The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 112 , Issue 3
Showing 1-21 articles out of 21 articles from the selected issue
  • Kaoru Omichi, Sumihiro Hase
    1992 Volume 112 Issue 3 Pages 303-305
    Published: 1992
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The existence of α-amylase (HXA) encoded by α-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, α-1, 4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting α-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl α-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl α-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic a-amylase (HPA, 1.11) and human salivary α-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary α-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.
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  • Setsuko Fujita, Kayo Maeda, Yuichiro Maéda
    1992 Volume 112 Issue 3 Pages 306-308
    Published: 1992
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    A 25 kDa fragment of β-type troponin T (β-TnT) was expressed in Escherichia coli, and its function as a component of the regulatory system for actomyosin ATPase was compared with that of the authentic counterpart, the full length α-TnT. The expressed species, designated as β-TnT (N'-208), consists of 208 residues. It lacks the entire variable region at the amino-terminus and, near the carboxyl-terminus, a segment of 14 residues is changed from the α-type to the β-type sequence. Functional tests indicated that the truncated β-TnT was not distinguishable from the full length α-TnT, suggesting that neither deletion of the variable N-terminal region nor alteration of the type has a significant effect on the regulatory action of TnT.
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  • Hiroshi Yokota, Akira Yuasa
    1992 Volume 112 Issue 3 Pages 309-313
    Published: 1992
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    Interactions between purified UDP-glucuronyltransferase from 3-methylcholanthrenetreated rat liver microsomes (named GT-1) and lysophosphatidylcholine, which is essential for expression of GT-1 activity, were examined. Phospholipid-free GT-1, which could not express its full activity [Yokota et al. (1988) J. Biochem. 104, 531-536], was activated fully by addition of lysophosphatidylcholine (0.04mM final concentration) into the assay medium. Lysophosphatidylcholine also protected GT-1 effectively against heat inactivation. Palmitoyllysophosphatidyicholine and stearoyllysophosphatidylcholine were most successful for the activation and stabilization of GT-1.On treatment of GT-1 with earboxypeptidase Y, the transferase was inactivated immediately, but the treatment in the presence of lysophosphatidylcholine affected the activity only a little. Lysophosphatidylcholine was also found to protect GT-1 against cleavage by carboxypeptidase Y. On treatment of GT-1 with trypsin or aminopeptidase T, the activity was lost and GT-1 protein could be digested even when lysophosphatidylcholine was present. It is suggested that UDP-glucuronyltransferase forms an active and stable conformation, in which the carboxyterminal region is protected against protease, with lysophosphatidylcholine.
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  • Yasushi Hamaguchi, Yoshiki Yamamoto, Hiroko Iwanari, Shingo Maruyama, ...
    1992 Volume 112 Issue 3 Pages 314-320
    Published: 1992
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    We have investigated whether Jx recombination signal sequence (RS) binding protein (RBP-Jx) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA. Murine RBP-Jx protein purified by Jx-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity. To obtain a large amount of purified RBP-Jx protein, recombinant RBP-Jx was synthesized in Escherichia coli as a fusion protein and also in silkworm cells. Although recombinant RBP-Jx produced in silkworm cells could bind Jx-RS, it failed to show either ligase or DNA bending activity. Since the DNA affinity-purified RBP-Jx has the ligase activity, the RBP-Jx protein may form a complex with a ligase in vivo. We have raised monoclonal antibodies against the RBP-Jx fusion protein which was synthesized in E. coli and unable to bind Jx-RS. Using the anti-RBP-Jx monoclonal antibody we have shown that the RBP-Jx protein is expressed ubiquitously in mammalian tissues. The ubiquitous expression of the RBP-Jx protein is consistent with the hypothesis that the RBP-Jx protein may have dual function [Furukawa et al. (1991) J. Biol. Chem. 266, 23334-23340].
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  • Takeshi Endo
    1992 Volume 112 Issue 3 Pages 321-329
    Published: 1992
    Released: November 18, 2008
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    Terminal differentiation of skeletal muscle cells is obligatorily accompanied by the expression of a battery of muscle-specific genes and cell fusion to form multinucleated myotubes. The transcription of some of the muscle-specific genes is activated by the products of myoD gene family including MyoD and myogenin. The mouse skeletal muscle cell line C2SVTts11, which is a clone of C2 cells transfected with SV40 T antigen genes (temperature-sensitive large T and wild-type small t) fused to metallothionein gene promoter, is prevented from differentiation when the large T is induced. If the large T is induced in the myotubes, which are preformed in the absence of large T expression, the terminally differentiated cells reenter the cell cycle. In good accordance with the induction of large T, endogenous c-jun but not c-fos or c-myc mRNA was induced, whereas the expression of myoD and myogenin was suppressed. Treatment of quiescent C2 cells with a tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, transiently induced c-jun and c-fos mRNAs, and temporarily deinduced myoD and myogenin mRNAs just after the expression of the protooncogenes. To ascertain whether c-jun induced by large T is sufficient to inhibit myogenic differentiation, c-jun cDNA was transfected into C2 cells. As the levels of exogenous c-jun expression were higher in the transfected clones, the cells expressed lower levels of myoD gene family and they formed fewer myotubes. Even the cells expressing the highest levels of exogenous c-jun, however, still formed small myotubes containing a few nuclei under differentiation conditions. These results suggest that large T inhibits myogenic differentiation by suppressing the expression of the members of myoD gene family, partly through inducing c-jun. In addition to this, other mechanisms seem to be required to achieve complete inhibition.
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  • Toyohiro Takehara, Kunio Matsumoto, Toshikazu Nakamura
    1992 Volume 112 Issue 3 Pages 330-334
    Published: 1992
    Released: November 18, 2008
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    Hepatocyte growth factor (HGF), a potent mitogen for mature hepatocytes, has been considered to act as a hepatotropic factor for liver regeneration. We examined the effect of HGF on albumin synthesis and DNA synthesis of adult rat hepatocytes cultured at various cell densities. HGF stimulated albumin synthesis of hepatocytes by 40-60% when they were cultured at higher cell densities such that there was tight cell-cell contact. But at lower cell densities HGF failed to stimulate albumin synthesis. In contrast, the stimulatory effect of HGF on DNA synthesis of hepatocytes was more potent at lower than at higher cell densities: HGF did not stimulate DNA synthesis of hepatocytes cultured at confluent cell density. Thus, HGF seems to stimulate both albumin synthesis and DNA synthesis of hepatocytes, in a reciprocal relationship depending on cell density. When the effects of various cytokines were examined, epidermal growth factor, transforming growth factor-α, and acidic fibroblast growth factor also stimulated albumin synthesis by 20-30%. However, transforming growth factor-β1, basic fibroblast growth factor, and interleukin-1β had no effect on albumin synthesis, while interleukin-6 inhibited it by 42%. Thus HGF was the most potent in stimulating albumin synthesis in these cytokines. Since HGF is markedly increased in the liver or plasma following various liver insults, HGF may be involved in liver regeneration through the potential to stimulate both cell growth and liver-specific functions such as albumin synthesis in a cell density-dependent manner.
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  • Kuniyo Inouye
    1992 Volume 112 Issue 3 Pages 335-340
    Published: 1992
    Released: November 18, 2008
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    It has been reported that neutral salts such as NaCl activate the thermolysin-catalyzed hydrolysis of substrates containing glycine at the P1 position (carboxylic side of the cleavage bond) [Holmquist, B. & Vallee, B. L. (1976) Biochemistry 15, 101-107]. In this paper, we demonstrate that high concentrations (1-4M) of neutral salts greatly enhance the thermolysin activity in both hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZAPM), a precursor of a peptide sweetener, aspartame, in which the L-aspartyl residue is the P1 residue. The enzyme activity is enhanced with an increase in salt concentration in a pseudo-exponential fashion. The degree of activation by salts was in the order LiCl>NaCl>KCl. The rate of ZAPM hydrolysis in the presence of 3.8M NaCl was 6-7 times higher than that in its absence, and 50 times or more activation is expected in saturated NaCl solution. The activation is brought about solely through an increase in the catalytic constant (kcat), and the Michaelis constant (Km) is not affected at all by the presence of NaCl.On mixing thermolysin with NaCl, a unique absorption difference spectrum suggesting a conformational change of the enzyme was observed. The intensity increased in a pseudo-exponential fashion with increase of NaCl concentration up to 3M, and this dependence is similar to that of the enzyme activity.
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  • Yen-Chywan Liaw, Shyh-Horng Chiou, Tschining Chang, Wen-Chang Chang
    1992 Volume 112 Issue 3 Pages 341-345
    Published: 1992
    Released: November 18, 2008
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    A systematic structural comparison of several carp γ-crystallins with high methionine contents was made by the secondary-structure prediction together with computer modelbuilding based on the established X-ray structure of calf γ-II crystallin. The overall surface hydrophilicity profile and the distribution of helices, β-sheets, and β-turns along the polypeptide chains are very similar among these carp γ-crystallins. In addition, their general polypeptide packing is close to the characteristic 2 domain/4 motif Greek key three-dimensional conformation depicted for the calf γ-II crystallin. Interestingly, most hydrophobic methionine residues are located on the protein surface with only a few buried inside the protein surface or in the interface between two motifs of each domain. The exposed hydrophobic and polarizable methionine cluster on the protein surface may have a bearing on the crystallin stability and dense packing in the piscine species, and probably also provides a malleable nonpolar surface for the interaction with other crystallin components for the maintenance of a clear and transparent lens.
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  • Masaru Nakamoto, Shyuichiro Matsubara, Teruo Miyauchi, Hiroya Obama, M ...
    1992 Volume 112 Issue 3 Pages 346-349
    Published: 1992
    Released: November 18, 2008
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    MK (midkine) and HB-GAM (heparin-binding growth-associated molecule) constitute a new family of heparin-binding growth differentiation factors. The modes of expression of MK and HB-GAM during mouse development were quantitatively examined by mRNA hybridization. The following three distinct patterns of expression were observed in the brain/head region. On the 11th-13th days of gestation, MK was intensely, but HB-GAM relatively weakly expressed; on the 15th-19th days, both MK and HB-GAM expression became weaker; and in the neonatal period, HB-GAM was intensely expressed and MK expression increased slightly. The level of HB-GAM expression was lower than that of MK in the whole embryo on the 11th to 13th days of gestation. HB-GAM mRNA was detected in the kidney of newborn and young mice, where MK was more highly expressed. The identity of the weakly expressed MK and HB-GAM signals was confirmed by means of the polymerase chain reaction in the neonatal brain (MK), the head of 13-day embryos (HB-GAM), and the kidney of 7-day-old mice (HB-GAM). In conclusion, MK and HB-GAM are frequently co-expressed in the same cells and anatomic regions of the fetus or new born mouse, while their modes of expression differ.
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  • Stefania Di Marco, Fritz Märki, Hans Hofstetter, Albert Schmitz, ...
    1992 Volume 112 Issue 3 Pages 350-354
    Published: 1992
    Released: November 18, 2008
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    Recombinant human Synovial fluid phospholipase A2 (rPLA2) and several variants with N-terminal sequences modified by addition or deletion of one or two amino acid residues (Ala or Met; Des-Asn1, Leu2) have been expressed in mammalian cells and in Escherichia coli, respectively, purified to homogeneity, and characterized. The observed values for the molecular mass of rPLA2 and variants are in complete agreement with the predicted values for a correctly folded structure containing seven disulfide bridges. Moreover, the relative proportions of the various types of secondary structures of the variants of rPLA2, as measured by CD spectroscopy, are similar to that found for native porcine pancreatic PLA2, indicating that the recombinant proteins are correctly folded. Enzymatic activities of rPLA2 with modified N-termini decreased to 1.3-0.005% of the activity of the mature rPLA2, emphasizing a key role of the N-terminus for catalytic activity.
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  • Shojiro Iwahara, Takashi Maeyama, Toshihiro Mishima, Takayuki Jikibara ...
    1992 Volume 112 Issue 3 Pages 355-359
    Published: 1992
    Released: November 18, 2008
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    Four novel oligosaccharide units were isolated from the acetolysis products of the acidic polysaccharide chain derived from the glycoproteins of Fusarium sp. M7-1. Their chemical structures were resolved mainly by 1H-NMR spectrometry in combination with methylation analysis and mass spectrometry. The results indicate that these oligosaccharide units originated from the side chains, GlcNAcα1→4GlcAα1→2(GlcNAcα1→4)GlcAα1→2Gal, GlcNAcα1→4GlcAα1→2 (GlcNAcα1→4) GlcAα1→2 (GlcNAcα1→4) GlcAα1→2Gal, ChN←P→ 6Manβ1→4GlcAα1→2Gal, and Manβ1→2 (ChN←P→6) Manβ1→4GlcAα1→2Gal linked together with the other units reported previously [Jikibara et al. (1992) J. Biochem. 111, 236-243] through β1→6galactofuranoside linkages in the acidic polysaccharide chain.
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  • Katsuki Yasufuku, Hiroshi Ohashi, Yumiko Katsuta-Enomoto, Takahiro Fuk ...
    1992 Volume 112 Issue 3 Pages 360-365
    Published: 1992
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    A fusion protein (pETB-42P), which encodes the 42-amino acid leader peptide and the 38-amino acid peptide of human big endothelin (ET)-1, was synthesized in Escherichia coli, isolated as inclusion bodies, and purified by DEAE-chromatography. Trypsin digestion of the purified pETB-42P gave big ET-1 (1-37) in a yield of 70%; then pepsin digestion of the purified big ET-1 (1-37) gave ET-1 (1-21) in a yield of 74% (overall yield: 52%). Sequential trypsin and pepsin digestions of the purified fusion protein in the same reaction vessel also allowed recovery of ET-1 in a yield of 60%. One milligram of ET-1 or 2.0mg of big ET-1 (1-37) was obtained from 1.8 liters of culture broth. Recombinant ET-1 thus obtained was identical to authentic ET-1 in terms of amino acid sequence and vasoconstrictor potency, and recombinant big ET-1 (1-37) had almost the same in vitro and in vivo biological activities as big ET-1 (1-38).
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  • Yukiko Konami, Kazuo Yamamoto, Toshiaki Osawa, Tatsuro Irimura
    1992 Volume 112 Issue 3 Pages 366-375
    Published: 1992
    Released: November 18, 2008
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    The complete amino acid sequence of 2-acetamido-2-deoxy-D-galactose-binding Cytisus scoparius seed lectin II (CSII) was determined using a protein sequencer. After digestion of CSII with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSII with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid residues of concanavalin A (Con A) involved in the metal binding site are highly conserved among those of CSII. A carbohydrate-binding peptide of CSII was obtained from the endoproteinase Asp-N digest of CSII by affinity chromatography on a column of GalNAc-Gel. This peptide was retained on the GalNAc-Gel column and was presumed to have affinity for the column. The amino acid sequence of the retarded peptide was determined using a protein sequencer. The retarded peptide was found to correspond to the putative metal-binding region of Con A. These results strongly suggest that this peptide represents the carbohydrate-binding and metal ion-binding sites of CSII.
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  • Sakae Shimizu, Tsuneo Imanaka, Tatsuya Takano, Shoji Ohkuma
    1992 Volume 112 Issue 3 Pages 376-384
    Published: 1992
    Released: November 18, 2008
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    Clofibrate increased oligomycin-resistant ATPase activity in peroxisomes more than 17-fold (5.15±0.71milliunits/mg protein) in rat liver. The activity was dependent on divalent cations (Mg2+>Ca2+) with an optimum pH of 7.5. This activity was partially inhibited by N-ethylmaleimide (NEM), 4, 4'-dithiocyanatostilbene-2, 2'-disulfonic acid (DIDS), silicotungstic acid (STA), and high concentrations of N, N'-dicyclohexylcarbodiimide (DCCD). Proteinase K digestion of intact peroxisomes severely reduced the NEM-sensitive activity, but little affected the NEM-resistant activity. NEM-sensitive and -resistant ATPases showed Km values for ATP of 780 and 73μM, respectively. The NEM-sensitive activity was inhibited completely by DIDS, 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl), tributyltin chloride (TBT), and quercetin, and partially by DCCD and STA, whereas the NEM-resistant activity was totally insensitive to these chemicals except for STA. These activities had unique requirements for divalent cations, anions, and substrates, respectively. They were partially separated by gel filtration chromatography and had molecular masses of 520 kDa (NEM-sensitive enzyme) and 450 kDa (NEM-resistant enzyme), respectively.
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  • Makoto Morita, Hiroshi Akanuma
    1992 Volume 112 Issue 3 Pages 385-388
    Published: 1992
    Released: November 18, 2008
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    The concentration of 1, 5-anhydro-n-glucitol (AG) was determined in various organs and tissues of normal rats and rats rendered diabetic with streptozocin, using an AG-assay method in which AG was extracted after acid hydrolysis of the whole tissues. The organs and tissues examined included skin, muscle, liver, and kidney. The plasma of control rats contained 3-12μg/ml of AG. In these rats, all the organs examined also contained AG at concentrations not much lower than that in the corresponding plasma, except for adipose tissues and testis, which have relatively small water spaces; the latter two contained AG at relatively low concentrations. In contrast, both the plasma and various organs of the diabetic rats contained only trace amounts of AG. The whole body perfusion of control rats depleted AG from most of the organs, the exception being spleen, the circulation system of which is known to have a structure that is difficult wash by means of perfusion. These observations indicated that AG readily diffused into the inter- and intra-cellular water spaces from the circulation. Accordingly, the plasma membranes of the cells in these organs were suggested to be permeable to AG.
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  • Akira Takeya, Osamu Hosomi, Nobuo Shimoda, Shin Yazawa
    1992 Volume 112 Issue 3 Pages 389-395
    Published: 1992
    Released: November 18, 2008
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    Human blood group O plasma was found to contain an N-acetylgalactosaminyltransferase which catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc to Galβ1→4Glc, Galβ1→4GlcNAc, asialo-α1, -acid glycoprotein, and Galβ1→4GlcNAcβ1→3Galβ1→4Glc-ceramide, but not to Galβ1→3GlcNAc. The enzyme required Mn2+ for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by β-N-acetylhexosaminidase and released N-acetylgalactosamine. Apparent Km values for UDP-GalNAc, Mn2+, lactose, N-acetyllactosamine, and terminal N-acetyllactosaminyl residues of asialo-α1-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5mM, respectively. Studies on acceptor substrate competition indicated that all the acceptor substrates mentioned above compete for one enzyme, whereas the enzyme can be distinguished from an NeuAcα2→3Galβ-1, 4-N-acetylgalactosaminyltransferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of N-acetylgalactosamine to lactose revealed that N-acetylgalactosamine had been transferred to the carbon-3 position of the β-galactosyl residue. Although the GalNAcβ1→3Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Galα1→4Gal β-1, 3-N-acetylgalactosaminyltransferase, which is involved in the synthesis of the major P antigen-active glycolipid, GalNAcβ1→3Galα1→4Galβ1→4Glc-ceramide. Hence, the GalNAcβ1→3Galβ1→4GlcNAc/Glc structure is synthesized by the novel Galβ1→4GlcNAc/Glcβ-1, 3-N-acetylgalactosaminyltransferase.
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  • Tsuyoshi Takasuka, Miki Adachi, Chikara Miyamoto, Yasuhiro Furuichi, T ...
    1992 Volume 112 Issue 3 Pages 396-400
    Published: 1992
    Released: November 18, 2008
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    The cloned cDNA genes for endothelin receptors ETA and ETB were expressed in COS cells, and the binding characteristics of the two receptors with three isopeptide ligands (ET-1, ET-2, and ET-3) were examined in detail. The results indicated that the stability of receptor-ET-1 complexes formed with ETA and ETB were significantly different from each other, while their affinities to ET-1 were similar. The preformed ETA-ET-1 complex readily dissociated upon SDS-PAGE, as did many of the other receptors so far studied, while the ETB-ET-1 complex survived SDS-PAGE when it was run at low temperature (_??_4°C). Clear differences in stability were also shown in comparative studies of acid treatment of the two types of complexes. Only the ETB-ET-1 complex was resistant to acid treatment (0.2M acetic acid, 0.5M NaCl), and its 50 kDa monomeric receptor-ligand complex remained intact. The ETB-ET-1 complex (50 kDa) formed at 4°C on the surface of COS cells, however, was susceptible to limited proteolysis at 37°C that reduced the molecular size of the complex to a distinct 35 kDa. No such size reduction was observed with the preformed ETA-ET-1 complex. The overall structure of two endothelin receptors, as deduced from the sequence of cloned cDNAs, is similar in many respects. However, the present findings demonstrate distinct differences in the biochemical nature of the two receptors, which suggest their distinct biological functions.
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  • Kouichirou Hori, Fumi Morita
    1992 Volume 112 Issue 3 Pages 401-408
    Published: 1992
    Released: November 18, 2008
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    F-Actin was digested with α-chymotrypsin in 6M urea, and two peptide fragments from subdomain 4 of actin molecule [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes K. C. (1990) Nature 347, 37-44] were purified by reverse-phase HPLC and Sephadex G-50 gel filtration. The peptide fragments were identified as segments from Arg-177 to Tyr-198 (2.6-kDa peptide) and from Ser-199 to Tyr-279 (9.1-kDa peptide). Their effects on actin polymerization induced by 50 or 100mM KCl were studied by measuring the increase in viscosity by the falling ball method. The 2.6-kDa peptide decreased the rate of actin polymerization and increased the critical concentration for the polymerization. Based on the atomic model of the actin filament [Holmes, K. C., Popp, D., Gebhard, W., & Kabsch, W. (1990) Nature 347, 44-49], the peptide is presumed to bind to the barbed end of the actin filament and inhibit the polymerization. By assuming that the peptide affected the rate of association of the actin monomer to the end of the actin filament, well-fitting curves for the polymerization kinetics were calculated. Computer-assisted results indicated that the dissociation constant of the 2.6-kDa peptide for F-actin is 200 to 260μM. In contrast, the 9.1-kDa peptide only slightly inhibited actin polymerization. These results suggest that the actin-actin interface in the region between Arg-177 and Tyr-198 has a stronger interaction than those between Ser-199 and Tyr-279. The amino acid sequence L-T-D-Y-L present in the 2.6-kDa segment is homologous to a common sequence in the F-actin capping domain of various actin-binding proteins.
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  • Hiroshi Okazaki, Chiemi Tani, Miyuki Ando, Kyoko Ishii, Sadahiko Ishib ...
    1992 Volume 112 Issue 3 Pages 409-413
    Published: 1992
    Released: November 18, 2008
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    A previously found proteinase possibly involved in the modification of hexokinase to eliminate the mitochondria-binding ability without appreciable change in the catalytic activity (called hexokinase-processing enzyme hereafter), was purified by sequential chromatographies from rat liver and its properties were examined. The hexokinase-processing enzyme had carbohydrate moieties as evidenced by adsorption on immobilized concanavalin A, and had a molecular weight of about 23, 000 as estimated by SDS-PAGE and gel filtration chromatography. Benzyloxycarbonyl-phenylalanyl-L-arginine-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA)-hydrolyzing activity was co-purified with this processing activity throughout the purification, while the hydrolyzing activity for benzyloxycarbonyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide (Z-Arg-Arg-MCA) was not. The processing activity, as well as Z-Phe-Arg-MCA hydrolyzing activity, was highly sensitive to cysteine proteinase inhibition, for example, by leupeptin and N-[N-3-(traps-carboxirane-2-carbonyl)-L-leucyl] agmatine (E-64). Furthermore, the enzyme preparation reacted with an antibody against cathepsin L purified from rat kidney. These results indicated that cathepsin L may be involved in the above-mentioned processing of hexokinase.
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  • Yasuhiko Yamamoto, Koji Iwafune, Riichirô Chûjô, Yos ...
    1992 Volume 112 Issue 3 Pages 414-420
    Published: 1992
    Released: November 18, 2008
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    1H-NMR spectra of deoxy myoglobins (Mbs) from shark (Galeorhinus japonicus), horse, and sperm whale have been studied to gain insights into their active site structure. It has been demonstrated for the first time that nuclear Overhauser effect (NOE) can be observed between heme peripheral side-chain proton resonances of these paramagnetic complexes. Val-E11 methyl and His-F8 CδH proton resonances of these Mbs were also assigned from the characteristic shift and line width. The hyperfine shift of the former resonance was used to calculate the magnetic anisotropy of the protein. The shift analysis of the latter resonance, together with the previously assigned His-F8 NδH proton resonance, revealed that the strain on the Fe-Nε bond is in the order horse Mb_??_whale Mb<shark Mb and that the hydrogen bond strength of the His-F8 NδH proton to the main-chain carbonyl oxygen in the preceding turn of the F helix is in the order shark Mb<horse Mb<whale Mb. Weaker Fe-porphyrin interaction in shark Mb was manifested in a smaller shift of the heme methyl proton resonance and appears to result from distortion of the coordination geometry in this Mb. Larger strain on the Fe-Nε bond in shark Mb should be to some extent attributed to its lowered O2 afnity (P50=1.1mmHg at 20°C), compared to whale and horse Mbs.
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  • Toshiko Kunikata, Hideki Yamano, Toshihiko Nagamura, Yasunori Nitta
    1992 Volume 112 Issue 3 Pages 421-425
    Published: 1992
    Released: November 18, 2008
    JOURNALS FREE ACCESS
    The hydrolysis of substrates (maltohentaose, maltopentaose, and maltotetraose) catalyzed by soybean β-amylase [EC 3. 2. 1. 2] at pH 5.4 and 25°C was followed by monitoring small changes in the quenching of fluorescence due to tryptophan residues by the stopped-flow method. By analysis of whole time course, the dissociation constants, Kds, of enzymesubstrate and enzyme-product complexes were reasonably evaluated; and the difference in fluorescence intensities per mol between the enzyme-complex (ES or EP) and the free enzyme, ΔF, was determined. The molecular activity, k0, was also determined by a new method of half time analysis. The Kds and k0 values are in good agreement with our kinetic data reported previously. The ΔFs of substrates were of smaller magnitude than those of products (G2 and G3), which means that the higher the binding affinity of the ligand is, the smaller the ΔF value is. This indicates that at least two tryptophan residues must be located in the active site if the enzyme is rigid, or that if there is only one, the active site must undergo a structural change caused by the binding of ligand.
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