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Volume 101 (1987)
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Volume 101, Issue 2
Displaying 1-29 of 29 articles from this issue
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Sachio MORIMOTO, Iwao OHTSUKI1987 Volume 101 Issue 2 Pages 291-301
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Ca2t+-sensitive ATPase activity of rabbit skeletal myofibrils disappeared completely after treatment with a solution containing CDTA, a strong divalent cation chelator, at a low ionic strength. A gel electrophoretic study revealed that all troponin C and about half of myosin light chain 2 were removed from the myofibrils by the CDTA treatment. The CDTA-treated myofibrils, when reconstituted with skeletal troponin C, showed almost exactly the same Ca2+- or Sr2+-sensitive ATPase activity as that of intact myofibrils. The CDTA-treated myofibrils reconstituted with porcine cardiac troponin C showed the same Ca2+- or Sr2+-sensitivity of the ATPase as that of porcine cardiac myofibrils. Sr2+-sensitivity relative to Ca2+-sensitivity was about ten times higher than, and the maximal slope of the activation curve was about half that of skeletal myofibrils. These findings indicate that these characteristic features of divalent cation regulation in the contraction of skeletal and cardiac muscles are determined solely by the species of troponin C. Bovine brain calmodulin hardly activated the ATPase activity of the CDTA-treated myofibrils even in the presence of Ca2+. Excess calmodulin, however, was found to give Ca2+- or Sr2+-sensitivity to the ATPase activity of the CDTA-treated myofibrils. Frog skeletal parvalbumins 1 and 2, even in excess, did not affect the ATPase activity of theView full abstractDownload PDF (2013K) -
Michio NAKAI, Shunji NATORI1987 Volume 101 Issue 2 Pages 303-309
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of the rapid inactivation of the cytotoxic activity produced by peritoneal macrophages stimulated with Sarcophaga lectin was investigated. Experiments using radioiodinated tumor killing factor (TKF) showed that the main reason for its inactivation was the rapid dissociation of its trimeric structure into 15-kDa subunits. This dissociation was found to proceed spontaneously in the culture medium irrespect of the presence of macrophages.View full abstractDownload PDF (1191K) -
Yasuo MUKOHATA, Manabu YOSHIDA1987 Volume 101 Issue 2 Pages 311-318
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe characteristics of ATP synthesis in cell envelope vesicles of Halobacterium halobiurn were further studied. The results confirmed the previous conclusion (Mukohata et al. (1986) J. Biochem. 99, 1-8) that the ATP synthase in this extremely halophilic archaebacterium can not be an ordinary type of F0F1-ATPase, which has been thought to be ubiquitous among all the aerobic organisms on our biosphere. The ATP synthesis was activated most in 1M NaCl and/or KCl, and at 40°C, and at 80mM MgCl2 where F0F1-ATPase loses its activity completely. The synthesis was negligible at 10°C, and at 5mM MgCl2. The Km for ADP was about 0.3mM in the presence of 20mM P1, 1M NaCl, 80mM MgCl2 and 10mM PIPES at pH 6.8 and 20°C. The ATP synthesis was not inhibited by NaN3 and quercetin (specific inhibitors for F0F1-ATPase) or vanadate (for E1E2-ATPase) or ouabain (for Na+, K+-ATPase) or P1, P5-di (adenosine-5') pentaphosphate (AP5A, for adenylate kinase). The ATP synthesis was not inhibited by modification (pretreatment) with NaN3 or 5'-p-fluorosulfonylbenzoyladenosine (FSBA). On the contrary, the ATP synthesis was rather non-specifically inhibited by N-ethylmaleimide (NEM), trinitrobenzenesulfonate (TNBS), phenylglyoxal, and pyridoxal phosphate. 7-Chloro-4-nitrobenz-2-oxa-1, 3-diazole (NBD-Cl) as well as N, N'-dicyclohexylcarbodiimide (DCCD) was found to be a specific inhibitor at least partly, because the NBD-Cl inhibition was partly prevented by ADP added to the modification mixture.View full abstractDownload PDF (601K) -
Yuji GOTO, Makoto TSUNENAGA, Yasushi KAWATA, Kozo HAMAGUCHI1987 Volume 101 Issue 2 Pages 319-329
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to understand the conformations and stabilities of the immunoglobulin domains, a derivative of the type-λ constant fragment in which the peptide bond Arg190-Ser191 is cleaved and a derivative of the type-κ constant fragment in which the peptide bond Arg142-Glu143 is cleaved were prepared by limited proteolysis with the proteinase from mouse submaxillary gland, endoproteinase Arg-C [EC 3. 4. 21. 40]. The cleaved peptide bond of each derivative is located in the loop formed by the intrachain disulfide bond and the two peptides formed are linked by the disulfide bond. The two nicked CL fragments did not assume any ordered conformation. On the other hand, a derivative in which the intrachain disulfide bond is reduced had a conformation very similar to that of the intact CL fragment, although the stability was considerably low. Since the entropy of the nicked fragment should be nearly the same as that of the reduced CL fragment in the unfolded state, a destabilizing effect of cleavage of the polypeptide bond in the folded state in addition to the entropic effect in the unfolded state seems necessary to account for the conformations of the nicked CL fragments.View full abstractDownload PDF (1740K) -
Masanobu KAWAKAMI, Toshio MURASE, Hiroko OGAWA, Shun ISHIBASHI, Natsuk ...1987 Volume 101 Issue 2 Pages 331-338
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTumor necrosis factor (TNF) has been reported to be identical to “cachectin, ” amonokine which we have previously proposed as a mediator of the enhanced catabolism observed in patients or animals responding to various invasive stimuli such as infections. Detailed quantitative studies were conducted on the effects of TNF on fatty acid metabolism in 3T3-L1 cells in order to explore the extent of the catabolic effects exerted by TNF compared with those by the crude cachectin. 3T3-L1 adipocytes responded to recombinant human TNF, showing a decrease in LPL activity and an increase in intracellular lipolysis. When TNF in the crude cachectin preparation was completely neutralized with anti-TNF antibody, about 75% of LPL suppression activity in the crude cachectin was absorbed, indicating that most of the mediator responsible for LPL suppression in the crude preparation is TNF. In contrast to the above effect on LPL, TNF markedly increased the lipolysis of stored fat in the cells. The effect on LPL was observed as early as 2 h after the addition of TNF, but enhancement of lipolysis required a time lag of at least 3 h before any increase of glycerol release became apparent. The effective concentrations of TNF for the stimulation of lipolysis were much higher (2.5 to 49 nM) than those for LPL suppression (50 pM to 50 nM), but both were in the same range as the concentration required for tumoricidal effect. These results demonstrate that cachectin is synonymous with TNF and that it plays an important role in the pathophysiology of deranged lipid metabolism through both suppression of LPL and enhancement of lipolysis in patients coping with invasive conditions such as infections.View full abstractDownload PDF (612K) -
Hiroaki SAWAI, Hideharu TAIRA, Kaichiro ISHIBASHI, Masao ITOH1987 Volume 101 Issue 2 Pages 339-346
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-Alanyltyrosine derivative of 2', 5'-tetraadenylate 5'-triphosphate, pppA2'p5'A2'-p5'A2'p5'A-β-Ala-Tyr was prepared by coupling of periodate-oxidized pppA2'p5'-A2'p5'A2'p5'A with β-alanyltyrosine methyl ester, followed by reduction with sodium cyanoborohydride. Its stability to 2', 5'-phosphodiesterase and phosphatase was investigated in mouse L cell extract. The 5'-triphosphate of the compound was cleaved gradually to form the 5'-dephosphorylated derivative, A2'p5'A2'p5'A2'p5'A-β-Ala-Tyr, followed by slow degradation of the 2', 5'-phosphodiester bond. On the other hand, pppA2'p5'A2'p5'A2'p5'A was hydrolyzed very quickly under the same conditions. The tetramer derivative bound tightly to the 2', 5'-oligoadenylatedependent endoribonuclease in rabbit reticulocyte lysate or mouse L cell extract and inhibited protein synthesis of mouse L cells more effectively than the unmodified 2', 5'-tetraadenylate 5'-triphosphate. The corresponding trimer derivative had slightly weaker activities than the unmodified trimer for binding to the endoribonuclease and for inhibition of protein synthesis. The compound, pppA2'p5'A2'p5'-A2'p5'A-β-Ala-Tyr, was iodinated easily at the tyrosine residue with125I, giving a high-specific-radioactivity derivative which was used as a radio-labeled probe in a radiobinding assay for 2', 5'-oligoadenylate.View full abstractDownload PDF (603K) -
Hitoshi AOSHIMA, Kenzi HORI, Akinori YAMAMOTO1987 Volume 101 Issue 2 Pages 347-355
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rate of slow Li+ influx and the fraction of active form of acetylcholine receptor (AChR) of Electrophorus electricus membrane vesicles at equilibrium between the active and desensitized forms of the receptor were measured in the presence of various concentrations of phenyltrimethylammonium (PTA) and nereistoxin (NTX), by a simple filtration assay and flame emission spectroscopy. The equilibrium constants of these ligands in the minimal model, which accounts for the AChRmediated ion flux, were estimated simply from these two measurements, since the equilibrium constants for acetylcholine (ACh) and carbamylcholine (Carb) estimated from two kinetic measurements agreed well with those estimated from five sophisticated kinetic measurements of AChR-mediated ion fluxes. PTA showed high potency but not high efficacy, and showed inhibition when large doses were applied. NTX showed both low potency and low efficacy and acted as an inhibitor when it was added with Carb. The apparent dissociation constants of these three agonists evaluated from the minimal model and the equilibrium constants agreed with those obtained by assay of inhibition of radiolabeled ligand binding.View full abstractDownload PDF (711K) -
Reiko IIDA, Toshihiro YASUDA, Koichiro KISHI1987 Volume 101 Issue 2 Pages 357-363
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUgl-Y is a glycoprotein that is detected in normal urine samples from young men and women aged 0 to 17 years. It was purified by ammonium sulfate precipitation and various column chromatographies including affinity chromatography using anti-adult urine antibody coupled to Sepharose 4 B. The homogeneity of the glycoprotein was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, column chromatography on Sephadex G-75, and the precipitation reaction with anti-Ugl-Y antibody. It was shown to have a molecular weight of 29, 000 by gel filtration, and to contain 5.2% neutral sugars (mannose and galactose) and 4% hexosamine (glucosamine). Amino acid analysis of the glycoprotein indicated high contents of acidic and hydroxylic amino acids. Its origin is unknown.View full abstractDownload PDF (972K) -
Kimiko SAITO-NAKATSUKA, Tetsuro YAMASHITA, Ichiro KUBOTA, Masao KAWAKI ...1987 Volume 101 Issue 2 Pages 365-376
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+-Transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum contains several SH groups which are reactive with N-ethylmaleimide (MaLNEt) at pH 7.0. The location of the one which is most reactive with MaLNEt (SHN, Kawakita et al. J. Biochem. 87, 609 (1980)) was identified on the amino acid sequence of the ATPase. SHN was labeled by reacting sarcoplasmic reticulum membranes with [14C] MalNEt to a labeling density of 1 mol/mol ATPase. [14C] MalNEt-labeled membranes were digested with thermolysin and 14C-labeled SHN peptides were fractionated by Sephadex LH-20 chromatography to give two major peaks of radioactivity. [14C]-MaINEt-labeled peptides were further purified to homogeneity by C18-reversed phase HPLC. Two radioactive peptides containing modified cysteine (Cys*), LeuGly-Cys*-Thr-Ser and Val-Cys*-Lys-Met, were finally obtained in roughly equal amounts and in reasonable recovery. Both of these sequences were found in the amino acid sequence of Ca2+-transporting ATPase (Brandl et al. Cell 44, 597 (1986)), and Cys344 and Cys364 were identified as the targets of MalNEt-modification. Thus, 0.5 mol/mol ATPase of each Cys residue actually reacted rapidly with MalNEt under the conditions leading to SHN-modification. Modification of either one with MalNEt may negatively affect the reactivity of the other. Both of the highly reactive SH groups are located in the neighborhood of Asp351, the phosphorylation site of ATPase.View full abstractDownload PDF (2080K) -
Tetsuro YAMASHITA, Masao KAWAKITA1987 Volume 101 Issue 2 Pages 377-385
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) aminonaphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+, Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 μM [14C] IAA at pH 7.0 and 30°C. One mole of IAA per mole of ATPase was bound in 6h without affecting the Ca2+-transport activity. [14C] IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys*674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys* denotes the [14C] IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 μM IAEDANS for 5 h, at pH 7.0 and 30°C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.View full abstractDownload PDF (1396K) -
Takeshi MIZUNO, Shoji MIZUSHIMA1987 Volume 101 Issue 2 Pages 387-396
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression of the ompC and ompF genes coding for the major outer membrane proteins, OmpC and OmpF, respectively, is known to be controlled by at least two regulatory genes, ompR and envZ, which together comprise a single ompB operon. We constructed chromosomal mutants with either ompR-envZ deletion or envZ deletion. Characterization of these deletion strains showed that the OmpR protein is necessary for transcription of the ompC and ompF genes, and the EnvZ protein is essential for normal regulation of the ompC and ompF expression, which is affected by the medium osmolarity. We also constructed several plasmids carrying different portions of the ompB operon. Characterization of these plasmids allowed us to identify the OmpR protein with an apparent molecular weight of 29 kilodaltons (kDa) and the EnvZ protein with an apparent molecular weight of 50 kDa. The initiation codon for EnvZ translation appeared to overlap with the termination codon for OmpR translation. It was also found that a truncated EnvZ polypeptide (44 kDa) which lacks the N-terminal 55 amino acid residues can complement the envZ deletion mutant. Based on these results, the structure and function of the ompB operon are discussed in relation to the regulation of ompC and ompF expression.View full abstractDownload PDF (2439K) -
Masaharu TAKIGAWA, Koji TAJIMA, Keisuke FUKUO, Hirota FUJIKI, Fujio SU ...1987 Volume 101 Issue 2 Pages 397-404
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS12-O-Tetradecanoylphorbol-13-acetate (TPA), a skin tumor-promoting phorbol ester, and teleocidin and aplysiatoxin, which are potent tumor promoters in mouse skin but are chemically unrelated to phorbol esters, induced change of cultured rabbit costal chondrocytes from a polygonal to a fibroblastic shape and inhibited glycosaminoglycan (GAG) synthesis and metachromatic matrix formation in these cells. The potencies of teleocidin and aplysiatoxin to inhibit GAG synthesis were almost the same as that of TPA. On the other hand, Tween 60 and cantharidin, weak mouse skin tumor promoters, phenobarbital, a liver tumor promoter, and saccharin, a bladder tumor promoter, had no effect on the morphology or GAG synthesis of cultured chondrocytes. Like TPA, teleocidin and aplysiatoxin increased DNA and RNA syntheses of chondrocytes. Parathyroid hormone (PTH) and dibutyryl cyclic AMP reversed the morphological and histochemical changes caused by a 4-day treatment with teleocidin or aplysiatoxin as well as with TPA, reversal being apparent after 2 days. PTH increased intracellular cyclic AMP after 2 min in chondrocytes pretreated with teleocidin or aplysiatoxin as well as with TPA. PTH also increased ornithine decarboxylase [ODC; EC 4. 1. 1. 17] activity in these chondrocytes after 4 h. These results show that retention of responsiveness to PTH is a typicalView full abstractDownload PDF (2245K) -
Maya ANDO-YAMAMOTO, Hideyuki HAYASHI, Toshihiro SUGIYAMA, Hiroyuki FUK ...1987 Volume 101 Issue 2 Pages 405-414
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxy-lyase, EC 4. 1. 1. 28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 μmol/min•mg protein) had a molecular weight of 100, 000 and gave a single band with a molecular weight of 50, 000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 μl of antiserum, respectively, were required for 50% precipitation of 2 nmol/ min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 μg of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.View full abstractDownload PDF (2566K) -
Kazuhito ROKUTAN, Keiichi KAWAI, Kozi ASADA1987 Volume 101 Issue 2 Pages 415-422
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSKetoacid oxidation in rat liver mitochondria was very sensitive to t-butyl hydroperoxide (t-BuOOH). Furthermore, 2-oxoglutarate and pyruvate each enhanced t-BuOOH-induced oxidative stresses of mitochondria, such as oxidation of pyridine nucleotides and GSH, inhibition of respiration with the other NAD-linked substrates, and peroxidation of mitochondrial lipids. We provide evidence that the t-BuOOH and ketoacid-induced effects are due to the failure of supply of NADH by 2-oxoglutarate dehydrogenase, and report the inactivation of the dehydrogenase in mitochondria by simultaneous addition of 2-oxoglutarate and t-BuOOH. Using the purified enzyme, we confirmed that t-BuOOH-induced inactivation of 2-oxoglutarate dehydrogenase was enhanced by its substrate and thiamine pyrophosphate protected the dehydrogenase from the inactivation. In contrast, succinate-dependent oxidation of mitochondria was not only scarcely affected by t-BuOOH, but also succinate protected against inactivation of 2-oxoglutarate dehydrogenase by t-BuOOH in mitochondria.View full abstractDownload PDF (634K) -
Eriko MATSUI, Kiyoshi OGURA, Shizuo HANDA1987 Volume 101 Issue 2 Pages 423-432
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNeutral and acidic glycolipids from the bovine pineal organ and neutral glycolipids from the bovine retina were characterized. The chemical structures of the isolated glycolipids were determined by means of carbohydrate analysis, methylation analysis, enzyme treatment, fatty acid analysis, long chain base analysis, mass spectrometry, NMR spectroscopy, and IR spectroscopy. GM3, GD3, and GT1 were the major bovine pineal organ gangliosides, GD3 accounting for 75% of the total gangliosides. Galactosylceramide, glucosylceramide, and lactosylceramide were found in both the bovine pineal organ and retina. Sulfatide was also present in both tissues. It had already been reported that the major bovine retina ganglioside was GD3 (Handa, S. & Burton, R. M. (1969) Lipids 4, 205-208). The glycolipid patterns of the two tissues were very similar to each other and quite different from those of other tissues.View full abstractDownload PDF (1882K) -
Ken-ichiro HIGASHI, Shigeo SUZUKI, Hiroshi FUJII, Yutaka KIRINO1987 Volume 101 Issue 2 Pages 433-440
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOptimal conditions for formation of giant liposomes and proteoliposomes were investigated. A suspension of small unilamellar vesicles made of various phospholipids in a buffer of 0-3M KCl, 0.1mM EDTA, and 20mM MOPS (pH 7.0) was subjected to a freeze-thaw treatment. Giant multilamellar liposomes of diameter ranging from 10 to 60 μm were found to form from phospholipid mixtures containing phosphatidylethanolamine as a major component and phosphatidylserine as a minor component. The concentration of KCl optimal for the giant vesicle formation was 30-500mM. By applying a patch-pipette to a giant liposome, suitable conditions for obtaining a high-resistance (giga-ohm) seal were sought. It was found that use of a patch-pipette of relatively small tip diameter (less than 1 μm), the presence of divalent metal cations in the suspension medium and inflation of vesicles in a hypotonic solution facilitated giga-seal formation. In a suspension of asolectin (soybean phospholipid) vesicles which had been subjected to the freeze-thaw treatment, giant unilamellar vesicles were found. They could be held on the tip of a suction pipette and impaled with a microelectrode filled with an EGTA solution. Small unilamellar proteoliposomes were prepared by the cholate-dialysis method from asolectin and sarcoplasmic reticulum vesicles, and were subjected to a freeze-thaw cycle. When the ratio of exogenous phospholipid to protein was larger than 10, giant multilamellarView full abstractDownload PDF (3608K) -
Yoshihiro FUKUMORI, Tateo YAMANAKA1987 Volume 101 Issue 2 Pages 441-445
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe electron transport system coupled to the oxidation of methylamine in Pseudomonas AM1 was investigated by reconstituting it from the highly purified components. A mixture of methylamine dehydrogenase, cytochrome cH and cytochrome c oxidase (=cytochrome aa3) actively oxidized methylamine (161 mol of O2 consumed/mol of heme a of cytochrome c oxidase•min). In this system, addition of amicyanin did not affect the oxygen consumption rate. The oxygen consumption rate of the cell-free extract prepared from the cells cultivated in a copper-deficient medium was directly proportional to the amount of amicyanin added, and extrapolation to zero copper concentration gave a value of 28 mol of O2 consumed/mol of heme a of cytochrome c oxidase•min. These results suggest that methylamine oxidation in the bacterium can occur at least to some extent without participation of amicyanin.View full abstractDownload PDF (388K) -
Shinobu IMAJOH, Hiroshi KAWASAKI, Koichi SUZUKI1987 Volume 101 Issue 2 Pages 447-452
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHigh-Ca2+-requiring calcium-activated neutral protease (mCANP), a dimeric enzyme composed of large (Mr=80, 000) and small (Mr=28, 000) subunits, is resistant to carboxypeptidase Y (CPase Y) in the absence of NaSCN. In the presence of 0.2M NaSCN, CPase Y digested mCANP, one or two amino acids being released from the COOH-termini of the large and small subunits, but no change occurred in the activity of the digested mCANP. In the presence of 1M NaSCN, 8-10 amino acids were released from the subunits by CPase Y, and the COOH-terminal potential Ca2+-binding sites of both subunits were destroyed. On digestion under these conditions, mCANP lost the ability to form a complex, and the proteolytic activity was not recovered even when the digested subunits were mixed with native subunits. These results suggest that the COOH-terminal regions of the two subunits of mCANP, which constitute the helical portions of the COOH-terminal E-F hand structures in both subunits, are essential for the subunit association and resulting proteolytic activity.View full abstractDownload PDF (399K) -
Fumio SAITOH, Haruki YAMAGUCHI1987 Volume 101 Issue 2 Pages 453-463
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteokeratan sulfate was extracted and purified from bovine corneal stroma and then characterized by chemical and biochemical analyses. It was fractionated into several fractions by affinity chromatography on a concanavalin A-Sepharose column or by hydrophobic chromatography on a phenyl-Sepharose column. These fractions differed widely from one another in carbohydrate content, though no significant differences of their amino acid compositions were observed. One fraction (ca. 25%, on a dry weight basis) tightly bound to a concanavalin A-Sepharose column, compared with another fraction (ca. 65%) weakly bound to the same column, was poor in galactose and N-acetylglucosamine, but contained mannose in a high proportion. Fractions (ca. 30%) tightly bound to a phenyl-Sepharose column, in contrast to the one (ca. 66%) weakly bound, had low carbohydrate contents, like the fraction tightly bound to a concanavalin A-Sepharose column. Additionally, the fractions tightly bound to these affinity columns exhibited strong inhibitory actions on erythrocyte-concanavalin A agglutination. To obtain further details of the carbohydrate moiety of the proteokeratan sulfate, an attempt was made to separate and characterize peptidokeratan sulfate and Asn-linked oligosaccharide derived from some proteokeratan sulfate fractions. The present work revealed that a) the proteokeratan sulfate contains keratan sulfate and high mannose-type oligosaccharide in an approximate chain number ratio of 3.5:1.0, b) the keratan sulfate content varies widely and the oligosaccharide content increases with decrease of the keratan sulfate content, and c) the protein core is homogeneous at least with respect to the amino acid composition.View full abstractDownload PDF (860K) -
Manfred FREIBURG1987 Volume 101 Issue 2 Pages 465-475
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the ciliate Tetrahymena pyriformis phosphorylation of RNA polymerase I [EC 2. 7. 7. 6] and of polymerase-associated polypeptides was investigated in growing and growth-arrested cultures which differ widely in their rates of rRNA synthesis. Several putative subunits of RNA polymerase I (of 180, 21.5, and 19.5 kDa) and a polymerase-associated polypeptide of 27 kDa were found to be phosphorylated, independent of the growth conditions. However, an additional enzyme-associated polypeptide of 26 kDa was intensively labeled with 32P only after arrestment of growth by starvation. The molar quantities of both phosphorylated, enzymeassociated polypeptides thereby did not differ in growing and growth-arrested cultures, and the specific 32P-labeling of cellular ATP remained nearly unchanged under the different culture conditions. These findings indicate a selective, reversible phosphorylation of the RNA polymerase I-associated 26 kDa polypeptide correlated with conditions of repressed rRNA synthesis induced by the starvation procedure. In vitro phosphorylation in macronuclei isolated from growing and growth-arrested cultures using [γ-32P] ATP revealed essentially the same pattern of labeling of the enzyme-associated polypeptides of 27 and 26 kDa as it was found in vivo.View full abstractDownload PDF (3551K) -
Makoto KAKITANI, Ben'ichiro TONOMURA, Keitaro HIROMI1987 Volume 101 Issue 2 Pages 477-484
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteractions of several amino acids and nucleotides with valyl-tRNA synthetase [EC 6. 1. 1. 91] (VRS) from Bacillus stearothermophilus were investigated using as a probe the ligand-induced quenching of protein fluorescence (λex=295 nm, λem=340 nm) of VRS. L-Valine, L-threonine, L-isoleucine, L-glutamic acid, L-leucine, and D-valine caused fluorescence quenching. Among them, L-threonine had a Kd value comparable to that for the cognate substrate, L-valine, but the other amino acids were bound more weakly as estimated by the fluorescence titration method. L-Alanine, L-histidine, and L-serine did not cause any fluorescence change. Among the nucleotides tested (ATP, ADP, AMP, GTP, ITP, CTP, and UTP), only ATP caused the fluorescence change. In the presence of an excess amount of ATP, only L-valine and L-threonine, among the tested amino acids, induced the fluorescence quenching, and the binding of L-valine was greatly favored under this condition. This is consistent with the results of the ATP-PP1 exchange reaction by VRS, in which only L-valine and L-threonine, of these 9 amino acids tested, could serve as substrates, and the Km value for L-valine was much smaller than that for L-threonine. Thus the binding of ATP to VRS enhances the substrate specificity of VRS towards amino acids. The binding stoichiometry deduced from equilibrium dialysis experiments for L-valine and ATP was found to be one when these substrates exist together, in contrast to our previous observation that there were two equivalent binding sites for each of L-valine and ATP when these substrates were added separately to the enzyme (Kakitani, M., Tonomura, B., & Hiromi, K. (1986) Agric. Biol. Chem. 50, 2437-2444).View full abstractDownload PDF (641K) -
Mitsunobu DOI, Masayuki TANAKA, Toshimasa ISHIDA, Masatoshi INOUE, Tak ...1987 Volume 101 Issue 2 Pages 485-490
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of [(4-bromo) Phe4, Met5] enkephalin (Tyr-Gly-Gly-(4-bromo)-Phe-Met) shows two independent molecular conformations. The molecules are arranged in parallel in a head-to-tail fashion and form an antiparallel β-sheet structure involving intermolecular hydrogen bonds. This dimeric β-structure is also observed in the [Met5] enkephalin crystal, in spite of their different crystal packing environments, which shows the energetic stability of this molecular conformation. The three-dimensional similarity between the dimeric β-structure and the β-turn form is discussed in the relation to the opioid δ and μ receptors.View full abstractDownload PDF (386K) -
Yasuyuki SUZUKI, Tadao ORII, Masaki TAKIGUCHI, Masataka MORI, Makoto H ...1987 Volume 101 Issue 2 Pages 491-496
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe biosynthesis of three major peroxisomal membrane polypeptides of rat liver was investigated. Total hepatic RNA extracted by the guanidinium/CsCl method from three control and three di (2-ethylhexyl) phthalate (a peroxisomal proliferator) fed rats was translated in vitro in a rabbit reticulocyte lysate protein-synthesizing system. Translation products were immunoprecipitated by the antibodies against peroxisomal membrane polypeptides, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and analyzed by fluorography. The in vitro trans-lation products of 70, 26, and 22 kDa peroxisomal membrane polypeptides were apparently of the same size as the respective mature polypeptides. The ratio of translatable mRNA levels for the 70, 26, and 22 kDa polypeptides in di (2-ethyl-hexyl) pbthalate-fed rats to those in control rats were 5.4, 11.4, and 2.7, respectively. The synthesis of these three polypeptides with the free polysome fraction from di (2-ethylhexyl) phthalate-fed rats was more active than that with the membranebound polysome fraction, whereas the synthesis of albumin with the free polysome fraction was 27% of that with the membrane-bound polysome fraction. These results indicate that the peroxisomal major membrane polypeptides are synthesized on free polysomes and transported to peroxisomal membrane without any apparent proteolytic processing, and that the induction of these polypeptides by administration of a peroxisomal proliferator corresponds well to the induction of the peroxisomal β-oxidation enzymes. The data also support the idea that peroxisomes are organized from pre-existing peroxisomes.View full abstractDownload PDF (1706K) -
Hitoshi SOHMA, Fumi MORITA1987 Volume 101 Issue 2 Pages 497-502
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe protein kinase that phosphorylates the regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was isolated from scallop smooth muscle (Sohma, H. & Morita, F. (1986) J. Biochem. 100, 1155-1163). The enzymatic properties of this kinase (aMK) were investigated using RLC-a as the substrate. The Km value for ATP was 6.5 μM in the presence of 27 μM RLC-a at pH 7.0, and that for RLC-a was 133 μM in the presence of 1mM ATP. The Vm value at saturation of both RLC-a and ATP was 0.25 s-1 at pH 7.0. The pH activity curve for aMK was bell-shaped with a maximum at around pH 7.8. The aMK activity was inhibited strongly by an increase in the KCl concentration. aMK required Mg2+, but was inhibited by high concentrations of Mg2+. The optimum activity was seen at 3 mM MgCl2. The mode of inhibition of the aMK activity by Ca2+ was studied. Assuming that the binding of Ca2+ to aMK induces the inhibition, the dissociation constant of Ca2+ was estimated to be 64 μM. aMK also phosphorylated LC20 of chicken gizzard myosin at a similar rate to that for RLC-a and the DTNB light chain of rabbit skeletal muscle myosin at a more lower rate. The helix and β-sheet contents of aMK were estimated to be 19 and 30%, respectively, from the CD spectrum.View full abstractDownload PDF (417K) -
Sachiko SEKI, Yuka HATTORI, Tetsuya HASEGAWA, Hiroki HARAGUCHI, Makoto ...1987 Volume 101 Issue 2 Pages 503-509
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNitrate reductase of Clostridium perfringens was purified by an improved method using immuno-affinity chromatography. The purified preparation contained Mo, Fe, and acid-labile sulfide; the Mo content was 1mol per mol and the Fe 3.7mol per mol of the enzyme. The inactive enzyme obtained from cells grown in the presence of tungstate did not hold Mo but contained 1mol of W. The content of Fe was not increased. The presence of molybdenum cofactor in the nitrate reductase was indicated by the formation of molybdopterin form A in the oxidation of the enzyme by iodine and by the complementation of NADPH-nitrate reductase with the heat-treated enzyme in the extract of Neurospora crassa nit-1. The Clostridium nitrate reductase had an absorption maximum at 279 nm and shoulders at 320, 380, 430, and 520 nm. This enzyme seems to contain an iron sulfur cluster since the reduced enzyme showed decreased absorption in visible region. The CD spectrum of the enzyme has a positive peak at 425 nm and negative ones at 310, 360, and 595 nm. It was compared with the CD spectrum of ferredoxin (2Fe-2S or 4Fe-4S cluster) and the nitrate reductase of Plectoneina boryanum.View full abstractDownload PDF (565K) -
Izumi KUMAGAI, Eisuke TAMAKI, Shinji KAKINUMA, Kin-ichiro MIURA1987 Volume 101 Issue 2 Pages 511-517
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn poly (A)+ RNA extracted from a lactating goat mammary gland, mRNA of about
750 nucleotides was shown to encode pre α-lactalbumin by using in vitro translation and immunoprecipitation. From the total poly (A)+ RNA, the cDNA library was constructed using the Escherichia coli plasmid pUC18; it was screened with the oligodeoxyribonucleotide probe corresponding to the amino acid sequence of Trp60-Gln65 of goat α-lactalbumin. A plasmid containing almost full-length cDNA of goat pre α-lactalbumin, pGLA-1, was identified. The cDNA insert of pGLA-1 comprises 727 base pairs and contains the signal peptide and mature protein sequence.View full abstractDownload PDF (1675K) -
Keiichi YAMAMOTO, Takamitsu SEKINE1987 Volume 101 Issue 2 Pages 519-523
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe subfragment-1-avidin complex, in which avidin is attached to a well defined thiol group called SH1, was purified by CM cellulose column chromatography or affinity chromatography using lipoic acid agarose. The interaction of the purified complex with F-actin was compared to that of normal subfragment-1 using chemical crosslinking and limited tryptic digestion techniques. It was found that the binding of avidin to SH1 lowered the extent of cross-linking between the subfragment-1 heavy chain and actin. The amount of the 175K product decreased to about 50% of the normal level and that of the 165K product decreased to about 35%. It was also found that the binding of avidin abolished the protective effect of F-actin on the 50K-22K junction of the S-1 heavy chain against tryptic attack. Since more than 95% of the S-1-avidin complex was attached to F-actin under our experimental conditions, these changes are due to an alteration of the S-1-actin interface. Considering the facts that SH1 is located on the side of S-1 facing the F-actin, in the tertiary structure, and is close to the cross-linked site and to the 50K-22K junction, in the primary structure, it is quite likely that avidin bound to SHE causes these effects by sterically preventing the close contact of S-1 and actin.View full abstractDownload PDF (2133K) -
Takaaki SATO, Hiroshi MATSUI, Susumu SHIBAHARA, Tsuyoshi KOBAYASHI, Ya ...1987 Volume 101 Issue 2 Pages 525-534
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe constructed several expression plasmids of human IL-2 gene, some of which directed high-level synthesis of mature IL-2 protein in E. coli. In all the plasmids reported here, we installed the E. coli trp promoter and SD sequence upstream of the IL-2 cDNA. When DNA sequences containing the ρ-independent transcription terminator such as those involved in the trpA and lpp gene were inserted downstream of the IL-2 cDNA sequence, the expression level of the IL-2 gene increased up to 5-fold. Moreover, the deletion of either the whole region including A-T and G-C tails or a part of the 3' non-coding sequence resulted in further increase of the expression of the IL-2 gene up to 500-fold. The mature IL-2 produced in E. coli exhibited biological and immunological activities indistinguishable from those of purified IL-2 from a human T cell line, Jurkat-111. The manipulations described here may be useful for the high-level expression of eukaryotic genes in E. coli.View full abstractDownload PDF (1226K) -
Satoshi NAKANISHI, Tokuzo NISHTNO, Jun NAGAI, Hirohiko KATSUKI1987 Volume 101 Issue 2 Pages 535-544
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnalysis of sterols of Saccharomyces cerevisiae mutants N3, N15, N26, and N3H, defective in sterol biosynthesis, was performed. Strains N3, N15, and N26 were isolated from their mother strain, M10, by screening with nystatin (Nagai et al. (1980) Mie Med. J. 30, 215-224), and strain N3H was isolated from N3 as a doubly-mutated strain. The main sterols of N3, N15, N26, and N3H were ergosta-7, 22-dienol, ergost-8-enol, cholesta-5, 7, 24 -trienol, and ergosta-7, 22, 24 (28) -trienol, respectively. The former three strains were characterized as defective in Δ5-desaturation, Δ8-Δ7 isomerization, and C-24 transmethylation. Strain N3H was found to be defective in Δ5-desaturation as well as in Δ24 (28)-reduction. However, the defect of N26 and N3H was suggested to be leaky, since small amounts of ergosterol and ergosta7, 22-dienol were found in these mutants, respectively. In N15, an accumulation (2% in total sterols) of the compound likely to be hydroxylated sterol was found. By aerobic adaptation of these strains, the accumulation of these strains, the accumulations of ergosta-7, 22-dienol (22mg/g dry cells), ergosta-7, 22, 24 (28) -trienol (24mg), ergosta-8, 24 (28) -dienol (18mg), and cholesta-8, 24-dienol (22mg) reached a maximum in N3, N3H, N15, and N26 after 20, 20, 30, and 30 h, respectively. These strains appear to be useful for making 14C-labeled and non-labeled preparations of the above sterols.View full abstractDownload PDF (665K)
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