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Volume 99 (1986)
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Volume 99, Issue 2
Displaying 1-33 of 33 articles from this issue
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Kei-ichi UCHIKAWA, Isao SEKIKAWA, Ichiro AZUMA1986 Volume 99 Issue 2 Pages 315-327
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStructural studies were carried out on the teichoic acids in cell walls of Listeria monocytogenes serotypes 3a, 4b, 4f, 6, and 7. The structure of the dephospho-rylated repeating units, obtained by treatment with 46% hydrogen fluoride or alkaline hydrolysis, was examined by methylation analysis, acetolysis, and 1H-NMR spectroscopy. The results of Smith degradation of the teichoic acids and 13C-NMR spectroscopy led to the following most likely structures of the repeating units of the teichoic acids: → 1-[N-acetylglucosaminyl (αl → 4)] ribitol-5-phosphate → for serotype 3a, → 4- [galactosyl (αl → 6)] [glucosyl (β1 → 3)]N-acetylglucosaminyl (β1 → 2) ribitol-5-phosphate → for serotype 4b, → 4- [galactosyl (αl → 6)] [N-acetylglucosaminyl (αl → 3)] N-acetylglucosaminyl (β1 → 2) ribitol-5-phosphate → for serotype 4f, → 4-N-acetylglucosaminyl (β1 → 4) ribitol-5-phosphate → for serotype 6, and → 1-ribitol-5-phosphate → for serotype 7. About 40% of the repeating units of the teichoic acid from serotype 4f were not substituted at C-3 of β-N-acetylglucosaminyl residues.View full abstractDownload PDF (1002K) -
Yasuzo NISHINA, Retsu MIURA, Hiromasa TOJO, Yoshihiro MIYAKE, Hiroshi ...1986 Volume 99 Issue 2 Pages 329-337
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResonance Raman (RR) spectra were obtained for the purple complexes of D-amino acid oxidase (DAO) with D-lysine or N-methylalanine. RR spectra of a complex of oxidized DAO with the oxidation product of D-lysine or D-proline were also measured. The isotope shifts of the observed bands of the purple complex with D-lysine upon 13C- or 15N-substitution of lysine indicate that the ligand is Δ1-piperideine-2-carboxylate. That the band at 1671 cm-1 for the purple intermediate with N-methylalanine shifts to 1666 cm-1 in D2O solution indicates that the imino acid, N-methyl-α-iminopropionate, has a protonated imino group. Many bands due to a ligand in the RR spectra of the complex of oxidized DAO with an oxidation product can be observed below 1000 cm-1, but no band for the purple complex is seen in this frequency region. The band associated with the CO2- symmetric stretching mode of the product, such as Δ1-piperideine-2-carboxylate or Δ1-pyrrolidine-2-carboxylate, complexed with the oxidized DAO shifts in D2O solution. This suggests that the product imino acid interacts with the enzyme through some proton(s).View full abstractDownload PDF (682K) -
Masayuki TAKAHASHI, Fumi MORITA1986 Volume 99 Issue 2 Pages 339-347
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn activating factor for the superprecipitation of actomyosin reconstructed from scallop smooth muscle myosin and rabbit skeletal muscle F-actin was purified from thin filaments of scallop smooth and striated muscles. Two components were obtained from the smooth muscle and one from the striated muscle. All three components similarly affected the actomyosin ATPase activity. According to the results of analysis involving double reciprocal plotting of the ATPase activity versus F-actin concentration, the activating factor for superprecipitation decreased the apparent dissociation constants of actomyosin about 30 to 110 times. The activation of the superprecipitation by the factor, therefore, may be due to the enhancement of the affinity between F-actin and myosin in the presence of ATP.
The activating factor was identified as tropomyosin based on it mobility on polyacrylamide gel electrophoresis and on the recovery of the Ca2+-sensitivity of purified rabbit skeletal actomyosin in the presence of troponin.View full abstractDownload PDF (1100K) -
Norifumi HIROTA1986 Volume 99 Issue 2 Pages 349-356
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMethyl-accepting chemotaxis proteins (MCPs) were solubilized from the membrane of thermophilic bacterium PS-3 in the presence of Triton X-100. The solubilized MCPs could be methylated and demethylated. Methylation of the solubilized MCPs reached a steady state, at which the methylation and demethylation rates were equal. The solubilized MCPs were purified by anti-MCPs Sepharose 4B column chromatography. The purified MCPs could also be methylated and demethylated without reconstituting them into liposomes. As suggested by the results of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified MCPs, ion-exchange chromatography showed that MCPs consisted of at least two components. Each component appeared on SDS gel electrophoresis as multiple bands in the 64K to 70K dalton range or in the 70K to 84K dalton range.
The initial rate and level of methylation of the solubilized MCPs were increased by the addition of attractants: glutamate, L-serine, L-aspartate, D-glucose, etc. The threshold of the glutamate concentration for this increase was about 10-7 M. The rate of demethylation was also increased by attractants.View full abstractDownload PDF (1993K) -
Tsutomu TAKAGI, Masaki SUGIURA, Masahiko KISUMI1986 Volume 99 Issue 2 Pages 357-364
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSArginine productivity of an arginine-producing mutant of Serratia marcescens decreased during successive batch culturing. The mutant grew more slowly than the parent strains in a minimal medium, and spontaneously produced derivatives that grew more rapidly than the mutant. A large majority of the derivatives required N-acetyl glutamate or arginine for growth, due to lack of N-acetylglutamate synthase, the argA gene product. The argA1 allele carried by the mutant was found to be relatively unstable. While the mutation rate in a stable argA mutant allele was less than 1×10-8 per cell per generation, that in the argA1 allele was 9×10-7. The instability of the arginine productivity, therefore, was owing to both a disadvantage of the mutant in growth and a high mutability in the argA1 allele. In addition to the auxotrophs, the unstable arginine-producing mutant spontaneously produced at low frequency stable arginine-producing derivatives; among them, AT428 formed N-acetylglutamate synthase with a reduced affinity for glutamic acid. The derivative showed restored capability for propagation, and stably produced a large amount of arginine in the presence of glutamic acid or fumaric acid. By transductional analysis, the derivative was found to have acquired in the argA allele an additional mutation leading to the reduced affinity independently of the original one leading to the feedback-resistant enzyme.View full abstractDownload PDF (620K) -
Tomoko NISHIMAKI, Hiroshi YAMANAKA, Michinao MIZUGAKI1986 Volume 99 Issue 2 Pages 365-374
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCrude extract of Escherichia coli was found to elongate medium chain acyl-CoA primers. The reaction products were fatty acids one or two C2 units longer than the primer. Acetyl-CoA acted as the condensing unit in this reaction, while malonyl-CoA did not. The optimal pH for the reaction was 5.0 in 0.1M citrate-phosphate buffer. NADH was the predominant electron donor for the incorporation of acetyl-CoA into fatty acids, and NADPH was one-third as effective as NADH at pH 5.0. Acyl carrier protein and cerulenin had no effect on the acetyl-CoA incorporation into the chain elongation products. Acyl-CoA compounds with medium carbon chain lengths proved to be the best as primers, and the maximum incorporation was observed with octanoyl-CoA. N-Ethylmaleimide and p-hydroxymercuri-benzoate blocked the chain elongation reaction by inhibiting either condensation or 3-ketoacyl reduction.View full abstractDownload PDF (733K) -
Maria Teresa VINCENZINI, Paolo VANNI, Eugenio GIACHETTI, Giorgio Maria ...1986 Volume 99 Issue 2 Pages 375-383
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIsocitrate lyase catalyzes the reversible cleavage of isocitrate into glyoxylate and succinate. The kinetic mechanism of bacterial isocitrate lyase has been reported to be ordered uni-bi. Moreover, it has been proposed that isocitrate lyase in higher plants may be switched on and off by a succinylation/desuccinylation mechanism. Similarly to bacterial citrate lyase, in which an acetylation/deacetylation mechanism is operative, succinylation might also play a role in the catalytic mechanism of plant isocitrate lyase. We have investigated the kinetic mechanism of isocitate lyase from Lupinus seeds. The results reported in this paper show that the system follows a preferentially ordered uni-bi pathway in which the succinate is released first. On the basis of our results and some other recently reported data, we conclude that it is unlikely that bacterial and plant isocitrate lyases have different catalytic mechanisms.View full abstractDownload PDF (650K) -
Keisuke FUKUO, Masaharu TAKIGAWA, Koji TAJIMA, Motomi ENOMOTO, Yuichi ...1986 Volume 99 Issue 2 Pages 385-396
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBoth retinoids and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate(TPA) inhibit expression of the differentiated phenotype by rabbit costal chondrocytes in culture, as judged by morphological changes and decreased sulfation of glycosaminoglycans (GAG). However, the inhibition of the differentiated phenotype of chondrocytes in TPA-treated cells is restored by parathyroid hormone (PTH), while the inhibition by retinoids is not [Takigawa et al. (1982) Mol. Cell. Biochem. 42, 145-153; Takigawa et al. (1983) Cell Differ. 13, 283-291]. In the present study, we examined the difference between TPA-treated chondrocytes and retinoic acid-treated chondrocytes to determine the mechanism of the restoration of the differentiated phenotype in de-differentiated cells treated with TPA.
PTH increased the activity of ornithine decarboxylase [ODC; EC 4.1.1.17], a rate limiting enzyme of polyamine biosynthesis, and proteoglycan synthesis in chondrocytes pretreated with TPA as well as in normal chondrocytes. The maximal stimulations of ODC activity and GAG synthesis were observed 4h and 24-36h, respectively, after addition of PTH. The dose-response curve for ODC induction by PTH was parallel to that of PTH-stimulated proteoglycan synthesis both in TPA-treated chondrocytes and in normal chondrocytes. PTH also increased the intracellular cyclic AMP level after 2 min in TPA-treated cells as in normal cells. Addition of dibutyryl cyclic AMP (DBcAMP) induced ODC and restored proteoglycan synthesis in TPA-treated cells. The dose-response curve for induction of ODC by DBcAMP was parallel to that of DBcAMP-stimulated proteoglycan synthesis in both TPA-treated chondrocytes and normal chondrocytes. On the other hand, the increases by PTH in the intracellular cyclic AMP level, ODC activity, and proteoglycan synthesis were inhibited in chondrocytes pretreated with a combination of TPA and retinoic acid as well as in those pretreated with retinoic acid alone. TPA stimulated the syntheses of DNA and RNA in chondrocytes but did not increase the cyclic AMP level or ODC activity. PTH and DBcAMP inhibited the syntheses of DNA and RNA both in TPA-treated cells and in normal cells. These results suggest that ODC induction mediated by elevation of cyclic AMP plays an important role in re-differentiation of de-differentiated cells pretreated with these agents.View full abstractDownload PDF (1867K) -
Manabu KUNIMOTO, Keiko MIYASAKA, Takashi MIURA1986 Volume 99 Issue 2 Pages 397-406
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts.
These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.View full abstractDownload PDF (3129K) -
Toshitsugu YUBISUI, Toshiyuki MIYATA, Sadaaki IWANAGA, Minoru TAMURA, ...1986 Volume 99 Issue 2 Pages 407-422
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of soluble NADH-cytochrome b5 reductase purified from human erythrocytes was determined. The enzyme, which contained 8 methionine residues, was cleaved by cyanogen bromide. The resulting nine peptides were separated by gel filtration and purified further by high-performance liquid chromatography. The purified peptides were sequenced by automated Edman degradation. Three large CNBr peptides, residues 1-101, 109-151, and 169-231, were further fragmented with trypsin, Staphylococcus aureus V8 protease or a lysyl endopeptidase of Achromobacter lyticus. The peptides obtained from the tryptic digest of citraconylated FAD-depleted apoprotein completed the alignments of the other peptides. The enzyme was composed of 275 amino acid residues. The 4 functionally important cysteine residues were located in the COOH-terminal portion. The molecular weight of the protein was calculated to be 31, 260 without FAD.
A prediction of the secondary structure was made by the method of Chou and Fasman. The protein was hydrophilic as a whole (43 % polarity), but some regions were rich in hydrophobic residues. From the sequence homology of this enzyme with the pyridine nucleotide-binding sites of other flavoproteins, three candidates for the FAD and NADH-binding domains were suggested.View full abstractDownload PDF (1121K) -
Kazuhiko SAEKI, Takahiko HARUNA, Tomisaburo KAKUNO, Jinpei YAMASHITA, ...1986 Volume 99 Issue 2 Pages 423-435
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was found that the cytoplasm of light-grown cells of Rhodospirillum rubrum could catalyze the reduction of methyl viologen (MV) (Em, 7=-0.44 V) by NADH and NADPH. In the present study, the enzyme capable of catalyzing MV reduction by NADH (NADH-MV reductase) was purified 1, 500-fold from an extract of cells with a yield of 4.4%. The purification procedure comprised (NH4)2SO4 fractionation, and chromatographies on Sepharose CL-6B, DEAE-Sepharose CL-6B, phenyl-Sepharose CL-4B, Blue-Cellulofine, and TSK-Gel G3000SW. Two NADPH-MV reductases were separated during the purification. The NADH-MV reductase obtained was nearly homogeneous, as judged on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The enzyme has a molecular weight of 220, 000 and an isoelectric point of 4.8; it is composed of four subunits with a molecular weight of 57, 000, and is bound with about 1mol FAD/mol subunit. The activity is optimum at pH 8. The Km values for NADH and MV are 115 μM and 1.3mM, respectively, with a molecular activity of 13, 000 min-1. The activity was stimulated 2.4-fold in the presence of 20-100mM ammonium ions. The enzyme also catalyzed the reduction of benzyl viologen, methylene blue and 2, 6-dichlorophenol-indophenol (Em, 7=-0.36, +0.011, and + 0.217V, respectively) at comparable rates. The ratios of the activity with NADH to that with NADPH were 80, 133, 41, and 5.5 with MV, benzyl viologen, methylene blue and 2, 6-dichlorophenolindophenol, respectively. The enzyme was significantly stable in the presence of both 5mM 2-mercaptoethanol and 20% (w/v) glycerol. The activity was not appreciably influenced by the presence of 2M urea, although the reagent caused dissociation to the subunits.View full abstractDownload PDF (943K) -
Tatsuko FURUYA, Yoshiyuki SUZUKI, Takashi MOMOI1986 Volume 99 Issue 2 Pages 437-443
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA highly sensitive microassay method and a microscale purification system were developed to isolate the residual acid β-galactosidase in GMi-gangliosidosis fibroblasts. The sensitivity of the microassay system, composed of a 96-well microplate and a microplate fluorometer, was 100-fold higher than that of the conventional system and the response was linear in the pmole range. Acid β-galactosidase was characterized as a thiol enzyme which was inactivated by a mercuric compound. This enzyme was completely adsorbed on an Hg-agarose column and was easily eluted from the column by 10mM 2-mercaptoethanol. The microscale purification system using Con A-Sepharose, PAT-Sepharose, and Hg-agarose column chromatography achieved 565- and 7, 970-fold purifications of acid β-galactosidase with an overall yields of 44% and 45% from normal and GM1-gangliosidosis fibroblasts, respectively. The purified enzyme fractions did not contain any other lysosomal enzyme activities except for a small amount of β-N-acetylhexosaminidase activity.View full abstractDownload PDF (470K) -
Israr Ahmad KHAN, Rashid ALI1986 Volume 99 Issue 2 Pages 445-452
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe antiferromagnetically spin-coupled Cu2+ pair present in the active center of tyrosinase was found to be indispensable for its catalytic function. However, the metal ion did not contribute to the conformational integrity or antigenicity of the enzyme molecule. Irradiation of tyrosinase with 254 nm light resulted in dose dependent, essentially irreversible losses of its catalytic and antigenic functions. The apparent first order rate constants for the two processes were 17.6×10-2 mini and 28.1×10-2 min-1, respectively. The approximately 1.6-fold difference between the two rate constants suggests that the sites of antigenic determinants in tyrosinase are distinguishable from the enzymic active site by their higher photosensitivity. Kinetic analysis of the data as to photoinactivation, and the UV induced losses of antigenicity and structural integrity revealed that UV radiation disrupts the shortrange noncovalent interactions occurring within the enzyme molecule. The disruption of the noncovalent interactions results in partial unfolding of the tyrosinase structure which in turn leads to the progressive loss of its catalytic activity and antigenicity. The anti-tyrosinase antibodies raised in rabbits were found to be directed against the native conformation of the enzyme. It is speculated that these antibodies might be useful in exploring the tyrosinase conformation and in studying the effects of various factors on the enzyme surface and molecular structure.View full abstractDownload PDF (1764K) -
Haruko KITAGAKI, Isamu MATSUMOTO, Nobuko SENO, Sumi NAGASE1986 Volume 99 Issue 2 Pages 453-458
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to elucidate the correlation between cell surface lectin binding sites and the degree of cell adhesiveness, quantitative lectin binding assays were performed using three types of rat ascites hepatoma cell lines (free cell, mixed cell, and islandforming cell types). The lectin binding site patterns showed no remarkable differences among the intact tumor cell lines, but treatment of the cells with L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or neuraminidase induced remarkable differences in the modulation of the number of lectin binding sites. TPCK-trypsin treatment caused a marked decrease in the number of peanut agglutinin binding sites on the island-forming and mixed cell types, concomitant with disaggregation of the cells, showing that trypsin sensitive binding sites are involved in the cell-cell adhesion. Neuraminidase treatment caused a decrease in wheat germ agglutinin binding sites and an increase in castor bean agglutinin binding sites, and these effects were greater for the free cell type. These results indicated that α-sialyl-β-D-galactosyl residues are more abundant on the cell surface of the free cell type than the other cell types. Therefore, it was suggested that electrostatic repulsion due to negative charges of the cell surface sialic acid contributes to the low cell adhesiveness of the free cell type.View full abstractDownload PDF (496K) -
Reiji YOSHINAKA, Mamoru SATO, Masao ITOKO, Michiaki YAMASHITA, Shizuno ...1986 Volume 99 Issue 2 Pages 459-467
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA collagenolytic enzyme was purified to homogeneity from the activated pancreatic extract of the catfish Parasilurus asotus by chromatography on DEAE-cellulose, hydroxylapatite, and Cellulofine columns. The molecular weight of the enzyme was estimated to be 29, 500 by SDS-polyacrylamide gel electrophoresis. The enzyme is capable of degrading native, reconstituted calf skin collagen fibrils at pH 7.5 and 37°C, and also of reducing the viscosity of native calf skin collagen at pH 7.5 and 20°C. The SDS-polyacrylamide gel electrophoresis of thermally denatured enzyme-collagen reaction mixtures showed that the enzyme can cleave peptide bonds in the non-helical and triple-helical regions of the collagen. The enzyme was inhibited by DFP, PMSF, soybean trypsin inhibitor, and chicken ovoinhibitor, but not by metal-chelating reagents EDTA, EGTA, o-phenanthroline, or L-cysteine. These results indicate that the enzyme is a unique collagenolytic proteinase belonging to the group of serine proteinases and is a new member of the class of pancreatic enzymes.View full abstractDownload PDF (1883K) -
Michinao MIZUGAKI, Mataichi SAGI, Hiroshi YAMANAKA, Hideki TAKAYAMA, M ...1986 Volume 99 Issue 2 Pages 469-476
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChain elongation of fusaric acid and related compounds in the presence of rat liver preparations was investigated by gas chromatography-mass spectrometry. The mitochondrial fraction catalyzed the elongation of the CoA esters of fusaric acid and 5-butyl-2-pyrimidinecarboxylic acid utilizing acetyl-CoA as a C2 donor. The microsomal fraction failed to afford elongation products. However, when the CoA ester of 3-(5-butyl-2-pyrimidinyl)-3-hydroxypropionic acid was incubated in the presence of the mitochondria) or the microsomal fraction, the corresponding αβ-unsaturated and saturated metabolites were identified in both cases, suggesting that the microsomal fraction could not catalyze the condensation or the keto-reduction of these heteroaromatic carboxylic acids.View full abstractDownload PDF (540K) -
Kazuhiro SHIMAZU, Miho KUWABARA, Michiko YOSHII, Kenji KIHIRA, Haruo T ...1986 Volume 99 Issue 2 Pages 477-483
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBile alcohols in bile, urine, and feces of a patient with cerebrotendinous xanthomatosis have been analyzed by a combination of capillary gas-liquid chromatography and mass spectrometry after fractionation into groups according to mode of conjugation. The presence of at least 18 bile alcohols, which were excreted mainly as glucurono-conjugates in bile and urine, and as unconjugated forms in feces, was demonstrated. The following bile alcohols were identified with certainty by direct comparison with reference compounds: 5β-cholestane-3α, 7α, 12α-triol; (23R)-5β-cholestane-3α, 7α, 12α, 23-tetrol; 5α- and 5β-cholestane-3α, 7α, 12α, 24-tetrols; 5α- and 5β-cholestane-3α, 7α, 12α, 25-tetrols; 27-nor-5β-cholestane-3α, 7α, 12α, 24, 25-pentol ; (22R)-5β-cholestane-3α, 7α, 12α, 22, 25-pentol; (23R)- and (23S)-5β-cholestane-3α, 7α, 12α, 23, 25-pentols; 3α, 12α, 25-trihydroxy-5β-cholestan-7-one; (24R)- and (24S)-5β-cholestane-3α, 7α, 1 2α, 24, 25-pentols; 5β-cholestane-3α, 7α, 12α, 25, 26-pentol. Although the bile alcohol profile in urine was quite different from those in bile and feces, the determination of urinary bile alcohols as well as of biliary and fecal bile alcohols could be used for diagnosis of cerebrotendinous xanthomatosis.View full abstractDownload PDF (593K) -
Kazuhiko SAEKI, Osamu ISHIKAWA, Toru FUKUOKA, Hajime NAKAGAWA, Yasushi ...1986 Volume 99 Issue 2 Pages 485-494
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPeroxidase was prepared from extracts of barley leaves and separated into seven components, different in pI. The purification procedure comprised two parts. The first part was based on the fact that all the components had practically the same molecular weights. It consisted of fractionations with acetone and ammonium sulfate, ion-exchange chromatographies on CM-cellulose and DEAE-Sepharose CL-6B, and molecular-sieve chromatography on Ultrogel AcA44; the components were all purified together to near homogeneity on sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis, and the procedure resulted in 1, 200-fold purification with a yield of 39%. The ion-exchange chromatographies were carried out under conditions such that the components would not be adsorbed. In the second part, the enzyme preparation was separated into the seven components by repeating isoelectric electrophoresis. Their isoelectric points (pI) were 6.3, 6.8, 7.4, 8.3, 8.5, 8.7, and 9.3. The components other than the pI 6.3 and 6.8 components were each purified to homogeneity in the electrophoresis.
The seven components thus prepared were the same in molecular weight on SDS-gel electrophoresis (44, 000) and showed absorption maxima at the same wavelengths (403, 496, and 534 nm), RZ (A403/A275, ) ranging from 2.09 to 2.81. Their protoheme IX contents were 0.81-1.07 mol/mol, and their true sugar contents 1526% (g/g). The amino acid compositions suggest that the five components described above are not real isoenzymes, but exhibit different pI values due to differences in glycosyl residue. The pI 9.3 component was crystallized in spite of its high sugar content.View full abstractDownload PDF (1114K) -
Hiroyuki MATSUYAMA, Yoichi KAWANO, Naotaka HAMASAKI1986 Volume 99 Issue 2 Pages 495-501
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe transport of phosphoenolpyruvate across the erythrocyte membrane was compared with the transport of inorganic phosphate in resealed ghosts of human erythrocytes. Two conditions were employed: in one, the external pH (pHe) was varied from 6.0 to 7.5 while internal pH (pHi) was maintained at 7.2 or 6.2, whereas in the other, the internal and external pH were adjusted to have the same value over a pH range of 6.0 to 7.5. At a constant pHi of 7.2, the pH profile for the transport of phosphoenolpyruvate was different from that of inorganic phosphate; both pH profiles were similar to those previously observed in intact erythrocytes (Deuticke, B. (1970) Naturwissenschaften 57, 172-179; Hamasaki, N., et al. (1978) Biochenn. J. 170, 39-46). In the absence of a transmembrane pH gradient, the pH profile for phosphoenolpyruvate transport was bell-shaped with the maximum at pH 6.8, and essentially the same pattern was seen with inorganic phosphate transport.
The different pH profiles obtained under the two conditions suggests that one or more residues exposed at the inner surface of the membrane participate in the transport of inorganic phosphate and phosphoenolpyruvate. One of the most likely candidates is a histidine residue of the transport protein.View full abstractDownload PDF (521K) -
Koshi SAITO, Kunihiro YAMAMOTO, Kazuko ANDO, Takeshi KUZUYA1986 Volume 99 Issue 2 Pages 503-511
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activity of I- counterflow was solubilized with n-octyl β-D-glucopyranoside from the thyroid plasma membranes and reconstituted into phospholipid vesicles made from soybean phospholipids by a detergent dilution procedure. The vesicles exhibited apparent “entrance” I- counterflow but no apparent Na+-dependent I- transport activity. The activity of I- counterflow was saturated by external I- at the concentration of 50-200μM, indicating the substrate specificity of the counterflow-mediating activity for I-. The optimal concentration of the detergent for solubilization was near 12.5 mg/ml, as reported in solubilization of some sugar transport carriers of E. coli. The counterflow of I- was inhibited by externally added SCN- but not by ClO4-. When solubilized protein was treated with 0.2-1 mM dithiothreitol and with 2-10 mM 2-mercaptoethanol prior to reconstitution, dosedependent inactivation of I- counterflow was observed. However, the activity of I- counterflow was fully preserved when solubilized protein was treated with these agents in the presence of high concentrations of I- (40 and 80 mM). In contrast, treatment with N-ethylmaleimide at a concentration of as high as I mM did not decrease the activity. In conclusion, it appears that the I- counterflow activity can be solubilized and reconstituted into phospholipid vesicles from the thyroid plasma membranes. The counterflow activity may be susceptible to disulfidereducing agents.View full abstractDownload PDF (714K) -
Noriko TAKAHASHI, Terumi SAITO, Yoshiko GODA, Kenkichi TOMITA1986 Volume 99 Issue 2 Pages 513-519
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn NADPH-dependent aldehyde reductase was purified from rat brain microsomes to electrophoretic homogeneity. The purified enzyme had a molecular weight of 75, 000 and reduced long chain fatty aldehydes such as octanal and hexadecanal with higher affinity (Km values of 0.21mM and 0.03mM, respectively) than for various artificial carbonyl compounds such as p-nitrobenzaldehyde and p-nitroacetophenone (Km values of 0.31mM and 1.4mM, respectively). The purified microsomal aldehyde reductase also showed NADPH-cytochrome c reductase activity, and it could not be distinguished from NADPH-cytochrome c reductase in molecular weight (75, 000), chromatographic behavior, electrophoretic mobility, or immunological properties. The solubilized microsomal fraction treated with steapsin lost the reductase activity for hexadecanal but not that for cytochrome c. These results suggest that the aldehyde reductase in brain microsomes is identical to NADPHcytochrome c reductase and that a hydrophobic portion of the NADPH-cytochrome c reductase is required for the reduction of hexadecanal.View full abstractDownload PDF (590K) -
Hiromichi KUMAGAI, Eisuke NISHIDA, Susumu KOTANI, Hikoichi SAKAI1986 Volume 99 Issue 2 Pages 521-525
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding of calmodulin to microtubule-associated proteins (MAPs) was analyzed by the equilibrium gel filtration method. The apparent dissociation constant (Kd) of calmodulin binding was found to be 2μm for tau, and 5μM for MAP2. These Kd values were similar to the Kd previously determined for calmodulin binding to tubulin. The inhibitory effect of increasing concentrations of calmodulin on the kinetics of microtubule assembly from tau and tubulin was not mimicked by decreasing the concentration of tau alone or tubulin alone. These results suggest that calmodulin inhibits microtubule assembly by its binding to both MAPS and tubulin.View full abstractDownload PDF (324K) -
Kazuko OGAWA, Tadao KONDO, Makoto KAWAKAMI, Shosuke TAKEMURA1986 Volume 99 Issue 2 Pages 527-530
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn unknown nucleoside in the first position of the anticodon of Torulopsis utilis tRNApro has been isolated. The UV, 1H NMR and secondary ion mass spectra indicated that this nucleoside is a uridine derivative, 5-carbamoylmethyluridine. The structure was completely established by comparison of the instrumental analysis results and chromatographic behavior of the isolated nucleoside with those of a synthetic sample.View full abstractDownload PDF (307K) -
Yukichi HARA, Junji YAMADA, Makoto NAKAO1986 Volume 99 Issue 2 Pages 531-539
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe possibility that H+ might substitute for Na+ at Na+ sites of Na+, K+-ATPase was studied. Na+, K+-ATPase purified from pig kidney showed ouabain-sensitive K+-dependent ATPase activity in the absence of Na+ at acid pH (H+, K+-ATPase). The specific activity was 1.1 μmol P1/mg/min at pH 5.7, whereas the specific activity of Na+, K+-ATPase was 14 μmol P1/mg/min at pH 7.5. The enzyme was phosphorylated from ATP in the absence of Na+ at the acid pH. The initial rate of the phosphorylation was also accelerated at the acid pH in the absence of Na+, and the maximal rate obtained at pH 5.5 without Na+ was 9% of the rate at pH 7.0 with Na+. The phosphoenzyme was sensitive to K+ but almost insensitive to ADP. The phosphoenzyme was sensitive to hydroxylamine treatment and the alpha-subunit of the enzyme was found to be phosphorylated. H+, K+-ATPase was inhibited as effectively as Na+, K+-ATPase by N-ethylmaleimide but was less inhibited by oligomycin or dimethyl sulfoxide. These results indicate that protons have an Na+-like effect on the Na+ sites of Na+, K+-ATPase and suggest that protons can be transported by the sodium pump in place of Na+.View full abstractDownload PDF (1260K) -
Kohji UENO, Nagahiko SAKUMA, Masanobu KAWAGUCHI, Takao FUJINAMI, Harum ...1986 Volume 99 Issue 2 Pages 541-547
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSelectivity factors (Vm/Km) for human and rat lecithin: cholesterol acyltransferases (LCAT) for the transfer of various acyl groups from the 2-position of phosphatidylcholine were determined. By multiplying these values by the proportions of acyl groups at the 2-position of phosphatidylcholine, one can predict the proportions of molecular species of cholesterol ester which will be synthesized by LCAT. In human subjects fasted overnight, the molecular composition of plasma cholesterol ester was found to reflect the LCAT selectivity relatively accurately. This result supports the concepts that hepatic acyl-CoA: cholesterol acyltransferase (ACAT) does not contribute significantly to the synthesis of plasma cholesterol ester and that removal of cholesterol ester from plasma is not selective with respect to molecular species under these conditions. In contrast to the results with humans, the molecular composition of plasma cholesterol ester formed in spontaneously hypertensive rats fed a high-cholesterol diet and then fasted overnight differs from that which is predicted from LCAT selectivity and the proportion of various fatty acids at the 2-position of phosphatidylcholine: these results suggest that cholesterol ester is formed mainly via the ACAT reaction.View full abstractDownload PDF (580K) -
Mi Young KANG, Shoji SHIMOMURA, Toshio FUKUI1986 Volume 99 Issue 2 Pages 549-559
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe selective inactivation of alcohol dehydrogenase by the inactivator found in the microsomal fraction of rice (Oryza sativa) seedlings growing in air (Shimomura, S. & Beevers, H. (1983) Plant Physiol. 71, 736-741; 742-746) was further studied. This inactivation was found to be essentially dependent on the presence of free fatty acids. The specificity for fatty acids and the inhibitory effects of imidazole, 2hydroxyfatty acids and dithiothreitol on the inactivation were all consistent with the properties of the fatty acid α-oxidation system in plants. Both O2 consumption and decarboxylation of fatty acid due to α-oxidation were also demonstrated in rice microsomes. When purified rice alcohol dehydrogenase was added to the α-oxidation system in rice microsomes, the decarboxylation of fatty acid was inhibited, and the cysteinyl residues of alcohol dehydrogenase were oxidized. The oxidation of two cysteinyl residues per monomer resulted in the complete inactivation of the enzyme. The activity of the inactivator in the isolated microsomes was gradually lost during storage and was rapidly lost upon heating. The inactivation of alcohol dehydrogenase was observed even when the enzyme was separated from microsomes by a dialysis membrane. These results indicate that the inactivation of alcohol dehydrogenase is closely related to fatty acid α-oxidation. We postulate that an intermediate of α-oxidation is the inactivator.View full abstractDownload PDF (883K) -
Takashi MORITA, Jun MIZUGUCHI, Shun-ichiro KAWABATA, Sadaaki IWANAGA1986 Volume 99 Issue 2 Pages 561-568
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt is known that protein S, a vitamin K-dependent plasma protein, isolated from a human source, gives a closely spaced doublet on sodium dodecyl sulfate-polyacryl-amide gel electrophoresis after reduction and that this heterogeneity in molecular size results from a limited proteolysis of protein S mediated by α-thrombin in human species. We found here that α-thrombin also rapidly converted single-chain bovine protein S to a nicked form, which consisted of the NH2-terminal segment containing γ-carboxyglutamic acid and the COOH-terminal large segment bridged by a disulfide linkage(s). These two segments were isolated and chemically characterized after S-alkylation of the nicked protein S. The results suggest that the α-thrombin-catalyzed hydrolysis of protein S probably occurs at a peptide linkage (Arg-Ser) located in the NH2-terminal portion. The conversion of single-chain protein S to the nicked form was also mediated by plasma kallikrein and plasmin, in addition to α-chymotrypsin and trypsin. However, the α-thrombin-catalyzed conversion did not occur when calcium ions were added to the reaction mixture.View full abstractDownload PDF (1850K) -
Makoto KANEDA, Akihiro SOBUE, Sugako EIDA, Naotomo TOMINAGA1986 Volume 99 Issue 2 Pages 569-577
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeven proteinases were isolated from the fruit of snake-gourd, Trichosanthes cucum-eroides Maxim. Their isozymes are all serine proteinases, and homologous in their respective molecular weights, amino acid compositions, and enzymatic properties. Their molecular weight was estimated to be about 50, 000. Using casein as a substrate, the maximum activity was found in the alkaline pH region. The optimum temperature using casein was 70°C at pH 7.3.
The enzymes were strongly inhibited by diisopropyl fluorophosphate and not inhibited by inhibitors of sulfhydryl or metalloproteases.
The reduced and S-carboxymethylated insulin B-chain was used as a substrate in an investigation of the specificity. The enzyme was found to have a wide specificity for this substrate but preferentially hydrolyzed the peptide bonds involving the carboxyl groups of charged amino acid such as S-cm-cysteine, glutamic acid, histidine, arginine, and lysine.
Experimental evidence indicated that the snake-gourd proteinases are similar in their properties to cucumisin, which is isolated from the sarcocarp of melon fruit.View full abstractDownload PDF (547K) -
Tsuneyoshi FUNAI, Arata ICHIYAMA1986 Volume 99 Issue 2 Pages 579-589
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA high-performance liquid chromatographic method was developed for the determination of glyoxylate in the liver. Alpha-keto acids in charcoal-treated acid-extract of the liver were converted to the corresponding 2, 4-dinitrophenylhydrazones and purified as the derivatives by successive extractions with ethyl acetate and sodium bicarbonate solution. The dinitrophenylhydrazones were then quantitatively converted to the corresponding substituted 2-hydroxyquinoxalines by reaction with o-phenylenediamine, followed by analysis by high-performance liquid chromatography with fluorescence detection. As a control to correct the recovery of tissue glyoxylate, an acid-extract of the liver prepared with the addition of standard glyoxylate (25-50 nmol/g wet weight of tissue) was simultaneously subjected to the analytical procedure. The maximum sensitivity of the glyoxylate measurement as 2-hydroxyquinoxaline (the quinoxaline derivative corresponding to glyoxylate) was defined as the peak area reading five times as high as the blank value obtained without sample and was approximately 10 pmol per injection.
Glyoxylate in the addition compound with tris (hydroxymethyl) aminomethane was quantitatively recovered as 2-hydroxyquinoxaline. The addition compounds of glyoxylate with bisulfite and cysteine did not react with 2, 4-dinitrophenylhydrazine under the conditions employed and were not detectable as glyoxylate by this method, while the adduct-forming substances added to the acid-extract of the liver did not interfere with the glyoxylate determination. No glyoxylate was detected when the liver extract had been incubated at neutral pH with a large excess of cysteine, indicating that little artificial production of glyoxylate occurred during the analytical procedure. Among 64 compounds tested for possible artificial production of glyoxylate or possible interference with the chromatographic determination of 2-hydroxyquinoxaline, p-hydroxyphenylpyruvate was the only compound which was converted to glyoxylate during the procedure. However, p-hydroxyphenylpyruvate was easily removed from the acid-extract of the tissue by charcoal treatment.
The amount of glyoxylate in the liver of fasted rat was measured by the present method to be approximately 5 nmol per g of wet weight.View full abstractDownload PDF (964K) -
Mari TOBA, Haruhiko MASAKI, Takahisa OHTA1986 Volume 99 Issue 2 Pages 591-596
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lysis genes of plasmids ColE2-P9 and CoIE3-CA38 were identified by DNA sequencing and electrophoretic analysis of the products of both wild type and artificially introduced ochre mutant genes. The E2 and E3 lysis genes had identical primary structures and were shown to encode 47 amino acids with a calculated molecular weight of 4, 861, which is much smaller than that proposed previously for the ColE3-CA38 lysis protein. They are homologous with ColDF13 gene H, except in their 3'-portions. The nine C-terminal amino acids of the E2 and E3 lysis proteins proved to be non-essential for the lysis phenotype.View full abstractDownload PDF (880K) -
Masuo MORISAKI, Yoshiko SONODA, Tomomi MAKINO, Noriko OGIHARA, Nobuo I ...1986 Volume 99 Issue 2 Pages 597-600
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeveral 15-oxygenated sterols were examined as to their inhibitory activity toward cholesterol synthesis from [24, 25-3H]-24, 25-dihydrolanosterol in the 10, 000×g supernatant fraction of a rat liver homogenate. At 40 μM, three 15α-hydroxylated compounds, 14α-ethylcholest-7-ene-3β, 15α-diol, 14α-methylcholest-7-ene-3β, 15α-diol, and lanost-7-ene-3β, 15α-diol, were found to be extremely potent inhibitors (more than 90% inhibition) of dihydrolanosterol metabolism. The inhibitory effect of the C-15 substituents appeared to be in the order of: 15α-hydroxyl>15-ketone>15β-hydroxyl.View full abstractDownload PDF (276K) -
Kazuhito TANIMOTO, Tomoyuki NISHIMOTO, Fumio SAITOH, Haruki YAMAGUCHI1986 Volume 99 Issue 2 Pages 601-604
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to facilitate the purification of 1, 2-α-mannosidase from an enzyme product of Aspergillus oryzae, we have devised a rapid and simple procedure. A partially purified enzyme preparation obtained from the A. oryzae enzyme product, by means of ammonium sulfate fractionation followed by CM-Sephadex C-50 chromatography, was subjected to affinity chromatography with baker's yeast mannan gel as an adsorbent. 1, 2-α-Mann osidase was retarded and well separated from the major protein peak on the affinity column. After a second affinity chromatography under the same conditions, 1, 2-α-mannosidase was finally purified 7, 500-fold with a 22.9% yield. The enzyme preparation thus obtained was quite suitable for the structural analysis of glycoconjugates.View full abstractDownload PDF (264K) -
Atsushi NAKAGAWA, Etsuro NAGASHIMA, Yoshiki HIGUCHI, Masami KUSUNOKI, ...1986 Volume 99 Issue 2 Pages 605-606
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome c553 from the sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki, has been crystallized. The combination of microdialysis and vapor diffusion allowed successful crystallization. The crystals were of good quality, and useful data were obtained that extended to the nominal resolution of 1.3Â. The space group is P43212 with cell dimensions of a=b=42.7 Å, c=103.4 Å. More than twenty heavyatom reagents were screened with the isomorphous replacement technique, and only the mersalyl derivative could be used for the phase determination. The single isomorphous replacement method combined with the anomalous scattering effect of the Hg-atom in mersalyl and the Fe-atom of the heme group was used for the phase determination.View full abstractDownload PDF (170K)
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