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Jun-Ichiro MURAYAMA, Motowo TOMITA, Akira HAMADA
1982 Volume 91 Issue 6 Pages
1829-1836
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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Crude glycophorin fraction was prepared from bovine erythrocyte membranes by extraction with lithium diiodosalicylate and partition in aqueous phenol. The crude fraction was further separated into three fractions by gel chromatography and ionexchange chromatography. The major fraction, designated glycophorin BA, accounts for 80% of the crude fraction. Its carbohydrate content was 79% by weight including galactose,
N-acetylgalactosamine,
N-acetylglucosamine and
N-glycolylneuraminic acid. Iodosobenzoate treatment of glycophorin BA produced two fragments; one is a highly glycosylated segment and the other a hydrophobic peptide. The amino-terminal sequence of the hydrophobic peptide representing the carboxylterminal half of glycophorin BA was determined.
The other two fractions, IIIa and IIlb, were still heterogeneous when analyzed by gel electrophoresis. Fraction IIIa contained two glycophorins having apparent molecular weights of 46, 000 and 42, 000, while fraction Illb contained a glycoprotein having a marked tendency to aggregate.
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Masayuki NOZAWA, Kazutaka TANIZAWA, Yuichi KANAOKA
1982 Volume 91 Issue 6 Pages
1837-1843
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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The kinetics of hydrolysis of “inverse substrates, ”
p-amidinophenyl alkanoates, catalyzed by urokinase, plasmin, kallikrein, and trypsins from various sources were studied. Dissociation constants of acyl enzyme-ligand complexes, which are a characteristic parameter of the reaction with “inverse substrates, ” were analyzed with a view to comparing the spatial requirements of active sites. It was concluded that the spatial restraint of the active site as regards coexistence of the acyl residue and specific ligand is strictest for hog pancreatic kallikrein and this restraint decreases in the following order: human urokinase; bovine plasmin, bovine and hog trypsins;
Streptomyces fradiae trypsin; and
Streptomyces griseus trypsin.
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Takayuki MIYANISHI, Tetsuo MAITA, Genji MATSUDA, Yuji TONOMURA
1982 Volume 91 Issue 6 Pages
1845-1853
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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Our laboratory has presented strong evidence for the nonidentical two-headed structure of skeletal muscle myosin. We previously showed that each of the two kinds of heads,
i.e., the burst head, which forms the myosin-P-ADP complex, and the nonburst head, which forms the myosin-ATP complex upon reaction with ATP, contains 1 mol of reactive lysine residue per mol which is modified rapidly with TNBS. We also found that in the presence of PP
1 only the reactive lysine residue in the burst head is modified with TNBS. Utilizing this phenomenon, we presented evidence [(1981)
J. Biochem.
89, 831-839] indicating that the chemical structures around the reactive lysine residues in the burst and the nonburst head are different.
In this study, we determined the amino acid sequence around the reactive lysine residues to demonstrate the nonidentical chemical structure of the two heads of skeletal muscle myosin. We found that the sequence around the reactive lysine residue in the burst head was ……Pro- Met-Asn-Pro-Pro-Lys-Tyr…… and the sequence in the nonburst head was ……Ser-Met-Asn-Pro-Pro-Lys-Tyr…… Thus, a proline residue located near the reactive lysine residue in the burst head was found to be replaced by a serine residue in the nonburst head.
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Mitsuo IKEBE, Hirofumi ONISHI, Yuji TONOMURA
1982 Volume 91 Issue 6 Pages
1855-1873
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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To determine whether the nonidentical two-headed structure of myosin is common to various kinds of muscle, we compared the stoichiometry in the reactions of ATP with chicken gizzard myosin and rabbit skeletal muscle myosin. We also compared the properties of acto-gizzard myosin ATPase with those of skeletal muscle actomyosin ATPase, to clarify the roles of the burst head and the nonburst head in muscle contraction. The findings obtained are as follows.
1. The maximum amount of total nucleotides (ATP+ADP) bound to HMM during the ATPase reaction was 2 mol/mol for both smooth and skeletal muscles. However, the amount of ADP bound to HMM increased linearly with increase in the ATP concentration, reaching the maximum level of 1 and 1.35-1.60 mol/mol HMM at 1 and 2 mol of ATP per mol of HMM for skeletal and smooth muscles, respectively. Also in the case of skeletal muscle, the ATP binding to HMM was observed only after saturation of the ADP binding with the maximum value of 1 mol/mol HMM, while in the case of smooth muscle, the amount of ATP bound to HMM was proportional to that of ADP at all the ATP concentrations tested.
2. The maximum amounts of P bound to HMM were the same as those of ADP, being 1 and 1.60 mol/mol HMM, respectively, for skeletal and smooth muscles.
3. The amounts of ATP required to induce maximum enhancement of the fluorescence intensity of HMM in the steady state in the presence of sufficient amounts of PK and PEP were 1 and 2 mol/mol HMM for skeletal and smooth muscles, respectively.
4. The ATPase activity of smooth muscle myosin was inhibited by trinitrophenylation, and the inhibition was prevented by the addition of PP
i. The amount of lysine residues involved in the ATPase inhibition was 2-3 mol/mol myosin.
5. The extent of dissociation of skeletal muscle actomyosin increased linearly, while that of acto-gizzard myosin (hybrid actomyosin reconstituted from gizzard myosin and skeletal muscle F-actin) increased sigmoidally with increase in the ATP concentration. The amounts of ATP required for the complete dissociation were 1 and 2 mol/mol myosin for skeletal muscle actomyosin and acto-smooth muscle myosin, respectively.
6. The amounts of ATP required for the maximum Mg
2+-ATPase activity were 1 and 2 mol/mol HMM for skeletal muscle acto-HMM and acto-smooth muscle thiophosphorylated HMM, respectively.
7. The Mg
2+-ATPase reaction of skeletal muscle acto-HMM in the presence of TM and TN exhibits a marked substrate inhibition upon removal of Ca
2+, and the extent of the inhibition is proportional to the amount of ATP bound to the nonburst head [Inoue, A. & Tonomura, Y. (1975)
J. Biochem.
78, 83-92]. In contrast to the above report, the Mg
2+-ATPase activity of acto-smooth muscle thiophosphorylated HMM measured in the presence of skeletal muscle TM and TN increased linearly both in the presence and absence of Ca
2+ with increase in the ATP concentration, reaching the maximum level when 2 mol ATP/mol HMM was added. The activity decreased markedly upon removal of Ca
2+, but exhibited no substrate inhibition.
The findings 1-4 clearly show that smooth muscle and skeletal muscle myosins have an identical and a nonidentical two-headed structure, respectively. The findings 5-7 also indicate that the burst head and the nonburst head are involved in the energy transduction and in the control by high concentrations of ATP in muscle contraction, respectively.
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Masao IWAMORI, Kenzo SAWADA, Yoshiko HARA, Minori NISHIO, Takashi FUJI ...
1982 Volume 91 Issue 6 Pages
1875-1887
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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By the application of a ganglioside-mapping technique, the lipid composition of bovine thyroid was analyzed systematically. The contents of cholesterol, lipidbound phosphorus and lipid-bound sialic acid in bovine thyroid were 5.10, 9.76, and 0.28 μmol/g of dry tissue, respectively, and the molar ratio of cholesterol, lipidbound sialic acid, and lipid-bound phosphorus was 52.4:2.9:100.0. The following phospholipids were contained in this order: phosphatidylcholine>phosphatidylethanolamine>sphingomyelin>phosphatidylserine and phosphatidylinositol>cardiolipin. When compared on a molar basis, the amount of total glycosphingolipids was only 3% of phospholipids. As the major neutral glycosphingolipids, ceramide glucoside, ceramide galactoside, ceramide lactoside, ceramide trihexoside and globoside were identified and the most abundant component was globoside (40% of total neutral glycosphingolipids). On the other hand, five molecular species of gangliosides were identified: GM 3, GM 1, fucosyl GM 1, GD 3, and GDla. Three types of GD 3 and GDla with a different sialic acid composition were recognized on the ganglioside-map and isolated in pure forms. GM 3 was the most abundant component, but the concentration of gangliosides with ganglio-
N-tetraose in bovine thyroid was higher than that of gangliosides with lactose. Also fucosyl GM 1 comprised 13% of total gangliosides. Thus, the high concentrations of gangliosides with ganglio-
N-tetraose and fucosyl GM 1 seemed to be characteristic of bovine thyroid glycosphingolipids. The glycosphingolipids contained in the following order: GM 3>GDla>globoside>GM 1>ceramide trihexoside>fucosyl GM 1>ceramide lactoside>ceramide glucoside>GD 3>ceramide galactoside.
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Yukiyasu TOYODA, Ichitomo MIWA, Jun OKUDA
1982 Volume 91 Issue 6 Pages
1889-1898
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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The enzyme mutarotase [aldose 1-epimerase, EC 5. 1. 3. 3] from hog kidney cortex was separated into four fractions (designated types I, II, III, and IV in order of elution) by column chromatography on DEAF-cellulose. Two major forms, types I and II, were purified to homogeneity as judged by polyacrylamide gel electrophoresis and isoelectric focusing on thin layer polyacrylamide gel.
Types I, II, III, and IV had isoelectric points of 5.78, 5.48, 5.23, and 5.10, respectively. The following physicochemical properties were common to all four types: molecular weight, 41, 000;
Km for α-D-glucose at pH 7.4 and 25°C, 19mM; optimum pH, 6.5-7.5; optimum temperature, 30-37°C; heat stability, up to 50°C.
On double immunodiffusion, the four types of mutarotase gave single precipitin lines, which fused completely with each other, against the antibody to purified type II enzyme.
Types I and II had an identical amino-terminal residue, arginine, and an identical carboxyl-terminal sequence, -(Phe-Phe-Ser-Val)-Val-Ala. The amino acid composition of type I was almost identical with that of type II. Very similar tryptic peptide maps were obtained from types I and II, with only a few points of variance.
These results suggest that the four types of hog kidney mutarotase are quite similar but not identical.
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Tadashi YOSHIMOTO, Daisuke TSURU
1982 Volume 91 Issue 6 Pages
1899-1906
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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A proline-specific dipeptidyl aminopeptidase was highly purified from cell-free extract of
Flavobacterium meningosepticum by a series of column chromatographies on DEAE-Sephadex A-50, Sephadex G-150, hydroxyapatite, and a second gel filtration on Sephadex G-150. The enzyme was most active at pH 7.4-7.8 for both Gly-Pro-β-naphthylamide (Gly-Pro-2-NNap) and Gly-Pro-
p-nitroanilide (Gly-Pro-pNA) and was stable between pH 7 and 9.5. The enzyme was markedly inhibited by diisopropylphosphofiuoridate (DFP) and mercury ion but not by sulfhydrylblocking reagents and metal chelators. The molecular weight of the enzyme was about 160, 000 as judged by the gel filtration method and the subunit molecular weight was estimated to be 75, 000 by sodium dodecyl sulfate (SDS)-gel electrophoresis, suggesting a dimeric form of the native enzyme. The isoelectric point was at pH 9.5. The enzyme hydrolyzed peptides and peptide amides at the carboxyl side of a proline residue penultimate to the amino-terminal amino acid, as did postproline dipeptidyl aminopeptidases from various mammals. However, antiserum raised against post-proline dipeptidyl aminopeptidase from porcine kidney did not cross-react with the
Flavobacterium dipeptidyl aminopeptidase.
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Michiyuki KASAI, Shizuo WATANABE
1982 Volume 91 Issue 6 Pages
1907-1915
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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In our previous report (
J. Biochem.
89, 87-101, 1981), it was shown that the contractile (ATPase and superprecipitation) activities of chicken-gizzard acto-phosphorylated myosin (acto-PM) both with and without tropomyosin (TM) were insensitive to Ca
2+ but were made sensitive to Ca
2+ by addition of rabbit skeletal troponin (and TM). In the present study, we were able to observe that the other three cases of Ca
2+-insensitive activities of acto-unphosphorylated myosin with TM (acto-UMTM) were also made sensitive to Ca
2+ by addition of troponin: (a) the activities in ATP concentrations as low as 1 μm, (b) those in MgCl
2 concentrations as high as 30mM and (c) those in ITP media.
We also studied the turbidity of gizzard myosin suspensions as a function of the ITP concentration in the presence of 10mM MgCl
2 We thus observed that the turbidity of UM suspensions and that of PM suspensions decreased as the ITP concentration increased to higher than approximately 0.3mM and to higher than approximately 1mM, respectively. Moreover, we observed that the ITPase activity of UM, that of acto-UM and its superprecipitation activity decreased when the ITP concentration increased to higher than approximately 0.3mM, and that the ITPase activity of PM, that of acto-PM and its superprecipitation activity decreased when the ITP concentration increased to higher than approximately 1mM. It appeared, therefore, that the decreases in the activities all resulted from dissociation of myosin filaments, which was indicated by the decrease in the turbidity of myosin suspensions.
On the other hand, as the MgCl
2 concentration increased, the ITP concentration required to decrease the turbidity of myosin suspensions increased, but the ITP concentration required for causing a decrease in the superprecipitation activity remained practically unaltered. It appeared therefore that dissociation of actomyosin into actin and myosin was responsible for the decrease in the contractile activities that was observed when the ITP concentration was higher than approximately 0.3mM or 1mM.
All the results described above were interpreted as indicating that actin-linked regulation of muscle contraction works only when myosin is in the form of filaments.
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Tsukasa SUGANO, Masakazu SHIOTA, Hiroaki KHONO, Masakazu SHIMADA
1982 Volume 91 Issue 6 Pages
1917-1929
Published: June 01, 1982
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The role of the cellular redox state in the control of gluconeogenesis was studied in hemoglobin-free perfused chicken liver, by fluorimetric measurement of the redox states of intracellular pyridine nucleotides.
The aminotransferase inhibitor, aminooxyacetate, completely inhibited gluconeogenesis from lactate in the perfused rat liver and to a small extent in the perfused chicken liver. In chicken liver, the highest rate of glucose production was seen with lactate, followed by fructose, pyruvate, and glycerol. When compared at 5mM, the rate of glucose production from pyruvate was only 10% of that from lactate. Glucose production from a pyruvate/lactate mixture decreased with increasing proportions of pyruvate, together with redox changes of pyridine nucleotides to a more oxidized state. Increased reduction of pyridine nucleotides upon infusion of ethanol was associated with an increased glucose production from pyruvate, and the increase was abolished during octanoate infusion. This abolishment was accompanied by an increase in the acetoacetate to β-hydroxybutyrate ratio with an oxidation of pyridine nucleotides. The octanoate-inhibited gluconeogenesis occurred at the higher lactate concentration (10mM) with a transient oxidation of pyridine nucleotides. No significant inhibition was observed at 1mM lactate, although an instant reduction of pyridine nucleotides was taking place. The rate of β-hydroxybutyrate generation during octanoate infusion was 2.2 times higher at 1mM than at 10mM lactate. The inhibitory effect of octanoate on gluconeogenesis was completely relieved by the addition of NH
4Cl.
The results demonstrate that the regeneration of NADH in the cytosol is limited in chicken liver, and that gluconeogenesis is regulated, in part, by alteration in the redox states of mitochondria and cytosol.
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Nobuhiko WAGAI, Toichiro HOSOYA
1982 Volume 91 Issue 6 Pages
1931-1942
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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An attempt was made to solubilize a peroxidase from the uterine tissue of estrogenprimed rats using various detergents, and the best result was obtained by incubation with 4% cetyltrimethylammonium bromide at 37°C for 60min. The solubilized material was then dialyzed and subjected to gel filtration on Sephacryl S-200 followed by CM-cellulose chromatography, resulting in a 50-250-fold increase in specific activity over the detergent extract. Some properties of the partially purified uterine tissue peroxidase were studied in comparison with those of other animal peroxidases. The absorption spectra, molecular weight, and isoelectric point are very similar to those of lactoperoxidase. However, the oxidation rates of various hydrogen donor substrates by the uterine peroxidase were not parallel to those of lactoperoxidase and other animal peroxidases and the affinities for cyanide and azide of these enzymes were somewhat different from each other. The uterine peroxidase was inhibited by histidine and excess hydrogen peroxide competitively with respect to guaiacol, suggesting an important role of an amino acid residue in the protein moiety.
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Koji FURUNO, Toyoko ISHIKAWA, Keitaro KATO
1982 Volume 91 Issue 6 Pages
1943-1950
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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Autolysosomes were isolated from rat livers treated with leupeptin by a combination of differential and Percoll density gradient centrifugation techniques. The purified autolysosome fraction was verified by morphological analysis to be highly purified and to contain contaminants which were scarcely detectable. The enrichment of the lysosomal enzyme activities in the purified autolysosomes over the homogenate was 12-, 14-, 22-, and 24-fold for β-glucuronidase, acid phosphatase, β-
N-acetylglucosaminidase, and cathepsin D, respectively. Measurement of the activity of the marker enzymes for various subcellular organelles also proved that the purified autolysosome fraction was essentially free from contamination by other organelles. When the autolysosomes isolated from rat livers treated with leupeptin for 1 h were disrupted by osmolysis, acid hydrolases were easily solubilized. Acid phosphatase, however, became membrane bound in the autolysosomes prepared at longer periods of time after the leupeptin treatment. The autolysosomes exhibited enhanced permeability of the membranes after a short duration of time after the leupeptin treatment (30 and 60min) and became stabilized later. These changes in the properties of the autolysosomes with time after the leupeptin treatment may be interpreted as meaning that progressive rearrangement of the lysosomal constituents occurred within the autolysosomes with time after the genesis.
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Toru SHIMIZU, Tsunenori NOZAWA, Masahiro HATANO
1982 Volume 91 Issue 6 Pages
1951-1958
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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The first application of deuterium nuclear magnetic resonance spectroscopy (
2H NMR) to fully deuterated pyridine (d
5-pyridine)-iron (III) porphyrin complexes is described.
(1) d
5-Pyridine gives very broad
2H NMR signals in the presence of hemin or horseradish peroxidase to which pyridine is hardly (or not) bound, probably due to relatively long electronic relaxation times of the high-spin ferric ions. d
5-Pyridine in the presence of horse-heart metmyoglobin gives resolved and less broadened
2H NMR signals, probably due to the relatively short electronic relaxation times of the ferric ion and/or to slow chemical exchange of the ligand.
(2) The three resonances of free d
5-pyridine coalesce into a single or a double resonance in the presence of a heme octapeptide prepared by trypsin digestion of
Candida krusei cytochrome
c. Nuclear spin-lattice and spin-spin relaxation times of the d
5-pyridine-heme octapeptide complex are markedly shorter than those of free d
5-pyridine. These findings are interpreted by the chemical exchange mechanism from the temperature dependences of the relaxation times. Thus, on certain assumptions the residence time of pyridine in the bound state and the exchange rate are estimated as _??_10
-3 s and _??_400 s
-1, respectively, at 25°C. Since
1H NMR signals of axial ligands of paramagnetic hemoproteins are hard to observe, the usefulness of deuterium magnetic resonance for investigating ligand exchange in the paramagnetic hemoproteins is emphasized.
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Atsushi HONDA, Masahiko IWAMA, Tetsuya UMEDA, Yo MORI
1982 Volume 91 Issue 6 Pages
1959-1970
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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After a dose of 10 fig of 6-aminonicotinamide (6-AN) was administered to day-4 chick embryo
in ovo, micromelia was obviously observed in the hind limbs of 7-day chick embryos. We examined the teratogenic mechanism of 6-AN by using the normal or micromelial hind limbs (buds) from day 5 to day 7, with special attention to the biosynthesis of glycosaminoglycan (GAG) and proteoglycan as an index of limb chondrogenesis.
The present study provides evidence for abnormalities in the levels of GAG or proteoglycan biosynthesis in the micromelial hind limbs (buds). 1) Both [
35S] sulfate and [
3H] glucosamine incorporation into GAG per 10 limbs or mg DNA of the micromelia were inhibited, suggesting a decrease of GAG synthesis. 2) The micromelial limbs synthesized low-sulfated chondroitin sulfate (chondroitin) as judged by the
35S/
3H ratio, the proportion of unsulfated disaccharide (
ΔDi-OS), and the result of cellulose acetate electrophoresis, although there were no significant differences in the approximate molecular size of
35S-chondroitin sulfates synthesized between the normal and micromelial limbs. 3) PAPS-synthesizing activity in the micromelial limbs was markedly inhibited, and this may result in the production of low-sulfated proteoglycan. 4) The transition from mesenchymal- to cartilagespecific proteoglycan synthesis did not appear in the micromelial limbs as judged by the sedimentation profiles. 5) 6-AN caused marked reductions in the oxygen consumption and ATP level of the micromelial limbs, thereby causing the defect in PAPS formation.
We suggest that these 6-AN-induced sequential molecular defects (the reduction of respiratory activity, ATP and PAPS level, and concomitant interference with GAG and proteoglycan biosynthesis) in the limbs (buds) during the critical period of limb morphogenesis must be major factors resulting in the cartilage growth retardation or disorder,
i.e., micromelia.
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Kozo YAMAMOTO, Jun HITOMI, Kanae KOBATAKE, Haruki YAMAGUCHI
1982 Volume 91 Issue 6 Pages
1971-1979
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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1, 2-α-Mannosidase was purified approximately 1, 400-fold from an enzyme product of
Aspergillus oryzae. The enzyme showed a single band in disc gel electrophoresis and the molecular weight was estimated to be about 49, 000 daltons by gel exclusion chromatography. The substrate specificity of the enzyme was examined with mannooligosaccharides, yeast mannan, glycopeptides, and a glycoprotein. The α-(1→2)-linking mannose residues located at the nonreducing-ends of the substrates were selectively removed by the enzyme, whereas
p-nitrophenyl α-D-mannopyranoside was completely stable to the enzyme. α-(1→2)-Linking mannose residues in intact bovine pancreatic ribonuclease B were also removed completely with the enzyme. The enzyme showed an optimum pH in the range of pH 4.9 to 5.3 and had a
Km value of 0.57mM with 1, 2-α-mannobiose. The present α-mannosidase was quite stable, and the activity was inhibited by D-mannono-γ-lactone and by heavy metal ions, including zinc ions.
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Haruo SUZUKI, Etsuko B. MUKOUYAMA
1982 Volume 91 Issue 6 Pages
1981-1994
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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The mechanism of inhibition of globin synthesis by poly (dT) was studied in the rabbit reticulocyte lysate system. When the lysate was incubated with [
14C] poly (dT), poly (dT) was found to bind with the native 40 S ribosomal subunit and the “supernatant factor.” But the binding to the native 40 S ribosomal subunits was not directly related to the poly (dT) inhibition.
Ribosomal subunits were prepared from rabbit reticulocytes and tested for their binding with poly (dT) and their effect on the poly (dT) inhibition. Poly (dT) was found to bind with the derived 40 S ribosomal subunit, but not with the derived 60 S subunit, and the poly (dT) inhibition was slightly reversed by the derived 40 S ribosomal subunit.
Under conditions such that the elongation of nascent chains was inhibited by sparsomycin, the formation of the 80 S/Met-tRNA
f complex was inhibited by poly (dT) and the inhibition was greater at high concentration of KOAc. However, the formation of the 40 S/Met-tRNA
f complex was inhibited to the same extent at 70mM and 200mM KOAc in the presence of GMPPCP.
A factor (TF) that reverses the poly (dT) inhibition was partially purified from the KCl-wash of rabbit reticulocyte ribosomes by ammonium sulfate fractionation, and Sephadex G-150 and DEAF-cellulose column chromatographies. From the Sephadex G-150 column chromatography of TF, the molecular weight of TF was estimated to be 81, 000-102, 000. TF reversed the poly (dT) inhibition of 80 S/[
3H]-mRNA/Met-tRNA
f complex or that of 40 S/[
3H] mRNA/Met-tRNA
f complex. TF bound to [
14C] poly (dT) or
3H-labeled globin mRNA. SDS/polyacrylamide slab gel electrophoresis of the complexes between the factor and [
14C] poly (dT) or [
3H]-mRNA showed common polypeptide bands of 22, 500, 25, 000, and 49, 000 daltons.
These data can be explained by assuming that poly (dT) binds to a factor which is required for the binding of 40 S/Met-tRNA
f complex with mRNA to form inactive complexes, and thus inhibits globin synthesis. The relationship between the poly (dT)-binding protein and known initiation factors is discussed.
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Shin-ichi NAKAYAMA, Masamatsu HOSHINO, Osamu TAKENAKA, Kenji TAKAHASHI ...
1982 Volume 91 Issue 6 Pages
1995-1998
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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ESR spectra of the carbonmonoxy, oxy, and deoxy derivatives of hemoglobin Izu [Hb Izu (Macaca): β 83 (EF 7) Gly→Cys] labeled at cysteine β 83 with maleimide spin label have been observed in the presence and absence of 2, 3-diphosphoglycerate and inositol hexaphosphate. The τ
c, values obtained from the spectra indicated that inositol hexaphosphate binds to all the derivatives of Hb Izu, but 2, 3-diphosphoglycerate only to the deoxy derivatives.
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Osamu OHARA, Sho TAKAHASHI, Tatsuo OOI, Yoshinori FUJIYOSHI
1982 Volume 91 Issue 6 Pages
1999-2012
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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Cross-linking experiments were performed on muscle skeletal actin, using imidoesters of various chain lengths. Chemical analyses on all products except one (derived from succinimidate) show evidence of the presence of intramolecular crosslinks in the molecule. The detailed properties of suberimidate-treated actin (SA) are as follows: SA contains nearly 1 mol of intramolecular cross-link per mol of actin and less than 15% of intermolecularly cross-linked products. Even at a low salt concentration, SA is polymeric, exchanges slowly its bound nucleotide with free nucleotides in solution, and shows an F-actin-type CD spectrum. Electron micrographs of SA reveal that SA exists actually as fibrous polymers in solutions of low ionic strength, although the fibers seem to be less rigid than those at high salt concentration. The F-form of SA at a high salt concentration is indistinguishable from intact F-actin. SA can bind heavy meromyosin and activate the ATPase of heavy meromyosin as observed for intact F-actin. Tropomyosin binds SA only at a high salt concentration. These results show that SA possesses the properties of F-actin even in media of low salt concentration, which are favorable for depolymerization of F-actin. Thus, we may infer that the conformation of SA is frozen in the F-state of actin by the introduction of intramolecular cross-links in the protein.
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Eiji SATO, Tadayoshi UEZATO, Michiya FUJITA, Kenji NISHIMURA
1982 Volume 91 Issue 6 Pages
2013-2019
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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The major gangliosides were isolated from small intestine of 2-week-old mice of C 3 H/He strain and identified as GM 3, GM 1, and GDla. These gangliosides characteristically contained a phytosphingosine moiety and α-hydroxy fatty acids. The gangliosides contained both
N-glycolylneuraminic acid and
N-acetylneuraminic acid. Gangliosides GM 3, GM 1, and GDla contents increased in mouse small intestine during the suckling period, and decreased after weaning. In contrast, neutral glycosphingolipids appeared after weaning, except for monohexosyl ceramide, which was present at a fairly constant level throughout postnatal life. The same results were obtained with mice of other strains and germ-free ICR mice, although such changes could not be observed in rats. These observations indicate that the developmental change in the composition of glycosphingolipids occurs in a species-specific manner.
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Yujiro HIGASHI, Yoshihiro SOKAWA
1982 Volume 91 Issue 6 Pages
2021-2028
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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By means of a new type of microinjection apparatus, which has a micropipette located in a hole through the optical axis of the condenser lens, we injected interferon (IFN) or 2', 5'-oligoadenylate (2-5 A) into mouse L cells, and observed their antiviral effects on the multiplication of vesicular stomatitis virus (VSV). After injection, cells were infected with VSV, and labeled with [
3H] uridine in the presence of actinomycin D. The proportion of cells infected with VSV which carried radioactive virus-RNA was determined by autoradiography. IFN introduced directly into L cells had no effect on the virus growth. This result supports the idea that IFN molecules exert their effect from outside the cell membrane without penetrating into the cytoplasm. 2-5 A, on the other hand, was able to inhibit the growth of VSV effectively when injected into L cells. The antiviral effect was dependent on the dose of 2-5 A injected, and moreover the effect was transient, since it disappeared completely after 24-h incubation.
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Yasuharu TANAKA
1982 Volume 91 Issue 6 Pages
2029-2037
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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The effects of polyamines on reactions catalyzed by bovine thymus poly (ADP-ribose) polymerase were examined under various conditions, and the following results were obtained. (1) Spermine and spermidine, and putrescine to a lesser degree can stimulate the synthesis of poly (ADP-ribose) covalently bound to the enzyme without Mg
2+ and histones. (2) A part of the above stimulation can be explained by the Mg
2+-sparing effect of polyamines. (3) The other part of the stimulation is shown to be through protection of the enzyme against the formation of an abortive complex of the enzyme and denatured DNA, which contaminates some native DNA preparations used for enzyme activation. Similar protection was shown earlier in this laboratory with histones. (4) Putrescine seems to lack this enzyme-protecting activity. (5) The polyamine effect observed in the Mg
2+-dependent reaction is variable depending on the DNA preparations used. (6) Chemical analysis shows that the average chain lengths of the products synthesized with Mg
2+ and spermine are similar, and the products are covalently bound to the enzyme, indicating that the reaction supported by polyamines is essentially the same as that by Mg
2+. (7) Under the histone H
1-dependent reaction conditions where ADP-ribosylation of histone H
1 is predominant, both Mg
2+ and polyamines are inhibitory on the reaction and both cations decrease the number of product molecules without affecting the size of the product. These data suggest that polyamines can at least partially replace Mg
2+ in terms of effect on the ADP-ribosylation reaction. The other effect of polyamines is the protection of the enzyme from abortive binding to denatured DNA, as has also been shown to occur with histones.
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Yuzuru ETOH, Hirofumi SHOUN, Takashi OGINO, Shizuo FUJIWARA, Kei ARIMA ...
1982 Volume 91 Issue 6 Pages
2039-2046
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL
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The location and state of an essential histidyl residue in a milk-clotting acid proteases, Mucor rennin, were investigated by NMR spectroscopy. Assignment of the C 2 H resonance peak of the essential histidyl residue was possible by comparison of the NMR spectrum of the native enzyme with that of the photo-oxidized enzyme. The pH titration curve for the chemical shift of the C 2 H proton showed two inflections, a major one with
pKa=7.4 and a minor one with
pKa =3.5 at 30°C. The major inflection, corresponding to an intrinsic protonation of the imidazole ring, shifted toward the alkaline side upon addition of acetyl pepstatin, an inhibitor specific for the acid protease. Modification of an essential carboxyl group in the enzyme with diazoacetyl-DL-norleucine caused disappearance of the minor inflection as well as an acidic shift of the major
pKa value. Perturbation effects on the C 2 H resonance of the lanthanide metals, Pr
3+, Eu
3+, and Gd
3+, suggested their selective binding to a carboxyl group and location of the bound metal atom close to the essential histidyl residue. All data suggested that the essential histidyl residue of Mucor rennin is located close to one of the two essential carboxyl groups in the catalytic site of the enzyme.
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Ikuo ASHIKAWA, Yoshifumi NISHIMURA, Masamichi TSUBOI, Kazutada WATANAB ...
1982 Volume 91 Issue 6 Pages
2047-2055
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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The fluorescence intensity and fluorescence lifetime of the tyrosine residues in calf thymus nucleosome core particles have been determined as functions of the ionic strength of the solvent. For interpreting the results, in the first approximation, the 30 tyrosine residues involved in the particle are classified into two groups. About 12 belong to class I; they are distributed in the protein core with an average distance of 2.0 nm from its center. In the intact particle (in 20mM to 0.4M salt solution), a Förster-type energy transfer is considered to take place from these class-I tyrosine residues to the DNA bases, but this no longer occurs on elevating the salt concentration to about 1.4M. The remaining tyrosine residues (about 18, called class II) are considered to be involved in hydrogen bonds or in some other intramolecular interactions in the intact core particles, so that their fluorescence is completely quenched. On elevating the salt concentration to 2.0M, this quenching is partially removed. Implications of these dynamic and static quenchings are discussed in terms of the structure of the core particle.
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Yoshimichi NAKATSU, Yusaku NAKABEPPU, Mntsiin SFKIGUCHI
1982 Volume 91 Issue 6 Pages
2057-2065
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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[
3H] Thymine-labeled poly (dA)•poly (dT) carrying many apyrimidinic (AP) sites has been prepared by treating an enzymatically synthesized poly (dA)•poly (dT, dU) with uracil-DNA glycosylase. Incubation of the polymer with a homogeneous preparation of T 4 endonuclease V resulted in conversion of the labeled material into acidsoluble forms. Native DNA with apurinic sites was also cleaved by the enzyme. Single-stranded polymers, poly (dT) carrying AP sites or poly (dT) with thymine dimers, were barely attacked by T 4 endonuclease V. The polymer whose aldehyde moieties at AP sites were reduced to alcoholic forms was not susceptible to the enzyme. The site of endonucleolytic cleavage was determined by using alternating copolymers whose phosphate groups were differentially labeled. The result is consistent with the view that T 4 endonuclease V cleaves a phosphodiester linkage on the 3'-side of AP sites, producing chains terminated at their 3'-ends with base-free deoxyribose and at their 5'-ends with phosphate.
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Satoko ISEMURA, Eiichi SAITOH, Kazuo SANADA
1982 Volume 91 Issue 6 Pages
2067-2075
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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Basic proline-rich peptides of human parotid saliva were fractionated and characterized. The amino acid sequence of one of the purified peptides, P-E, was determined to be SPPGKPQGP_??_PQGGNQPQG_??_PPPPGKPQG_??_PPQGGNRPQ_??_PPPPGKPQG_??_PPQGDKSRS_??_R.
The results demonstrate the repetitiveness of the partial sequence within the molecule and the occurrence of structures common to those of other salivary polypeptides such as P-C and Protein C.
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Sadao WAKABAYASHI, Hideki TAKEDA, Hiroshi MATSUBARA, Chong H. KIM, Tso ...
1982 Volume 91 Issue 6 Pages
2077-2085
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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A heme-not-containing protein was isolated from cytochrome
c1 preparation by gel filtration after carboxymethylation and citraconylation. The amino acid sequence of this protein was determined by the analyses of tryptic and chymotryptic peptides as well as by solid-phase sequence analysis. It consisted of 78 amino acid residues and the molecular weight was calculated to be 9, 175. This protein contained a high proportion of glutamic acid and glutamine (27% of the total residues) but no methionine, isoleucine, tyrosine, and tryptophan. The most notable feature was an acidic cluster of 8 consecutive glutamic acid residues near the amino (N)-terminus. The secondary structure was predicted to have a high proportion of helical content.
The amino acid composition and N-terminal sequence of a protein independently prepared from bovine heart mitochondria, which is essential to the formation of the cytochrome
c1-
c complex, suggested that this colorless factor and the present heme-not-containing protein are identical. Evidence shows that another protein, called the non-heme protein, isolated from “two-band” cytochrome
c1 preparation is also the same protein as that presented in this paper.
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Hiroshi YANAGAWA, Yumiko MAKINO, Kazuki SATO, Masato NISHIZAWA, Fujio ...
1982 Volume 91 Issue 6 Pages
2087-2090
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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In the course of a study of chemical evolution in the primeval sea, a novel reaction of carbonyl compounds with ammonia was found. Glyoxylic acid reacted with ammonia to afford
N-oxalylglycine, which gave glycine in a 3-20% yield after 6 N HCl hydrolysis. Similarly, glyoxylic acid was treated with methylamine to give
N-oxalylsarcosine, which afforded sarcosine in a 9-12% yield upon hydrolysis. Pyruvic acid reacted with ammonia to give
N-acetylalanine, which gave alanine in a 1-4% yield upon hydrolysis. These reactions provide a novel and facile route to α-amino acids and their derivatives. A mechanism for the reactions is proposed.
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Mieko OGURO, Hiroshi NAGANO
1982 Volume 91 Issue 6 Pages
2091-2094
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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Isolated HeLa cell nuclei were treated with NaCl at various concentrations and inhibition by aphidicolin of DNA synthesis in the treated nuclei was studied. The inhibition was either noncompetitive or of the mixed type with respect to each dNTP when the nuclei were treated with NaCl at concentrations lower than 0.08M. However, aphidicolin was a competitive inhibitor with respect to dCTP and a noncompetitive or mixed type inhibitor with respect to the other 3 dNTPs when they were treated with NaCl at concentrations higher than 0.1M. These results suggest the presence of nuclear factor (s) responsible for the changes in the inhibitory mode of aphidicolin on endogenous nuclear DNA synthesis.
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Susumu KANDA, Keizo INOUE, Shoshichi NOJIMA, Hideo UTSUMI, Herbert WIE ...
1982 Volume 91 Issue 6 Pages
2095-2098
Published: June 01, 1982
Released on J-STAGE: November 18, 2008
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When an aqueous solution of a spin-labeled “two tail” gangliosidoid was incubated with liposomes or sheep erythrocytes, the broad single resonance line in the ESR spectrum disappeared and a signal showing an anisotropic motion appeared, indicating that the spin-labeled “two tail” gangliosidoid in the micellar state was transferred to the lipid phase of the acceptor membranes. The transfer was temperature- and time-dependent, irrespective of the acceptor membranes, indicating that the rate of transfer is determined by the escape of monomers from the micelles.
The kinetics and temperature-dependence of the association of ganglioside II
3NeuAc-GgOse
4Cer with sheep erythrocytes was very similar to that of the “two tail” gangliosidoid, indicating that parts of ganglioside II
3NeuAc-GgOse
4Cer could be incorporated into the lipid phase of membranes
via a similar mechanism.
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