All issues
Volume 98 (1985)
- Issue 6 Pages 1427-
- Issue 5 Pages 1147-
- Issue 4 Pages 863-
- Issue 3 Pages 585-
- Issue 2 Pages 269-
- Issue 1 Pages 1-
Volume 98, Issue 3
Displaying 1-30 of 30 articles from this issue
- |<
- <
- 1
- >
- >|
-
Katsuhiko BABASAKI, Toshifumi TAKAO, Yasutsugu SHIMONISHI, Kiyoshi KUR ...1985 Volume 98 Issue 3 Pages 585-603
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubtilosin A, a new antibiotic produced by Bacillus subtilis 168, was extracted from culture medium with n-butanol and purified to homogeneity by a combination of gel filtration and thin-layer chromatography. The yield was 5.5mg from a liter of culture. It had bacteriocidal activity against some gram-positive bacteria. Amino acid analysis and mass spectrometry showed that it was a peptide with a molecular weight of 3398.9, consisting of 32 usual amino acid and some non-amino acid residues. Its amino- and carboxyl-termini were blocked. By analysis of the fragments obtained by partial acid hydrolysis, as well as by chymotryptic and thermolysin digestions of reduced and S-carboxymethylated samples and Achromobacter protease I digestion of performic acid-oxidized samples, the amino acid sequence was determined to be as follows: X-Gly-Leu-Gly-Leu-Trp-Gly-Asn-Lys-Gly-Cys-Ala-Thr-Cys-Ser-Ile-Gly-Ala-Ala-Cys-Leu-Val-Asp-Gly-Pro-Ile-Pro-Asp-Glx-Ile-Ala- Gly-Ala. The analyses of cross-linking structures revealed that there were linkages between the amino- and carboxyl-termini and between the Cys-19 and the Glx-28 residues through an unknown residue with a residue weight of 163. Consequently, subtilosin A was deduced to be a cyclic peptide antibiotic with a novel cross-linking structure.
The production of subtilosin A begins at the end of vegetative growth and finishes before spore formation. Studies on the correlation between the production of subtilosin A and spore formation with decoyinine in the original strain and in asporogenous mutants of B. subtilis 168 suggested that there was no close correlation between the two phenomena. The production of subtilosin A was repressed by inhibitors of protein and RNA synthesis in contrast to that of many other antibiotic peptides, suggesting that it is synthesized by the mechanism of usual protein synthesis.View full abstractDownload PDF (1611K) -
Takashi MITSUYAMA, Shinsei GASA, Takayuki NOJIMA, Naoyuki TANIGUCHI, A ...1985 Volume 98 Issue 3 Pages 605-613
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSActivator protein for galactosylceramide sulfatase (GSase) was purified from human liver. The activator has an approximate molecular weight of 22, 000, is glycoprotein in nature, and is most probably a trimer consisting of an 8, 000 dalton monomer. Monospecific rabbit antiserum raised against the activator strongly inhibited the activity of the activator. In the presence of a 10-fold or more excess of galactosylceramide sulfate (GS) on a molar basis, GS binding to the GSase activator occurred, and was saturated at an equimolar ratio.
Binding studies on the GSase activator were conducted using affinity chromatography on derivatives of GS as ligands, and gel filtration of mixtures containing glycolipids and the activator. A “GS-acid” derivative, which was prepared by oxidative cleavage of sphingosine moiety in GS, and a sulfonamide derivative of GS as ligands still retained affinity for the GSase activator, while a hydrophobic ligands, an aminohexyl group did not bind completely the activator. A ligand of “galactosylceramide-acid” had weak affinity for GSase activator. These results suggest that the sulfate group and one of the two hydrocarbon chains in GS are not essential for the binding of the activator. The affinity of galactosylceramide for the GSase activator was confirmed by the detection of the lipid-protein complex on gel filtration. The activator weakly stimulated porcine GM1-β-galactosidase activity.View full abstractDownload PDF (1225K) -
Shizuo NAKAJIN, Masayuki TAKAHASHI, Kimio HIGASHIYAMA, Masato SHINODA1985 Volume 98 Issue 3 Pages 615-620
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFormation of androstadienone or androstadienol, a Δ16-C19-steroid, from progesterone or pregnenolone is catalyzed by the so-called Δ16-C19-steroid synthesizing enzyme in the pig testicular microsomes. The enzyme activity was also present in the testicular microsomes prepared from neonatal pig. The enzyme activity was considerably inhibited by CO, and such cytochrome P-450 inhibitors as SU 8000, SU 10603, and metyrapone. Δ16-C19-Steroid synthesizing enzyme activity was extracted from the testicular microsomes by sodium cholate in potassium phosphate buffer, pH 7.4, containing EDTA and dithiothreitol, and the solubilized enzyme activity was partially purified by DEAE-cellulose column chromatography. It was shown by reconstitution of the enzyme activity that Δ16-C19-steroid synthesizing enzyme is a cytochrome P-450-linked oxygenase system dependent on cytochrome P-450-reductase and cytochrome b5. In particular, cytochrome b5 was an essential component for the activity of Δ16-C19-steroid synthesizing enzyme.View full abstractDownload PDF (522K) -
Miyoko OKUDA, Kikuo ARAKAWA1985 Volume 98 Issue 3 Pages 621-628
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of bradykinin-potentiating activity of [des-Proline3]-bradykinin, a kinin originally generated from human plasma protein by trypsin, was studied in terms of its inhibitory actions on angiotensin-converting enzyme and kininase II prepared from rat lung. The results were compared with those obtained with Captopril.
[Des-Pro3]-bradykinin was found to have a potent inhibitory action against angiotensin-converting enzyme with a K1 of 4.5×10-12M, which is approximately 7 times more potent than Captopril. It was also inhibitory to kininase II with a K1 of 4×10-11M, which is approximately 2, 300-fold more potent than Captopril.
The pattern of inhibition was purely competitive with increased apparent Km but no change in apparent Vmax for both angiotensin-converting enzyme and kininase II. This is in contrast to Captopril, which showed a mixed competitive and noncompetitive type of inhibition with increased apparent Km and decreased Vmax for both enzymes.
Such a potent inhibitory activity of [des-Pro3]-bradykinin or Arg-Pro-Gly-Phe-Ser-Pro-Phe-Arg is noteworthy, and accordingly we propose the name “converstatin” for this peptide.View full abstractDownload PDF (591K) -
Hiroko NARA-INUI, Hideo AKUTSU, Yoshimasa KYOGOKU1985 Volume 98 Issue 3 Pages 629-636
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe circular dichroic (CD) spectra of natural DNAs (from Cl. perfringens, T 2 phage, calf thymus, E. coli, and M. lysodeikticus) as well as duplexes of synthetic DNAs (poly (dA)•poly (dT), poly (dA-dT), and poly (dG-dC)) were measured in water-ethanol mixtures with 0.3mM NaCl. A conformational change from the B to the A form was observed for the natural DNAs on adding ethanol. The ethanol concentration that induces the transition and the extent of the change in the CD spectrum are different for the five natural DNAs depending on their GC contents. The higher the GC content is, the more easily the transition to the A form takes place. The results indicate that the GC content of a DNA is an important factor for induction of the B-A transition. The results for the synthetic DNAs show that their properties cannot be inferred by simple extrapolation of those of natural DNAs. Coexisting ions and the molecular weight of a DNA were also found to affect the induction of the B-A transition.View full abstractDownload PDF (649K) -
Toichiro HOSOYA, Ikuro SATO, Yoshio HIYAMA, Hirokatsu YOSHIMURA, Hiroo ...1985 Volume 98 Issue 3 Pages 637-647
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA peroxidase assay method (Mini assay method) which is applicable for a minute amount (as small as a few mg) of thyroid tissue was developed, employing guaiacol or iodide as the second substrate. This method is a modification of the previous one (Ordinary assay method): the volume of the reaction mixture was reduced to about one-tenth with prior solubilization of the enzyme. The correlation between the Mini assay and Ordinary assay methods, and between the guaiacol and iodide assays by both methods were satisfactorily good, but the iodine content of thyroglobulin was found to be not directly correlated to the peroxidase activities. Proteinbased specific activities of peroxidase from normal human thyroid tissue were about 0.030 guaiacol units/mg protein and 0.0066 iodide units/mg protein, which were slightly higher than those of porcine thyroid tissue. The Mini assay method developed in the present study was used for the determination of peroxidase activity in a small amount (1-8mg) of thyroid tissue obtained by means of a needle biopsy from patients with thyroid disorders. One specimen (goitrous cretinism) showed no peroxidase activity in both the guaiacol and iodide assays, and three specimens (two chronic thyroiditis, one familial nontoxic goiter) possessed no ability to catalyze the oxidation of iodide in spite of the high reactivity towards guaiacol, suggesting the presence of an abnormal peroxidase in these tissues.View full abstractDownload PDF (1041K) -
Yoshiko MINAMI, Sadao WAKABAYASHI, Setsuko IMOTO, Yoshimoto OHTA, Hiro ...1985 Volume 98 Issue 3 Pages 649-655
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMarchantia polymorpha ferredoxin was purified by DE-52 and Sephadex G-75 column chromatographies to homogeneity. The complete amino acid sequence of the carboxymethylated (Cm) ferredoxin was determined by conventional methods to be as follows. Thr-Phe-Lys-Val-Thr-Leu-Asn-Thr-Pro-Thr-Gly-Gln-Ser-Val-Ile-Asp-Val-Glu-Asp-Asp-Glu-Tyr-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Ala-Gly-Leu-Ser-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Val-Thr-Ala-Gly-Glu-Val-Asp-Gln-Ser-Asp-Glu-Ser-Phe-Leu-Asp-Asp-Asp-Gln-Met-Asp-Glu-Gly-Tyr-Val-Leu-Thr-Cys-Ile-Ala-Tyr-Pro-Thr-Ser-Asp-Leu-Thr-Ile-Asp-Thr-His-Gln-Glu-Glu-Ala-Leu-Ile. The total number of amino acid residues was 95 and the molecular weight was calculated to be 10, 174, excluding iron and sulfur atoms. The distribution of the four cysteine residues chelating the two iron atoms was identical to those of other [2 Fe-2 S] ferredoxins. The relationship between M. polymorpha and other plants was discussed in terms of plant phylogeny.View full abstractDownload PDF (492K) -
Tohru YOSHIOKA, Hiroko INOUE, Takeshi KASAMA, Yousuke SEYAMA, Shigeru ...1985 Volume 98 Issue 3 Pages 657-662
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have found that arachidonic (20:4) acid is indetectable in phosphatidylinositol and diacylglycerol extracted from Drosophila heads. After careful examinations of the lipid extraction processes and fatty acid detection system (gas-liquid chromatography), we excluded the possibility of the oxidation of polyunsaturated fatty acids or of having overlooked a trace amount of the fatty acid. The precursors of arachidonic, dihomo γ-linolenic (20:3), and γ-linolenic (18:3) acid, were also indetectable in these lipids. On the basis of these results, it appears that the arachidonic acid cascade is essentially absent in Drosophila head, including the brain and compound eyes.
Since arachidonic acid is considered to be a key molecule in phosphatidylinositol turnover in the brain, it is of interest that Drosophila brain and eyes do not require arachidonic acid for their functions.View full abstractDownload PDF (470K) -
Tomoko TAKAHASHI, Keiko KATO, Yoshio IKEGAMI, Masachika IRIE1985 Volume 98 Issue 3 Pages 663-671
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree forms of glucoamylase [EC 3. 2. 1. 3] of a Rhizopus sp., Gluc1 (M. W. 74, 000), Gluc2 (M. W. 58, 600), and Gluc3 (M. W. 61, 400), which have similar pH optima and specific activities towards soluble starch were studied as to their behavior towards raw starch. The pH optima for raw starch digestion were different, that is, 4.5 for Gluc1 and 5.0 for both Gluc2 and Gluc3. All the enzymes digested raw starch almost completely but at quite different rates; Gluc2 and Gluc3 which lack the N-terminal portions of Gluc, were 22 and 25 times less effective, respectively, for raw starch digestion than Gluc1. Of the three enzymes, only Gluc1 tightly bound to raw starch. Binding of Gluc1 to raw starch occurred pH-dependently with a broad pH optimum of 4.5-5.5, but temperature and ionic strength affected it only slightly and little, respectively. The binding constant of Gluc1 for raw starch at pH 5.0 and 4&C was estimated to be 1.2×105M-1. Fragment H (M. W. 16, 700), presumably released from the N-terminal part of Gluc1 not only bound to raw starch itself but also inhibited the binding of Gluc1 to raw starch. pap-Glue (M. W. 57, 000) and chymo-Gluc (M. W. 64, 000), which are papain- and chymotrypsinmodified Gluc1, respectively, and lack the N-terminal portions of Gluc1, resembled Gluc2 and Gluc3 in raw starch binding as well as digestion. All these results indicate that Gluc1 has a raw starch-binding site, different from the active center, in the N-terminal region. Various substrates and analogs inhibited the binding of Gluc1 to raw starch, presumably due to steric hindrance.View full abstractDownload PDF (804K) -
Saul YEDGAR, Ben-Ami SELA1985 Volume 98 Issue 3 Pages 673-680
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe possibility that liver cell membrane is modified in hyperlipidemic state was studied using nephrotic hyperlipidemic rats. Liver cells of normal and nephrotic rats were isolated and subjected to labeling of cell surface components using lactoperoxidase catalyzed radioiodination. The labeling of total surface lipids of hepatocytes of nephrotic rats was about five times higher than that of normal ones and was particularly higher in glycosphingolipids. Cultivation of the isolated hepatocytes as primary cultures reduced drastically labeling of surface lipids in liver cells of both nephrotic and normal rats and abolished the differences observed in liver cells of the two types. Determination of cell associated gangliosides, showed that the level in nephrotic rat hepatocytes was only 35% higher than that of normal rats. Yet, in both types of liver cells 24 h cultivation decreased markedly the ganglioside content. However, similar to the effect observed in hyperlipidemic rats, supplementing the culture medium with very low density lipoproteins (VLDL) increased considerably the ganglioside level of cultured hepatocytes. These treatments did not affect the activity of enzymes involved in the synthesis of gangliosides. It is suggested that ganglioside content in liver cell membrane is modulated in the hyperlipidemic state.View full abstractDownload PDF (1626K) -
Morio UMENO, Motoaki ANAI, Makoto SASAKI, Yasuyuki TAKAGI1985 Volume 98 Issue 3 Pages 681-685
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSrecBC DNase of Escherichia coli has been purified from the transformant, HB 101/ pFS 11-04 (recB+ recC+), by successive ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, hydroxyapatite chromatography, DNA cellulose affinity chromatography, and second DEAE-cellulose chromatography. The purified enzyme was obtained in an overall yield of 3%. The enzyme protein appeared as a single pure component on native polyacrylamide gel electrophoresis. The purified enzyme was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and two-dimensional electrophoresis. The results show that recBC DNase consists of two nonidentical subunits with molecular weights of 125, 000 and 135, 000, and isoelectric points of 5.6 and 5.7, respectively.View full abstractDownload PDF (1158K) -
Norihisa KIKUCHI, Kiyoshi NAGATA, Nobuo YOSHIDA, Michio OGAWA1985 Volume 98 Issue 3 Pages 687-694
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour forms of pancreatic secretory trypsin inhibitor (PSTI; A1, A2, B, and C) were purified from human pancreatic juice. According to sequence results, the primary structure of B was different from that reported earlier (Greene, L. J., et al. (1976) Method Enzymol. 45, 813-825) at two positions, i.e. Asn21→Asp21, Asp29→Asn29. A1 and A2 were deamidated forms of B judging from peptide mappings with Staphylococcus aureus V 8 protease. Gln45 in B was replaced by Glu in A1 and Gln51 in B was replaced by Glu in A2. C was an inhibitor lacking five amino acid residues from the amino terminal of B. B and C inhibited human cationic trypsin activity stoichiometrically with similar dissociation constants, but A1 and A2 showed poorer trypsin inhibitory activity than B and C.View full abstractDownload PDF (1302K) -
Hitoshi YAGISAWA, Yukiko YAMASHITA, Shunichi YAMAGISHI, Hiroyuki SUGIY ...1985 Volume 98 Issue 3 Pages 705-711
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported that muscarinic acetylcholine receptors (mAChRs) from porcine brains are glycoproteins. When porcine brain membranes were solubilized with digitonin or 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS), approximately 20% of the receptors were solubilized, most (90 or more) of which bound to Sepharose 4 B conjugated with wheat germ agglutinin (WGA). In contrast, when membranes were solubilized with Lubrol PX, a much larger fraction (approximately 60%) of the receptors were solubilized. However, about a third of this solubilized receptor population remained unbound to WGA-Sepharose even in the presence of an excess amount of the lectin-Sepharose. These results suggested a structural heterogeneity of the mAChR in terms of its carbohydrate moiety.
The effects of lectins on the ligand binding properties of mAChRs were also studied. WGA or concanavalin A (ConA) was found to cause a 2- to 3-fold increase in the affinity of membrane-bound receptors to an antagonist [3H] quinuelidinyl benzylate ([3H] QNB) without affecting the maximum number of sites, whereas the lectins had no significant effects on the binding of the agonist [3H] cis-methyldioxolane. When the membranes were dissolved with detergents, lectin did not increase the [3H] QNB affinity: These lectins caused an approximately 2 fold decrease in the affinity of digitonin-solubilized receptors for [3H] QNB. Thus the lectins exert differential effects on agonist and antagonist binding to the brain membrane mAChRs, most likely by modulating some intermolecular interactions.View full abstractDownload PDF (610K) -
Tiee Cherng SHIEH, Shuji TANAKA, Hiroshi KIHARA, Motonori OHNO, Satoru ...1985 Volume 98 Issue 3 Pages 713-721
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme bearing thrombin-like specificity has been purified to homogeneity from the venom of Trimeresurus flavoviridis (the Habu snake). The enzyme is a monomer with a molecular weight of 23, 500 as determined by analytical gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The protein contains approximately 210 amino acid residues and has a relatively high content of aspartic acid and glutamic acid. The isoelectric point was 4.8 and the extinction coefficient at 280 nm for a 1% solution was 11.5. The enzyme acted directly on fibrinogen to form a fibrin clot with 2.0 NIH units. Analysis by high performance liquid chromatography of enzyme-treated fibrinogen revealed the release of a peptide identical in composition to thrombin-induced fibrinopeptide A, but no peptide corresponding to fibrinopeptide B was detected. The enzyme showed esterase and amidase activities on synthetic substrates containing arginine. The enzyme exhibited higher activity toward tosyl-L-arginine methyl ester (TAME) but 6-times lower activity toward benzoyl-L-arginine p-nitroanilide when compared with bovin thrombin. The esterase activity was inhibited by diisopropylfluorophosphate and at a slower rate by phenylmethanesulfonyl fluoride, but was least affected by tosyl-L-lysine chloromethyl ketone, showing that the enzyme is a serine protease like thrombin. The enzyme showed a bell-shaped pH dependence of kcat/Km for hydrolysis of TAME, with a maximum around pH 8.5.View full abstractDownload PDF (1400K) -
Shoko MIYAZAWA, Takashi HASHIMOTO, Sadaki YOKOTA1985 Volume 98 Issue 3 Pages 723-733
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe identity of long-chain acyl-CoA synthetase in microsomes, mitochondria, and peroxisomes of rat liver was examined by using the antibody raised against a purified preparation of the microsomal enzyme. The enzyme activities of these three organelles and the purified microsomal enzyme were titrated by the antibody in a very similar fashion when the activity was measured in terms of palmitoyl-CoA synthetase activity. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitates and by Western blot analysis that the enzymes of all three organelles consisted of a polypeptide with the same molecular weight as that of the purified enzyme, and that the specific enzyme activity of the antigenic protein in all three subcellular compartments was nearly the same. The presence of other palmitoyl-CoA synthetase activity in these organelles could not be confirmed. Immunocytochemical study to locate the antigenic site with protein A-gold complex showed that the gold particles were closely associated with the membranes of these organelles.
The cell-free translation product in a rabbit reticulocyte lysate protein-synthesizing system and the subunit of the mature enzyme labeled with [35S] methionine in the liver slices exhibited the same mobility as the subunit of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme in microsomes, mitochondria, and peroxisomes was labeled at nearly the same rate when the liver slices were incubated with [35S] methionine.View full abstractDownload PDF (4545K) -
Yumiko EMOTO, Takanori KAWAMURA, Katsuhisa TAWADA1985 Volume 98 Issue 3 Pages 735-745
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur previous work showed that the active site heterogeneity in heavy meromyosin (HMM) becomes evident when highly reactive SH-groups in HMM are modified by thimerosal (Kawamura, Higuchi, Emoto, & Tawada (1985) J. Biochem. 97, 1583-1593). The heterogeneity was revealed by “affinity-labeling” analysis with vanadate plus ADP, which was developed in the previous paper. To see whether this heterogeneity is due to the head-head interaction or two different alkali light chains present in HMM, we carried out similar studies with myosin subfragment-1 (S 1) and one of the isozymes, S 1 (A 1), which contains only the alkali light chain 1, and obtained essentially the same results as those previously obtained with HMM. The S 1 results are easily explained by the same hypothesis previously used for explaining the HMM results: SH-modified S 1 or S 1(A 1) contains two kinds of active site in a 1:1 ratio with almost the same ATPase activity: one hydrolyzes ATP by a mechanism giving a protein Trp fluorescence enhancement, whereas the other hydrolyzes ATP by another mechanism giving no fluorescence enhancement.View full abstractDownload PDF (917K) -
Satoshi ISHII, Hidemi ISHII, Tetsuya SUGA1985 Volume 98 Issue 3 Pages 747-755
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHepatic peroxisomes and mitochondria from 20-day-old chick embryo were separated by sucrose density gradient centrifugation and the characteristics of carnitine acyltransferases in these organelles were studied. The carnitine acyltransferase activities in peroxisomes were increased markedly by the treatment of chick embryo with clofibrate, while those in mitochondria did not change. In the liver of clofibratetreated chick embryo, approximately 50% of total liver carnitine palmitoyltransferase (CPT) activity was present in the peroxisomal fraction. Peroxisomal CPT activity was easily solubilized, in contrast with mitochondrial CPT. The solubilized protein solutions from isolated peroxisomes and mitochondria were separately chromatographed on a column of Blue Sepharose CL-6 B after the gel filtration on Sephadex G-25. Peroxisomal CPT was completely bound to a Blue Sepharose CL-6 B column and was eluted below 0.25M KCl, whereas mitochondrial CPT was not retained on the column. The substrate specificity profile of peroxisomal CPT with long-chain acyl-CoAs (C8 to C18) was similar to that of mitochondrial CPT, and the apparent Km value of peroxisomal CPT for palmitoyl-CoA was 5.2 μM, being similar to that of mitochondrial CPT.
It is concluded that carnitine long-chain acyltransferase, which is different from mitochondrial CPT and is induced by clofibrate treatment, is present in peroxisomes of chick embryo liver.View full abstractDownload PDF (810K) -
Megumi NAKAMURA, Mitsushi INOMATA, Masami HAYASHI, Kazutomo IMAHORI, S ...1985 Volume 98 Issue 3 Pages 757-765
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn endogenous inhibitor of calcium-activated neutral protease (CANP), which was isolated from rabbit skeletal muscle with chemically drastic pretreatments, comprised major (high-molecular-weight form, HMW-inhibitor) and minor (low-molecular-weight form, LMW-inhibitor) components. HMW-inhibitor was purified to homogeneity using FPLC and preparative electrophoresis. The purified inhibitor appeared as a single protein with a molecular weight of 110, 000 on SDS-polyacrylamide gel electrophoresis, and a molecular weight of 210, 000 on gel filtration. It was therefore presumed that the inhibitor is a dimer protein under native conditions. It contained large amounts of glutamic acid, alanine, and proline, and small amounts of aromatic amino acids, showing an amino acid composition similar to that of LMW-inhibitor. HMW-inhibitor inhibited CANPs with both low (m-type) and high μ-type) Ca2+-sensitivity but had no effect on any other proteases examined. It was demonstrated that the inhibition was due to the formation of a stoichiometric complex between rabbit mCANP and inhibitor subunit in the ratio of five to one. These results suggest that HMW-inhibitor might have several reactive sites per molecule and that LMW-inhibitor subunit might be a proteolytic fragment of HMW-inhibitor containing an active site.View full abstractDownload PDF (1457K) -
Yoko YANO-TOYOSHIMA1985 Volume 98 Issue 3 Pages 767-779
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe biochemical properties of 21 S dynein derived from sea urchin sperm flagella and of its components dissociated by low salt treatment were studied. SDS-urea gel electrophoresis and two-dimensional gel electrophoresis showed that the 21 S dynein preparation contains two distinct heavy chains. These two heavy chains, termed Aα and Aβ, had apparently the same molecular weight of 500, 000 but showed different mobilities on SDS-urea gels. The isoelectric points of Aα and Aβ heavy chains were 5.7 and 5.2, respectively, in the presence of urea. Proteolytic digestion patterns of these two heavy chains were clearly different, but the amino acid compositions were similar.
Low salt treatment and sucrose density gradient centrifugation could partially separate the components of 21 S dynein into two fractions: the one with larger sedimentation coefficient contained the Aα heavy chain, and the other with smaller sedimentation coefficient contained the Aβ heavy chain and three intermediate chains. These two fractions showed distinctly different kinetic properties, and thus may play different roles in dynein-microtubule interaction.View full abstractDownload PDF (4077K) -
Tadashi OGISHIMA, Yoshiie OKADA, Tsuneo OMURA1985 Volume 98 Issue 3 Pages 781-791
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIsolated bovine adrenal cortex mitochondria imported in vitro synthesized pre-P-450 (SCC) and processed it to the mature form. Partial radio-sequencing of the processed P-450 (SCC) gave a result identical with that for authentic P-450 (SCC). Rat liver mitochondria also imported pre-P-450 (SCC) and processed it to the mature form, whereas bovine heart mitochondria were unable to import and process pre-P-450 (SCC) although both mitochondrial preparations imported and processed preadrenodoxin. The pre-P-450 (SCC) processing activity of bovine adrenal cortex mitochondria was associated with the matrix side surface of the inner membrane. The processing protease could be solubilized by sodium cholate and partially purified by ammonium sulfate fractionation. The partially purified processing protease cleaved pre-P-450 (SCC) at the correct position. It was also active in processing pre-P-450 (11 β) but inactive toward pre-adrenodoxin. Bovine heart mitochondria lacked the processing activity to pre-P-450 (SCC).
The localization of pre-P-450 (SCC) and mature P-450 (SCC) in bovine adrenal cortex mitochondria was examined. Mature P-450 (SCC) processed by the mitochondria was found associated with the matrix-side surface of the inner membrane, which is the correct location of P-450 (SCC) in the cell. In the presence of o-phenanthroline, pre-P-450 (SCC) was imported into the organelles without being processed and remained soluble in the matrix. The incorporation of newly processed mature P-450 (SCC) into the inner membrane was also observed when pre-P-450 (SCC) was incubated with inner membrane vesicles. Mature P-450 (SCC) generated in vitro from pre-P-450 (SCC) by the partially purified processing protease was incorporated not only into the inner membrane vesicles but also into bovine adrenal cortex microsomes. These findings suggested that the processing of pre-P-450 (SCC) occurred prior to the incorporation of mature-P-450 (SCC) into the inner membrane.View full abstractDownload PDF (7791K) -
Nobuo IDA, Masanobu TOKUSHIGE1985 Volume 98 Issue 3 Pages 793-797
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN-(7-Dimethylamino-4-methylcoumarynyl) maleimide (DACM), a fluorescent reagent for sulfhydryl groups, was employed to determine the functionally essential cysteine residues in aspartase from Escherichia coli. Analysis of the tryptic peptides containing DACM-labeled residues by reverse phase HPLC revealed that Cys-140 and Cys-430 were selectively modified, among 11 residues whose loci were recently determined by a DNA sequencing study (Takagi, J. S., et al. (1985) Nucl. Acids Res. 13, 2063-2074). When the modification was carried out in the presence of Mg2+ and L-aspartate, the enzyme activity remained unchanged and no cysteine residue was modified. This suggests that two cysteine residues are located at the L-aspartate binding site and that at least one of them is involved in the catalytic reaction.View full abstractDownload PDF (379K) -
Masahiro SHIRAKAWA, Yasushi KAWATA, Sang Jong LEE, Hideo AKUTSU, Fumio ...1985 Volume 98 Issue 3 Pages 799-805
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInstitute for Protein Research, Osaka University The tyrosine residues of λ cro repressor were partially nitrated with tetranitromethane under mild conditions. After digestion by Achromobacter protease I, the extent of nitration was determined by HPLC and amino acid analysis. Tyr 26 was most easily nitrated and Tyr 51 followed it. Tyr 10 was resistant to nitration. By comparison of the proton magnetic resonance spectrum of the partially nitrated cro protein with the above result, the aromatic proton resonances of the tyrosine side chains could be assigned to individual tyrosine residues. The extent of nitration is parallel to the accessibility to a flavin dye as measured by photo CIDNP (chemically induced dynamic nuclear polarization).View full abstractDownload PDF (496K) -
Shigeo NAKAJO, Kazuyasu NAKAYA, Yasuharu NAKAMURA1985 Volume 98 Issue 3 Pages 807-813
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties of a protein kinase-substrate complex precipitated with Ca2+ from the cytosol of AH-66 hepatoma cells were characterized. The endogenous phosphorylation reaction of the complex was little affected by addition of histone, cyclic nucleotides, Ca2+-calmodulin, or Ca2+-phospholipid but was increased about two-fold by addition of casein. The complex contained several phosphate acceptor proteins with molecular weights ranging from 74, 000 to 13, 000 as analyzed by two-dimensional gel electrophoresis. These phosphate acceptor proteins were specifically concentrated in the complex. The protein kinase in the complex was purified by successive chromatography and proved to be casein kinase 2.View full abstractDownload PDF (2156K) -
Kenji TAKAHASHI1985 Volume 98 Issue 3 Pages 815-817
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of ribonuclease T1 was reinvestigated over the entire molecule by manual Edman degradation of performic acid-oxidized RNase T1 and some of its tryptic and chymotryptic peptides. The validity of the sequence was confirmed except for the sequence Pro-Gly-Ser at positions 71-73. This sequence should be revised to Gly-Ser-Pro.View full abstractDownload PDF (224K) -
Koukichi MARUYAMA, Hiroshi KITAMURA1985 Volume 98 Issue 3 Pages 819-824
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMechanisms of growth inhibition by propionate on the growth of Rhodopseudomonas sphaeroides were studied.
Partially purified pyruvate dehydrogenase complex (PDC) from R. sphaeroides was inhibited by propionyl-CoA, one of the metabolic intermediates of propionate, while propionate itself did not inhibit the enzyme. This suggests that the inhibitor of the growth in vivo is not propionate but propionyl-CoA. The inhibition by propionyl-CoA was competitive with respect to coenzyme A concentration. The K1 value for propionyl-CoA was 0.84mM.
Addition of NaHCO3 which restored the growth of this bacterium in the presence of propionate, increased the rate of propionate incorporation by 1.7-fold and decreased the intracellular level of propionyl-CoA by half. These findings suggest that HCO3- ion lowers the level of propionyl-CoA by accelerating its carboxylation reaction, which is catalyzed by propionyl-CoA carboxylase.
Effects of NaHCO3 and acetate on the growth restoration were also studied by the use of propionyl-CoA carboxylase-deficient mutants. NaHCO3 did not restore the growth of the mutants, indicating an essential role of propionyl-CoA carboxylase on the restoration of growth by NaHCO3 as suggested above.
Addition of acetate restores the growth of the mutants in the presence of propionate. Acetate probably restores the growth by supplying acetyl-CoAView full abstractDownload PDF (534K) -
Junichi MATSUSHITA, Yoji KATO, Kazuo MATSUDA1985 Volume 98 Issue 3 Pages 825-832
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo components of α-D-xylosidase (α-D-xylosidase I and II) were detected in the culture filtrate of Aspergillus nigher grown in a medium containing Sanzyme 1000-treated Glyloid 2 A. The major component (α-D-xylosidase I) was purified to an electrophoretically pure state. The purified enzyme showed approximately 540-fold increase in specific activity over the original culture filtrate.
The purified enzyme was shown to be an oligomeric protein consisting of four subunits, each of which had a molecular weight of 123, 000. The enzyme showed the highest activity at pH 2.5-3.0 and 45&C, and was stable in the pH range from 3.0 to 7.0 and at the temperatures up to 60&C. The isoelectric point of this enzyme was pH 5.6. The purified enzyme was highly specific for p-nitrophenyl α-D-xylopyranoside and isoprimeverose (6-O-α-D-xylopyranosyl-D-glucopyranose). The apparent Km and Vmax values of the enzyme for p-nitrophenyl α-D-xylopyranoside and isoprimeverose were 10.5mM and 40.8 μmol/min/mg protein, and 2.2mM and 30 μmol/min/mg protein, respectively. The purified enzyme could also split off the α-D-xylopyranosyl residue on the non-reducing terminal of the backbone of oligoxyloglucans such as α-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-{[α-D-xylopyranosyl-(1→6)-]-β-D-glucopyranosyl-(1→4)-}2-D-glucopyranose.View full abstractDownload PDF (2004K) -
Hiroko TODA, Michio YAZAWA, Fumio SAKIYAMA, Koichi YAGI1985 Volume 98 Issue 3 Pages 833-842
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of calmodulin from wheat germ was determined by isolating and sequencing the cyanogen bromide and tryptic peptides. The protein consisted of 149 amino acid residues and its amino (N)-terminus was blocked with an acetyl group. Wheat germ calmodulin lacked tryptophan and contained 1 mol each of histidine, tyrosine, cysteine, and Nε-trimethyllysine residues per mol of the protein. A comparison of its amino acid sequence with that of bovine brain calmodulin indicated that there were eleven amino acid subsitutions other than amide assignments, two insertions and one deletion of amino acid residues in wheat germ calmodulin.View full abstractDownload PDF (837K) -
Leif HÅKANSSON, Pam FREDMAN, Lars SVENNERHOLM1985 Volume 98 Issue 3 Pages 843-849
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSImmune complexes in the serum of tumor-bearing patients were adsorbed from whole blood or plasma on a protein A-Sepharose column. The adsorbed material was eluted, precipitated and analyzed for gangliosides. All precipitates obtained from eight patients at different treatment occasions contained gangliosides at concentrations varying from 0.1 to 12.2 nmol sialic acid/mg protein. The compositions of gangliosides were similar among the patients, regardless of the type of cancer, and quite different from that of normal serum. Most (75-85% of total sialic acid) belonged to the gangliotetraose series, of which 26-33% was GM1, 26-34% GD1a, 8-17% GD1b, and 5-13% GT1b. However, the dominant ganglioside in normal serum, GM 3, was present in only trace amounts, which ruled out a nonspecific adsorption of serum ganglioside by protein A-Sepharose. Similar results were obtained for whole blood and plasma treatments, and these results suggest a specific interaction between gangliosides of the gangliotetraose series and serum immunoglobulins, either by the gangliosides acting as antigens and forming immune complexes or by their binding to already formed complexes.View full abstractDownload PDF (612K) -
Osamu TARUTANI, Nobuo UI1985 Volume 98 Issue 3 Pages 851-857
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAffinity chromatography on a concanavalin A (con A)-Sepharose column is a potentially useful for the isolation of whole thyroglobulin (Tg) at least from normal thyroid tissue. In addition to being a simple procedure for the isolation of Tg, large amounts of Tg can be applied to the column and recovered in good yield with a buffer containing MeG.
In gradient elution with buffer containing increasing amounts of MeG, a single but broad peak was obtained, without separation into subfractions. However, a hemagglutination-inhibition test showed that the Tg preparation eluted early from the column had less affinity for con A than the Tg preparation eluted later, suggesting a heterogeneous distribution of carbohydrate moieties among Tg preparations.
When human Tg from thyroid tumor was applied to the column, tumor Tg partly passed through the column without being adsorbed. This unadsorbed Tg showed a very low affinity for lectins, con A and wheat germ agglutinin (WGA), as determined by a double diffusion reaction in agar gel. In contrast to this fraction, the Tg adsorbed on the con A-gel column showed a very strong affinity for WGA, differing from Tg of normal thyroid tissue. Therefore, tumor Tg preparation appears to have an abnormally modified carbohydrate structure, at least in part. The higher affinity for WGA (with a specificity for N-acetylglucosamine) seen in adsorbed Tg could be due to a larger amount of GlcNAc residues which bind irregularly in the carbohydrate moiety of tumor Tg.View full abstractDownload PDF (1254K) -
Hiroshi UEMURA, Yoshifumi JIGAMI, Hideaki TANAKA, Nobumasa TOSHIMITSU, ...1985 Volume 98 Issue 3 Pages 859-862
Published: 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeveral plasmids carrying different length of the 5' flanking region of yeast (Saccharomyces cerevisiae) enolase 1 gene (ENO1) which is fused in frame to the Escherichia coli lacZ gene were constructed by recombination in vitro. Promoter activity of ENO1 was assayed by measuring β-galactosidase activity of the fused gene product. Comparison of the promoter activity of these plasmids suggests that the sequences required for a strong promoter activity lie within the DNA segment -724 to -353 base pairs (bp) upstream from the start of ENO1 coding sequence. The nucleotide sequence of this region was determined.View full abstractDownload PDF (388K)
- |<
- <
- 1
- >
- >|