巻号一覧
98 巻, 5 号
選択された号の論文の29件中1～29を表示しています
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Toshifumi YUUKI, Takako NOMURA, Hidetoshi TEZUKA, Akio TSUBOI, Hideo Y ...1985 年 98 巻 5 号 p. 1147-1156
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe gene coding for the heat-stable and pH-stable α-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1, 948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis α-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55, 200. The amino acid sequence of B. licheniformis α-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus α-amylase and relatively heat-unstable Bacillus amvloliquefaciens α-amylase, respectively. Nevertheless, several regions of the α-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.抄録全体を表示PDF形式でダウンロード (1597K) -
Karl MUELLER, Manfred FREIBURG, Gudrun HOYER-FEIGE1985 年 98 巻 5 号 p. 1157-1167
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA procedure for the separation and purification of DNA-dependent RNA polymerases [EC 2. 7. 7. 6] from macronuclei of Tetrahymena pyriformis is described. We have used it to isolate and characterize the class I enzyme. RNA polymerase J was identified by its resistance against α-amanitin and its location in nucleoli. The purified enzyme consists of at least 12 major subunits with approximate molecular weights of 180, 000, 118, 000, 37, 500, 36, 000, 29, 000, 27, 500, 20, 000, 18, 500, 15, 600, 14, 500, 13, 500, and 12, 600. Chromatography on DEAE-Sephadex separated two forms of RNA polymerase I which differed in the presence of an additional polypeptide of 25 kDa. Independently of this polypeptide, the enzyme was found to segregate on DNA cellulose into a binding and a non-binding fraction. This type of heterogeneity was found to be unrelated to differences in molar ratios or molecular weights of the enzyme subunits. The catalytic properties of all enzyme subfractions were very similar and complied with the general characteristics of RNA polymerase I [cf. Roeder, R. G. (1976) in RNA Polymerase (Losick, R. & Chamberlin, M., eds.) pp. 285-329, Cold Spring Harbor Publ. Co., New York].抄録全体を表示PDF形式でダウンロード (1689K) -
Michael E. BREIMER, Gunnar C. HANSSON, Hakon LEFFLER1985 年 98 巻 5 号 p. 1169-1180
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーTotal non-acid and acid glycolipid fractions were isolated from epithelial cell scrapings and the non-epithelial residue of a human upper ureter. The glycolipid fractions were structurally characterized as total mixtures by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy. Selected structural information was also obtained on binding of monoclonal antibodies and bacteria to the thinlayer chromatograms. The major epithelial cell glycolipids were Glcβl-lceramide (75%), dihexosylceramide (10%) and NeuAcLacceramide (10%). In addition, 8 minor glycolipids belonging to the blood group P, Lewis and ABO systems were identified. The major glycolipids of the non-epithelial residues were mono- and dihexosylceramides together with globotriaosyl- and globotetraosylceramides. The epithelial mono- and diglycosylceramide compounds had an unusual ceramide composition with mainly C18 and C20 trihydroxy long chain bases in combination with C22-C24 hydroxy fatty acids in contrast to the non-epithelial glycolipids which contained mainly C18 dihydroxy long chain bases in combination with C16-C24 non-hydroxy fatty acids.抄録全体を表示PDF形式でダウンロード (1883K) -
Yasuko KAWAMURA-KONISHI, Haruo SUZUKI1985 年 98 巻 5 号 p. 1181-1190
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーBinding of hemin to globin was studied in the presence of 25mM caffeine by measuring CD and optical absorption changes in the Soret region. CD and optical absorption spectra after mixing equimolar amounts of hemin and globin were the same as those of ferric hemoglobin. In contrast, addition of excess globin to hemin formed a complex that was distinguishable from ferric hemoglobin in terms of the CD and optical absorption spectra. By comparing the spectra of the complex with those of various hemoglobin derivatives, it was concluded that the complex was globin which carried a heroin exclusively on the a chain. This means that the α chain of the globin molecule has a greater affinity for hemin than the β chain, as observed by other investigators using hemin-cyanide.
The rate of binding of hemin to globin was estimated by the use of CD and optical absorption stopped-flow apparatus. The rate of hemin binding to the α chain of globin was obtained by mixing hemin and excess globin, and that to the β chain was obtained by mixing equimolar concentrations of hemin and globin. The results showed that (1) hemin was bound to the α chain in the globin molecule to form a transient intermediate, followed by its transformation into another intermediate, (2) the transformation was the rate-limiting step, and (3) the fl chain in the globin molecule had a greater affinity for heroin after hemin binding to the α chain than before.抄録全体を表示PDF形式でダウンロード (734K) -
Takehiko KOIDE, Shoji ODANI, Teruo ONO1985 年 98 巻 5 号 p. 1191-1200
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe simple and simultaneous purification of histidine-rich glycoprotein (HRG) and antithrombin III (AT III) from human plasma and gross structural characterization of HRG have been performed. The purification method consists of two chromatographic procedures using heparin-agarose and DEAE-Sephadex. The yields of HRG and AT III were 22mg and 70mg, respectively, from 1 liter of plasma. The purified HRG is a single-chain polypeptide with a molecular weight (Mr) of 75, 000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, indicating it was the native form of this protein. Cyanogen bromide cleavage of HRG, followed by analysis of the amino acid composition and determination of the amino-terminal sequence of each purified cyanogen bromide fragment established that the gross structure of HRG consisted of three cyanogen bromide fragments; an amino-terminal CN-50 kDa fragment (Mr 50, 000) and a carboxy-terminal small fragment of eight amino acids, and a CN-30 kDa fragment (Mr 30, 000) between them. As to the amino acid composition of the CN-30 kDa fragment, it had an unusually high content of histidine (25 mol%), suggesting the presence of a histidine-rich region (s) in the carboxy-terminal half of the molecule. These results together with our previous results (Koide, T., Odani, S., & Ono, T. (1982) FEBS Lett. 141, 222-224) and those of Morgan (Morgan, W. T. (1985) Biochemistry 24, 1496-1501) imply that HRG is composed of at least two domains with distinct functional properties; i.e. an aminoterminal domain with heparin-binding ability and a carboxy-terminal domain with heme- and divalent metal-binding abilities.抄録全体を表示PDF形式でダウンロード (1538K) -
Ichiro TANII, Masanori OSAFUNE, Toshiaki ARATA, Akio INOUE1985 年 98 巻 5 号 p. 1201-1209
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーMyosin was purified rapidly from the nematode Caenorhabditis elegans by an improved method. Crude actomyosin was extracted from the worms at low ionic strength. Paramyosin was removed by repeating the precipitation of myosin filaments in the presence of Mg2+ and the dissolution of them in 0.6 M NaCl. Actin was removed by ultracentrifugation in the presence of Mg-ATP and finally by column chromatography on DEAE-cellulose. This method gave a good yield of myosin (20-30mg from 50g wet weight of worms), and its EDTA (K+)-ATPase activity was about 3-fold higher than that of myosin prepared by the method of Harris and Epstein (1979). ATP hydrolysis by nematode myosin showed an initial P1-burst due to formation of the myosin-phosphate-ADP complex. Tryptophan fluorescence of myosin was enhanced about 8% by ATP. The relationship between the structure and function of myosin is discussed based on the above results and the amino acid sequences of myosins from rabbit skeletal muscle and Caenorhabditis elegans.抄録全体を表示PDF形式でダウンロード (1562K) -
Kouji MATSUOKA, Toshiyuki KUREBAYASHI, Kinuko KIMURA1985 年 98 巻 5 号 p. 1211-1219
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe glutamine synthetase from Bacillus cereus IFO 3131 was purified to homogeneity. The enzyme is a dodecamer with a molecular weight of approximately 600, 000, and its subunit molecular weight is 50, 000. Both Mg2+ and Mn2+ activated the enzyme as to the biosynthesis of L-glutamine, but, unlike in the case of the E. coli enzyme, the Mg2+-dependent activity was stimulated by the addition of Mn2+. The highest activity was obtained when 20mm Mg2+ and 0.5mm Mn2+ were added to the assay mixture. For each set of optimal assay conditions, the apparent Km values for glutamate, ammonia and a divalent cation•ATP complex were 1.03, 0.34, and 0.40mm (Mn2+:ATP=1:1); 14.0, 0.47, and 0.91mm (Mg2+:ATP=4:1); and 9.09, 0.45, and 0.77mm (Mg2+:Mn2+:ATP=4:0.2:1), respectively. At each optimum pH, the Vmax values for these reactions were 6.1 (Mn2+-dependent), 7.4 (Mg2+-dependent), and 12.9 (Mg2+ plus Mn2+-dependent) μmoles per min per mg protein, respectively. Mg2+-dependent glutamine synthetase activity was inhibited by the addition of AMP or glutamine; however, this inhibitory effect was suppressed in the case of the Mg2+ plus Mn2+-dependent reaction. These results suggest that the activity of the B. cereus glutamine synthetase is regulated by both the intracellular concentration and the ratio of Mn2+/Mg2+ in vivo. Also in the present investigation, a potent glutamine synthetase inhibitor (s) was detected in crude extracts from B. cereus.抄録全体を表示PDF形式でダウンロード (2246K) -
Tatsuzo FUJII, Akira TAMURA, Tsunehiko YAMANE1985 年 98 巻 5 号 p. 1221-1227
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリー14C-Labeled phosphatidylcholine (PC) and lysophosphatidylcholine (lysoPC) species with two homologous saturated acyl chains and of a saturated acyl chain of various lengths, respectively, were each incorporated into the outer leaflet of the membrane lipid bilayer of intact human erythrocytes, and the transbilayer movement into the inner leaflet during incubation at 37°C of the lipid-loaded erythrocytes was followed.
The labeled PC and lysoPC molecules present in the outer leaflet were extracted with egg-yolk PC liposome suspension and BSA solution, respectively, and the amount which moved into the inner leaflet during the incubation was measured by determining the residual amount of the labeled lipid in the membrane. Translocation of lysoPC molecules was also measured by assaying the decrease in the amount of the added labeled lysoPC in the membrane during the incubation on the basis of the previously reported fact that lysoPC molecules are all converted metabolically to PC or glycerylphosphorylcholine plus fatty acid as soon as they are translocated from the outer to the inner leaflet.
Every lipid tested showed significant transbilayer movement during the course of the incubation for up to 10 h. With the C8, C10, and C12 species of PC the rate of the transbilayer movement increases with decreasing acyl chain length. The same is true with the C14, C18, and C18-lysoPC species.抄録全体を表示PDF形式でダウンロード (551K) -
Motohiro MATSUURA, Akihiro YAMAMOTO, Yasuhiko KOJIMA, J. Yuzuru HOMMA, ...1985 年 98 巻 5 号 p. 1229-1237
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーAnalogues of the nonreducing sugar part of lipid A were chemically synthesized and tested for biological activities such as Limulus amebocyte lysate gelation, interferon- and tumor necrosis factor-induction, lethal toxicity in galactosamine-sensitized mice, and pyrogenicity.
A 4-O-monophosphorylglucosamine derivative possessing 2-N-3-tetradecanoyl-oxytetradecanoyl and 3-O-tetradecanoyl groups (GLA-27) exhibited all activities tested except for pyrogenicity. Alteration of the acyl substituents or dephosphorylation as well as acylation or phosphorylation of the 6-OH caused most activities of GLA-27 to diminish or disappear altogether. On the other hand, the biological activities expressed by GLA-27 were not significantly affected even when the glucosamine backbone was changed to 1-deoxy type, epimer type at C-3 (allose form), or 3-amino type.
These results indicate that the acyl substituents and the phosphorylation positions rather than the backbone structures in these partial structure analogues of lipid A affect the expression of biological activities of endotoxin. The results also clearly indicate that some biological activities of endotoxin can be expressed separately from pyrogenicity.抄録全体を表示PDF形式でダウンロード (669K) -
Hideaki WATANABE, Eiichi ANDO, Kazuko OHGI, Masachika IRIE1985 年 98 巻 5 号 p. 1239-1245
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーIn order to estimate the size of the active site of guanylic acid specific RNases (RNase T1 from Aspergillus oryzae and RNase St from Streptomyces erythreus) and guaninepreferential RNase (RNase Ms from A. saitoi), the depolymerization reaction of oligoinosinic acid, (Ip)nI>p, having various chain lengths was studied. The kinetic parameters for depolymerization of oligoinosinic acids, (pKm, log V and log V/Km) by the three RNases increased with increase of the chain length of the substrates, and became almost constant at n=2 or more. Thus, the size of the active site of RNase T1, RNase St, and RNase Ms was estimated to be three nucleotides in length.抄録全体を表示PDF形式でダウンロード (507K) -
Kenji NISHIMURA, Akira NAKAMURA1985 年 98 巻 5 号 p. 1247-1254
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA new sensitive assay method for sphingoids, 4-D-hydroxysphinganine, sphingosine, and sphinganine, involving reversed phase HPLC was described. Chromatography of p-N-nitrophenylacetyl derivatives of the sphingoids showed sufficient resolution, and each peak showed a linear relationship between molar quantity and area response, from 10 pmol to 10 nmol. The method was applied to the analysis of long chain bases in intestinal glycolipids from embryonic and adult Japanese quails. At the embryonic stage of 12 days' incubation, 4-D-hydroxysphinganine accounted for about 40% of the long chain bases of glycolipids, a concentration comparable to that in adult tissue. Egg yolk, which is an exclusive exogenous nutrient mixture supplied by the mother, contained only a few percent of 4-D-hydroxysphinganine as a glycolipid component. While, [3H] palmitic acid administered to embryos was incorporated into 4-D-hydroxysphinganine as well as sphingosine and sphinganine. These results suggest that sphingoids of intestinal glycolipids including 4-D-hydroxysphinganine are synthesized in the tissue, excluding the possibility of their exogenous origin in the embryonic tissue.抄録全体を表示PDF形式でダウンロード (556K) -
Akihiko MORIYAMA, Takashi KAGEYAMA, Kenji TAKAHASHI, Makoto SASAKI1985 年 98 巻 5 号 p. 1255-1261
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA pepsinogen C-like acid protease zymogen was found in Japanese monkey prostate extract and seminal plasma by means of the double immunodiffusion method using rabbit anti-pepsinogen C antiserum, and was purified from the prostate by a combination of ammonium sulfate fractionation, DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and immunoadsorption to an anti-pepsinogen C column. The zymogen was purified 6, 400-fold in a yield of 13.1%. The purified zymogen gave a single band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The zymogen was converted to the active form by acid treatment at pH 2.8 for 4 h with concurrent reduction of the molecular weight from 41, 000 to 36, 000. By the double immunodiffusion method, prostate pepsinogen C-like acid protease zymogen, pepsinogen C, lung procathepsin D-II, and their active forms gave a single, fused precipitin line in agar plate with anti-pepsinogen C antiserum, which did not react with cathepsin D and pepsinogen A. Furthermore, the optimal pH of 2.5-3.0, the effect of pepstatin on the activity, and the amino acid compositions were also in good agreement among these three zymogens, showing that they are very similar protease zymogens.抄録全体を表示PDF形式でダウンロード (1804K) -
Ulla CHRISTENSEN, Setsuko ISHIDA, Shin-ichi ISHII, Yukio MITSUI, Yoich ...1985 年 98 巻 5 号 p. 1263-1274
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーStreptomyces subtilisin inhibitor (SSI), a dimeric protein that strongly inhibits subtilisins, was shown to form tight inhibitory complexes with Streptomyces griseus proteases A and B (SGPA and SGPB). The apparent dissociation constants of the SGPA-SSI and SGPB-SSI complexes were found to be orders of magnitude less than those of subtilisin-SSI complexes. Using the known atomic coordinates for SGPA and SSI, the highly complementary nature of the surface geometries of the two proteins was confirmed by a computer graphics study, which led to a proposed structure for the SGPA-SSI complex. Kinetic studies further suggested that the SSI dimer can bind two molecules of either SGPA or SGPB, and the 2:1-com-plexes (consisting of one inhibitor dimer and one enzyme molecule) apparently possess lower intrinsic dissociation constants than the 2:2-complexes. It was also shown that both of SGPA and SGPB are inhibited by both soybean trypsin inhibitor (Kunitz) and bovine pancreatic trypsin inhibitor (Kunitz), but far less strongly than by SSI.抄録全体を表示PDF形式でダウンロード (1264K) -
Large Scale Isolation and Some Properties of AGY-Specific Serine tRNA from Bovine Heart MitochondriaTakuya UEDA, Takahisa OHTA, Kimitsuna WATANABE1985 年 98 巻 5 号 p. 1275-1284
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA method was developed for large scale isolation of AGY-specific serine tRNA (tRNASerAGY) from bovine heart mitochondria. By this method, 5 A260 units of tRNASerAGY were recovered from 6.3kg of bovine hearts.
The nucleotide sequence was identical to that reported previously. tRNASerAGY showed abnormal melting profiles, as was predicted from its unique primary sequence. Its secondary and/or tertiary structure was analyzed by nuclease digestion method. It was suggested that three extra base pairs could occur in the anticodon stem region, with one adenosine residue protruding. The T loop was quite sensitive to nuclease S1, suggesting that the T loop doesn't interact with other regions. This finding is consistent with the model proposed by Sundaralingam (1980).
tRNASerAGY was aminoacylated in vitro with only mitochondrial enzyme but not with the enzymes from E. coli and yeast. The aminoacylation rate of tRNASerAGY with mitochondrial enzyme was much faster than that of cytosolic tRNASerUCN, perhaps reflecting differences due to the presence and absence of the D arm of the tRNAs.抄録全体を表示PDF形式でダウンロード (3107K) -
Masayuki MASUKO, Hidekazu IWASAKI, Takeshi SAKURAI, Shinnichiro SUZUKI ...1985 年 98 巻 5 号 p. 1285-1291
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe effects of freezing on Alcaligenes sp. nitrite reductase [nitric-oxide: ferricyto-chrome c oxidoreductase, EC 1. 7. 2. 1] dissolved in sodium phosphate (pH 7.2) were investigated. The nitrite reductase was gradually activated with time in the frozen state, resulting in an increase in its activity of 2.5-4.5 times. The final freezing temperature influenced the enzyme activation, maximal activation being observed at around -20&C. All the enzymatic activities that the nitrite reductase is known to catalyze were enhanced by freeze-thawing.
The activation was followed by neither association-dissociation nor any gross conformational change of the enzyme molecule, but was accompanied by an increase in the fluorescence intensity of 2-p-toluidinonaphthalene-6-sulfonate used as a hydrophobic probe. The results are consistent with the hypothesis that the activation of the NiR is due to a limited conformational change of the enzyme molecule, particularly in the hydrophobic region.
The mechanism of the activation of NiR by freeze-thawing is discussed, in comparison with the mechanisms of inactivation by freeze-thawing of many enzymes reported by previous workers.抄録全体を表示PDF形式でダウンロード (545K) -
Hideyoshi YOKOSAWA, Shogo ENDO, Yoshitaka OHGAKI, Jun-ichi MAEYAMA, Sh ...1985 年 98 巻 5 号 p. 1293-1299
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーHydrolysis of substance P and nine kinds of substance P analogs by angiotensinconverting enzyme highly purified from rat lung was examined by using amino-group fluorometry and high-performance liquid chromatography. The enzyme hydrolyzed substance P and several analogs, notwithstanding that they did not contain free C-terminal residues. The analyses of cleavage products separated by high-performance liquid chromatography indicated that the enzyme hydrolyzed substance P and its analogs mainly at the bond between Phe8-Gly9 and also at another bond, possibly between Gly9-Leu10, to a lesser extent by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action. The analogs that had D-amino acid residues substituted at the presumed cleavage sites were scarcely hydrolyzed. It was further found that (Pyr6)-fragment (6-11) was hydrolyzed by the enzyme more efficiently than the other fragment-type analogs and was cleaved at a single bond by the endopeptidase activity of the enzyme. Therefore, this fragment was used as a substrate in order to characterize the endopeptidase activity of the enzyme by employing fluorometry. The activity was dependent on chloride ion, and was inhibited by captopril, MK-421, and EDTA. Thus, the endopeptidase activity of the enzyme showed properties similar to those of the dipeptidyl carboxypeptidase activity of the enzyme.抄録全体を表示PDF形式でダウンロード (559K) -
Masao SHIOTA, Tasuku NAKAJIMA, Atsuko SATOH, Mariko SHIDA, Kazuo MATSU ...1985 年 98 巻 5 号 p. 1301-1307
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA mutant of Saccharomyces cerevisiae defective in the cell wall β-glucan structure was obtained. The mutant cells are extremely sensitive to (β1-3)-glucanase digestion and mild alkali treatment. Structural analysis revealed that the alkali-insoluble, skeletal glucan from wild type cells contains two components, a (β1-3) linked glucan with a laminated structure, and a highly branched glucan containing predominantly (β1-6) linkages. The mutant cells lack the latter component.抄録全体を表示PDF形式でダウンロード (1130K) -
Nobuko KAWASAKI, Toshisuke KAWASAKI, Ikuo YAMASHINA1985 年 98 巻 5 号 p. 1309-1320
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーConglutinin is a bovine plasma protein which mediates the agglutination of the sensitized erythrocyte-solid phase iC 3 b complex (conglutination). The serum mannan-binding protein (MBP) is a lectin specific for mannose and N-acetylglucosamine. Since conglutination was shown to be inhibited specifically by N-acetylglucosamine [Leon, M. A. & Yokohari, R. (1964) Science0 143, 1327-1328], the possibility was raised that conglutinin might be a bovine serum MBP. The present study, undertaken to solve this problem, revealed that bovine plasma contained an MBP besides conglutinin. These two proteins were very similar in their chemical and physicochemical properties as well as binding specificity. Both bound with high affinity (Kd=10-8M) to glycoproteins terminated with mannose and/or N-acetylglucosamine residues in the presence of calcium, although conglutinin preferred N-acetylglucosamine rather than mannose. They were multimeric proteins of large molecular size (over 1, 000, 000 daltons, and approximately 600, 000 daltons for conglutinin and MBP, respectively) and consisted of a single kind of subunit with molecular weight of around 45, 000. The MBP was shown to have a collagenlike structure in the molecule, as was recently reported for conglutinin [Davis, A. E., III & Lachmann, P. J. (1984) Biochemistry 23, 2139-2144]. Despite these similarities, the MBP and conglutinin were immunochemically distinct, and the MBP did not show any conglutination activity.抄録全体を表示PDF形式でダウンロード (2567K) -
Hiroyuki SADANO, Tsuneo OMURA1985 年 98 巻 5 号 p. 1321-1331
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーDetermination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450 (PB-1), and P-450 (MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of δ-amino [14C] levulinic acid (ALA) into the heme of P-450 (PB-1) or P-450 (MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome.
When heme-labeled cytosol prepared from [14C] ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450 (PB-1), whereas the incorporation into P-450 (MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913.
Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.抄録全体を表示PDF形式でダウンロード (921K) -
Makoto TAKANO, Masa-aki TAKAHASHI, Motohisa OOBATAKE, Kozi ASADA1985 年 98 巻 5 号 p. 1333-1340
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーTo reveal the molecular orientation of plastocyanin (PC) on spinach thylakoid membranes, the position of Lys residues modified by acetic anhydride was compared between thylakoid-bound PC and the isolated one. Digestion of the isolated PC by a trypsin yielded a peptide map with seven spots prior to the acetylation of the protein; none of the spots appeared after the isolated PC was acetylated. On the other hand, there were two spots on the peptide map of the PC acetylated when it was bound to the thylakoids. Those spots were revealed by their amino acid compositions to correspond to the peptide fragments Phe 82-Lys 95 and Val 96-Asn 99. Thus, the Lys residues 81 and 95 of the thylakoid-bound PC were not acetylated. These results suggest that the PC molecule binds to the thylakoids with a specific region including the Lys's 81 and 95 in contact with the membranes. The Lys's 81 and 95 are located near Tyr 83, which has been thought to be the delivery site of electrons from the Cu2+ center.抄録全体を表示PDF形式でダウンロード (634K) -
Sadao MATSUURA, Takashi SUGIMOTO, Shizuaki MURATA, Yoko SUGAWARA, Hito ...1985 年 98 巻 5 号 p. 1341-1348
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe structure of (-)-tetrahydrobiopterin, the cofactor for phenylalanine, tyrosine, and tryptophan hydroxylases, was determined as (6 R, 1' R, 2' S)-6-(1', 2'-dihydroxypropyl)-5, 6, 7, 8-tetrahydropterin by X-ray crystallographic analysis. The CD spectra of (-)-tetrahydrobiopterin exhibits a negative Cotton effect at 290-240 nm in pH 3.2 solution. The relationship of the negative Cotton effect and the 6 R configuration was supported by the application of CD analysis with 2-methyltetrahydronaphthalene as a model compound. The stereochemistries at the C (6) position of various biologically active 6-substituted tetrahydropterins were determined from the CD spectra. The relative configuration of two asymmetric centers, C (6), and C (1'), was determined by HPLC analysis on a strong cation exchange column.抄録全体を表示PDF形式でダウンロード (489K) -
Hideo SAWADA, Akira HARA, Toshihiro NAKAYAMA, Kazunori SEIRIKI1985 年 98 巻 5 号 p. 1349-1357
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーKinetic and physicochemical properties of hamster liver diacetyl reductase have been examined. The results of kinetic studies on the reduction of diacetyl and NADPH to acetoin and NADP+ suggest that the reaction follows an Ordered Bi Bi mechanism in which NADPH binds first before diacetyl. The enzyme is a tetrameric glycoprotein of single subunits of a molecular weight of 23, 500 with a sedimentation coefficient of 6.0 S. The enzyme does not contain Zn, Cu, or Fe. The amino acid composition revealed an unusually low proportion of proline residues (0.9%). p-Chloromercuriphenylsulfonate and phenylglyoxal inactivated the enzyme, but the presence of NADPH prevented the loss of activity due to thiol and arginine modification. The enzyme transferred the pro 4 S hydrogen atom of NADPH to the substrate and the binding of the enzyme to NADPH resulted in a red shift of the ultraviolet absorption spectrum of the cofactor.抄録全体を表示PDF形式でダウンロード (1550K) -
Danièle BOUHOURS, Jean-Frangois BOUHOURS1985 年 98 巻 5 号 p. 1359-1366
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーNeutral glycosphingolipids were isolated from the colon of rats between birth and adulthood. The glycolipid concentration was stable during this period. Epithelial cells of the adult colon contained three times more glycolipids than the whole organ. The distribution pattern underwent only minor modifications during development. Free ceramide contributed for 23-27% of the total neutral sphingolipids at all ages. In 6-day-old rats, it was constituted of nonhydroxylated fatty acids linked to C18-sphingenine (57.3% of the bases), C18- and C20-4 D-hydroxysphinganine (24.2 and 14.0% of the bases, respectively). This composition was essentially maintained during development. Glucosylceramide was the major glycolipid at all ages (40-50% of the total neutral sphingolipid content). At birth, 40% of its fatty acids were 2-hydroxylated and 93% of the bases were C18-4D-hydroxysphinganine. In adult epithelial cells, 75% of the fatty acids were 2-hydroxylated and C18- and C20-4 D-hydroxysphinganine contributed for 66 and 25% of the bases, respectively. A transient increase of the contribution of nonhydroxylated fatty acids and C18-sphingenine was observed during the first week of life. C20-4 D-hydroxysphinganine, which was characterized by gas-liquid chromatography of its aldehydes after periodate oxidation and of its N-acetyl O-trimethylsilyl derivatives, appeared after birth and reached 20% of the bases after two weeks. These findings are another example of the specificity of the lipidic part of glucosylceramide during the ontogenic differentiation.抄録全体を表示PDF形式でダウンロード (2276K) -
III. Elucidation of the Structure of Ganglioside GM 3 LactoneRobert K. YU, Theodore A. W. KOERNER, Susumu ANDO, Herbert C. YOHE, Ja ...1985 年 98 巻 5 号 p. 1367-1373
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーGanglioside GM 3 lactone (1) was prepared in 95% yield from the parent ganglioside by incubation at 25&C for 4 days in glacial acetic acid. Inspection of the 500 MHz proton NMR spectra of 1 and its precursor in dimethylsulfoxide-d6 deuterium oxide at 30&C revealed a large deshielding (+1.42 ppm) of the H-2 resonance of the galactosyl residue. This suggests that 1 must be the lactone formed by esterification of the sialic acid carboxyl group with the C-2 hydroxyl of the galactosyl residue. Consideration of all the NMR data leads to a specific structure proposal in which 1 has a highly rigid structure. Interesting features of the structure include a hydrophobic inner surface and a semicircular outer edge of seven-oxygen atoms, which may have physiological importance.抄録全体を表示PDF形式でダウンロード (1418K) -
Arata ICHIYAMA, Eiko NAKAI, Tsuneyoshi FUNAI, Toshiaki ODA, Ritsuko KA ...1985 年 98 巻 5 号 p. 1375-1385
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーIn order to establish a standard procedure for the spectrophotometric determination of urinary and plasma oxalate with oxalate oxidase (Laker, M. F., et al. (1980) Clin. Chem. 26, 827-830; Sugiura, M., et al. (1980) Clin. Chim. Acta 105, 393-399) and to define the limitations of the method, the procedures and reactions involved in the assay have been examined. Among the chromogenic hydrogen donors for peroxidase tested, a combination of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and sodium N-sulfopropylaniline (HALPS) was found to be best for the oxalate determination under the conditions used. Urine contained substance (s) which were inhibitory to the measurement of hydrogen peroxide by the peroxidase-catalyzed oxidative condensation of MBTH and HALPS, but they were largely removed by charcoal treatment at pH 5.6 without significant loss of oxalate. Deproteinization of plasma was carried out by ultrafiltration through a membrane cone (Centriflo CF-25) at neutral pH. The plasma oxalate ultrafiltrability under the conditions employed was calculated to be approximately 95%. A standard assay system for oxalate in these urine and plasma samples was then set up based on a series of studies on the reactions involved in the assay. In the case of normal plasma, however, the absorbance change was very small due to the low concentration of oxalate, and in addition, pretreatment of plasma with excess oxalate decarboxylase followed by the ultrafiltration and oxalate determination did not abolish completely the oxalate oxidase-dependent absorbance increase. It was concluded that the enzymic method was useful for the assay of urinary oxalate and in detecting elevated levels of plasma oxalate such as those in hemodialysis patients but was not sensitive enough to determine accurately the normal or decreased level of oxalate in plasma. The apparent concentration of oxalate in normal human plasma was measured in this work as 3.5±0.8 μM (mean±S. D., n=8), and this result was interpreted to mean that the concentration of plasma oxalate was less than approximately 3.5 μM, as estimated by the present method.抄録全体を表示PDF形式でダウンロード (979K) -
Shinobu INOUE, Seiichi HASHIDA, Takeyuki KOHNO, Koichiro TANAKA, Eiji ...1985 年 98 巻 5 号 p. 1387-1394
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーA micro-scale method for the conjugation of affinity-purified Fab' to β-D-galactosidase from Escherichia coli is described. Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab')2 and applied to a column of human chorionic gonadotropin-Sepharose 4 B, followed by elution at pH 2.5. The affinity-purified anti-human chorionic gonadotropin F (ab')2 was mixed with non-specific goat F (ab')2 (0.5mg) as a carrier, reduced with 2-mercaptoethylamine to split F (ab')2 to Fab' and conjugated to β-D-galactosidase using N, N'-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-β-D-galactosidase conjugate was separated from non-specific goat Fab'-β-D-galactosidase conjugate and unconjugated β-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4 B using 4M urea. The amount of the affinity-purified conjugate obtained was 56-69 pg. The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification.
This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4M urea.抄録全体を表示PDF形式でダウンロード (682K) -
Yoshihiro JINNO, Seiji MATUO, Hisayuki NOMIYAMA, Kazunori SHIMADA, Ich ...1985 年 98 巻 5 号 p. 1395-1403
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーThe structure of the 5' end region of the human argininosuccinate synthetase (AS) gene was analyzed in detail. The 5' flanking region up to 350 nucleotides from the putative transcription initiation site is unusually G+C rich (80%). Three repeats of a CCGCCC sequence and one complementary GGGCGG sequence are present in this region. It contains no CCAAT box-like sequence, but does contain one typical TATA box and a 20 bases-long inverted repeat sequence that could form a stem and loop structure just upstream from the TATA box. An octanucleotide sequence, AGAAGTGA, found in the 5' flanking region of the AS gene is also commonly present in those regions of the two other human genes expressed mainly in the liver. These structural features of the AS gene indicate that it shares common structures with two completely different groups of genes, i.e. tissue-specific genes and housekeeping ones. Moreover, two enhancer core-like sequences, alternating purine-pyrimidines tracts and two independent Alu type highly repetitive sequences are apparent in the first intron of the AS gene.抄録全体を表示PDF形式でダウンロード (2132K) -
Katsuyuki IMAI1985 年 98 巻 5 号 p. 1405-1416
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーLysosomal membrane fractions were prepared from lysosomes of mouse liver by freeze-thawing in a hypotonic buffer: 54% of β-glucosidase [EC 3. 2. 1. 45] in lysosomes was associated with the membrane fractions, whereas 96% of β-glucuronidase [EC 3. 2. 1. 31] was recovered in the soluble fractions of lysosomes. β-Glucosidase was solubilized by pH 9.5 treatment or by Triton treatment of membranes. The enzyme solubilized with alkali and concentrated with (NH4)2SO4 was rapidly inactivated in a solution of pH 9.5, but could be protected against inactivation by acidic detergent. Gel filtration analysis indicated that β-glucosidase was in an aggregated form at neutral pH and could be disaggregated by alkali and detergents. The enzyme dissociated with detergents also showed a higher activity than the alkalitreated enzyme. These results suggested that β-glucosidase is a peripheral enzyme bound to acidic lipids in membranes. β-Glucosidase was purified to apparent homogeneity by (NH4)2SO4 fractionation and chromatographies with Sephacryl S-300, hydroxylapatite and cation exchangers in the presence of detergents. The catalytic activity of the purified enzyme was maximally stimulated by phosphatidylserine and heat-stable protein in the presence of a low concentration of Triton X-100. The stimulation was mainly due to an increase in Vmax.抄録全体を表示PDF形式でダウンロード (1377K) -
Kuniaki MUKAI, Toshiyuki MIYAZAKI, Sadao WAKABAYASHI, Seiki KURAMITSU, ...1985 年 98 巻 5 号 p. 1417-1425
発行日: 1985年
公開日: 2008/11/18
ジャーナル フリーPurified bovine heart two-band cytochrome c1 subcomplex was dissociated by treatment with p-chloromercuribenzoic acid (pCMB) into its heme subunit and a colorless subunit called hinge protein, which is essential for the formation of cytochrome c1-c complex. The subcomplex was found by titration to react with 4 mol of pCMB per mol of cytochrome c1. The contents of mercury of the dissociated heme subunit and the hinge protein were 3 and 1 mol per mol of polypeptide, respectively. These results, together with the sequence analysis, indicated that the three cysteine residues in cytochrome cl heme subunit not involved in heme-binding existed in free thiol form. One of the five cysteine residues in the hinge protein was in free form and four in two disulfide bonds. The dissociated hinge protein was digested with staphylococcal protease and the cysteine-containing peptides were separated by reversedphase high-performance liquid chromatography (HPLC). The content of mercury and the result of performic acid oxidation of cystine peptides revealed that Cys-30 existed in free thiol form and two disulfide bridges were formed between Cys-24 and Cys-68 and between Cys-40 and Cys-54. The conformation of the hinge protein was predicted to be composed largely of either two-α-helical or four-α-helical conformation with the amino (N)-terminal 20 residues being in a random structure.抄録全体を表示PDF形式でダウンロード (1413K)
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