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Volume 99 (1986)
- Issue 6 Pages 1551-
- Issue 5 Pages 1289-
- Issue 4 Pages 993-
- Issue 3 Pages 607-
- Issue 2 Pages 315-
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Volume 99, Issue 3
Displaying 1-45 of 45 articles from this issue
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Tsuneo NAKAMURA, Akinori NAITO, Yukio TAKAHASHI, Hiroshi AKANUMA1986 Volume 99 Issue 3 Pages 607-613
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacteria which grow on 1, 5-anhydro-D-glucitol (AG) were isolated from soil. One such strain showing the highest AG-assimilating activity was further characterized and identified as a new strain of the Pseudomonas family (named Pseudomonas sp. NK-85001). A subcellular membranous fraction obtained from this strain catalyzed the oxidation of AG to 1, 5-anhydro-D-fructose. This oxidation reaction consumed molecular oxygen as the terminal electron acceptor. The AG-oxidizing activity was further purified after solubilization. The AG oxidation catalyzed by this solubilized enzyme utilized molecular oxygen only in the presence of an electron mediator such as 2, 6-dichlorophenolindophenol or phenazine methosulfate. Thus, the enzyme was suggested to be a dehydrogenase rather than an oxidase. The solubilized enzyme preparation also showed a strict substrate specificity. The observed specificity indicated that application of the enzyme for AG assay in clinical samples might be possible.View full abstractDownload PDF (1206K) -
Motokazu FUJIWARA, Shigehiro MORIKAWA, Shinkichi TANIGUCHI, Keiichiro ...1986 Volume 99 Issue 3 Pages 615-625
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEffects of the calmodulin antagonists chlorpromazine, trifluoperazine, and N-(6aminohexyl)-5-chloro-l-naphthalenesulfonamide on phospholipid metabolism were examined in rabbit platelets using [3H] serine, [3H] ethanolamine, [3H] choline, and [3H] glycerol. All these drugs markedly stimulated the incorporation of [3H] serine into phosphatidylserine. On the other hand, these drugs had only a slight effect on the rate of incorporation of [3H] ethanolamine and [3H] choline into the corresponding phospholipid. When [3H] glycerol was used as a precursor of the phospholipids, 3H-labeled phospholipids were mainly composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Although the phosphorus content of phosphatidylserine was about 40% of that of phosphatidylcholine in rabbit platelets, the amount of phosphatidylserine labeled with [3H] glycerol was less than 2%, of that of the labeled phosphatidylcholine, and calmodulin antagonists slightly stimulated the incorporation of [3H] glycerol into phosphatidylserine. Treatment with calmodulin antagonists caused a marked decrease in the content of endogenous free serine with concomitant increase in the contents of endogenous free ethanolamine and choline. On the other hand, the contents of other free amino acids, including essential and non-essential amino acids, were unchanged. These results suggest that the calmodulin antagonists we used did not affect de novo synthesis of phosphatidylserine, but did stimulate the serine phospholipid base-exchange reaction in rabbit platelets.View full abstractDownload PDF (857K) -
Masachika IRIE, Hideaki WATANABE, Kazuko OHGI, Masatomi HARADA1986 Volume 99 Issue 3 Pages 627-633
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C] iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-SerTyr.... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-AspTyr-Ala..... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M.
The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.View full abstractDownload PDF (510K) -
Nobuo NAKANISHI, Hiroyuki HASEGAWA, Shozo YAMADA, Miki AKINO1986 Volume 99 Issue 3 Pages 635-644
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new type of dihydropteridine reductase [EC 1.6.99.10], which is specific for NADPH as the substrate in the reduction of quinonoid-dihydropterin to tetrahy-dropterin, was purified to homogeneity from bovine liver and human liver. The molecular weight of the enzyme was determined to be 65, 000-70, 000. The enzyme was composed of two subunits with identical molecular weight of 35, 000; the amino terminal residue was determined to be valine. The isoelectric point of the enzyme was 7.05. The physicochemical properties of this enzyme were quite different from those of bovine liver NADH-specific dihydropteridine reductase [EC 1.6.99.7]. NADPH-specific dihydropteridine reductase did not cross-react with an antiserum raised against the NADH-specific dihydropteridine reductase, nor did the latter enzyme react with an antiserum to the former enzyme, indicating that the two enzymes have no common antigenic determinants. NADPH-specific dihydropteridine reductase from human liver was shown to have properties similar to those of the bovine liver enzyme.View full abstractDownload PDF (1811K) -
Nobuo NAKANISHI, Hiroyuki HASEGAWA, Miki AKINO, Shozo YAMADA1986 Volume 99 Issue 3 Pages 645-652
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe catalytic properties of a new type of dihydropteridine reductase, NADPH-specific dihydropteridine reductase [EC 1.6.99.10], from bovine liver, were studied and compared with those of the previously characterized enzyme, NADH-specific dihydropteridine reductase [EC 1.6.99.7]. With quinonoid-dihydro-6-methylpterin, approximate Km values of NADPH-specific dihydropteridine reductase for NADPH and NADH were estimated to be 1.4 μM and 2, 900 μM, respectively. The Vmax values were 1.34μmol/min/mg with NADPH and 1.02 μmol/min/mg with NADH. With NADPH, the Km values of the enzyme for the quinonoid-dihydro forms of 6-methylpterin and biopterin were 1.4 μM and 6.8 μm, respectively. The enzyme was inhibited by its reaction product, NADP+, in a competitive manner, and the inhibition constant was determined to be 3.2 μM. The enzyme was severely inhibited by L-thyroxine and by 2, 6-dichlorophenolindophenol.View full abstractDownload PDF (596K) -
Thomas N. SATO, Masao HAYASHI1986 Volume 99 Issue 3 Pages 653-661
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have investigated whether species-specific epitopes of human fibronectin are localized at a specific domain of fibronectin using rabbit polyclonal antibodies. Tryptic fragments of human fibronectin were tested for reactivity with anti-human fibronectin antibody, which had been previously absorbed with other animal fibro-nectins to establish species specificity. Human-specific epitopes were found to be present on 75, 000, 65, 000, and 42, 000 dalton fragments. The 42, 000-dalton fragment shares almost all the epitopes with the 75, 000 and 65, 000 dalton fragments. It does not promote BHK cell spreading, whereas the 75, 000 and 65, 000 dalton fragments do. The amino acid sequence from the amino terminus of the 42, 000-dalton frag-ment is Asp/Gly-Gln/Val-?-Ile-Val-, which is almost identical to the sequence Asp-Gln-Cys-Ile-Val- located in the carboxyl terminal 1/3 of the collagen-binding domain of human fibronectin (Kornblihtt et al. (1985) EMBO J. 4, 1755-1759). These results suggest that human fibronectin bears human-specific epitopes mainly on the amino-terminal half of domain 4 (Hayashi & Yamada (1983) J. Biol. Chem. 258, 3332-3340) located between the collagen and cell binding domains almost at the center of the fibronectin polypeptide. The domain specific for human fibronectin may be a general species-specific domain of animal fibronectins.View full abstractDownload PDF (2677K) -
Koichi NISHIGAKI, Yuzuru HUSIMI, Misa TSUBOTA1986 Volume 99 Issue 3 Pages 663-671
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDenaturant gradient gel electrophoresis performed at low temperature was shown to be able to detect the mobility change corresponding to the denaturation of the secondary structure of single-stranded(ss)DNA. Mobility transitions observed were determined to correspond to the melting of local structures, since both denaturants and temperature were verified to have similar effects on the mobility transition of single-stranded DNAs as on that of double-stranded DNAs. In this study it was found that point mutations can effectively change the secondary structures of ssDNA and RNA, and that the method adopted here is very sensitive to such alterations. The validity of the method was supported by a computer analysis of the secondary structure of DNA.View full abstractDownload PDF (2300K) -
Yasuzo NISHINA, Hiromasa TOJO, Kiyoshi SHIGA1986 Volume 99 Issue 3 Pages 673-680
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPicolinate binds to a reduced form of D-amino acid oxidase, and the complex formed has a broad absorption band around 600nm as in the case of the purple intermediate of the enzyme with a substrate. The dissociation constant at 25°C was 35μM at pH 7.0. The pH dependence (pH 8.3-pH 6.4) of the dissociation constant indicates that one proton is associated with the complex formation, and picolinate protonated at the N atom binds to the reduced enzyme. Resonance Raman spectra of the complex support that picolinate in the complex is a cationic form protonated at the N atom. Nicotinate also binds to the reduced enzyme, but isonicotinate does not.View full abstractDownload PDF (609K) -
Toshiya ENDO, Hyung Suk KIM, Nobuyo MAEDA, Nobuo TAMIYA, Tatsuo MIYAZA ...1986 Volume 99 Issue 3 Pages 681-691
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe 270-MHz proton NMR spectra of the unique long neurotoxins bearing Phe-25, Astrotia stokesii b (As b) and Astrotia stokesii c (As c) from Astrotia stokesii, and Acanthophis antarcticus b (Aa b) from Acanthophis antarcticus, have been analyzed. The aromatic proton resonances of Phe-25 in As b and Aa b were assigned on the basis of the nuclear Overhauser effects observed on irradiation of slowly exchanging amide protons. Phe-25 was found to be involved in hydrophobic interactions with Ile/Val-42, Ala-46 and Ile-58 in As b and As c, and with Ala-46 and Val-58 in Aa b. These hydrophobic interactions, instead of the hydrogen bond between Tyr-25 and Glu-42 found in other neurotoxins, appear to be important for maintenance of the biologically active tertiary structure. The pH dependency of the chemical shift and intensity of the Trp-72 N-1 proton resonance of As b indicates that the indole ring is not fully exposed to the solvent and that the extra tail segment of this long neurotoxin interacts with the main part of the molecule.View full abstractDownload PDF (830K) -
Ken-ichiro TASHIRO, Teruo IWAMASA, Hisao KATO, Shigenori OGATA, Motoak ...1986 Volume 99 Issue 3 Pages 693-701
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcid α-glucosidase [EC 3.2.1.3] was purified from pig liver by a procedure including Sephadex G-100 affinity chromatography. Electrophoresis on SDS-polyacrylamide gel of the purified enzyme indicated the presence of two components with molecular weights of 73K and 64K. The two components of the enzyme were completely separated, in reasonable yield, by chromatography on a DEAE-5PW column. Both components catalyzed the hydrolysis of the α-1, 4 and α-1, 6 linkages of glycogen, maltose, isomaltose, dextrin, and a synthetic glucoside at acid pH. The pH optima of both components were 4.3 for maltase and glucoamylase, and 4.8 for isomaltase and dextrinase. But as to the activity on 4MU-α-GIc, the pH optimum of the larger component was 4.8 and that of the smaller component 5.3. The Km values of both components for 4MU-α-Glc, maltose, glycogen, isomaltose, and dextrin were 1.0×10-4 M, 9.1×10-3 M, 16.7 mg/ml, 6.7×10-2 M, and 12.5 mg/ml, respectively. Erythritol, Tris, and turanose inhibited the two components competitively.The Kl values of the larger component were 5.0×10-2 M, 13.3×10-3 M, and 3.2×10-3 M, and those of the smaller component were 2.5×10-2 M, 6.1×10-3 M, and 4.7×10-3 M, for erythritol, Tris, and turanose, respectively.View full abstractDownload PDF (2447K) -
Toshikatsu NAKABAYASHI, Hiroh IKEZAWA1986 Volume 99 Issue 3 Pages 703-712
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrom various rat organs, alkaline phosphodiesterase I was liberated by the action of phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis. Especially, a large amount of alkaline phosphodiesterase I was released from slices of small intestine, testis, lung, and kidney, but not from pancreas and liver. The release of the enzyme induced by phospholipase C was dependent on, or proportional to, the reaction time and the concentrations of the phospholipase C and the weight of the slices of small intestine or testis. Furthermore, little enzyme was released from the homogenate of pancreas. These results suggest an important role of phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membranes of rat small intestine and pancreas.
The alkaline phosphodiesterase I released from slices of rat small intestine and testis had a molecular weight of about 240, 000, and was activated by Mg2+ and Ca2+ but inhibited by EDTA. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9, having the Km values of 0.36mM (small intestine) and 0.25mM (testis). The intestinal enzyme differed from the testis enzyme in pI values, thermostability, and Arrhenius plot having a single breakpoint.View full abstractDownload PDF (705K) -
Yasuhiro SASAKI, Naoya KOJIMA, Yukiya KOIKE, Yoshio ARAKI, Eiji ITO1986 Volume 99 Issue 3 Pages 713-722
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStructural studies were carried out on two kinds of teichuronic acid-glycopeptide complexes (designated as TU-GP-I and TU-GP-II) isolated from lysozyme digest of N-acetylated cell walls of Bacillus megaterium AHU 1375 by ion-exchange chromatography and gel chromatography. TU-GP-I, accounting for about 25% of thecell walls, contained N-acetylmannosaminuronic acid, N-acetylglucosamine, glucose, galactose, glycerol, and phosphorus in an approximate molar ratio of 1:1:2:1:0.5:0.5, together with small amounts of glycopeptide components. TU-GP-II, accounting for about 9 % of the cell walls, contained glucuronic acid, glucose, and fucose in a molar ratio of about 2:1.5:1, together with small amounts of glycopeptide components.
The results of analyses involving Smith degradation, chromium oxidation, methylation, acetolysis, and 1H-NMR measurement led to the conclusion that the polysaccharide chain of TU-GP-I comprised repeating units, → 6) Glc (α1 → 3) -ManNAcUA (β1 → 4) [Gal (αl → 3) ] [Glc (β1 → 6) ] GIcNAc(β1 →. About half of the repeating units were substituted by glycerophosphoryl residues at C-6 of the β-glucosyl residues linked to the N-acetylglucosamine residues. By means of a similar procedure, the polysaccharide chain of TU-GP-II was shown to comprise repeating units, → 4) GIcUA (αl → 3) GIcUA (αl → 3) Glc (α1 → 3) Fuc (α1 →, of which about half were substituted by α-glucosyl residues at C-3 of the 4-substituted glucuronosyl residues.View full abstractDownload PDF (849K) -
Shintaro KIKUCHI, Takashi KUSAKA1986 Volume 99 Issue 3 Pages 723-731
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe supernatant fraction after 105, 000× g centrifugation of an extract of sonically disrupted cells of Mycobacterium smelt matis catalyzed the desaturation of lignoceroyl-CoA to a _??_15 -monounsaturated derivative in the presence of molecular oxygen and NADPH. This desaturation system was separated by ammonium sulfate fraction-ation, gel filtration, DEAE-cellulose column chromatographies, and affinity column chromatography on immobilized dye, into three components; a NADPH-oxidase, a ferredoxin-containing fraction and a desaturase, all of which were required for the reconstituted desaturation system for lignoeeroyl-CoA. This system was in-hibited by FMN and ferrous ions but not by KCN. All of these features clearly distinguish this system from the previously known fatty acid desaturation systems of various origins.View full abstractDownload PDF (632K) -
Osamu OHARA, Mikio TAMAKI, Etsuo NAKAMURA, Yuji TSURUTA, Yoko FUJII, M ...1986 Volume 99 Issue 3 Pages 733-739
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe performed molecular cloning of the cDNAs that encode dog and rat pancreaticphospholipases A2 to predict the primary structures of these enzymes. The deduced amino acid sequences exhibited a highly conservative feature which is common to a group of pancreatic phospholipases A2 from various animal species. Furthermore, the structures of the signal sequences of dog and rat pancreatic phospholipases A2 were predicted, although the assignment of the positions cleaved post-translationally is only tentative at the present time.View full abstractDownload PDF (469K) -
Toshiyuki SAKAKI, Kenji OEDA, Yoshiyasu YABUSAKI, Hideo OHKAWA1986 Volume 99 Issue 3 Pages 741-749
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe recombinant plasmids pAMCl and pJMCl were constructed; the former contained the cytochrome P-450MC (P-450MC) cDNA expression unit consisting of yeast alcohol dehydrogenase I (ADH) promoter, rat P-450MC cDNA and ADH terminator, and the Leu 2 marker gene, and the latter contained the same expression unit and the leu 2-d gene. Saccharomyces cerevisiae AH22 cells transformed with each of the recombinant plasmids were examined for plasmid copy number, P-450MC mRNA level, P-450MC content, and monooxygenase activity. The S. cerevisiae AH22/pJMCl cells contained about 2-fold higher levels of the plasmid, P-450MC mRNA, and P-450MC than the AH22/pAMCl cells. Monooxygenase activity towards 7-ethoxycoumarin and acetanilide of the AH22/pJMCl cells was 1.7-fold and 1.5-fold higher than that of the AH22/pAMCl cells, respectively, whereas the activity of the AH22/pAMCl cells towards 7-ethoxycoumarin and acetanilide was more than 1, 000-fold and 10-fold higher than that of the control AH22/pAAH5 ells which contain no P-450MC cDNA, respectively. Therefore, it is likely that monooxygenase activity of the AH22 cells carrying rat P-450MC cDNA was ap-proximately proportional to the expression level of P-450MC cDNA.View full abstractDownload PDF (1587K) -
Noriko SHIMIZUI, Takashi OBINATA1986 Volume 99 Issue 3 Pages 751-759
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe actin concentration and monomer-polymer ratio in developing chicken skeletal muscle were determined by means of a DNase I inhibition assay. The concentration of G-actin in embryonic muscle was much higher than the critical concentration for polymerization of purified actin. As muscle development progressed, the amount of total actin remarkably increased, whereas the concentration of G-actin markedly decreased, and finally in adults reached the critical concentration for polymerization of purified actin. When the monomeric actin in the soluble fraction of embryonic muscle was purified, the critical concentration for polymerization of the embryonic actin decreased to the same value as that of adult skeletal muscle actin. On the other hand, there was no difference between the crude and purified actin in the type of actin. They consisted of α-, β-, and γ-actins; their amounts were in the order, β> γ > α. Furthermore, polymerization of the monomeric actin in the soluble fraction of embryonic muscle was induced by the addition of myosin or HMM. The large amount of monomeric actin in the embryonic skeletal muscle may be due to the presence of some factor(s) which inhibits actin polymerization and also to an insuf- ficiency of myosin.View full abstractDownload PDF (2653K) -
Yuhei MORITA, Hiroyuki IWAMOTO, Shigeo AIBARA, Takashi KOBAYASHI, Eiic ...1986 Volume 99 Issue 3 Pages 761-770
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyeloperoxidase was purified from normal human leukocytes in a crystalline state. Two types of crystals were obtained by the batchwise and dialysis crystallization methods, one of which had a bipyramidal shape belonging to the orthorhombic system. Three multiple forms of human myeloperoxidase were separated from the crystalline enzyme by CM-Sepharose chromatography with sodium chloride gradient elution. These three multiple forms were found to have very similar enzymatic, spectroscopic, and chemical properties. However, slight differences were observed in their amino acid compositions and the molecular weights of their large subunits determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Hemi-myeloperoxidase was prepared from holo-myeloperoxidase by reduction with dithiothreitol and modification with iodoacetamide, and the molecular shape of the holo- and hemi-enzymes were determined by analytical ultracentrifugation. The axial ratios were calculated to be 2.4-3.5 for the bolo-enzyme and 2.9-3.1 for the hemi-enzyme. These results suggest that the shapes of the two enzymes are more spherical in solution than the proposed structural model previously reported.View full abstractDownload PDF (1179K) -
Takeshi KASAMA, Yousuke SEYAMA1986 Volume 99 Issue 3 Pages 771-775
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe developed a simple quantitative procedure for cholestanol in serum involving reversed phase thin layer chromatography. This procedure was satisfactory with regard to the linearity of the calibration curve in the range of 100ng to 1, 000ng, recovery and reproducibility. Only 100μl of serum was needed for determination of the cholestanol concentration. Prior to thin layer chromatography, cholesterol was converted to α- and β-epoxides with m-chloroperbenzoic acid, which were clearly distinguishable from cholestanol on TLC. Detection of sterols was performed by spraying with phosphomolybdic acid solution. Quantification of cholestanol was carried out with a TLC scanning densitometer. When serum cholestanol in cerebrotendinous xanthomatosis (CTX) patients was quantified by TLC, GC-MS, and GC, the correlation among the three methods was found to be approximately 1:1:1. It was found that the present method was useful for the primary diagnostic screening of CTX because of its simplicity and because many samples could be analyzed at one time.View full abstractDownload PDF (1261K) -
Morio SETAKA, Noriko SATOH, Toshihide KOBAYASHI, Toshio HONGO, Takao K ...1986 Volume 99 Issue 3 Pages 777-783
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA phospholipid bilayer membrane was spread from an organic solvent solution between a polyacrylamide gel surface and an aqueous buffer solution. The membrane was quite similar to the conventional black lipid membrane, but was of a large size and was stable since it was supported on the gel surface.
Bacteriorhodopsin, impregnated into the membrane, generated membrane potential and current upon illumination. The induced current was large, and this was attributed to the large area of the present membrane. Remarkable responses of the light-induced potential and current were also observed with a thick layer of organic solvent containing phospholipids. The effects of applied membrane potential, carbonylcyanide-m-chlorophenyl hydrazone (CCCP) and gramicidin were examined on these photoresponses. Steady-state current, which is due to protons flowing through the membrane, was enormously enhanced by applying membrane potential opposite to the photopotential or by adding gramicidin to the membraneforming solution.View full abstractDownload PDF (513K) -
Atsushi TAKEDA, Shu KOBAYASHI, Hiroyuki KAJI, Yoshio AOKI, Tatsuya SAM ...1986 Volume 99 Issue 3 Pages 785-791
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conformational changes of the papain molecule on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2)) from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(l)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuc- cinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinaseinhibitors.View full abstractDownload PDF (503K) -
Shiroki YAGISAWA, Hideaki TANIMORI, Tsunehiro KITAGAWA1986 Volume 99 Issue 3 Pages 793-802
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method for determining antigen-antibody binding constants by using enzyme-labeled antigens has been developed. In the measurement, enzyme-labeled and unlabeled antigens (Ag∗ and Ag) were allowed to compete in binding to the antibody (Ab) under conditions where Ag∗ <<Ab <<Ag. The data were analyzed according to a new theory developed for the analysis of competitive binding of two ligands to a heterogeneous receptor. The theory indicates that the binding degree of a labeled ligand measured at various concentrations of the receptor can be used to prepare a standard curve relating the binding degree of the labeled ligand and the average of the concentrations of the free receptor components which are in binding equilibrium with another unlabeled ligand. For homogeneous receptors, the method gives usual binding constants for the unlabeled ligand, but for heterogeneous receptors, it gives a new type of average binding constant for the unlabeled ligand in which the contribution of each receptor component is amplified in proportion to its affinity against the labeled ligand. This average binding constant was named the “affinity-average binding constant.”
A rabbit anti-blasticidin S (BLS) antiserum analyzed by the present method using β-galactosidase-labeled BLS as the labeled ligand was found to be fairly homogeneous with respect to the affinity and to have a binding constant of 1.48± 0.24 (S. D.) × 108 M-1 for unlabeled BLS.View full abstractDownload PDF (708K) -
Yasuki NONAKA, Shigeru FUJII, Toshio YAMANO1986 Volume 99 Issue 3 Pages 803-814
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNADPH-adrenodoxin oxidoreductase was titrated with NADPH under anaerobic conditions. As the amount of added NADPH was increased to a ratio to the reductase of 1:1, a broad absorbance band from approximately 500 to 900 nm, which is attributed to a charge transfer complex, increased and then sharply decreased after the 1:1 ratio was attained. Concomitant with the decrease in the charge transfer band, a peak at 575 nm with a shoulder at 635 nm increased, indicating the formation of a semiquinone. This showed clearly that a semiquinone was formed only when more than the stoichiometric amount of NADPH (It is meant by “the stoichiometric amount of NADPH” that the molar ratio of NADPH to adrenodoxin reductase is equal to one, that is, NADPH/FAD bound to the reductase=1.) was added. The semiquinone band reached its maximum with an approximately 3- fold excess of NADPH over the reductase, and then gradually decreased. Concurrent with the decrease in absorbance of both the charge transfer complex and the semiquinone, the reaction mixture was bleached, indicating that a pale colored species was produced. 1H NMR studies suggested that the pale colored species was a complex of fully reduced adrenodoxin reductase and NADPH, and that the semiquinone also bound 1 mol of the pyridine nucleotide per mol of the reductase. These data suggest that the semiquinone state of the reductase is observable only when a complex between NADPH and the enzyme in the flavin semiquinone is formed.View full abstractDownload PDF (961K) -
Eiji ITAGAKI1986 Volume 99 Issue 3 Pages 815-824
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA steroid monooxygenase from cells of a fungus, Cylindrocarpon radicicola ATCC 11011, grown in the presence of progesterone has been purified by affinity chromatography on a pregnenolone-Sepharose column. The obtained enzyme was gel electrophoretically homogeneous and exhibited a molecular weight of about 115, 000. SDS-gel electrophoresis revealed that the enzyme consisted of two equal-sized subunits with a molecular weight of 56, 000.
Sedimentation equilibrium analysis at 20°C indicated that the enzyme protein behaved as a mixture of monomeric and dimeric subunit species. The enzyme contained one molecule of FAD in each subunit and exhibited absorption maxima at 375 and 440 nm.
The monooxygenase catalyzed a Baeyer-Villiger type oxidation, i.e., oxygenative esterification of C21-20-ketosteroid to form an acetate ester of C19- 17β-hydroxysteroid with consumptions of NADPH and molecular oxygen. The enzyme displayed a wide substrate specificity toward C21-20-ketosteroids, while it strictly required NADPH as the external electron donor in a ratio of 1:1:1 for ketosteroid: NADPH: molecular oxygen. Kinetic study showed the enzyme to have very high affinity for progesterone.View full abstractDownload PDF (798K) -
Eiji ITAGAKI1986 Volume 99 Issue 3 Pages 825-832
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA steroid monooxygenase of Cylindrocarpon radicicola was found to catalyze oxygenative lactonization of 17α-etosteroid, androstenedione, to yield D-hmo-17α-oxasteroid, testololactone, i.e., the androstenedione monooxygenase reaction, in addition to catalyzing the progesterone monooxygenase reaction. The reaction product was identified by TLC, GLC, and mass spectrometry.
The oxygenation proceeded with unitary stoichiometry for 17-ketosteroid, NADPH, and molecular oxygen, indicating that it is a typical monooxygenase reaction of the external electron donor type. The enzyme catalyzed successively the side chain cleavage reaction of 17α-hydroxy-20-ketosteroid to produce its 17-ketoerivative and the lactonization of the product.
The effects of pH and of the concentration of substrate steroids on the androstenedione monooxygenase reaction were different from those on the progesterone onooxygenase reaction. Progesterone is a strong and competitive inhibitor of he lactonization of 17-ketosteroids. The steroid monooxygenase is concluded to ave the activities of both oxygenative esterification of 20-ketosteroids and oxygenative lactonization of 17-ketosteroids.View full abstractDownload PDF (563K) -
Teruhiro TAKABE, Kiyoshi TAKENAKA, Hajime KAWAMURA, Yoshitaka BEPPU1986 Volume 99 Issue 3 Pages 833-840
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA modified form of the Debye-Marcus equation relating electron transfer rate constants to charges on proteins and distances of electron transfer has been applied to the reaction of chemically modified cytochrome ƒ, in which positively charged amino groups are replaced with negatively charged carboxyl groups. The rate of electron transfer from reduced cytochrome ƒ to ferricyanide decreased with increasing ionic strength when the native and singly substituted cytochrome ƒ were used, although a sharp decrease was observed in the former case. When doubly or more than triply substituted cytochrome ƒ was used, the rate of electron transfer was almost constant or increased with increasing ionic strength, respectively. The kinetic-ionic strength effects on this reaction can be well explained by the Debye-Marcus equation in which the charge and radius of the protein are treated as variable parameters. The results show the importance of local positive charges of about 2.0 on native cytochrome ƒ and effective radius of about 11 Å of cytochrome ƒ for the electron transfer to ferricyanide. Since the net charge on the native cytochrome ƒ is negative and the calculated radius of the protein is 22.8 Å, the above results indicate that positive charges on the electron transfer site control the electrostatic interactions in this reaction. Previously reported data which had been analyzed by using the total net charge and full radius of the protein, were also well explained by the local charge and effective radius of the protein. Therefore, it may be said that for the application of the Debye-Marcus equation to biological electron transfer reactions, the charge and radius of the protein should be treated as variable parameters although most previous workers used the full protein radius and total net charge.View full abstractDownload PDF (589K) -
Tetsuya KAMATAKI, Kaori MAEDA, Miki SHIMADA, Ryuichi KATO1986 Volume 99 Issue 3 Pages 841-845
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16α-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16α-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment.
These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.View full abstractDownload PDF (398K) -
Takanori NAKAMURA, Toshihiro HORIUCHI, Takashi MORITA, Sadaaki IWANAGA1986 Volume 99 Issue 3 Pages 847-857
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn intracellular clotting factor, factor B, which is closely associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus), was purified and characterized. The purified preparation gave a single band (Mr=64, 000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while three bands (Mr=64, 000, 40, 000, and 25, 000) were detected on SDS-PAGE after reduction, This preparation was converted by limulus clotting factor C to an activated form, factor B, with Mr = 56, 000 consisting of a heavy chain (Mr=32, 000) and a light chain (Mr=25, 000) bridged by disulfide linkage(s). The factor B, which was produced separately by treating the partially purified factor B with factor C, was also purified. It gave a single band on unreduced SDS-PAGE and two bands on reduced SDS-PAGE. The purified factor B had Mr = 56, 000 consisting of a heavy chain (Mr=32, 000) and a light chain (Mr = 25, 000). These results indicated that the purified factor B zymogen is a mixture of single-chain and two-chain forms, both of which have the same molecular weight of 64, 000, and that these two forms are converted to factor B by factor C. The diisopropyl phosphorofluoridate-sensitive site of factor B was found in the heavy chain. The reconstitution studies using purified factor C, factor B, proclotting enzyme and coagulogen in the presence of lipopolysaccharide indicated that factor B is an essential component to complete sequential activation of the limulus clotting system, and that it specifically activates proclotting enzyme to the active clotting enzyme.View full abstractDownload PDF (785K) -
Takashi OHYAMA, Kazuya NISHIKAWA, Shosuke TAKEMURA1986 Volume 99 Issue 3 Pages 859-866
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide sequence of T. utilis tRNATyr has been modified to have a deletion or substitution of the“conserved” nucleotide sequence Gm18-G19 in the D-loop by enzymatic procedures in vitro. Conformations of the variant tRNAs were analyzed by measuring melting profiles and electrophoretic mobilities in “native” polyacrylamide gels, and by examining the RNase T1 digestion patterns in sequencing gels. The results obtained shed light on the importance of the interaction between the sequence Gm18-G19 and nucleotides in the TΨC-loop (probably Ψ57-C58) for the maintenance of the total conformation of tRNATyr in solution. The association of D-loop and Tt'C-loop regions in the variant tRNATyrs is slightly relaxed even at room temperature and melting occurred at temperatures higher than 40°C. The relationship between the tertiary structure of the variant tRNA and its aminoacylation capacity was assayed at various temperatures. The results indicate that highly ordered tertiary structure is needed for tRNATyr to be fully aminoacylated.View full abstractDownload PDF (2406K) -
Hiroshi NAKADA, Takaya SAWAMURA, Tadayoshi OKUMURA, Yutaka TASHIRO1986 Volume 99 Issue 3 Pages 867-876
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt has been reported that a rat asialoglycoprotein receptor is composed of three polypeptide chains with molecular masses of 43, 54, and 64 kilodaltons (43, 54, and 64-Kd forms) and that the first has a different primary structure from the latter two forms. Incorporation of [3H] leueine into these forms showed that no precursor-product relationship is found between the 54-Kd and 64-Kd forms. The half-life of the 43-Kd form (25 h) was shorter than those of the 54-Kd and 64-Kd forms (66 and 70 h, respectively). Glycopeptides of the three forms were prepared from rat livers previously labeled in vivo with [3H]glucosamine. Gel filtration analysis of the glycopeptides before and after endo H treatment revealed that they were all resistant to endo H. Alkali treatment did not change the elution position appreciately. These results indicate that the three molecular forms contained only complex oligo-saccharide chains.
The receptor was prepared from rat livers previously treated with tunicamycin in vivo and subjected to SDS-PAGE. A distinct band with a molecular mass of 33 Kd was observed. The receptor was also immunoprecipitated from rat hepatocytes in primary culture previously labeled with [35S]methionine and analysed by SDS-PAGE and fluorography. In addition to the major 43-Kd form, a band with a molecular mass of 41 Kd was found and tunicamycin treatment gave rise to a 33-Kd component, which is in good agreement with the receptor purified from tunicamycin treated rats. It is suggested that the 43-Kd form is synthesized as a 33 Kd polypeptide, cotranslationally glycosylated to form the 41 Kd component and hen processed to the final 43-Kd form. We also think that the 43-Kd form could bind to asialoorosomucoid-Sepharose 4B without its carbohydrate chains.View full abstractDownload PDF (2347K) -
Hiroshi NAKADA, Sanae MATSUMOTO, Yutaka TASHIRO1986 Volume 99 Issue 3 Pages 877-884
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmyloid P component was isolated from rat liver and serum; its properties, biosynthesis, and glycosylation in the liver were investigated. The molecular weights of intracellular and serum amyloid P component were estimated to be 28, 000 and 30, 500, respectively. The two forms were immunologically identical, and kinetic study revealed a clear precursor-product relationship between them. The total mRNA was prepared from rat liver with or without turpentine treatment, and the RNA content of amyloid P component was estimated by the incorporation of [35S] methionine into the in vitro translation products. The turpentine treatment induced a marked increase in the level of translatable mRNA of the amyloid P component (_??_46 fold), while the serum level of the protein elevated only moderately (_??_1.7 fold). Most of the intracellular amyloid P component was sensitive to endo H. Various subcellular fractions were prepared from rat livers previously labeled in vivo with [35S] methionine. The protein prepared from the rough and smooth microsomes and heavy Golgi fraction were all sensitive to endo H, whereas that from the light Golgi fraction was a mixture of forms sensitive to and resistant to endo H. This result suggests that the processing of the mannosyl oligosaccharide chains and the subsequent addition of terminal sugars to convert the liver amyloid P component (28, 000 daltons) to serum counterpart (30, 500 daltons) were performed in the trans-Golgi region just before secretion of the amyloid P component.View full abstractDownload PDF (3759K) -
Kenshi OBARU, Makoto FUKUDA, Shuichiro MAEDA, Kazunori SHIMADA1986 Volume 99 Issue 3 Pages 885-894
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA clone inducible by either a tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), or a T-cell mitogen, phytohemagglutinin (PHA), was isolated from a cDNA library constructed from the poly(A)+RNAs of TPA- and PHA-stimulated human tonsillar lymphocytes, and was named pLD78. Stimulation of the tonsillar lymphocytes with either TPA or PHA increased the amount of pLD78-specific RNA by about 10-fold, and simultaneous stimulation with TPA and PHA, by at least 30-fold. Analysis of the pLD78 cDNA sequence revealed that it codes for a polypeptide consisting of 92 amino acid residues, including a putative signal sequence. Moreover, the sequence of the 5' flanking region of the nuclear DNA encoding for the pLD78 cDNA showed a significant homology with the corresponding regions of the human interleukin 2 and immune interferon genes.View full abstractDownload PDF (3131K) -
Motoo MATSUDA, Takahyro NOMURA, Tadanori KAMEYAMA1986 Volume 99 Issue 3 Pages 895-900
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn attempt was made to immunochemically and biochemically purify and characterize the U1-snRNP(s) of mouse embryonal carcinoma cells.
The results obtained by RNA analysis of U1-snRNP(s) purified immunochemically from embryoid bodies, F9 cells and PYS-2 cells indicated that the U1-snRNP(s) in these cells consisted of U1a-snRNP and U1b-snRNP. The proportion of U1a-snRNP to U1b-snRNP was also found to be high in the embryoid bodies and F9 cells. The U1a-snRNP predominance in U1-snRNP population was also detected in PYS-2 cells.
The immunochemically purified U1-snRNP population from liver nuclei of 129 syngeneic male mouse (129/sv), a host mouse for transplantable tetratocarcinoma OTT6050, and ICR male mouse, contained approximately equal levels of the two U1-snRNP species (U1a- and U1b-snRNP).
Partially purified U1-snRNP from embryoid bodies was also obtained by elu-
tion from a DEAE-Sepharose column at around 0.18M NH4Cl or by fractionation by 5-20% linear sucrose gradient centrifugation. The electrophoretic RNA profiles of the partially purified U1-snRNP of embryoid bodies were similar to those obtained immunochemically.View full abstractDownload PDF (2110K) -
Retsu MIURA, Toshio YAMANO, Yoshihiro MIYAKE1986 Volume 99 Issue 3 Pages 901-906
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHeterogeneity of brewer's yeast old yellow enzyme (OYE) was found by anion-exchange high-performance liquid chromatography (HPLC) as well as by 13C-NMR spectroscopy of [4a-13C] FMN reconstituted into apo OYE. Though the OYE sample prepared according to the conventional procedure gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the OYE sample was found to consist of five species on anion-exchange HPLC. The 13C-NMR spectrum of the [4a-13C] FMN-reconstituted OYE gave multiple peaks corresponding to 4a-13C. This multiplicity indicates that this OYE preparation possesses heterogeneity in the environment surrounding FMN, i.e., the active site of OYE. The different species of OYE were separately obtained by preparative HPLC on an anion-exchange column. These species as well as the unresolved sample showed identical mobility on SDS-PAGE and similar but slightly different NADPH oxidase activities. This heterogeneity was shown not to have resulted from proteolytic modification during the conventional purification procedure, which includes autolysis of the yeast cells, since the enzyme extracted by mechanical destruction of the yeast cells in the presence of various protease inhibitors exhibited identical heterogeneity. The pure OYE forms obtained by preparative anion-exchange HPLC are homogeneous in the flavin environment as revealed by a single 13C-NMR signal for the [4a-13C] FMN-reconstituted species.View full abstractDownload PDF (412K) -
Retsu MIURA, Toshio YAMANO, Yoshihiro MIYAKE1986 Volume 99 Issue 3 Pages 907-914
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe 31P- and 13C-NMR spectra of old yellow enzyme (OYE) were measured. The 31P-NMR signal of FMN bound to apo OYE-I, one of the pure forms of OYE, was observed at a substantially lower field compared to that of free FMN. While the 31P-signal of free FMN is pH-titratable with a pK value of about 6.5, which corre-sponds to the monoanion-dianion transition of the phosphate group, the 31P-signal of FMN bound to OYE-I shows no pH-dependence at pH 5-9, indicating that the phosphate group of FMN bound to OYE-I is fixed in the dianionic form in the pH region of 5-9. Apo OYE (0), i.e., the OYE preparation obtained by the conventional method, was reconstituted with [2-13C] FMN or [4, l0a-13C2] FMN, while apo OYE-I was reconstituted with [4a-13C] FMN. The 13C-NMR spectra of these reconstituted OYE species were measured in the absence and presence of phenolic compounds which form complexes with OYE. Each 13C-signal of the 13C-labeled FMN became broader in the bound state compared to the free state, indicating restriction of flavin mobility in the bound form. Complex formation of the reconstituted OYE species with p-bromophenol did not shift the 10a-13C signal but shifted the 2- and 4-13C signals slightly upfield, whereas the 4a-13C signal was shifted significantly upfield in the complexed form. This complex-induced upfield shift of the 4a-13C signal was measured with various p-substituted phenols. An approximately linear negative relationship was observed between the complex-induced upfield shift and the value of the Hammett para constant of the substituent of the phenolic compound. We interpret the results as indicating that the complex of OYE and a phenolic compound is a charge transfer complex and that the oxygen of phenolate anion is the charge-donating site.View full abstractDownload PDF (595K) -
Sei-ichi TANUMA, Kohtaro KAWASHIMA, Hiroyoshi ENDO1986 Volume 99 Issue 3 Pages 915-922
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe acceptor proteins for (ADP-ribose)n, were investigated by using nuclei or chromosomes isolated from specific phases of the cell cycle of HeLa S3 cells. Analysis of HMG proteins and histone H1 by acetic acid/urea polyacrylamide gel electro-phoresis demonstrated that the (ADP-ribosyl)n, -ation of HMG 14 and 17 and histone H1 increased by 12- and 5-fold, respectively, in the metaphase chromosomes as compared with that in the G1 phase cell nuclei. The degree of (ADP-ribosyl)n, -ation of these proteins in the S phase cell nuclei was as low as that in GI phase cell nuclei. In the G2 phase cell nuclei, the degrees of (ADP-ribosyl)n, -ation of HMG 14 and 17 and histone H1 were about 5- and 2-fold greater, respectively, as compared with that in the G1 phase cell nuclei. The (ADP-ribosyl)n, -ation of HMG 1 and 2 was constant through the cell cycle except for a slight decrease in the S phase. The data may imply that the (ADP-ribosyl)n, -ation of HMG 14 and 17 and histone H1 is linked to chromatin structural changes in mitosis.View full abstractDownload PDF (643K) -
Hideki IMAI, Shu-ichi HIRAI, Hirofumi HIRONO, Tamio HIRABAYASHI1986 Volume 99 Issue 3 Pages 923-930
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTroponin T from fast muscle of chicken legs was found to be composed of about 40 kinds of isoforms by two-dimensional polyacrylamide gel electrophoresis in conjunction with immunoblotting tests with an antiserum to chicken breast muscle troponin T. Almost all of the isoforms were found in the myofibril preparation and troponin preparation from the leg muscle, and they showed complex-forming ability with troponin I and troponin C. These isoforms existed in most of the fast muscle except pectoralis and posterior latissimus dorsi muscles, and they changed in composition during development. The breast muscle troponin T also showed different types of isoforms in the period soon after hatching. Since proteolysis was completely inhibited during two-dimensional gel electrophoresis and since the many isoforms were observed consistently in various muscles of chicken leg, they are most probably products of mRNAs generated by differential gene splicing.View full abstractDownload PDF (3680K) -
Yasushi TANIGUCHI, Koji AKASAKA, Tsugio SHIROYA, Hiraku SHIMADA1986 Volume 99 Issue 3 Pages 931-938
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtease activity was extracted from sea urchin sperm with 1% Triton X-100 and partially purified by DEAE-cellulose and Sephadex G-100 chromatography. The enzyme preferentially degraded histone H1, while showing only a weak activity toward other histones. Heat-denatured casein and bovine serum albumin were not digested by this enzyme under the present experimental conditions. This protease hydrolyzed only Boc-Val-Leu-Lys-MCA among various peptidyl-MCAs. The optimal pH ranged from 7 to 11. Its molecular weight was about 41, 000. Among various known inhibitors of proteases, only o-phenanthroline effectively inhibited the activity. The enzyme was stimulated by Zn2+ or Co2+. It was inactivated by o-phenanthroline but could be reactivated by the addition of Zn2+ or Co2+. Therefore, this protease seems to be a metalloprotease dependent on Zn2+ or Co2+. The insensitivity of this enzyme to phosphoramidon and its very restricted substrate specificity suggest that this enzyme is very different from other metallopro-teases described hitherto.View full abstractDownload PDF (1236K) -
Mayumi SHIBUYA, Mitsuko OKADA1986 Volume 99 Issue 3 Pages 939-944
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDL-[14C] Leucine or L-[3H] leucine was injected intraperitoneally into pyridoxine-deficient and control rats, and the subsequent incorporation of the radioactivities into aspartate aminotransferase (AspAT) isozymes and the total soluble protein in the liver was measured. AspAT in the cytosol (AspATc) was separated into 3 subfractions with different characteristics on chromatofocusing. The results showed that in the liver of pyridoxine-deficient rats, the syntheses of all 3 subfractions of AspATc and degradation of AspATc (total) were increased, but that the syntheses and degradation of the total soluble protein and mitochondrial AspAT (AspATm) were not much different from those in control rats. The half-lives of soluble protein and AspATm were calculated to be 3.26-3.72 and 5.02-6.67 days, respectively, in both groups, and that of AspATc in control liver was found to be 4.78 days. The rate of degradation of AspATc in pyridoxine-deficient rat liver could not be calculated, because its kinetics were very complicated; there were apparently at least 2 components with different rates of degradation. Thus pyridoxal 5'-phosphate (PLP) apparently affects both the synthesis and degradation of AspATc, but does not affect the turnover of AspATm in rat liver.View full abstractDownload PDF (437K) -
Hiroshi OHKAWA, Nobuko OHISHI, Kunio YAGI1986 Volume 99 Issue 3 Pages 945-951
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrom a survey of flavin compounds using a high performance liquid chromatograph equipped with a fluorescence detector, 8α-hydroxyriboflavin, its derivative, riboflavin, FMN, and FAD were found in rat organs. The derivative of 8α-hydroxyriboflavin was purified from the kidney sequentially by DEAF column chromatography, paper chromatography, and high performance liquid chromatography, and was determined to be 8α-hydroxyriboflavin 5'-phosphate. The amount of 8α-hydroxyriboflavin in the brain, heart, liver, and kidney of the rat was calculated to be 0.010, 0.026, 0.172, and 0.103 nmol per g of wet tissue, respectively. The amount of 8α-hydroxyriboflavin 5'-phosphate in the brain, heart, and liver was less than 0.002 nmol, while that in the kidney was 0.330 nmol, per g of wet tissue. On the other hand, we could not detect 7α-hydroxyriboflavin in the organ extracts, even though it was excreted into human urine along with 8α-hydroxyriboflavin. This is the first reported occurrence of 8α-hydroxyriboflavin and 8α-hydroxyriboflavin 5'-phosphate in higher animal organs.View full abstractDownload PDF (449K) -
Teruhide YAMAGUCHI, Mizuho KANEDA, Katsuko KAKINUMA1986 Volume 99 Issue 3 Pages 953-959
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo laboratories (Borregaard et al. (1984) J. Biol. Chem. 259, 47; Ohno et al. (1985) J. Biol. Chein. 260, 2409) have reported that a b-type cytochrome (b558) was trans-located into plasma membranes from specific granules in activated neutrophils. In an attempt to examine the cytochrome b translocation, porcine neutrophils were activated by treatment with surface-active agents such as myristate (MA) and phorbol myristate acetate (PMA), and then the postnuclear supernatants of both activated and unactivated cells were fractionated by Percoll density gradient centrifugation with a Zonal rotor. In activated neutrophils, high O2- generating activity was found in the plasma membrane fraction, which showed a peak of Na, K-ATPase activity as a marker enzyme. Cytochrome b558 was recovered 74 to 78% in the plasma membrane fraction and 14 to 16% in granules in either activated or unactivated cells. No change in specific content of cytochrome b558 was observed in plasma membranes before and after activation of cells. Furthermore, in both activated and unactivated cells, vitamin B12-binding protein, a specific granule marker, was mainly found in the bottom fractions and scarcely at all in plasma membranes. These results suggest that no translocation of cytochrome b558 occurs during activation of neutrophils.View full abstractDownload PDF (576K) -
Isamu YAMAMOTO, Hidehito SHIMIZU, Takashi TSUJI, Makoto ISHIMOTO1986 Volume 99 Issue 3 Pages 961-969
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160, 000, and two minor bands. The molecular weight of the enzyme was determined to be 160, 000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-I mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mm, 0.12 mm, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).View full abstractDownload PDF (1151K) -
Yasuhiro OHASHI1986 Volume 99 Issue 3 Pages 971-979
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWith the use of a reconstituted poly (ADP-ribosyl) ating enzyme system and three purified nucleases, micrococcal nuclease (MN), bull seminal RNase (BS RNase) and Ca2+, Mg2+-dependent endonuclease (BS DNase), as model acceptor proteins for ADP-ribose, the effect of ionic strength on the modification reaction was examined in detail. When these three nucleases were extensively poly(ADP-ribosyl)ated in this system at a low ionic strength (5 mm Tris), they were all inhibited by about 80 and the chain length of the polymer covalently bound to the nucleases was 13 to 23 ADP-ribose units. The observed inhibition was markedly prevented by increasing the ionic strength in the reaction mixture with a concomitant decrease in the polymer size bound to the nucleases. The NaCl concentrations required for decreasing the extent of the inhibition to half of the maximum were calculated to be 20, 50, and 100 mm for MN, BS RNase, and BS DNase, respectively. These values are similar to the NaCl concentrations required for decreasing the average chain lengths of the polymer to half, suggesting that the length of polymer is closely correlated to the extent of inhibition of these nucleases. DNA-binding affinities of these nucleases, expressed in terms of the NaCl concentrations required for eluting the enzymes from DNA-cellulose, were 140, 280, and 340 mm for MN, BS RNase, and BS DNase, respectively. Considering that maintainance of a ternary complex of poly (ADP-ribose) synthetase, acceptor and DNA may be essential for the modification reaction, the relatively strong salt effect observed in the modification of MN may be explained by its low DNA-binding affinity.View full abstractDownload PDF (1364K) -
Tatsuya TSURUMI, Koichiro MAENO, Yukihiro NISHIYAMA1986 Volume 99 Issue 3 Pages 981-984
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRestriction enzyme HindIII digestion of the whole genome of herpes simplex virus type 2 strain 186 yielded 10 DNA fragments with molecular weights ranging from approximately 22×106 to 1.2×106, which were cloned into the Hindlll site of bacterial plasmid pACYC 184. The cloned fragments were identified by hybridization to HSV-2 virus DNA and by double digestion with restriction endonucleases. The recombinant plasmids, even if they carried DNA sequences with molecular weights of more than 107, were efficiently replicated in E. coli HB101.View full abstractDownload PDF (718K) -
Fusako USUKI, Shoichi ISHIURA, Hideo SUGITA1986 Volume 99 Issue 3 Pages 985-988
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have defined one type of acid α-glucosidase and two types of neutral α-glucosidases from quail skeletal muscle on the basis of differences in the elution patterns on a DEAE-cellulose column. The appearance of the two neutral α-glucosidase isoenzymes was age-dependent. A decrease in acid α-glucosidase activity was demonstrated in Japanese quails with glycogenosis type II. The characteristics of these three α-glucosidase isoenzymes are described.View full abstractDownload PDF (326K) -
Saori TAKAHASHI, Akiko IRIS, Yoshiaki KATAYAMA, Keiichi ITO, Yoshihiro ...1986 Volume 99 Issue 3 Pages 989-992
Published: March 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe N-terminal amino acid sequences of human urinary prokallikrein and kallikrein have been determined. Their amino acid sequences are as follows. Prokallikrein: (H2N)-Ala-Pro-Pro-5-Ile-Gln-Ser-Arg-Ile1-Val-Gly-Gly-Trp5-Glu-( )-Glu-Gln-His10-Ser-Gln-Pro-Trp-Gln15-Ala-Ala-Leu-Tyr-His20-Phe-Ser-Thr-. Kallikrein: (H2N)-Ile1-Val-Gly-Gly-Trp5-Glu-( )-Glu-Gln-His10-Ser-Gln-Pro-Trp-Gln15-Ala-Ala-Leu-Tyr-His20-Phe-Ser-Thr-Phe-Gln25-. The results showed that prokallikrein comprises an additional seven amino acids at the amino terminus of the kallikrein, of which the sequence is (H2N)Ala-Pro-Pro-Ile-GIn-Ser-Arg (COOH).
Comparison of the structure of this peptide with those of other proteins revealed extensive sequence identity with the propeptide portions of rat and mouse tissue kallikreins, that were predicted from the preproenzyme-encoded nucleotide sequences. The amino acid sequence of the peptide was also highly homologous to that of the propeptide portion of EGF-binding protein, that was predicted from the nucleotide sequence, and that of the a-subunit of NGF. The N-terminal amino acid sequence of kallikrein was completely identical to the reported one (Lottspeich, F., et al. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1947-1950) and shows considerable amino acid sequence homology with the porcine and rat pancreatic kallikreins. As far as the present results are concerned, it is strongly indicated that the inactive kallikrein in human urine is a tissue type prokallikrein which is activated on the release of the N-terminal peptide consisting of seven amino acids.View full abstractDownload PDF (312K)
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