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Volume 109 (1991)
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- Issue 2 Pages 199-
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Volume 109, Issue 2
Displaying 1-27 of 27 articles from this issue
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Jing Xu, Tairo Oshima, Masasuke Yoshida1991 Volume 109 Issue 2 Pages 199-203
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the previous paper [Xu, J., Oshima, T., & Yoshida, M. (1990) J. Mol. Biol. 215, 597-606], we reported that phosphofructokinase from Thermus thermophilus is allosterically inhibited by phosphoenolpyruvate, which induces dissociation of the active four-subunit enzyme into an inactive two-subunit form. When T. thermophilus was cultured in a glucose-containing medium, another phosphofructokinase (PFK2) appeared in addition to the reported one (PFK1). The molecular weights of the native PFK2 molecule (132, 000) and its subunit (34, 500), which are slightly smaller than those of PFK1, suggest that PFK2 is also composed of four identical subunits. However, the hyperbolic kinetics and molecular form of PFK2 are not affected at all by phosphoenolpyruvate. The NH2-terminal amino acid sequences of subunits of PFK1 and PFK2 revealed that they are composed of very similar but different polypeptides.View full abstractDownload PDF (855K) -
Tai Heon Yoon, Natsuko Mori, Kiyoshi Kitamura, Shun Ishibashi, Hitoshi ...1991 Volume 109 Issue 2 Pages 204-210
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSix mouse monoclonal antibodies against rabbit apolipoprotein E (apo E) have been developed. Of these monoclonal antibodies, clone 5 revealed a high affinity for purified apo E, very low density lipoprotein (VLDL) and β-VLDL. This monoclonal antibody was used to prepare an immunoaffinity column. Coupled to Sepharose 4B, this antibody allowed complete removal of lipoproteins containing apo E from plasma of New Zealand white (NZW) rabbits; 62, 46, 14, and 3% of VLDL-, IDL-, LDL-, and HDL-protein, respectively, were bound to the anti-apo E affinity column. The bound VLDL was significantly rich in free cholesterol (FC) and cholesteryl esters (CE) relative to the unbound VLDL, whereas bound IDL, LDL and HDL were significantly rich in FC only. All of the bound fractions were characterized by significantly increased ratios of FC/phospholipids (PL). These results indicate that the two lipoprotein populations with and without apo E have different lipid compositions. The relatively high content of cholesterol in lipoproteins containing apo E suggests a contribution of apo E to plasma cholesterol transport.View full abstractDownload PDF (895K) -
Akio Sugihara, Tadaaki Tani, Yoshio Tominaga1991 Volume 109 Issue 2 Pages 211-216
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22, 000, and pI of 5. 1. The optimal pH at 30°C, and optimal temperature at pH 5. 6 were 5. 5-7. 2, and 60°C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad, and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.View full abstractDownload PDF (1405K) -
Tomofumi Kurokawa, Yukio Toyoda, Susumu Iwasa1991 Volume 109 Issue 2 Pages 217-222
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1.View full abstractDownload PDF (686K) -
Yoshio Nakano, Kinuko Kimura1991 Volume 109 Issue 2 Pages 223-228
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe report the overexpression, purification, and properties of the regulatory protein, G1nR, for glutamine synthetase synthesis of Bacillus cereus. The protein was found to be a dimer with a molecular weight of approximately 30, 000, and its subunit molecular weight was 15, 000 in agreement with that (15, 025) of deduced amino acid sequence of G1nR. The purified G1nR protein bound specifically to the promoter region of the glnRA operon of B. cereus and Bacillus subtilis. The binding of the G1nR protein to the DNA fragment was enhanced by the presence of glutamine synthetase, the product of glnA, of B. cereus or B. subtilis, although the affinity of the G1nR protein for DNA was not affected in the presence of glutamate, glutamine, Mg2+, Mn2+, or ammonia. These results indicate the existence of an interaction between G1nR and glutamine synthetase, and support the hypothesis that the regulation of glnA expression requires both G1nR protein and glutamine synthetase in Bacillus.View full abstractDownload PDF (1990K) -
Haruhiko Tokuda, Osamu Kozawa, Masahiro Yoneda, Yutaka Oiso, Kensuke T ...1991 Volume 109 Issue 2 Pages 229-233
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn cloned osteoblast-like cells, MC3T3-E1, prostaglandin E2 (PGE2) stimulated the forma-tion of inositol phosphates in a dose-dependent manner in the range between 10 nM and 10μM. Pertussis toxin inhibited the effect of PGE2 dose-dependently in the range between 1μg/ml and 1μg/ml. In the cell membranes, pertussis toxin catalyzed ADP-ribosylation of a protein with an Mr of about 40, 000. Pretreatment of membranes with 10μM PGE2 in the presence of 2.5 mM MgCl2 and 100μM GTP markedly attenuated this pertussis toxin-catalyzed ADP-ribosylation of the protein in a time-dependent manner. G12 was detected in these cells by immunoblotting with purified anti-G12αantibodies. The results indicate the possible coupling of PGE2 signalling with pertussis toxin-sensitive GTP-binding protein, which is probably G12, in osteoblast-like cells.View full abstractDownload PDF (887K) -
Shin-ya Ohki, Sakae Tsuda, Shigeki Joko, Michio Yazawa, Koichi Yagi, K ...1991 Volume 109 Issue 2 Pages 234-237
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmide proton exchange rates of Ca2+-saturated calmodulin and Ca2+-saturated calmodulin-mastoparan complex were studied by 1H NMR spectroscopy. Exchange rates of G1y25, G1y61, G1y98, G1y134, 11e27, Ile100, and Asn137 were determined for Ca2+-saturated calmodulin and for Ca2+-saturated calmodulin-mastoparan complex, and were found to be less than 10-4 s-1. All these residues of which the amide proton resonances appear at lower fields were considered to form hydrogen bonds, based on the results of X-ray analysis. Exchange rates of I1e27 and Asn137 became an order of magnitude smaller when mastoparan bound to Ca2+-saturated calmodulin, while those of the four glycines and Ile100 did not change appreciably. The reduction in accessibility of Asn137 to water cased by mastoparan binding suggests that a part of the mastoparan binding site is probably located in or near the hydrophobic cluster of the C-terminal-half domain. The reduction in accessibility of 11e27 also suggests that another part of the mastoparan binding site is located in or near the hydrophobic cleft of the N-terminal-half domain.View full abstractDownload PDF (393K) -
Masatoshi Mita, Tatsuo Harumi, Norio Suzuki, Nobuo Ueta1991 Volume 109 Issue 2 Pages 238-242
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSea urchin spermatozoa obtain energy for movement through oxidation of endogenous phospholipids, particularly phosphatidylcholine (PC). This study was undertaken to determine the localization of PC available for utilization in energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Following incubation with sea water, the PC content in sperm heads decreased significantly, while that in sperm tails did not change. PC was abundant in sperm heads, particularly the midpieces. PC composed of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in the midpieces PC to be unsaturated. Phospholipase A2 activity was also distributed in sperm heads, particularly the midpieces. It thus appears that PC as a substrate for energy metabolism is located in the midpieces of sea urchin spermatozoa.View full abstractDownload PDF (1968K) -
Kumiko Ohkubo, Shigenori Ogata, Yoshio Misumi, Noboru Takami, Yukio Ik ...1991 Volume 109 Issue 2 Pages 243-250
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA glycoprotein with Mr 63, 000 purified from rat serum was found to inhibit trypsin activity but not chymotrypsin or elastase activity, resembling contrapsin purified from mouse serum. To obtain further information on the molecular structure, a cDNA clone (λCPi-21) for this contrapsin-like protease inhibitor was isolated from a rat liver cDNA library. The 1.6-kb cDNA insert contained an open reading frame that encodes a 416-residue polypeptide (CPi-21), in which the first 29 residues were suggested to comprise a signal peptide by comparison with the NH2-terminal sequence of the purified protein. The predicted structure also contained other peptide sequences determined by Edman degradation. Four potential N-linked glycosylation sites were found in the molecule, presumably accounting for the larger molecular mass of the mature form. Further screening of the cDNA library with a Pst-Xbal fragment (302 bp) of λCPi-21 as a probe yielded two other cDNA clones (λCPi-23 and λCPi-26), which encode 413-residue and 418-residue polypeptides, respectively. A comparison of their amino acid sequences revealed that CPi-21 has 89 and 71% homology with CPi-23 and CPi-26, respectively. The primary structure of each of the three proteins has about 70% homology with that of mouse contrapsin, in contrast to 43-46% homology with that of rat αl-protease inhibitor. These results indicate that all the CPi proteins presented here belong to a subfamily of “serpins” of which mouse contrapsin was the first member to be identified.View full abstractDownload PDF (1280K) -
Yoichi Nakajima, Kunie Nakamura1991 Volume 109 Issue 2 Pages 251-255
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe most reactive single thiol group of rabbit skeletal muscle phosphofructokinase per protomer was modified with the following thiol reagents: iodoacetamide, iodoacetate, 2-hydroxyethyl disulfide, 3, 3'-dithiodipropionate, and glutathione disulfide. As a result of the modification, there was increase in not only the apparent activation constants of activating monovalent cations, NH4+ (about 3-, 9-, 12-, 20-, and 30-fold, respectively) and K+ (about 3-, 10-, 15-, 17-, and 20-fold, respectively), but also the apparent Km for ATP (about 3-, 10-, 15-, 100-, and 20-fold, respectively) without any significant change in maximum velocity or apparent Km, for fructose 6-phosphate in the presence of high concentrations of NH4+. These results suggest that modification of the thiol group destabi-lizes the enzyme-monovalent cation-MgATP complex proposed by Suelter [Science (1970) 168, 789-795], causing an apparent loss in catalytic activity.View full abstractDownload PDF (513K) -
Yoshihiko Igarashi, Kazumoto Kimura, Akihiko Kajita1991 Volume 109 Issue 2 Pages 256-261
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOxygen equilibrium curves of the giant hemoglobin from the earthworm Eisenia foetida were determined at various concentrations of cations. Using the Adair model of 12 oxygenation steps, we succeeded in fitting the data better than the simple concerted model (MWC model). Analysis of the Adair constants (K1 to K12) indicated that the increase in oxygen affinity occurs in the last six steps (K7 to K12) of the oxygen binding and that it is enhanced by increase in Ca2+ concentration. The Hill coefficient (nmax) at pH 7.5 attained a maximum value of 9.76 at 20 mM CaCl2. In the presence of physiological levels of Ca2+(5 mM), the Bohr effect was similar to that seen in vertebrates. The data were consistent with the release of two Bohr protons being accompanied by the oxygen-linked binding of one Ca2+. Mg2+ and Na+ exerted a similar effect on the hemoglobin, though to a lesser extent. The stoichiometry of Ca2+ binding of the hemoglobin revealed the presence of two classes of binding sites, of which the affinities are high (Ka =8.8×103±103 M-1) and low. The number of high affinity sites per heme was found to be 0.3, comparable to the number of oxygen-linked Ca2+ binding sites.View full abstractDownload PDF (776K) -
Kazuyuki Tao, Kozo Makino, Shuji Yonei, Atsuo Nakata, Hideo Shinagawa1991 Volume 109 Issue 2 Pages 262-266
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Escherichia coli oxyR gene is required for the induction of a regulon that is inducible by hydrogen peroxide and confers resistance to oxidative stresses. We constructed a plasmid system that greatly overproduced OxyR protein and purified the protein. OxyR protein specifically bound to the upstream regulatory regions of the oxyR and katG genes as demonstrated by the gel-retardation assay and the DNase I footprinting experiment, and activated the transcription initiation of the katG gene in vitro. Using a plasmid carrying an oxyR'-'lacZ fusion gene, we studied the regulation of oxyR expression in vivo. The expression of oxyR was not induced by the treatment with a low concentration of hydrogen peroxide which induces the genes in the oxyR regulon. The expression of the oxyR'-'lacZ gene was higher in an oxyR-deletion strain than in the oxyR+ strain, and was repressed by overexpressing the OxyR protein. These results suggest that OxyR protein functions as a repressor for oxyR, in addition to its known function as a transcriptional activator for the genes in the oxyR regulon.View full abstractDownload PDF (3671K) -
Koichiro Omori, Kyoko Omori, Takashi Morimoto, Tatsuyoshi Takada, Masa ...1991 Volume 109 Issue 2 Pages 267-275
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe N-terminal half of the β-subunit of rat brain Na, K-ATPase was expressed in HeLa cells transfected with the plasmid pSV2TlineoβN containing the truncated β-subunit cDNA to study the assembly and transport of α-β complex. Immunoprecipitation from extracts of metabolically labeled transformed cells demonstrated that the truncated, β-subunit polypeptide (βN) was neither transported to the plasma membrane nor assembled into an α-β complex with the endogenous α-subunit. Cell fractionation experiments showed that the βN truncated subunit remained unassembled within rough microsomes, suggesting that it never exited from the endoplasmic reticulum (ER). The assembly of the endogenous α-and β-subunits in the βN-expressing cells was significantly inhibited compared with control cells or with the transformants that did not express the βN. These results suggest that the N-terminal portion of the β-subunit interferes with the normal assembly of the endogenous complex which normally takes place in the ER.View full abstractDownload PDF (3141K) -
Tsutomu Kodaki, Kohei Hosaka, Jun-ichi Nikawa, Satoshi Yamashita1991 Volume 109 Issue 2 Pages 276-287
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe yeast phosphatidylethanolamine methylation pathway is encoded by two structural genes, PEM1 and PEM2. The abundance of their transcripts was coordinately repressed by myo-inositol and choline. The most upstream transcriptional start sites for PEM1 and PEM2 were mapped at positions -142 and -42 relative to their first ATG codons, respectively. Promoter deletion analysis defined the 5' boundary of the regulatory region of PEM1 between -336 and -332 and that of PEM2 between -177 and -158. The 38-bp sequence between -336 and -299 from PEM1 and the 48-bp sequence between -177 and -130 from PEM2 conferred regulated transcription upon an upstream- activation-sequence-deficient test gene, CYC1-lacZ. Comparison of these two regions revealed the presence of a common octameric sequence, 5'-CATRTGAA-3', which occurred twice in the 38-bp PEM1 regulatory region and once, followed by the 5'-AAACCCACACATG-3' GRFI site, in the 48-bp PEM2 regulatory region. When synthesized chemically and placed in front of CYC1-lacZ, a single copy of CATATGAA directed a rather low level of gene expression, but multiple copies produced high-level expression. In both cases, gene expression was sensitive to myo-inositol and choline. The synthesized GRFI site directed considerable but constitutive lacZ expression. When used in conjunction with CATATGAA, synergistic, regulated gene expression was obtained. Hence CATRTGAA was concluded to play an important role in the myo-inositol-choline regulation of PEM1 and PEM2. Binding proteins to these sequences were demonstrated by electrophoretic mobility shift assay. Protein binding to CATRTGAA was not competitive with binding to the GRFI sequence, and vice versa. CATRTGAA was also found in the upstream regions of other genes encoding phospholipid-synthesizing enzymes, such as choline kinase, phosphatidylserine synthase, and myo-inositol-1-phosphate synthase, known to be repressed by myo-inositol and choline.View full abstractDownload PDF (3538K) -
Ken Karasawa, Ichiro Kudo, Tetsuyuki Kobayashi, Hiroshi Homma, Nobuyos ...1991 Volume 109 Issue 2 Pages 288-293
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAfter screening 900 E. coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity. Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed approximately 11.4 times higher lysophospholipase L1 activity than that of the parental strain. Starting from those overproducing strains, the lysophospholipase L1 was purified to near homogeneity by sequential use of ammonium sulfate fractionation, Sephacryl S-300, DEAE-cellulose, hydroxyapatite and Sephacryl 5-200 column chromatographies. The apparent molecular weight of the purified lysophospholipase L1 was estimated to be 20, 500-22, 000 both by SDS-poly-acrylamide gel electrophoresis and by gel permeation chromatography. The specific activity of the homogeneous lysophospholipase L1 was 10, 400 nmol/min/mg protein when 1-acyl-sn-glycero-3-phosphoethanolamine was used as the substrate. The amino acid sequence of the amino-terminal portion of purified lysophospholipase L1 was determined and was different from that of lysophospholipase L2, which had previously been purified from the envelope fraction of E. coli strains bearing its cloned structural gene, pldB [Karasawa, K., Kudo, I., Kobayashi, T., Sa-eki, T., Inoue, K., & Nojima, S. (1985) J. Biochem. 98, 1117-1125]. The gene responsible for overproduction of lysophospholipase L1 activity was designated as pldC (phospholipid degradation C). Its restriction enzyme map was also different from that of cloned pldB. These results further confirmed that, in E. coli, there are two lysophospholipases with distinct characteristics.View full abstractDownload PDF (1944K) -
Soichi Arai, Hirohito Watanabe, Hiroto Kondo, Yasufumi Emori, Keiko Ab ...1991 Volume 109 Issue 2 Pages 294-298
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOryzacystatin, a cysteine proteinase inhibitor occurring in rice seeds, contains a particular glycine residue (Gly5) near the NH2-terminal position, and the sequence Gln53-Val54-Val55-Ala56-Gly57 in a central part of the molecule. Both are conserved among most members of the cystatin superfamily. We have found from Escherichia coli expression studies that the NH2-terminal 21 residues of oryzacystatin are not essential for its papain-inhibitory activity, and that the conserved pentapeptide region may be indispensable [Abe, K., Emori, Y., Kondo, H., Arai, S., & Suzuki, K. (1988) J. Biol. Chem. 263, 7655-7659]. Here we present more detailed data based on quantitative analyses of the inhibitory activities of NH2-and COOH-terminally truncated oryzacystatin and site-directed mutants at the Gln-Val-Val-Ala-Gly region. The data indicate the following results. (1) The truncated mutants lacking the NH2-terminal 21 residues or the COOH-terminal 11 residues exhibit potent papaininhibitory activity equivalent to the activity of wild oryzacystatin. (2) However, neither the mutant lacking the NH2-terminal 38 residues nor that lacking the COOH-terminal 35 residues is completely able to inhibit papain. (3) Site-directed mutants at the Gln residue of the Gln-Val-Val-Ala-Gly region have drastically reduced papain-inhibitory activities: the Gln→Pro mutant is completely inactive and the Gln→Leu mutant has an approximately 150 times higher K1 value than wild-type oryzacystatin. On the other hand, mutations at the central Val residue of the Gln-Val-Val-Ala-Gly region result in moderate effects on activity: the Val→Gly mutant is as active as wild oryzacystatin, and the Val→Asp mutant has a 40 times higher K1 value than the wild type. (4) The completely inactive Gln→Pro mutant is unable to bind to a Cm-papain column, suggesting that the affinity site of oryzacystatin is also its reactive site. These results clearly indicate that the ln-Val-Val-Ala-Gly sequence of the cystatin molecule is the primary region of interaction ith the cysteine proteinase and is thus responsible for the inhibitory activity.View full abstractDownload PDF (2135K) -
Kazuya Taniguchi, Toshio Sasaki, Eiji Shinoguchi, Yoshiko Kamo, Eiichi ...1991 Volume 109 Issue 2 Pages 299-306
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOligomycin reduced the fluorescence intensity of an N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM) probe at Cys-964 of the α-chain of pig kidney Na+, K+-ATPase with increase in the concentration of Na+ with a Hill coefficient of nh=0.77 with Kh=231 mM. The maximum fluorescence decrease was around 80% of the value observed after accumulation of ADP-sensitive phosphoenzyme (E1P) in the presence of 2M Na+. The addition of Mg2+ and ATP with Na+ or choline chloride to give the same final ligand concentration to the Na+-enzyme-oligomycin complex formed with 16 mM Na++1, 984 mM choline chloride or 2M Na+ induced rapid phosphorylation (20 or 21/s) and slower fluorescence decrease (12.1±1.2 or 10.1±3.2/ s). These additions to the Na+-enzyme complex formed under the former or the latter conditions induced slow phosphorylation (13/s) prior to a much slower fluores-cence decrease (3.4±0.3 or 8.6±0.7/s). The addition of Ca2+ and ATP to these enzyme complexes induced rapid fluorescence changes (21-11/s) followed by one order of magni-tude slower rates of phosphorylation (1.5-1.3 s). These data suggest that the decrease in BIPM fluorescence induced by ATP with Ca2+ or with Mg2+, reflects the change of the Na+ binding state before or after the formation of E1P, respectively.View full abstractDownload PDF (1093K) -
Kazuki Kitamura, Yusuke Mizuno, Akio Sasaki, Akira Yasui, Shigeru Tsui ...1991 Volume 109 Issue 2 Pages 307-310
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit lα has been isolated from a rat kidney λgt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase lα from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type lα protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2Aα protein phosphatase. Thus, the protein sequence of type lα protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of A1113 were remarkably increased when compared to those of normal livers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.View full abstractDownload PDF (421K) -
Kikuko Takeuchi, Kastuhito Takahashi, Masahiro Abe, Wataru Nishida, Ku ...1991 Volume 109 Issue 2 Pages 311-316
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalponin is an actin binding protein found in the smooth muscle cells of chicken gizzard. The localization of the protein was examined in bovine platelets, mouse fibroblasts, and the smooth muscle cells of the bovine aorta. Immunoblotting of whole platelet lysates revealed that the antibody to chicken gizzard calponin recognized two proteins with apparent molecular masses of 37 and 23 kDa in the resting state and an additional high-molecular-weight component (approximately 40 kDa) in the activated state. The localizations of calponin and caldesmon, and the correlation of their localizations with that of the actin cytoskeleton were analyzed by immunofluorescence microscopy using appropriate anti-bodies and rhodamine-phalloidin. In resting bovine platelets, calponin exhibited the same distribution as actin filaments, which are organized in a characteristic wheel-like struc-ture. A similar distribution was observed with the anti-caldesmon antibody. Co-localizations of calponin and actin were shown in activated platelets and along stress fibers of both fibroblasts and smooth muscle cells. These results suggest not only a cytoskeletal role associated with microfilaments but also a regulatory role of these proteins for actin-myosin interaction.View full abstractDownload PDF (3736K) -
Ritsuko Sakaguchi, Masato Katahira, Yoshimasa Kyogoku, Satoshi Fujii1991 Volume 109 Issue 2 Pages 317-327
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conformations of drug-free d(GGGGCCCC)2 and the chromomycin-d (GGGGCCCC)2 complex in aqueous solution were studied by NMR spectroscopy. The present study has indicated that free d (GGGGCCCC)2 takes the B form in solution, although it takes the A form in the crystalline state. The NMR spectrum of the complex indicated that chromomycin binds as a symmetry-related dimer to the minor groove of the central four residues of d (GGGGCCCC)2. The drastic conformational change in the central four residues of d (GGGGCCCC)2 on going from the B form family to the A form was demonstrated by the characteristic NOEs and coupling patterns. The change seems to be indispensable for accommodation of the bulky chromomycin dimer in the minor groove. On the basis of the intermolecular NOEs between chromomycin and d (GGGGCCCC)2, the structure of the complex has been constructed and refined by energy minimization.View full abstractDownload PDF (1164K) -
Tur-Fu Huang, Joen-Rong Sheu, Che-Ming Teng, Shun-Wen Chen, Chen-Sheng ...1991 Volume 109 Issue 2 Pages 328-334
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTriflavin, an antiplatelet peptide containing Arg-Gly-Asp, purified from Trimeresurus licwoviridis venom, inhibits aggregation of human platelets stimulated by a variety of agonists. It blocks aggregation through interference with fibrinogen binding to its ecific receptor on the platelet surface membrane in a competitive manner, but it has no apparent effect on intracellular events, such as thromboxane B2 formation, phosphoinositides breakdown and intracellular Ca2+ mobilization of thrombin-activated platelets. In this study, we determined the complete sequence of triflavin, which is composed of a single polypeptide chain of 70 amino acids. Its sequence is rich in cysteine and contains Arg-Gly-Asp at residues 49-51 in the carboxy-terminal domain. Triflavin shows about 68% identity of amino acid sequence with trigramin, which is a specific antagonist of the fibrinogen receptor associated with glycoprotein IIb/IIIa complex. [125I] Triflavin binds to unstimulated and ADP-stimulated platelets in a saturable manner and its IC, values are estimated to be 76 and 74nM, respectively; the corresponding numbers of binding sites are 31, 029 and 34, 863 per platelet, respectively. [19] Triflavin binding is blocked by Gly-Arg-Gly-Asp-Ser in a competitive manner. EDTA, the Arg-Gly-Asp-containing peptides (in-cluding naturally occurring polypeptides, trigramin and rhodostomin), and monoclonal antibody, 7E3, raised against GP IIb/IIIa complex, inhibit [125I] triflavin binding to unstimulated and ADP-stimulated human platelets. In conclusion, triflavin specifically binds to fibrinogen receptor associated with GP IIb/IIIa complex and its binding site is located at or near GP IIb/IIIa complexoverlapping with those of 7E3 and another Arg-Gly-Asp-containing polypeptide, rhodostomin. The Arg-Gly-Asp sequence of triflavin plays an important role in mediating the binding of triflavin towards GP IIb/IIIa complex.View full abstractDownload PDF (889K) -
Toshiaki Arata1991 Volume 109 Issue 2 Pages 335-340
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPolymerization of G-actin in the presence of salt and phalloidin was blocked by treatment of G-actin with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) (designated as m-actin). The actin dimer produced by chemical crosslinking of F-actin with N, N' -p-phenylenedimaleimide did not polymerize and was still dimeric or tetrameric after further treatment with MBS (designated as d-actin). The m- and d-actins retained the ability to bind to myosin heads with apparent dissociation constants of 3-8×10-6 and 3-5×10-7M, respectively. d-Actin formed a 1:1 actin monomer-myosin head complex. However, m-actin formed a 2:1 m-actin-head complex, suggesting the presence of at least two latent actin-binding sites on a myosin head. ATP weakens only 2-to 6-fold the binding of these complexes. One of two m-actins on a myosin head was replaced by d-actin. Native F-actin blocked the binding of both m- and d-actins to myosin heads in the presence and absence of ATP, although the affinities of myosin head for MBS-treated actins and F-actin are similar in the presence of ATP. These results suggest that there are at least three actin binding sites on a myosin head: one is responsible for binding of F-, m-, and d-actins, the second for binding of F- and m-actins, and the third for binding of F-actin at least in the presence of ATP. F-Actin binding to the third site may in some way block the first and second binding sites.View full abstractDownload PDF (2058K) -
Hiromitsu Ikeda, Nobuvoshi Esaki, Shunji Nakai, Keiji Hashimoto, Shini ...1991 Volume 109 Issue 2 Pages 341-347
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcyclic monoterpene primary alcohol: NADP+ oxidoreductase, a key enzyme in the iosynthesis of monoterpene alcohols in plants, is unstable and has been only poorly characterized. However we have established conditions which stabilize the enzyme from Rauwolfia serpentina cells, and then purified it to homogeneity. It is a monomer with a molecular weight of about 44, 000 and contains zinc ions. Various branched-chain allylic primary alcohols such as nerol, geraniol, and 10-hydroxygeraniol were substrates, but ethanol was inert. The enzyme exclusively requires NADP+ or NADPH as the cofactor. Steady-state kinetic studies showed that the nerol dehydrogenation proceeds by an ordered Bi-Bi mechanism. NADP+ binds the enzyme first and then NADPH is the second product released from it. Gas chromatography-mass spectrometric analysis of the reaction products showed that 10-hydroxygeraniol undergoes a reversible dehydrogenation to produce 10-oxogeraniol or 10-hydroxygeranial, which are oxidized further to give 10-oxogeranial, the direct precursor of iridodial. The enzyme has been found to exclusively transfer the pro-R hydrogen of NADPH to neral. The N-terminal sequence of the first 21 amino acids revealed no significant homology with those of various other proteins including the NAD (P)+ -dependent alcohol dehydrogenases registered in a protein data bank.View full abstractDownload PDF (769K) -
Kenzi Suzuki, Tomoharu Gomi, Eiji Itagaki, Toshio Kaidoh1991 Volume 109 Issue 2 Pages 348-353
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSalicylate hydroxylase [EC 1. 14. 13. 1] from Pseudomonas putida catalyzed the formation of catechol from substrate analogues such as o-nitro-, o-amino-, o-iodo-, o-bromo-, and o-chloro-phenol by removing the ortho-substituted groups. They are converted into nitrite, ammonia, and halide ions, respectively. Kinetic parameters of these reactions were determined by spectrophotometric and polarographic methods. Hydroxylation of o-nitro-or o-iodophenol proceeds with the unusual stoichiometry of 2: 1:1 for consumed NADH, O2-uptake, and catechol formed. Other ortho-substituted phenols examined also gave the same results. Like salicylate, these substrates perturb the absorption spectrum of salicylate hydroxylase in the visible region, indicating the formation of enzyme•substrate complexes. Titration experiments with ortho-substituted phenols gave the dissociation constants of the complexes. The complexes were quantitatively reduced with NADH or dithionite without detectable formation of the intermediates. The fact that one atom of 18O2 was incorporated into the produced catechol in hydroxylation of o-nitrophenol indicates that the reaction is of monooxygenase nature. It is concluded that salicylate hydroxylase cleaves the C-N and C-X bonds of ortho-substituted phenols.View full abstractDownload PDF (666K) -
Yasuo Suzuk, Hiroshi Nishi, Kazuya Hidari, Yoshio Hirabayashi, Makoto ...1991 Volume 109 Issue 2 Pages 354-360
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new monoclonal antibody (NS24) directed to the N-acetylneuraminylα2-3Ga1β1- 4G1cNAc residue in type II sugar chain of N-acetylneuraminyllactoneotetraosylceramide [sialylparagloboside, IV3(NeuAc)nLc4Cer] was prepared by hybridoma technique. Lipo-somes composed of dipalmitoylphosphatidylcholine, cholesterol, IV3 (NeuAc) nLc4Cer, and lipopolysaccharides from Salmonella minnesota R595 were used for immunization with IV3 (NeuAc) nLc4Cer isolated from human erythrocytes. This method allowed the fusion of spleen cells of immunized mouse with myeloma cells only three days after immunization. NS24 reacted specifically to both naturally occurring and chemically synthesized IV3-(NeuAc)nLc4Cer, whereas it has no reactivity to structurally related gangliosides, such as IV6(NeuAc)nLc4Cer, N-glycolylneuraminyl α2-3 1actoneotetraosylceramide [IV3(NeuGc)-nLc4Cer], i-active ganglioside [VI3 (NeuAc) nLc6Cer], I-active ganglioside [VIII3 (NeuAc)-VP(NeuAc)IV6kladoLc8Cer], GM4 (NeuAc), GM3 (NeuAc), GM3(NeuGc), GM1b(NeuAc), GD3-(NeuAc), other ganglio-series gangliosides, sulfatide, and paragloboside (nLc4Cer). Syn-thetic N-acetylneuraminylα2-3lactotetraosylceramide [IV3 (NeuAc) Lc4Cer] and its asi-alo-derivative (Lc4Cer) carrying type I sugar chain also showed no reaction with NS24. One to 100 pmol of IV3 (NeuAc) nLc4Cer was detected dose-dependently by a thin-layer chro-matography/enzyme immunostaining procedure. Human gastric carcinomas showed positive reactions with NS24 immunochemically and histochemically. NS24 reacted preferentially with poorly differentiated adenocarcinomas rather than well differentiated ones.View full abstractDownload PDF (2027K) -
Tomoko Sonoda, Masanori Taira, Sumio Ishijima, Toshiharu Ishizuka, Tai ...1991 Volume 109 Issue 2 Pages 361-364
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESScDNA clones for human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) were isolated from a glioblastoma cell line MGC 1 cDNA library. The longest clone contained 2, 075 base pairs (bp) almost covering the 2.3-kb mRNA and the base sequence of the coding region (954 bp) had a 92.0% sequence homology with that of rat PRS I cDNA. The deduced amino acid sequences were identical between human and rat PRS I. This perfect conserva-tion has heretofore not been reported for other enzymes involved in nucleotide metabolism and glycolysis. A comparison with other isoforms of this enzyme, PRS II and PRS III, showed that the human PRS I was 79.9 and 92.2% homologous in the coding sequence and 95.3 and 94.0% in the deduced amino acid sequence to human PRS II and PRS III, respectively. The high value of the synonymous difference between PRS I and PRS II cDNAs places their time of divergence long before that of the radiation of mammals. Based on the evolutionary rate of amino acid substitution, the PRS I and II genes probably diverged about 760 million years ago.View full abstractDownload PDF (510K) -
Kohichi Okada, Tsutomu Ohta, Susumu Hirose1991 Volume 109 Issue 2 Pages 365-369
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the presence of a molar excess of eukaryotic DNA topoisomerase II and an appropriate concentration of dextran sulfate, relaxed closed circular DNA is converted to a negatively supercoiled form. The reaction is dependent on ATP. Neither adenosine 5'-[β, γ-thrimido]- triphosphate nor adenosine 5'-[γ-thio]triphosphate can substitute for ATP. The negative supercoils formed are relaxed by subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of a nicked circular DNA in the presence of DNA topoisomerase II and dextran sulfate but in the absence of ATP causes a small decrease in the linking number. These results suggest that when an appropriate concentration of dextran sulfate is present, the binding of a molar excess of eukaryotic DNA topoisomerase II constrains a small number of negative supercoils in DNA, which in turn generate unconstrained negative supercoils at the expense of ATP.View full abstractDownload PDF (2547K)
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