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Volume 103 (1988)
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- Issue 5 Pages 741-
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Volume 103, Issue 5
Displaying 1-35 of 35 articles from this issue
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Daisuke Kohda, Nobuhiro Go, Kyozo Hayashi, Fuyuhiko Inagaki1988 Volume 103 Issue 5 Pages 741-743
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe tertiary structure of mouse epidermal growth factor (EGF) in solution (28°C, pH 2.0) was studied by two-dimensional NMR spectroscopy. Proton-proton distance constraints derived from NOESY spectra were used to construct a mechanical molecular model of mouse EGF, which was subsequently checked by means of a preliminary distance geometry calculation. The chain-folds in the two structural domains of mouse EGF were very similar to those previously reported (Montelione et al. (1987) Proc. Natl. Acad. Sci. U. S. 84, 5226-5230). However, the relative orientations of the two domains were different. Because we could assign much more inter-domain NOEs, the relative orientations of the two domains were well determined in our model. The hollow between the two domains may function as a binding site for the EGF receptor.View full abstractDownload PDF (402K) -
Teruo Miyazawa, Keiichi Yasuda, Kenshiro Fujimoto, Takashi Kaneda1988 Volume 103 Issue 5 Pages 744-746
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA chemiluminescence-high performance liquid chromatography (CL-HPLC) system was newly developed and used for the hydroperoxide-specific determination of phosphatidyl-choline hydroperoxide (PCOOH) in human plasma. The method involves separation of phosphatidylcholine derivatives from plasma lipids by normal phase HPLC and subsequent detection of hydroperoxide-dependent chemiluminescence (CL) of PCOOH. CL was produced through luminol oxidation during the reaction of the hydroperoxide and cytochrome c-heme. The high specificity for the hydroperoxide allows the sensitive assaying of a large PCOOH range over a concentration range of 50-2, 000 pmol of hydroperoxide-O2. Using this method, the occurrence of PCOOH in normal human plasma was strongly suggested and was confirmed quantitatively.View full abstractDownload PDF (321K) -
Shozo Shoji, Akira Tashiro, Yukiho Kubota1988 Volume 103 Issue 5 Pages 747-749
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSN-Myristoyl (N-Myr-) glycinal (aminoacetoaldehyde, GO) diethylacetal (A), which is abbreviated as N-Myr-GOA, and other N-Myr-compounds (N-Myr-Gly-GOA, N-Myr-Gly-Gly-GOA, and N-Myr-Gly-Gly-Gly-GOA) were newly synthesized and then employed for NH2-terminal antimyristoylation of structure proteins in the human T-cell leukemia virus (HTLV-I) and the human immunodeficiency virus (HIV). The protein myristoylation of structure proteins p 19gag, of HTLV-I, and p 17gag, of HIV, was determined separately, using radiolabeled myristic acid, in vitro. The radiolabeled proteins, after immunoprecipitation with an antiserum to adult T-cell leukemia (ATL) or the anti-p 17gag monoclonal antibody of HIV, were identified as p 19gag of HTLV-I and p 17gag of HIV by fluorography after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The protein myristoylation was resistant to NH2OH-treatment. Of the N-Myr-compounds tested, N-Myr-GOA remarkably prevented the myristoylation of p 19gag and p 17gag, but N-Myr-Gly-Gly-GOA and N-Myr-Gly-Gly-Gly-GOA did not.View full abstractDownload PDF (1474K) -
Makoto Miyata, Mitsukuni Yasui, Toshiaki Arata, Akio Inoue1988 Volume 103 Issue 5 Pages 750-754
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe rates of the elementary steps of the actomyosin ATPase reaction were measured using the myosin subfragment-1 of porcine left ventricular muscle. The results could be explained only by the two-route mechanism for actomyosin ATPase (Inoue, Shigekawa, & Tonomura (1973) J. Biochem. 74, 923-934), in which ATP is hydrolyzed via routes with or without accompanying dissociation of actomyosin. The dependence on the F-actin concentration of the rate of the acto-S-1 ATPase reaction in the steady state was measured in 5mM KCl at 20°C. The maximal rate, Vmax, and the dissociation constant for F-actin of the ATPase, Kd, were 3.0 s-1 and 2.2mg/ml, respectively. The Kd value was almost the same as that determined from the extent of binding of S-1 with F-actin during the ATPase reaction. The rate of recombination of the S-1-phosphate-ADP complex, S-1ADPP, with F-actin, vr, was lower than that of the ATPase reaction in the steady state. Thus, ATP is mainly hydrolyzed without accompanying dissociation of acto-S-1 into S-1ADPP and F-actin. In the cardiac acto-S-1 ATPase reaction, the rate of the ATPase reaction in the steady state and that of recombination of S-1ADPP with F-actin were about 1/5 those of the skeletal acto-S-1 ATPase reaction. Under more physiological conditions, i.e., 0.1M KCl and 30°C, the apparent second-order rate constant for the recombination of cardiac S-1ADPP with F-actin (0.022 s-1×(mg/ml)-1) was much smaller than that in the case of skeletal S-1 (0.57 s-1×(mg/ml)-1). The ATP hydrolysis through the dissociation of actomyosin into MADPP+ F-actin and their recombination may not be coupled with muscle contraction, since the velocity and the tension on cardiac muscle contraction do not differ so much from those of rabbit skeletal muscle.View full abstractDownload PDF (569K) -
Ken-ichiro Takamiya1988 Volume 103 Issue 5 Pages 755-758
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe presence of ubiquinol: cytochrome c2 oxidoreductase was shown in the membranes from a photosynthetic bacterium, Rhodopseudomonas palustris. Some properties of the enzyme in situ were investigated. The optimal pH of this enzyme activity was 7.0 in the intact membranes. The activity was inhibited by both antimycin and myxothiazol. Maximal activity (Vmax) was 3-4 mol cytochrome c (c2) reduced/mol cytochrome c1•s. Apparent activity of the enzyme with horse heart cytochrome c as the electron acceptor decreased as the concentration of salts in the reaction mixture increased, whereas when R. palustris cytochrome c2 was used as the electron acceptor, the activity increased as the concentration of salts increased. Moreover, the activity of the enzyme did not depend on the species or concentration of anions but on both the concentration and valency of the cations of the salts. These salt effects were thought to be due to the change of effective concentration of cytochrome molecules caused by cations near the membrane surface, which was net negatively charged. Apparent Km for ubiquinol-1 was about 80 μM irrespective of the species of cytochrome and the presence of salts.View full abstractDownload PDF (379K) -
Mode of Activation and Kinetic Properties of Three Distinct Forms of Protein Kinase C from Rat BrainKazuo Sekiguchi, Masanori Tsukuda, Katsuhiko Ase, Ushio Kikkawa, Yasut ...1988 Volume 103 Issue 5 Pages 759-765
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThree types of protein kinase C, designated types I, II, and III, were purified from rat brain cytosol, and have been shown to correspond to the cDNA clones γ, β, and α, respectively. Their relative activities in the whole brain tissue were roughly 26, 49, and 25% with H 1 histone as a substrate. Type II enzyme was an unequal mixture of two subspecies (roughly 1:7) encoded by βI and βII sequences which differ from each other only in a short range of their carboxyl-terminal end regions. Although the three types have closely similar structures, they showed slightly different modes of activation and kinetic properties. Type I enzyme was less sensitive to diacylglycerol but was significantly activated by low concentrations of free arachidonic acid. Type II enzyme exhibited substantial activity without elevated Ca2+ levels, and responded well to diacylglycerol and, to some extent, arachidonic acid. The type III enzyme responded to diacylglycerol as well as to arachidonic acid. The mode of activation of the enzyme by arachidonic acid required elevated levels of Ca2+ but not phospholipid. In the presence of phospholipid, phorbol esters could activate all three types in a manner similar to diacylglycerol. Among various phospholipids tested, phosphatidylserine was the most effective for all three types. Type III enzyme was most sensitive to 1-stearoyl-2-arachidonylglycerol for activation. Conversely, type I enzyme was activated most efficiently by synthetic permeable diacylglycerols, such as 1, 2-didecanoylglycerol and 1, 2-dioctanoylglycerol. Many heavy metal ions exerted variable and distinct effects on the catalytic activities of these three types. Zn2+ inhibited all types when added in the presence of Ca2+, whereas in the absence of added Ca2+ this metal ion could activate type I but not type II or III enzyme. It is concluded that the three types of protein kinase C show subtle individual characteristics, and possibly play distinctly different roles in the regulation of neuronal functions.View full abstractDownload PDF (2115K) -
Tatsuya Samejima, Yuko Tamagawa, Yoshifusa Kondo, Akira Hachimori, Hir ...1988 Volume 103 Issue 5 Pages 766-772
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSChemical modifications by photooxidation in the presence of rose bengal (RB) and with tetranitromethane (TNM) were carried out to elucidate the amino acid residues involved in the active site of inorganic pyrophosphatase (pyrophosphate phosphohydrolase) [EC 3. 6. 1. 1] from Escherichia coli Q 13. The photooxidation caused almost complete inactivation, which followed pseudo-first-order kinetics depending on pH and concentration of RB. The presence of Mg2+ or complex between Mg2+ and substrate or substrate analogues, imidodiphosphate and sodium methylenediphosphate, gave partial protection against the photoinactivation, whereas the substrate alone showed no protective effect. The enzyme was almost completely inactivated by chemical modification with TNM, depending upon the concentration of TNM. The amino acid analyses and enzyme activity measurements revealed that 2 histidyl residues among 5 photooxidized residues and 2 tyrosyl residues per subunit were essential for the enzyme activity. The circular dichroism (CD) spectra in the far ultraviolet region showed no significant alteration during these two modifications, indicating that the polypeptide chain backbone of the enzyme remained unaltered. However, the modifications altered considerably the CD bands in the near ultraviolet region and the fluorescence spectra, indicating that subtle change in conformation had occurred in the vicinity of the active site in the enzyme molecule. These results strongly suggest that histidyl and tyrosyl residues may be involved in the active site or be located in the vicinity of the active site and seem to participate in the mechanism of stability against heat inactivation.View full abstractDownload PDF (825K) -
Kou Hayakawa, Jun Oizumi1988 Volume 103 Issue 5 Pages 773-777
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe effects of a disulfide reducing agent and sulfhydryl blocking agents on the biotinidase activity in human serum and on the purified biotinidase have been extensively studied by using a newly developed HPLC assay method. This HPLC method directly measures the product (p-aminobenzoate, PAB), and is not interfered with by sulfhydryl-reactive agents. Further, because the substrate solution of this HPLC assay method contains only substrate (biotin 4-amidobenzoate) and phosphate buffer, accurate studies on the effects of sulfhydryl blocking reagents on the purified enzyme could be performed. Biotinidase activities in human sera (n=83) were always enhanced by 2-mercaptoethanol (ME). The optimum concentration was found to be 1mM. The degree of activation was variable (100 to 400% of the original) depending on the serum sample. Sulfhydryl blocking reagents such as organic mercurials were tested on fresh serum and purified enzyme. Mercuric agents were found to inhibit the activity of fresh serum and purified enzyme at 0.05 and 0.005mM, respectively. Sulfhydryl alkylating agents, N-ethylmaleimide (NEM) and dithiobis (2-nitro) benzoic acid (DTNB), inhibited 100 and 64% of the activity of the purified enzyme at 0.1 and 1.0mM, respectively. However, lower concentrations (less than 5 nM) of organic mercurials and mercuric ion exhibited a slight enhancement (20-30%) of the activity of the purified enzyme. These results indicate the presence of an essential sulfhydryl residue at the active center. The enzyme contains 2.5 sulfhydryls per molecule, as determined by using Ellman's assay method. Serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) did not inhibit the enzyme activity at 0.05mM or higher concentration. The presence of a serine residue in the active site is not essential in human serum biotinidase.View full abstractDownload PDF (607K) -
Minoru Takita, Shiro Ikawa, Yoshio Ogura1988 Volume 103 Issue 5 Pages 778-786
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe effects of bile duct ligation on bile acid and cholesterol metabolism were examined in male Wistar strain rats. Quantitative and qualitative changes of bile acids and cholesterol in serum and urine occurred; β-muricholic acid predominantly increased in serum and urine and the ratio of urinary cholic acid and β-muricholic acid changed from about 5:3 on day 1 to about 1:8 on day 5 under biliary obstruction. The form of the increased urinary bile acids was mainly taurine-conjugated and partly sulfated. Under conditions of bile duct ligation on day 5, 14C-labeled 3 β-hydroxy-5-cholenoic, lithocholic, and chenodeoxycholic acids were intragastrically administered to the rats after pretreatment with antibiotics and the metabolites of these three acids were investigated. 3 β-Hydroxy-5-cholenoic acid was most efficiently converted to β-muricholic acid. The present study strongly suggested the presence of an alternative metabolic pathway induced by bile duct ligation, which caused the change in composition of urinary bile acids, and especially the marked increase in β-muricholic acid formation. A possible alternative pathway for bile acid biosynthesis under biliary obstruction in rats is postulated.View full abstractDownload PDF (1124K) -
Atsumi Yamaguchi, Hiroshi Azuma, Satomi Sekizaki, Hidenori Suzuki, Ken ...1988 Volume 103 Issue 5 Pages 787-791
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWhen 50 μM cetiedil alone was added to a platelet suspension, increase in Na+ content, decrease in K+ content, and depolarization of platelet membrane were observed without change in the intracellular concentration of free Ca2+ ( [Ca2+]1) or in the morphology of platelets. The cetiedil-induced depolarization was attenuated by the reduction of extracellular sodium concentration, while sodium transport inhibitors such as procaine and tetrodotoxin failed to modify the depolarization. On the other hand, thrombin caused such changes in platelets as increases in Na+ content, 22Na space and [Ca2+]1, decrease in K+ content, and membrane depolarization. All these changes caused by thrombin were inhibited by cetiedil. It is suggested that cetiedil brought the increased ion transport and subsequent partial depolarization, which might lead to modification of the reaction of platelet membrane induced by thrombin.View full abstractDownload PDF (1563K) -
Tsukasa Seya, Shigeharu Nagasawa1988 Volume 103 Issue 5 Pages 792-796
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe third component of human complement, C 3 is composed of two disulfide-bridged polypeptide chains of Mr 120, 000 (α chain) and Mr 70, 000 (β chain). C 3 has a thioester bond that serves as a binding site for targets when C 3 is activated. Heat treatment of C 3 induces autolytic peptide bond cleavage at the thioester site in the a chain as well as rupture of the thioester bond. The α chain fragments are linked to each other and β chain via disulfide bonds. This study, however, documented that prolonged heating gave rise to liberation of several fragments including β and the larger fragment of α chain. Using a fluorescent thiol reagent and [14C] iodoacetamide, we analyzed thiol residues present on each fragment, and elucidated that the thiol residue exposed by rupture of the thioester bond shifts in turn to another fragment resulting in the liberation of the fragments. The results were compatible with those on C 4, and suggested that the generated thiol residue induces thiol-disulfide interchange reaction. On heating of plasma, fragments of C 3 were not released, while the cleavage of the α chain occurred more effectively. The heated C 3 (56°C, 15min) became insusceptible to C3b inactivator (I) and factor H, suggesting that additional conformational change is accompanied with cleavage of the thioester bond.View full abstractDownload PDF (4347K) -
Yoshiyuki Horio, Tatsuya Tanaka, Masato Taketoshi, Fujio Nagashima, Su ...1988 Volume 103 Issue 5 Pages 797-804
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESScDNA clones for rat cytosolic aspartate aminotransferase (cAspAT, L-aspartate:2-oxoglutarate aminotransferase) [EC 2. 6. 1. 1] were isolated from a rat cDNA library, and the primary structure of the gene for cAspAT was deduced from its cDNA sequence. Rat cAspAT consists of 412 amino acids and its molecular weight is 46, 295. The deduced amino acid sequence of rat cAspAT was compared with the sequences of AspATs from other species. The degree of sequence identities of rat/mouse cAspAT, rat/pig cAspAT, rat/chicken cAspAT, rat/pig mAspAT, and rat/Escherichia coli AspAT were 97.1, 89.6, 81.7, 48.1, and 41.2%, respectively. A coding region of rat cAspAT eDNA was inserted into E. coli expression vector pUC9, and enzymatically active cAspAT was expressed as a β-galactosidase-cAspAT hybrid protein. This hybrid protein represented about 18% of the soluble proteins in E. coli and its kinetic propertis were comparable with those of cAspAT preparations purified from rat liver.View full abstractDownload PDF (3686K) -
Rat Cytosolic Aspartate Aminotransferase: Regulation of Its mRNA and Contribution to GluconeogenesisYoshiyuki Horio, Tatsuya Tanaka, Masato Taketoshi, Toshihiko Uno, Hiro ...1988 Volume 103 Issue 5 Pages 805-808
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSInduction of cytosolic aspartate aminotransferase (cAspAT) was observed in rat liver on administration of a high-protein diet, or glucagon and during fasting. The enzyme activity in the liver of rats given 80% protein diet or glucagon injection during starvation increased to 2- to 2.4-fold that in the liver of rats maintained on 20% protein diet, with about 2-fold increases in the levels of hybridizable cAspAT mRNA, measured by blot analysis using the cloned rat cAspAT cDNA as a probe. No increae in the enzyme was detected in kidney, heart, brain, or skeletal muscle. The activity of mitochondrial aspartate aminotransferase (mAspAT) did not increase. Induction of cAspAT was observed when glucose metabolism tended toward gluconeogenesis. The physiological function of the induction of cAspAT is considered to be to increase the supply of oxaloacetate as a substrate for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) [EC 4. 1. 1. 32] for gluconeogenesis.View full abstractDownload PDF (2914K) -
Akihito Hattori, Koui Takahashi1988 Volume 103 Issue 5 Pages 809-814
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSUsing polyclonal antibodies against paratropomyosin, which is believed to modify the actin-myosin interaction in postrigor skeletal muscles, we studied the localization of paratropomyosin in chicken breast muscle myofibrils. Intact myofibrils stained with fluorescent antibodies showed that paratropomyosin was exclusively located at the A-I junction region of sarcomeres. In stretched myofibrils (3.7 μm in sarcomere length), the approximate width of the fluorescent stripes and their relation to the A band remained constant. Removal of the A band from myofibrils led to loss of stainability. During postmortem storage of muscles, on the other hand, paratropomyosin was translocated from its original position at the A-I junction region onto thin filaments. The translocation of paratropomyosin was successfully induced with a calcium ion concentration of 10-4M in the presence of protease inhibitors. We therefore conclude that in postrigor muscles, paratropomyosin is released from the A-I junction region following the increase in the sarcoplasmic calcium ion concentration to 10-4M, and then binds to thin filaments, which results in weakening of rigor linkages formed between actin and myosin.View full abstractDownload PDF (5757K) -
Ryohei Yanoshita, Ichiro Kudo, Koichi Ikizawa, Hyuen Wook Chang, Susum ...1988 Volume 103 Issue 5 Pages 815-819
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe examined the substrate specificity of PAF-degrading enzymes from various sources using platelet activating factor (PAF) and its synthetic analogs. The results were as follows: 1) Tissue-originated acetylhydrolases, such as rat kidney soluble enzyme, deacetylated 1S-methyl-1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (1S-Me-PAF) slightly more rapidly than PAF, whereas plasma acetylhydrolase hydrolyzed PAF more effectively than 1S-Me-PAF. 2) Rat polymorphonuclear leukocytes, monocytes, and lymphocytes homogenates showed an appreciable acetylhydrolase activity, the substrate specificity of which resembled that of the plasma enzyme. 3) Pleural exudates in an experimental pleurisy induced in rats by carrageenan contained an acetylhydrolase activity, the properties of which were similar to those of the plasma enzyme. 4) An extracellular phospholipase A2 activity, which was also observed in the pleural exudate and required Ca2+ ion for maximum activity, seemed not to participate in the deacetylation of PAF, since addition of EDTA did not affect the PAF deacetylation catalyzed by the pleural exudate. These findings indicate that the inactivation reaction of PAF present in the extracellular space is mainly catalyzed by plasma acetylhydrolase, which yields lysoPAF.View full abstractDownload PDF (640K) -
Peptide Boronic Acids, Substrate Analogs, Inhibit Chymase, and Histamine Release from Rat Mast CellsYasuhisa Kato, Hiroshi Kido, Naomi Fukusen, Nobuhiko Katunuma1988 Volume 103 Issue 5 Pages 820-822
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPeptide boronic acids, such as methoxysuccinyl-Ala-Ala-Pro-(L) boro-Phe-OH, its pinacol ester, and t-butyloxycarbonyl-Phe-Pro-(L) boro-Phe-pinacol, inhibited the activity of chymase from connective tissue mast cells approximately 40- to 80-fold more than atypical chymase from mucosal mast cells, and did not inhibit trypsin. Only peptide boronic acids containing “L” forms of boronic acids were inhibitory. The K1 values of these peptide boronic acids for chymase were in the 60-170 nM concentration range, like those of the natural inhibitors tested, but all the natural inhibitors tested except Eglin C and chymostatin inhibited both chymase and trypsin. Thus these peptide boronic acids should be useful for selective inhibition of chymase with less inhibitory activity for atypical chymase and without inhibition of trypsin. These peptide boronic acids markedly inhibited histamine release induced by anti-rat immunoglobulin E, suggesting that chymase in connective tissue mast cells plays some role in the process of histamine release. These peptides are assumed to be therapeutically useful for treatment of allergic inflammations catalyzed by chymase.View full abstractDownload PDF (388K) -
Katsuyoshi Nakazato, Takaki Yamamura, Kazuo Satake1988 Volume 103 Issue 5 Pages 823-828
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe study of guanidine-HCl or thermal denaturation of diferric ovotransferrin (Fe2Tf) has revealed a simultaneous unfolding of the two domains of the protein (Ikeda et al. (1985) FEBS Lett. 182, 305-309). In urea denaturation of Fe2Tf, however, two distinct steps of unfolding were observed in the urea concentration range from 4.5 to 9M at pH 8.0 and 37°C by measuring the residual iron-bound protein (absorbance at 465 nm) and the remaining folded structures (circular dichroism at 222 nm). From a study of urea denaturation of partially iron-saturated Tf whose iron preferentially occupied the N-domain, it was found that the first and the second steps of denaturation corresponded to those of the N-terminal (4.5-6M urea) and C-terminal domains (over 7M urea), respectively. The N-domain of Fe2Tf was selectively unfolded in 7M urea and digested with trypsin to provide an iron-bound C-terminal fragment (42 kDa) in good yield (about 80% of theoretical). The kinetic analysis of the decrease in A465 of Fe2Tf in 9M urea showed that the N-domain unfolded 3×102 times faster than the C-domain. With partially iron-saturated Tf, the decrease of A465 in 9M urea also proceeded in a biphasic manner and the ratio, the decrement in A465 of the rapid phase/the decrement in A465 of the slow phase, gave the value of iron distribution as Fe at the N-site/Fe at the C-site.View full abstractDownload PDF (1649K) -
Jun Kino, Eijiro Adachi, Tsuyoshi Yoshida, Keisuke Nakajima, Toshihiko ...1988 Volume 103 Issue 5 Pages 829-835
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSWe have produced four monoclonal antibodies against type IV collagen obtained from human placenta. An antibody with a high titer by ELISA, named JK-199, reacted not only with type IV collagen in the triple-helical conformation but also with thermally denatured chains. After affinity chromatography on JK-199 antibody-coupled resin, the amino acid composition and CD spectrum of the affinity-purified peptides from the crude pepsin extract of human placenta were typical of those of human type IV collagen in the triple-helical conformation. On SDS-polyacrylamide gel electrophoresis, the purified protein showed only one broad band with a molecular weight of approximately 260, 000 before reduction and six smaller peptide bands after reduction. On immunoelectroblotting, JK-199 reacted with all six peptide bands. Immunohistochemically, typical basement membranes were exclusively and strongly stained with JK-199 on frozen sections of PLP-fixed human placentas without any enzymatic pretreatment in the routine immunoperoxidase method. Judging from these findings, it is concluded that the epitopes of type IV collagen that reacted with JK-199 are exposed on the surface of basement membranes. This antibody should be useful for identification of type IV collagen in normal or pathological basement membranes or other structures.View full abstractDownload PDF (3669K) -
Kazuo Ishii, Haruo Okajima, Youji Okada, Hiroshi Watanabe1988 Volume 103 Issue 5 Pages 836-839
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSMature salmon roe lipids were found to consist of triacylglycerols (63%), phospholipids (30%), sterols (4.2%), steryl esters (0.7%), and other minor components. In the steryl esters and phospholipids, furan fatty acids were detected instead of the triacylglycerols of the testes lipids in male fish. The representative 12, 15-epoxy-13, 14-dimethyleicosa-12, 14-dienoic acid (F6) amounted to 3.8% and 0.6% of the total fatty acids in each fraction, respectively. However, the absolute amount of the acid in the phospholipid was much more than that contained in the steryl esters. The characteristic distribution of the furan acids found in the phospholipids was common to the steryl esters in the liver. Large amounts of furan acids were contained in phosphatidylcholine (PC) rather than in phosphatidylethanolamine. For positional analysis of furan fatty acids in PC, furan-containing species in the molecule were concentrated fourteenfold by using selective hydrogenation and repeated silica gel column chromatography. A series of furan fatty acids in PC was found to be exclusively linked to the sn-1 position. The amount of the acids in the roe phospholipids was comparable with that in the testes triacylglycerols. The physiological roles of furan fatty acids are discussed.View full abstractDownload PDF (393K) -
Keiko Nohara-Uchida, Kunimitsu Kaya1988 Volume 103 Issue 5 Pages 840-842
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSTaurolipids A and B, which are detergent-type compounds isolated from protozoan Tetrahymena cells, were demonstrated to inhibit strongly the activity of Clostridium perfringens sialidase. On addition of 280 pmol of taurolipid B to 20 mU of the enzyme, the sialidase activity was decreased to 7% of the original activity at pH 5.1 as the optimum pH. The inhibition was non-competitive. Effective inhibition was observed at the acidic region from the isoelectric point of the sialidase, and at a low ionic strength. Both the long chain acyl and sulfonic acid groups of taurolipids were required for the inhibition of the sialidase activity. A mechanism is postulated for the inhibition.View full abstractDownload PDF (327K) -
Minoru Yamanoue, Koui Takahashi1988 Volume 103 Issue 5 Pages 843-847
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSParatropomyosin is a myofibrillar protein believed to weaken rigor linkages formed between actin and myosin. Using glycerinated fibers of rabbit psoas muscles, we studied the effect of paratropomyosin on the weakening of rigor linkages, which was followed in terms of the increase in sarcomere length of rigor-shortened muscles. The rigor tension developed was reduced to about 65% of the initial value within 10min after the addition of purified paratropomyosin, whereas it remained constant for at least 3.5 h in control fibers. Paratropomyosin showed a stronger effect on the increase in sarcomere length of passively stretched fibers, which developed weaker rigor-tensions. The purpose of our research was to establish a rigor solution which would best permit the observation of the workings of paratropomyosin. The most successful rigor solution contained 0.2-0.25M KCl, pH 5.5, at 5-10°C. Under these conditions, the sarcomere length was easily increased from 2.4 to 3.6 μm, if rigor-contracted fibers were passively stretched after the addition of purified paratropomyosin. Because the experimental conditions coincide well with those of postmortem muscles, it is very probable that paratropomyosin plays an important role in restoration of the sarcomere length of rigor-shortened muscles, resulting in tenderization of meat during postrigor ageing.View full abstractDownload PDF (626K) -
Shigeharu Takai, Toshikazu Nakamura, Nobuhiko Komi, Akira Ichihara1988 Volume 103 Issue 5 Pages 848-852
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSAddition of epinephrine to primary cultured adult rat hepatocytes stimulated their DNA synthesis dose-dependently, especially in presence of insulin and epidermal growth factor. This effect of epinephrine was strongly inhibited by an α1-antagonist, prazosin, but not by a β-antagonist, propranolol, and was also slightly inhibited by an α2-antagonist, yohinbin. These results indicate that the stimulation of DNA synthesis of hepatocytes by epinephrine is mediated predominantly by an α1-action. 12-o-Tetradecanoylphorbol-13-acetate (TPA) or Ca2+-ionophore A-23187 stimulated DNA synthesis of Swiss 3T3 cells, but did not induce DNA synthesis of hepatocytes either singly or in combination. The fact that pretreatment of hepatocytes with TPA caused down-regulation of the stimulatory effect of epinephrine on DNA synthesis of hepatQcytes within 15min suggested that the effect of epinephrine on hepatocytes is mediated by its a, receptor and that TPA activated protein kinase c in the hepatocytes. Addition of dibutyryl cGMP did not induce DNA synthesis of hepatocytes. Therefore, the α1-action of epinephrine that induce stimulation of DNA synthesis of primary cultured adult rat hepatocytes was apparently not mediated by either activation of phospholipid-dependent protein kinase or Ca2+ mobilization. Possible alternative mechanism was discussed.View full abstractDownload PDF (562K) -
Shin-ichi Hayashi, Tsuneo Omura, Takeshi Watanabe, Kyuichiro Okuda1988 Volume 103 Issue 5 Pages 853-857
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSPolyclonal antibody elicited in a rabbit against purified cytochrome P-450cc25, which catalyzes 25-hydroxylation of vitamin D3, inhibited not only 25-hydroxylation of cholecalciferol and 1α-hydroxycholecalciferol, but also 16α- and 2α-hydroxylation of testosterone catalyzed by the purified P-450cc25 preparation. Antibody inhibition experiments with microsomes revealed that most 16α- and 2α-hydroxylation of testosterone and most 25-hydroxylation of cholecalciferol by male rat liver microsomes were catalyzed by P-450cc25. In order to examine the identity of cholecalciferol 25-hydroxylase and testoster-one 16α-hydroxylase, monoclonal antibodies recognizing three different epitopes of P-450cc25 were prepared from hybridoma clones produced by fusion of mouse myeloma cells (P3X63Ag8U1) with the spleen cells of immunized BALB/c mouse. All of these monoclonal antibodies inhibited both 25-hydroxylation of la-hydroxycholecalciferol and 16α-hydroxylation of testosterone by purified P-450cc25. These observations suggested that immunochemically indistinguishable form (s) of cytochrome P-450 catalyzed both reactions.View full abstractDownload PDF (1581K) -
Shin-ichi Hayashi, Ken-ichirou Morohashi, Hidefumi Yoshioka, Kyuichiro ...1988 Volume 103 Issue 5 Pages 858-862
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSRat cytochrome P-450 (M-1) cDNA was expressed in Saccharomyces cerevisiae TD 1 cells by using a yeast-Escherichia coli shuttle vector consisting of P-450 (M-1) cDNA, yeast alcohol dehydrogenase promoter and yeast cytochrome c terminator. The yeast cells synthesized up to 2×105 molecules of P-450 (M-1) per cell. The microsomal fraction prepared from the transformed cells contained 0.1 nmol of cytochrome P-450 per mg of protein. The expressed cytochrome P-450 catalyzed 16α- and 2α-hydroxylations of testosterone in accordance with the catalytic activity of P-450 (M-1), but did not hydroxylate vitamin D3 or 1α-hydroxycholecalciferol at the 25 position. The expressed cytochrome P-450 also catalyzed the oxidation of several drugs and did not show 25-hydroxylation activity toward 5β-cholestane-3α, 7α, 12α-triol. However, it cross-reacted with the polyclonal and monoclonal antibodies elicited against purified P-450cc25 which catalyzed the 25-hydroxylation of vitamin D3. These results indicated that P-450 (M-1) cDNA coded the 2α- and 16α-hydroxylase of testosterone, and that these two positions of testosterone are hydroxylated by a single form of cytochrome P-450. Vitamin D3 25-hydroxylase and testosterone 16α-and 2α-hydroxylase are different gene products, although these two hydroxylase activities are immunochemically indistinguishable.View full abstractDownload PDF (1472K) -
Shin-ichi Hayashi, Emiko Usui, Kyuichiro Okuda1988 Volume 103 Issue 5 Pages 863-866
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCholecalciferol 25-hydroxylase was partially purified by polyethylene glycol fractionation and chromatographies on octylamino-Sepharose and hydroxylapatite columns starting from the liver microsomes of female rats, and compared with P-450cc25 purified from the liver microsomes of male rats (Hayashi, et al. (1986) J. Biochem. 99, 1753-1763). On octylamino-Sepharose 4 B column chromatography, most of the activity was recovered in the fraction eluted with 0.08% Emulgen 913 in the case of the male enzyme, whereas the female enzyme was recovered in the fraction eluted with 0.2% Emulgen. Anti-cc25 antibodies against purified male P-450cc25 inhibited the 25-hydroxylation activity of male polyethylene glycol (PEG) fraction and partially purified male enzyme, but did not inhibit the activities of the corresponding female fractions. The antibodies formed a single precipitation line with male P-450cc25, but did not form a precipitation line with partially purified female 25-hydroxylase on immuno-diffusion. These observations indicated that the vitamin D3 25-hydroxylase in female rat liver microsomes is a different entity from that of male rats.View full abstractDownload PDF (1682K) -
Tanetoshi Koyama, Ichirou Yoshida, Kyozo Ogura1988 Volume 103 Issue 5 Pages 867-871
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSAn undecaprenyl diphosphate synthase fraction, which was free of other prenyltransferases and was active without the addition of detergent or phospholipid, was obtained by Sephadex G-100 chromatography of cell-free extracts of Micrococcus luteus B-P 26 cells. The addition of small amounts of Triton X-100 to this fraction caused a marked loss of the enzyme activity, but the activity was gradually restored as further detergent was added. When the enzyme fraction was chromatographed on DEAF-cellulose, the synthase was partially purified, but the activity was not detected unless assayed with addition of the detergent or a lipid fraction of this bacterium. Among the three phospholipids isolated from this bacterium, cardiolipin and phosphatidylglycerol had a marked effect in activating lipid-depleted undecaprenyl diphosphate synthase, but O-lysyrphosphatidylglycerol, which occurs prominently in this bacterium, had little effect.View full abstractDownload PDF (506K) -
Sumiko Odani, Naoya Kenmochi, Kikuo Ogata1988 Volume 103 Issue 5 Pages 872-877
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe amino acid compositions of 24 proteins of 40 S ribosomal subunits of Artemia sauna cysts were determined and compared with those of rat liver. The basic proteins of A. sauna 40 S ribosomes were separated by two-dimensional polyacrylamide gel electrophoresis and extracted with 70% formic acid. Samples were freed from contaminants by gel-filtration through a high-performance liquid chromatography column. Amino acid compositions were determined for individual proteins by pre-column derivatization with N, N-dimethylaminoazobenzenesulfonyl chloride followed by reverse phase high-performance liquid chromatography. The similarity of amino acid compositions between A. sauna and rat liver 40 S ribosomal proteins was evaluated by the method of Cornish-Bowden (Cornish-Bowden, A. (1980) Anal. Biochem. 105, 233-238), and possible relationships between A. sauna and rat were detected for 16 protein species (S2, S3, S4, S6, S7, S8, S15a, 516, S17, and S18, strongly related and S14, 515, 520, 523, S24, and S26, weakly related), indicating a conservative nature of eukaryotic ribosomal proteins.View full abstractDownload PDF (628K) -
Isao Fujii, Yutaka Ebizuka, Ushio Sankawa1988 Volume 103 Issue 5 Pages 878-883
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSAn enzyme activity which catalyzes the ring cleavage of the anthraquinone questin to form benzophenone desmethylsulochrin was found in the cell-free extract of Aspergillus terreus, a (+) -geodin producer. The product was identified as desmethylsulochrin by high-resolution mass spectroscopy and chemical carrier dilution analysis. The enzyme showed an absolute requirement of NADPH and molecular oxygen. Therefore, the enzyme, named questin oxygenase, was considered to be classified as a monooxygenase. The optimum pH was around 7.5. The enzyme was very unstable and lost its activity completely after storage overnight at 4°C in 0.05M phosphate buffer, pH 7.5. The instability of the questin oxygenase was partially overcome by the addition of polyols and the non-ionic detergent Tween 80 to the buffer. By DEAE-cellulose column chromatography, two protein fractions, named DE-I and DE-II, were obtained. Neither fraction reacted with questin by itself. However, the combination of DE-I and DE-II reconstituted the questin oxygenase system to convert questin to desmethylsulochrin. This result suggested that the system is not a simple combination of oxygenase and hydrolase, but requires some additional fator(s) such as electron transfer protein.View full abstractDownload PDF (609K) -
Masashi Fukuzawa, Hiroshi Ochiai1988 Volume 103 Issue 5 Pages 884-888
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSThe preparation and properties of monoclonal antibodies against carbohydrate-binding proteins (discoidin I and discoidin II) in the cellular slime mold, Dictyostelium discoideum are described. Monoclonal antibody (mAb) ndI, II-1 bound both discoidins I and II specifically. mAb nI-1 and mAb dI-1 bound only discoidin I but their binding specificities were different: nI-1 recognized the native form and dI-1 the denatured form. mAb dII-1 bound only denatured discoidin II. In preliminary work mAbs dII-1 and nI-1 were found to be useful for localizing discoidins I and II immunohistochemically.View full abstractDownload PDF (2669K) -
Etsuko Yasugi, Takeshi Kasama, Yousuke Seyama1988 Volume 103 Issue 5 Pages 889-893
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSA large amount of branched long chain bases was detected in the cerebrosides of guinea pig Harderian gland. The long chain bases of cerebrosides were analyzed by GLC as trimethylsilyl derivatives. The branched long chain bases were separated into four peaks (I, II, III, IV) according to the number of carbon atoms and the position of branching. In the present work, the structures of long chain bases in the four peaks were analyzed by GLC and GC-MS after conversion of them to aldehydes, alcohols, and fatty acids. Furthermore the main component of long chain bases (Peak II) was isolated by HPLC as N-acetyl derivatives and analyzed by NMR. The structures of branched long chain bases in Peaks 1, II, III, and IV are as follows. Branched long chain bases of Peak I are 2-amino-10- (main component), 2-amino-9-, and 2-amino-8-methylhexadecane-1, 3-diol. Branched long chain bases of Peak II also consist of a mixture of 2-amino-10-, 2-amino-9-, and 2-amino-8-methyl-beptadecane-1, 3-diol. The branched long chain base of Peak III is 2-amino-10-methyl-octadecane-1, 3-diol, while that of Peak IV is 2-amino-16-methyloctadecane-1, 3-diol. Among these branched long chain bases, 10-methylsphinganines are dominant though the chain lengths are different. These branched long chain bases, in which the substituted positions exist in the middle part of aliphatic chain (10-, 9-, or 8-methylsphinganine) are novel long chain bases in mammals.View full abstractDownload PDF (539K) -
Shinobu Sueyoshi, Kazuo Yamamoto, Toshiaki Osawa1988 Volume 103 Issue 5 Pages 894-899
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSCarbohydrate binding specificity of a lectin, allo A, isolated from a beetle (Allomyrina dichotoma), was investigated by means of lectin affinity chromatography. Sialylated complex-type and hybrid-type oligosaccharides/glycopeptides, and sialyllactose were retained by the column, whereas desialylated ones were retarded but not retained by the column. The association constants of allo A for biantennary oligosaccharides from human serum transferrin, determined by frontal analysis, were 8.0×105 M-1, 4.5×105 M-1, and 2.5×105 M-1 for disialo-, monosialo-, and asialo-oligosaccharides, respectively. Removal of the β-galactose residues markedly reduced the association constant to 3.5×103 M-1. Furthermore, allo A was found to have no affinity for mucin-type glycopeptides carrying the sialylated Gal β1→3 GalNAc sugar sequence (Ka:3.5×103 M-1). The results of this study indicated that allo A strongly binds to the trisaccharide structure, NeuAc α 2-3 (6) Gal-β1-4GlcNAc, and that its binding potency is affected by the inner core structures of oligosaccharides and glycopeptides, because the presence of a bisecting N-acetyl-glucosamine residue and an α-fucose residue linked to the innermost N-acetylglucosamine residue reduced the association constants for oligosaccharides and glycopeptides.View full abstractDownload PDF (685K) -
1988 Volume 103 Issue 5 Pages 900a
Published: 1988
Released on J-STAGE: June 30, 2008
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1988 Volume 103 Issue 5 Pages 900b
Published: 1988
Released on J-STAGE: June 30, 2008
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1988 Volume 103 Issue 5 Pages 900c
Published: 1988
Released on J-STAGE: June 30, 2008
JOURNAL FREE ACCESSDownload PDF (47K) -
1988 Volume 103 Issue 5 Pages 900d
Published: 1988
Released on J-STAGE: June 30, 2008
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