galactoside-binding protein highlights a potentially novel mechanism for regulating the immune response and points to a rational basis for the postulated immunomodulatory properties of this protein family.View full abstractDownload PDF (6260K) -
Hajime Julie Yuasa, Shigeru Sato, Hiroaki Yamamoto, Takashi Takagi1997 Volume 122 Issue 2 Pages 374-380
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe solitary ascidian Halocynthia roretzi possesses three types of muscle: the larval tail striated muscle, the adult heart striated muscle, and the adult body wall smooth muscle. The troponin complex is observed in all types of muscle, and the isoform sequences and expression patterns of two of the three troponin components, troponins C and T, have been reported. In this study, we have determined cDNA sequences of the three TnI isoforms from H. roretzi. One of the three isoforms (adult TnI), expressed in adult body wall smooth muscle and heart muscle, was composed of 173 amino acids, being similar to vertebrate fast and slow skeletal TnIs in length. The other two isoforms (larval TnIα and TnIβ) were isolated from a cDNA library of larvae. Both larval TnIs were composed of 142 amino acids, with truncation amounting to ca. 30 amino acid residues at the C-termini. These larval TnIs are the smallest known TnIs. The position of the last intron of these TnIs was also determined. When compared with vertebrate TnI genes, the last intron of the ascidian adult TnI gene is located at 6 nucleotides downstream, and the introns of the two larval TnIs are positioned at 9 nucleotides upstream. These results suggest that H. roretzi TnI is encoded by at least three genes.View full abstractDownload PDF (1797K) -
Hisashi Hashimoto, Yoshiyuki Matsuo, Yoshihiro Yokoyama, Haruhiko Toyo ...1997 Volume 122 Issue 2 Pages 381-386
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo distinct stress-activated protein kinase (JNKa and b) cDNAs were isolated from a carp ovary cDNA library. These cDNAs contained a full-length open reading frame encoding 427 amino acid residues with a predicted mass of 48.6 kDa. The deduced amino acid sequences of JNKa and b were 95.8% identical, with 18 residues replaced, and showed a high degree of sequence similarity to mammalian JNK/SAPK subgroup including the common dual phosphorylation motif of TPY. By Northern blot analysis, the carp JNKs were found to be abundant in the brain and ovary. Detailed study by RT-PCR assay revealed ubiquitous expression of JNKb, although expression of JNKa was specific to the brain and ovary. The high level expression of both JNKa and b in the ovary implies that JNKs play an important role in egg maturation or ectogenetic early development.View full abstractDownload PDF (4791K) -
Kazunari Matsumura, Makoto Komiyama1997 Volume 122 Issue 2 Pages 387-394
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLanthanide (III) ions have shown enormous catalyses for the hydrolysis of the phosphodiester linkages in RNA, indicating their high potential for versatile applications to biotechnology and molecular biology. The activity monotonically increases with increasing atomic number in the lanthanide series, the last three ions (Tm3+, Yb3+, and Lu3+) being the most active. Non-lanthanide metal ions are virtually inactive. The pseudo first-order rate constant for the hydrolysis of adenylyl (3'-5') adenosine (ApA) by LuCl3 (5 mmol•dm-3) at pH 7.2 and 30°C is 1.9×10-1min-1 (the half-life is only 3.6min), corresponding to 108-fold acceleration. The product is an equimolar mixture of adenosine and its 2'- or 3'-monophosphate without any byproducts. The 2', 3'-cyclic monophosphate of adenosine is not accumulated much in the reaction mixture. Lanthanide ions also efficiently hydrolyze oligoribonucleotides without a specific base-preference. In ApA hydrolysis by NdCl3 and GdCl3, the dependence of the hydrolysis rate on either the pH or concentration of the metal salt coincides fairly well with the corresponding profile of the equilibrium concentration of the bimetallic hydroxo-cluster [M2(OH)2]4+ (M=metal ion). Both the formation of the pentacoordinated intermediate and its decomposition are greatly promoted by lanthanide ions. A catalytic mechanism in which two metal ions (or their coordination water) in these tetracationic hydroxo-clusters show acid/base cooperation is proposed.View full abstractDownload PDF (2065K) -
Giuseppe Graziano, Francesca Catanzano, Angelo Riccio, Guido Barone1997 Volume 122 Issue 2 Pages 395-401
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe existence of cold denaturation is now firmly demonstrated by its direct observation for several globular proteins in aqueous solution. But the physico-chemical explanation of this intriguing phenomenon is still unsatisfactory. In this paper we deepen our understanding of cold denaturation by taking advantage of the theoretical model developed by Ikegami and using thermodynamic data on the transfer to water of liquid N-alkyl amides. The analysis leads to the conclusion that the presence of water is fundamental to determine the existence of cold denaturation due to its strong energetic interaction with the amino acid residues previously buried in the protein's interior.View full abstractDownload PDF (995K) -
Wataru Nishii, Tomonari Muramatsu, Yoshiyuki Kuchino, Shigeyuki Yokoya ...1997 Volume 122 Issue 2 Pages 402-408
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteolytic trimming of isoprenylated proteins, including Ras, at the C-terminal CAAX motifs is a key event in their activation. However, the protease responsible for the proteolysis has not been well characterized yet. In this study, we established a novel assay method for the enzyme using a fluorescent substrate, dansyl (Dns)-KSKTKC (S-farnesyl)-VIM, with which we can assess the proteolytic activity with high sensitivity and more easily than by the former assay methods using radio-labeled substrates. Using this assay method, we purified the protease 104-fold from bovine brain microsomal membranes by Sepharose CL-6B gel filtration and DE-52 chromatography. The partially purified enzyme was shown to be an endoprotease specific to the farnesylated peptide and to have a Km value of 1.0μM for Dns-KSKTKC (S-farnesyl) VIM. o-Phenanthroline and zinc chloride strongly inhibited the activity. Interestingly, however, m- and p-phenanthrolines were as effective as o-phenanthroline, indicating that the inhibition by o-phenanthroline is not simply due to its chelating action. The molecular mass of the protease was deduced to be 480 kDa by gel filtration. The enzymatic activity was lost during further attempts at chromatographic purification, but was partially recovered by mixing the chromatographic fractions which had apparently lost the activity. These results suggest that this protease consists of multiple subunits.View full abstractDownload PDF (896K) -
Shoji Ando, Toshihiko Ikuhara, Tomoko Kamata, Yasuharu Sasaki, Shin-ic ...1997 Volume 122 Issue 2 Pages 409-414
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo examine structural features of proline which are essential for the proline-directed phosphorylation by cdc2 kinase or cdk5, we prepared the peptide representing the cdc2 kinase phosphorylation site at Ser-55 in vimentin [Ser-Leu-Tyr-Ser-Ser-Ser55-Pro56-Gly-Gly58-Ala-Tyr-NH2], the peptide containing arginine in place of Gly-58, and their derivatives containing various N-methylamino acids or proline homologs in place of Pro-56, and tested them as substrates for the kinases. While substitution of the proline by proline homologs (L-pipecolic acid or L-azetidine-2-carboxylic acid) increased the Km value 2- to 4-fold at utmost, substitution by N-methylamino acids (sarcosine, L-N-methylalanine, L-N-methylvaline, or L-N-methylleucine) increased the Km value 7- to 40-fold for cdc2 kinase. For cdk5, these substitutions led to parallel effects on the Km value to those found for cdc2 kinase; cdk5 recognized the peptides with a proline specificity similar to that for cdc2 kinase. These results suggest that the pyrrolidine ring of proline is important for substrate recognition by cdc2 kinase or cdk5. Molecular dynamics and molecular mechanics simulations indicated that the pyrrolidine ring of proline is optimal to stabilize a β-turn at the phosphorylation site and that the Km values of the peptides for the enzymes might be related to the probability of the turn structure. The results obtained here also suggest that the pyrrolidine ring of proline is required to maintain a high Vmax value for cdc2 kinase or especially for cdk5. These will aid in designing specific substrates or inhibitors for cdc2 kinase or cdk5.View full abstractDownload PDF (840K) -
Hiro Nakamura, Keitarou Saiki, Tatsushi Mogi, Yasuhiro Anraku1997 Volume 122 Issue 2 Pages 415-421
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome bo from Escherichia coli belongs to the heme-copper terminal oxidase superfamily and functions as a redox-driven proton pump. In the present study, we examined the functional roles of the cyoABCDE genes, which encode cytochrome bo. We expressed the cyoABCDE genes in minicells using pTTQ18 derivatives and identified subunits II, I, III, and IV of the oxidase complex and heme O synthase as polypeptides with molecular weights of 33, 500, 75, 000, 20, 500, 12, 000, and 28, 000, respectively. The expression level of heme O synthase (CyoE) was much lower than those of the oxidase subunits and seems to be controlled just tightly enough for the incorporation of heme O into the oxidase complex. To facilitate functional analysis of the gene products, we developed a single copy expression vector pHNF2, a derivative of the F-sex factor. Genetic complementation tests showed that deletions in each gene resulted in nonfunctional enzymes. Western blotting analysis indicated that the expression levels of subunits I and II were not affected by the deletions in the other cyo gene products. However, spectroscopic analyses of the mutant membranes revealed that all the deletions perturbed or eliminated the redox metal centers in subunit I. Present findings suggest that subunits II, III, and IV of the oxidase complex are required for the assembly of the metal centers in subunit I.View full abstractDownload PDF (3544K) -
Masato Kawasaki, Tatsushi Mogi, Yasuhiro Anraku1997 Volume 122 Issue 2 Pages 422-429
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump. Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and CuB, and serves as a reaction center of the oxidase complex. This work focuses on the functional and structural roles of 14 charged amino acid residues that are conserved in subunit I of the heme-copper terminal oxidases. Substitutions of Lys55, Tyr173, Asp188, Asp256, Arg481, and Arg482 by neutral amino
acid residues did not affect the catalytic activity and spectroscopic properties of the cytoplasmic membranes. In contrast, genetic complementation tests indicated that replacements of Arg80, Asp135, Arg257, Glu286, Tyr288, Lys362, Asp407, and Glu540 resulted in nonfunctional enzymes. The R80Q mutation caused loss of a diagnostic peak for low-spin heme b in the 77 K redox difference spectrum. The K362Q, D407N, and E540Q mutations affected the CO-binding by the heme-copper binuclear center. The D135N, R257Q, E286Q, and Y288F mutations specifically eliminated the CuB center from the oxidase complex, whereas the E286D mutant did not show significant perturbations on the redox metal centers even though it was still inactive. Based on these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved charged amino acid residues in subunit I of the heme-copper terminal oxidases.View full abstractDownload PDF (1100K) -
Tomoyasu Hirano, Tatsushi Mogi, Motonari Tsubaki, Hiroshi Hori, Yutaka ...1997 Volume 122 Issue 2 Pages 430-437
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome bo-type ubiquinol oxidase in Escherichia coli belongs to a superfamily of the heme-copper respiratory oxidases and catalyzes the redox-coupled proton pumping. Previous studies [Y. Orii, T. Mogi, M. Sato-Watanabe, T. Hirano, and Y. Anraku (1995) Biochemistry 34, 1127-1132] suggest that it requires chloride ions for the facilitated heme b-to-heme o intramolecular electron transfer. To extend our previous studies on chloride binding by bo-type ubiquinol oxidase, we prepared two kinds of chloride-bound enzymes, UQO-412 and UQO-409, and a chloride-depleted enzyme, UQO-407, and examined their spectroscopic and enzymatic properties. UQO-412, which exhibits the Soret peak at 412nm in the air-oxidized state, was obtained by purification with anion-exchange liquid chromatography, and UQO-409 was derived from UQO-412 by extensive washing and showed a 3-nm blue shift. UQO-407 was obtained from UQO-409 by omitting chloride ions from buffers throughout purification and showed a further blue shift in the Soret peak and the pronounced chloride-sensitive EPR signals at g=6 and g=3.15, which are attributable to spin-spin exchange interaction at the binuclear center. Kinetic studies on chloride binding by UQO-407 revealed the presence of a chloride-binding site with a Kd value of 3.5mM. Flow-flash experiments demonstrated that the heme b-to-heme o electron transfer was perturbed in both UQO-409 and UQO-407, although steady state enzyme activities of three UQOs were indistinguishable. The present studies demonstrated that the E. coli bo-type ubiquinol oxidase is endowed with a novel chloride-binding site which controls the electromagnetic state of the heme-copper binuclear center. Further, we suggest that the intramolecular electron transfer in the enzyme requires diffusible molecules other than the bound chloride ion.View full abstractDownload PDF (1054K) -
Akihiko Tsuji, Chiemi Hine, Yasuhiro Tamai, Kayoko Yonemoto, Kazushige ...1997 Volume 122 Issue 2 Pages 438-452
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPACE4 (_??_aired basic _??_mino acid _??_leaving _??_nzyme) is a member of a family of the mammalian kexin-like proprotein convertases containing a subtilisin-like catalytic domain. Previously we reported seven isoform mRNAs of PACE4 that vary in size and 3'-coding sequence [A. Tsuji et al. (1994) Biochem. Biophys. Res. Commun. 200, 943-950; K. Mori et al. (1997) J. Biochem. 121, 941-948]. To determine the origin of these isoforms, the entire human PACE4 gene has been isolated as a set of overlapping genomic DNA fragments, and analyzed by restriction enzyme digestion and nucleotide sequence determination. The human PACE4 gene spans at least 250 kb and is distributed over 25 exons that range in size from 39 to 1, 422 base pairs. Human PACE4 gene is the largest kexin-like proprotein convertase gene reported to date. The most striking feature of its genomic structure is the size of the introns and the number of exons, although the general organization of signal peptide, propeptide, and catalytic domains, which are conserved in this family, is very similar to that reported for other kexin-like protease genes. The structural analysis of PACE4 genomic DNA indicates that multiple PACE4 transcripts are produced as a consequence of alternative RNA splicing events, including exon skipping, and differences in the usage of the inner 5'-splicing donor and polyadenylation sites. A major transcriptional start site was detected 314 bp upstream from the ATG translational start site by primer extension analysis. Sequence analysis of the 5'-flanking region revealed that PACE4 gene lacks TATA and CCAAT boxes in the proximal upstream region of the start site, although potential binding sites for several transcription factors including SP1, AP1, AP2, PEA3, Ets-1, GHF (growth hormone factor)-1, CREB (cyclic AMP response element binding protein), and basic helix-loop-helix proteins, were present. An unusual sequence of six tandem repeats of a nonadecamer (GGCCTGGGGGTTCACCTGC) containing an E box is found in the 5'-flanking region. These results suggest that PACE4 is not a constitutive gene product and its expression is regulated by various transcription factors.View full abstractDownload PDF (4449K) -
Tohru Takada, Kazuhiro Toriyama, Hisako Muramatsu, Xiao Jun Song, Shuh ...1997 Volume 122 Issue 2 Pages 453-458
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMidkine (MK) is a retinoic acid-inducible heparin-binding cytokine. In the inflammatory synovitis of rheumatoid arthritis and osteoarthritis, MK was detected in synovial fluid, synoviocytes, and endothelial cells of new blood vessels. Normal synovial fluid and noninflammatory synovial tissue did not contain detectable MK. Therefore, MK showed inflammation-associated expression in these cases. Furthermore, MK was found to promote chemotaxis of neutrophils in the range of 10ng/ml. The mode of action of MK was found to be haptotactic; the substrate-bound form of MK was the active one. MK is also known to promote fibrinolysis. These activities of MK are in agreement with the modes of MK expression in various pathological statuses, and thus MK is proposed to be an important molecule regulating inflammatory responses.View full abstractDownload PDF (2048K) -
Takaaki Akaike, Katsuhisa Inoue, Tatsuya Okamoto, Hirohito Nishino, Ma ...1997 Volume 122 Issue 2 Pages 459-466
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNitrosothiols (RS-NOs) appear to be critically involved in various signal transduction mechanisms. We describe here a specific and highly sensitive quantification method for RS-NOs by using high performance liquid chromatography (HPLC) combined with a flow reactor system. RS-NOs were applied to an HPLC system of C18-reverse phase or a gel filtration column and eluted with 10mM sodium acetate buffer (pH 5.5) plus 0.5mM diethylenetriamine pentaacetic acid with or without either 0-7% methanol or 0.15M NaCl. The eluate from the HPLC column was mixed with a solution containing 1.75mM HgCl2 or 1.75mM CuSO4 for RS-NO decomposition in a reaction coil via a three-way connector. NO2-generated via the metal-induced RS-NO decomposition was then reacted with Griess reagent, which was infused through a second three-way connector, yielding a diazocompound detected at 540nm. In a separate experiment, a copper particle-loaded column was used for RS-NO degradation instead of the metal-ion flow reactor. In all RS-NOs tested, i. e., nitrosoglutathione (GS-NO), nitroso-L-cysteine, and nitrosoalbumin, the nitrosogroup was converted to NO2- by the Hg2+-reaction system as well as copper-loaded column, and the recovery was almost 100%. The Cu2+-solution flow reaction system, however, yielded only 30% recovery of RS-NOs as NO2-. Also, the RS-NOs could be identified at nanomolar concentrations: detection limit, 3.0nM in a 150-μl aliquot. These RS-NOs showed well-resolved elution profiles even in the presence of NO2- and NO3-. More importantly, biological generation of GS-NO was quantitatively demonstrated with RAW264 cells in culture incorporating free GSH in the medium. In conclusion, our novel RS-NO assay will be useful to examine the formation and functions of RS-NOs in biological systems.View full abstractDownload PDF (974K) -
Kazuo Watanabe, Kazunaga Yazawa, Kiyosi Kondo, Akihiko Kawaguchi1997 Volume 122 Issue 2 Pages 467-473
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe fatty acid synthesis systems of a Shewanella sp., strain SCRC-2738, that produces a large amount of eicosapentaenoic acid were investigated. Two kinds of fatty acid synthesis system, de novo synthesis and chain elongation ones, were detected in the cytosol. The de novo synthesis system required an acyl carrier protein, and produced palmitoyl- and palmitoleoyl-acyl carrier proteins as final products. The chain elongation system also required an acyl carrier protein, and produced an acyl-acyl carrier protein as a product, using palmitoyl-, palmitoleoyl-, stearoyl-, and oleoyl-CoAs as primers but not eicosanoylor eicosenoyl-CoA. There were an anaerobic pathway and an aerobic desaturation one for the production of unsaturated fatty acids. Eicosapentaenoic acid seemed to be produced through the aerobic desaturation pathway and not through the anaerobic one, since the latter pathway produced n-7 type monoenoic fatty acids, which are different from eicosapentaenoic acid in the position of the double bond. The desaturase utilized an acyl-acyl carrier protein as a substrate, and this activity increased in the presence of ferredoxin and ferredoxin NADP+ reductase. Thus, Shewanella sp., strain SCRC-2738, has novel characteristics as to both fatty acid chain elongation and desaturation systems.View full abstractDownload PDF (2846K) -
CD9 Antigen Interacts with Heparin-Binding EGF-Like Growth Factor through Its Heparin-Binding DomainTakahiko Sakuma, Shigeki Higashiyama, Shigeto Hosoe, Seiji Hayashi, Na ...1997 Volume 122 Issue 2 Pages 474-480
Published: 1997
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHeparin/heparan-sulfate proteoglycan (HSPG) binds to heparin-binding epidermal growth factor-like growth factor (HB-EGF) through its heparin-binding domain (HBD), which consists of 21 amino acid residues (P21). The CD9 antigen also interacts with a membraneanchored form of HB-EGF (proHB-EGF) and enhances its juxtacrine activity. The CD9 antigen potentiates the juxtacrine activity of both proHB-EGF and proamphiregulin, but has no effect on proTGF-α. While both HB-EGF and amphiregulin contain an HBD, TGF-α does not. This suggests that the HBD of HB-EGF is also involved in CD9 antigen binding. Mutant CHO cells which lack HSPG recovered their capacity to bind to immobilized P21 when transfected with CD9 antigen cDNA. This binding was competitively inhibited by heparin in a dose-dependent manner. The interactions between synthetic peptides corresponding to the extracellular domain of CD9 antigen and the immobilized P21 were analyzed with surface plasmon resonance. The 119VIKEVQEFYKDTYNKLKTKD138 sequence of the CD9 antigen is thought to represent the binding site for HB-EGF. The kD values for heparin/P21 and 119V-D138/P21 were (2.82±0.10)×10-8M and (3.71±0.71)×10-6M, respectively. These results suggest that the 119V-D138 sequence of the CD9 antigen is the site which interacts with the HBD and may play an essential role in the upregulation of the juxtacrine activity of proHB-EGF.View full abstractDownload PDF (917K)
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