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Volume 105 (1989)
- Issue 6 Pages 855-
- Issue 5 Pages 671-
- Issue 4 Pages 491-
- Issue 3 Pages 339-
- Issue 2 Pages 149-
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Volume 105, Issue 4
Displaying 1-31 of 31 articles from this issue
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Hideo Maeda, Hiroyo Nakamura, Shozo Kobori, Mitsuko Okada, Hironori Ni ...1989 Volume 105 Issue 4 Pages 491-493
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo types of apoE alleles from a genomic DNA library of a subject with the apoE2/E5 phenotype have been cloned. One of these alleles encodes an apoE isoprotein having cysteine, arginine, and cysteine at positions 112, 145, and 158 of mature apoE isoproteins, respectively, indicating an ε apoE2 allele. The other allele encodes a different isoprotein, having cysteine, arginine, and arginine at each of the above positions, respectively. In addition, this allele has a base substitution (G→A) which causes the substitution of lysine for glutamic acid at position 3 near the NH2-terminus of the mature apoE. This allele is a new variant, the ε apoE5 allele.View full abstractDownload PDF (301K) -
Reiko Nakayama, Kunihiko Saito1989 Volume 105 Issue 4 Pages 494-496
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1-O-Alk-1'-enyl-2-O-acetyl-glycerophosphocholine (vinyl form of PAF) was found with PAF in perfused rat and guinea pig hearts. The main molecular species of the vinyl form of PAF, after separation by reverse phase HPLC, were identified as 1-O-hexadec-, -octadec-, and-octadecen-1'-eny1-2-O-acetyl-GPCs (16:0, 18:0, and 18:1 vinyl forms of PAF) by mass spectrometry. The amounts of the predominant 16:0 species in rat and guinea pig hearts, respectively, were 46.4 and 22.5 ng per mg lipid-phosphorus of the original heart phospholipids.View full abstractDownload PDF (350K) -
Tadashi Ogishima, Fumiko Mitani, Yuzuru Ishimura1989 Volume 105 Issue 4 Pages 497-499
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo distinct forms of cytochrome P-45011β, with apparent molecular weights of 48, 500 (48.5K) and 49, 500 (49.5K), have been isolated from bovine adrenocortical mitochondria. Their amino acid sequences up to the 19th position from the N-terminus were only different at the 6th position (Val and Ala for the 48.5K and 49.5K enzymes, respectively). Each sequence was assignable to a distinct cDNA clone for cytochrome P-45011β (Kirita, S., et al. [1988] J. Biochem. 104, 683-686), indicating that the two proteins originate from different genes in bovine adrenocortical cells. Both forms of cytochrome P-45011β were capable of catalyzing aldosterone synthesis as well as the 11β- and 18-hydroxylation of 11-deoxycorticosterone. Thus, at least two distinct cytochrome P-45011β species exist in the adrenal cortex and participate in steroidogenesis.View full abstractDownload PDF (363K) -
Hiroaki Miyajima, Toshiaki Oda, Arata Ichiyama1989 Volume 105 Issue 4 Pages 500-504
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStudies were performed in the rat liver to examine whether or not insulin as well as glucagon causes the induction of mitochondrial serine:pyruvate aminotransferase (SPTm) [EC 2.6.1.51] and if so, whether the mechanisms of induction are similar or different for the two hormones. Not only glucagon but also insulin induced SPTm. Cell-free translation assaying and RNA blot analysis showed that both hormones cause an increase in the hepatic level of mRNA for the precursor of SPTm. Their effects were virtually additive, and the time course of the increase in the mRNA level differed between the hormones. The maximal increase induced by glucagon was observed 3.5 h after the hormone injection while that by insulin was found after 6h. The increase in the mRNA due to insulin was completely inhibited by the co-administration of cycloheximide, while that due to glucagon was not. The finding suggests that a newly synthesized, insulin-dependent protein (s) is involved in the regulation of the mRNA level by insulin. On the other hand, hydrocortisone treatment selectively suppressed the increase in the mRNA due to glucagon. These data indicate that the synthesis of the mRNA for SPTm is regulated by glucagon and insulin through different mechanisms. The size of the hormone-induced mRNA for SPTm gradually decreased with time, but the cell-free translation products did not exhibit size alteration. RNase H digestion to remove the poly (A) tail of the mRNA indicated that shortening of the poly (A) sequence might be responsible for the time-dependent size alteration of the mRNA.View full abstractDownload PDF (3472K) -
Tze-Kuei Chiou, Takashi Matsui, Shoji Konosu1989 Volume 105 Issue 4 Pages 505-509
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn aminopeptidase was isolated from a soluble fraction of Alaska pollack roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The molecular weight of the enzyme was estimated to be 125, 000 and 105, 000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The pH optimum and temperature optimum were 7.2 and 35°C, respectively. The purified enzyme hydrolyzed various α-aminoacyl β-naphthylamides and cleaved L-Ala-β-naphthylamide most rapidly. Both a sulfhydryl group and a divalent metal ion are essential for activity; however, the enzyme was inhibited when incubated with divalent metal ions. Puromycin, chelating agents, and thiol reagents were effective inhibitors. The enzyme was also inhibited by L-amino acids, in particular glutamic acid. Thus, the Alaska pollack roe aminopeptidase resembles soluble alanyl aminopeptidase [EC 3.4.11.14].View full abstractDownload PDF (514K) -
Toshiyuki Nishio, Minoru Kamimura, Masakazu Murata, Yoshiyasu Terao, K ...1989 Volume 105 Issue 4 Pages 510-512
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMicrobial lipase-catalyzed transesterification between vinyl acetate and (RS)-2-octanol or (RS)-1-phenylethanol was investigated in a reaction system without additin of aqueous or organic solvents. From a screening test with various lipases, it was found that the enzymes from Pseudomonas species could efficiently catalyze the reaction, and R-enantiomers of the racemic alcohols were preferentially esterified by them. Enantiomeric purities of the optically active alcohols (S) and esters (R) obtained from (RS)-1-phenylethanol by the stereoselective transesterification of these lipases were all more than 95%.View full abstractDownload PDF (1759K) -
Masato Nakai, Toshiharu Hase, Hiroshi Matsubara1989 Volume 105 Issue 4 Pages 513-519
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA major 70 kDa protein of the yeast mitochondrial outer membrane is coded by a nuclear gene, synthesized on cytoplasmic ribosomes, and transported to the mitochondrial outer membrane. In order to investigate in detail the information necessary for localizing the 70 kDa protein at the outer membrane, we have examined the intracellular and intramitochon-drial location of fusion proteins which consist of various lengths of the amino-terminal region of the 70 kDa protein with an enzymatically active β-galactosidase. The results indicate that the extreme amino-terminal 12 amino acids of the 70 kDa protein function as a targeting sequence, whereas the subsequent uncharged region (up to residue 29) is necessary for “stop-transfer” and “anchoring” functions. Moreover, we have found that a fusion protein which contained the amino-terminal 19 amino acids of the 70 kDa protein is localized on the outer membrane as well as in the matrix space. Changes in the dual localization of this fusion protein accompanied its overproduction or expression in a respiration-deficient yeast mutant.View full abstractDownload PDF (841K) -
Hiroshi Mizushima, Ichiro Kudo, Kazuhiko Horigome, Makoto Murakami, Ma ...1989 Volume 105 Issue 4 Pages 520-525
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was reported previously that rat platelets release phospholipase A2 upon in vitro stimulation by thrombin, ADP, or A23187 (Horigome, K., Hayakawa, M., Inoue, K., & Nojima, S. (1987) J. Biochem. 101, 53-61). Secretion of phospholipase A2 was also observed with rabbit platelets. Rabbit platelets seem to release phospholipase A2 upon stimulation in vivo, because the rabbit plasma taken immediately after intravenous injection of PAF contained an appreciable level of phospholipase A2 activity and fewer platelets. Rabbit platelet phospholipase A2 released in vitro was purified by column chromatography using Sepharose CL-4B conjugated with anti-rat platelet derived phospholipase A2 monoclonal antibody, followed by reversed-phase HPLC. The purified enzyme was subjected to structural analysis by HPLC peptide mapping and primary sequence determination of the separated peptides. Based on the homology with rat platelet secretory phospholipase A2 (Hayakawa, M., Kudo, I., Tomita, M., Nojima, S., & Inoue, K. (1988) J. Biochem. 104, 767-772), a partial primary structure (62 amino acid residues) of the rabbit enzyme was tentatively determined; the two sequences were highly homologous (72%). The rabbit sequence was also nearly identical to that of rabbit ascitic fluid phospholipase A2, which was determined by Forst et al. (Forst, S., Weiss, J., Elsbach, P., Maraganore, J. M., Reardon, I., & Heinrikson, R. L. (1986) Biochemistry 25, 8381-8385). Phospholipase A2 from the membrane fraction of rabbit platelets was also purified; it had the same characteristics and the same amino-terminal sequence as the purified secretory enzyme. Secretory and membrane-bound phospholipase A2 of rabbit platelets may in fact be identical.View full abstractDownload PDF (684K) -
Do Ik Lee, Hiroshi Hirai, Shunji Natori, Kazuhisa Sekimizu1989 Volume 105 Issue 4 Pages 526-528
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new nucleolytic activity that causes restricted digestion of U6 RNA was found in a nuclear extract of Ehrlich ascites tumor cells. This nucleolytic activity specifically degrades U6 RNA in the vicinity of its 3'-end with accumulation of a discrete sized degradation product of RNA of 90-95 nucleotides. Since this degradation product was not digested further by the nuclease under these conditions, this trimming of U6 RNA is supposed to be a biologically meaningful reaction. This nucleolytic activity required Mg2+, and was inhibited by Zn2+ or Ca2+.View full abstractDownload PDF (359K) -
Koui Takahashi, Akihito Hattori1989 Volume 105 Issue 4 Pages 529-536
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relationship between the ultrastructure and protein components of Z-disks was studied using psoas muscles of rabbit and breast muscles of chicken. Glycerinated fiber bundles of these muscles were treated with a solution containing 0.1mM Ca2+ to induce a structural weakening of Z-disks non-enzymatically. During the treatment, clear geometrical configurations of Z-filaments could be observed along with removal of amorphous matrix under an electron microscope. Even after a prolonged treatment for 14 d in which all Z-disks were entirely weakened, entangled Z-filaments were left in the original region of the disks. Immunoelectron microscopic observations showed that antibodies againstα-actinin bound to the entangled Z-filaments, forming dense lines at the position of the original Z-disks. On SDS-PAGE of Z-disk substances, α-actinin remained unchanged and Mr 75, 000 and 55, 000 proteins were removed during the Ca2+ -treatment. We therefore conclude that α-actinin is a component of Z-filaments, and that the amorphous matrix is composed at least of these Mr 75, 000 and 55, 000 proteins.View full abstractDownload PDF (7373K) -
Eriko Hatada, Masakazu Hasegawa, Jun Mukaigawa, Kazufumi Shimizu, Ryuj ...1989 Volume 105 Issue 4 Pages 537-546
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe established a quantitative hybridization system by which three types of influenza yin, RNAs (vRNA, mRNA, and cRNA) for the 8 genome segments were measured individuall As the hybridization probes, 32P-labeled RNAs of both plus and minus polarity wet produced employing an SP-6 transcription system and used in a large molar owes; sufficient to overcome complementary RNAs present in the viral RNA samples. Employin the system, we studied the control of the synthesis of each viral RNA species in MDCK cell infected with A/Udorn/72 (H3N2). Our new observations were as follows. 1) Segment specific transcription was observed at the primary transcription. 2) Replication of the viru genome began simultaneously for all segments. No delay was observed in the replication c the segments carrying late genes. 3) In addition to control at the transcriptional levels, th expression of viral late genes was regulated at some post-transcriptional step(s). Thes results are not compatible with the concepts reported previously, and lead us to propos unique regulations operating on the expression of the viral late genes.View full abstractDownload PDF (6969K) -
Shigeto Yamamoto, Sumihiro Hase, Shigeharu Fukuda, Osamu Sano, Tokuji ...1989 Volume 105 Issue 4 Pages 547-555
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInterferon-γ produced by the human myelomonocyte cell line HBL-38 contained galactose, mannose, fucose, N-acetylglucosamine, and N-acetylneuraminic acid as sugar components. Sugar chains were liberated from interferon-γ by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine under new reaction conditions in which no sialic acid residue was hydrolyzed. The pyridylamino (PA-) derivatives of the sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Seven major PA-sugar chains were isolated and the structure of each purified PA-sugar chain was identified by stepwise exoglycosidase digestion and 500-MHZ 1H-NMR spectroscopy. The results indicated that the structures of the major PA-sugar chains were of the biantennary type, to which 0 to 2mol of fucose and 1 to 2mol of N-acetylneuraminic acid were linked as shown below.View full abstractDownload PDF (985K) -
Nobuhisa Iwata, Norihisa Inazu, Tetsuo Satoh1989 Volume 105 Issue 4 Pages 556-564
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo carbonyl reductases have been highly purified from rat ovary to apparent homogene-ity. Though they have similarities in terms of molecular weight (33, 000), substrate specificities, inhibitor sensitivities, amino acid composition, and immunological properties, they differed in pI values (6.0 and 5.9). Both enzymes reduced aromatic aldehydes, ketones, and quinones at higher rates, compared to prostaglandins and 3-ketosteroids, whereas they showed higher affinity for prostaglandins and 3-ketosteroids. The enzymes also catalyzed oxidation of the 9-hydroxy group of prostaglandin F2α. Moreover, they showed the remarkable characteristic of catalyzing the reduction of not only the 9-keto group of prostaglandin E2 but also the 15-keto group of 13, 14-dihydro-15-ketoprostaglandin F2α. Both enzymes were inhibited by SH-reagents, quercitrin, indomethacin, furosemide, and disulfiram. The results of immunoinhibition, using antibody against the purified enzymes, indicated that the enzymes were solely responsible for the overall catalytic activities of prostaglandin E series reduction, as well as 13, 14-dihydro-15-ketoprostaglandin F2α reduction and prostaglandin F2α oxidation in rat ovarian cytosol. Western-blot analysis revealed that immunoreactive proteins were present in adrenal gland and various reproductive tissues except uterus of rats.View full abstractDownload PDF (2827K) -
Takehiro Miyake, Seiji Inoue, Kiyoshi Ikeda, Keizo Teshima, Yuji Samej ...1989 Volume 105 Issue 4 Pages 565-572
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe phospholipase A2 of Trimeresurus flavoviridis was found to show monomer-dimer equilibria. Under conditions where the enzyme exists predominantly in the monomeric form, the chemical reaction rate of p-bromophenacyl bromide (BPB) with the catalytic group, His 48, was studied at 25°C and ionic strength 0.2 by measuring the residual enzymic activity using a fluorescent substrate, 1, 2-bis [4-(1-pyreno) butanoyl]-sn-glycero-3-phosphorylcholine (diPBPC). The pH-dependence curve of the reaction rate for the intact enzyme was practically the same as that for the modified enzyme, in which the N-terminal α-NH2 group had been selectively converted into an α-keto group. The pH-dependence curves were monophasic (sigmoidal) with a midpoint at pH 7.53, which corresponds to the pKa value of His 48. The pH dependences of the binding constants of Ca2+ to the intact and the α-NH2 modified enzymes were also studied at 25°C and ionic strength 0.2 by measuring the changes in the tryptophyl fluorescence and/or aromatic CD spectra. The pH-dependence data for the modified enzyme were interpreted in terms of participation of Asp 49 (pKa 5.40) and His 48 (pKa 7.53), assuming that the protonation of Asp 49 competes with the Ca2+ binding. The pH-dependence data for the intact enzyme were similarly interpreted in terms of participation of the α-NH2 group (pKa 9.40) in addition to that of Asp 49 (pKa 5.40) and His 48 (pKa 7.53). The enzyme dimerization was found to reduce the binding constant of Ca2+ to the enzyme at neutral and acidic pH values, suggesting that the way in which one enzyme molecule is brought into contact with the other is unfavorable to the Ca2+ binding. This result seems compatible with the recent X-ray crystallographic data on the dimeric phospholipase A2 of Crotalus atrox (Brunie et al. (1985) J. Biol. Chem. 260, 9742-9749), which belongs to the same family, Crotalidae, as T. flavoviridis.View full abstractDownload PDF (947K) -
Yasunori Nitta, Yukihiro Isoda, Hiroko Toda, Fumio Sakiyama1989 Volume 105 Issue 4 Pages 573-576
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSoybean β-amylase was modified with 2, 3-epoxypropyl α-D-[U-14C]glucopyranoside ([14C]α-EPG), a radioactive affinity-labeling reagent for β-amylase, until it lost 95% of its enzyme activity. After S-carboxymethylation at pH 8.0 of SH groups, the modified enzyme was digested at pH 7.0 with Achromobacter protease I and the digest was fractionated by reverse-phase HPLC. A radioactive peptide was finally isolated and its amino acid sequence was determined to be 181Leu-Gly-Pro-Ala-Gly-Glu186. Radioactivity derived from [14C]-α-EPG was found exclusively at Glu-186, the γ-carboxyl group of which is esterified with the affinity label. It was concluded that the carboxylate of Glu-186 is a functional group at the catalytic site of soybean β-amylase.View full abstractDownload PDF (459K) -
Tomoko Hayashi, Hiroaki Hayashi, Koichi Iwai1989 Volume 105 Issue 4 Pages 577-581
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of a high mobility group (HMG) nonhistone chromosomal protein of the ciliated protozoan Tetrahymena pyriformis (GL strain) was determined. This protein was extracted with 0.5M HCIO4 together with histone H1 (molar ratio 1:1) from the whole histone extract, then purified by gel filtration and reverse-phase HPLC. The HMG protein showed a single electrophoretic band on SDS gel electrophoresis. The amino acid sequence was determined by Edman degradation of intact protein, BrCN fragments, and their staphylococcal protease and tryptic peptides. Thus the total sequence, consisting of 99 amino acid residues and having a molecular weight of 11, 626, was completely determined. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that this HMG protein was phosphorylated at two positions, each 6-7%, and contained 0.15mol phosphate/mol protein. This Tetrahymena HMG is rather similar to the central part of vertebrate HMG 1 in terms of the amino acid sequence and the hydropathy profile.View full abstractDownload PDF (485K) -
Tohru Funahashi, Shinji Yokoyama, Akira Yamamoto1989 Volume 105 Issue 4 Pages 582-587
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen human apolipoprotein E (apoE), which forms a self-associated tetramer in an aqueous solution, bound to the surface of triolein/phosphatidylcholine microemulsion with a particle diameter of 26 nm, it became monomeric on the lipid particle surface without strong evidence for its accumulation on a particular particle that might be expected from its tetramer formation in the aqueous phase. ApoE in the form of the self-associated tetramer did not inhibit binding of human low density lipoprotein (LDL) to its receptor on cultured human skin fibroblast. LDL binding was inhibited only when apoE was bound to the lipid particle surface. The affinity of the apoE-containing lipid particle to the LDL receptor was of the same order as that of LDL on the basis of particle molarity when the surface of the particle was covered with apoE up to 40 to 50% of the saturation level. When the particle was covered more with apoE, the affinity increased by some 20 times. Since the surface of the lipid particle was saturated with 7 apoE molecules, the particle seemed to require to have at least 4 apoE molecules on its surface in order to obtain high binding affinity to LDL receptor.View full abstractDownload PDF (1884K) -
Yumiko Iwahashi, Akio Hitoshio, Nobuyuki Tajima, Taro Nakamura1989 Volume 105 Issue 4 Pages 588-593
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAt least two enzymes that phosphorylate diphosphopyridine nucleotides were detected in Saccharomyces cerevisiae: NADH-specific kinase was localized exclusively in the mitochondria, and NAD+-specific kinase was distributed in the microsomal and cytosol fractions but not in the mitochondria. The identity of NAD+ kinase detected in the two fractions remains equivocal. NADH kinase was highly purified 1, 041-fold from the mitochondrial fraction. The Km values for NADH and ATP were 105μM and 2.1mM, respectively. The relative molecular mass was estimated to be 160, 000 by means of molecular sieve chromatography. From inactivation studies with SH inhibitors and protection by NADH, it was demonstrated that a cysteine residue is involved in the binding site of NADH.View full abstractDownload PDF (708K) -
Takayuki Hara, Tokuji Kimura1989 Volume 105 Issue 4 Pages 594-600
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA stable covalent complex was prepared by cross-linking adrenodoxin reductase with adrenodoxin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent com-plex was purified extensively until free components were removed completely. The major component of the complex had a molecular weight of 63 kDa, which corresponds to a 1:1 stoichiometric complex between adrenodoxin reductase and adrenodoxin. NADPH-cytochrome c reduction activity of the covalent complex was comparable to that of an equimolar mixture of adrenodoxin reductase and adrenodoxin (native complex), and the NADPH-ferricyanide reduction activity of the complex was equal to that of the native one. In contrast to the native complex, the covalent complex produced much less superoxide upon NADPH-oxidation, and the covalent complex was found to be more stable than the native complex, suggesting that the complex state is more favorable for catalysis. From these results, we conclude that the adrenodoxin molecule does not need to dissociate from the complex during electron transfer from NADPH to cytochrome c.View full abstractDownload PDF (1993K) -
Takayuki Hara, Tokuji Kimura1989 Volume 105 Issue 4 Pages 601-605
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to elucidate the mechanism of the electron transfer reaction of mitochondrial steroid hydroxylase, the reduction reaction of cytochrome P-450scc (F-450scc) catalyzed by covalently cross-linked complexes between adrenodoxin reductase (AR) and adrenodoxin (AD) was studied. The reduction rate with the covalent AR-AD complex was very slow (0.030min-1, as the flavin turnover number) compared with the reduction catalyzed by AR and AD (4.6min-1). When free AD was added to the reaction mixture containing the AR-AD complex, the rate increased about 30 times. The AD dimer [(AD)2], and a complex between AR and the AD dimer [AR-(AD)2] were then prepared. The Vmax for the P-450scc reduction activity of AR with (AD)2 was 50% of that of AR with AD. The Km value for the total concentration of AD in the P-450scc, reduction reaction mixture containing AR and (AD)2 was found to be the same as that in the reaction mixture containing AR and AD. P-450scc reduction by AR-(AD)2 was about 5 times faster than that by AR-AD. The addition of free AD to the AR-(AD)2 complex enhanced the P-450scc reduction about 30 times. AR-AD and AR-(AD)2 were able to reduce external AD, cytochrome c, and acetylated cytochrome c. From these results, it is concluded that organized complexes consisting of AR and AD are more active in the presence of free AD than in its absence, in terms of the reductive activity toward P-450scc. It was confirmed that the properties of P-450scc reduction by covalent complexes differ from those of cytochrome c reduction: the addition of free AD stimulates the reduction of P-450scc whereas it does not stimulate the reduction of cytochrome c.View full abstractDownload PDF (1983K) -
Hideo Takeshima, Junji Inokoshi, Yoshio Takada, Haruo Tanaka, Satoshi ...1989 Volume 105 Issue 4 Pages 606-610
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme, tentatively termed aculeacin A acylase, useful in preparing deacylated peptides which are used as starting material for semisynthetic antifungal antibiotics, was purified from the culture filtrate of Actinoplanes utahensis NRRL12052. The purification involved ultrafiltration and column chromatographies on DEAE-cellulose, hydroxyapatite, and Butyl-Toyopearl 650M. The purified enzyme was composed of two dissimilar subunits with molecular weights of 55, 000 and 19, 000. The subunits were dissociated in the presence of 0.1% SDS or 6 M guanidine hydrochloride; the dissociation accompanied loss of acylase activity. The enzyme was fully active at pH 7.0 and at 60°C. Its pI was estimated to be above 10.25. The Km and Vmax for aculeacin A were 1.53 mM and 39.7 μmol/ min/mgprotein, respectively.View full abstractDownload PDF (2935K) -
Shuichi Onodera, Hirokazu Matsui, Seiya Chiba1989 Volume 105 Issue 4 Pages 611-618
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acid α-glucosidase was purified from rabbit liver by fractionation with ammonium sulfate, and chromatographies on Sephadex G-100, CM-Toyopearl, Toyopearl HW-55F, and Toyopearl HW-65F column. The resulting preparation showed a single band on polyacryl-amide disc gel electrophoresis. The molecular weight was estimated to be 1.03×104 by SDS-disc electrophoresis. The optimum pH was found to be 4.7. The α-glucosidase showed relatively high activity not only toward maltose but also toward α-glucans, such as shellfish glycogen, soluble starch, β-limit dextrin, amylopectin, and amylose. The Km values for maltose and shellfish glycogen were 2.1 and 16mM (the concentration of non-reducing glucose units), respectively, and the ratio of maximum velocities of hydroly-sis of the two substrates was 100:133. The nature of the active site catalyzing the hydrolyses of maltose and shellfish glycogen was investigated by electrophoresis in the presence of urea and by kinetic methods. The purified enzyme was not separated into two components, maltase and glycogen hydrolase, in the electrophoretic gel containing 3M urea, contrary to the report by Belenki and Rosenfeld ((1972) Biochem. Biophys. Res. Commun. 46, 443-448). In experiments with mixed substrates of maltose and glycogen, the kinetic features agreed very closely with those theoretically predicted for a single site mechanism. The essential ionizable groups, 1 (on the acidic side) and 2 (on the alkaline side), were identified as -COO- and -COOH for the hydrolysis of both substrates. Cations, Na+, K+, Mg2+, were about equally effective for the stimulation of enzyme action on maltose and glycogen. Tris, turanose, erythritol, and methyl α-glucoside inhibited the hydrolyses of both substrates competitively. From these results, rabbit liver acid α-glucosidase was concluded to attack maltose and glycogen at a single active site.View full abstractDownload PDF (1804K) -
Keishiro Wada, Masaaki Onda, Hiroshi Matsubara1989 Volume 105 Issue 4 Pages 619-625
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree ferredoxin isoproteins (R-Fd A, R-Fd B-1, and R-Fd B-2) were purified from white roots of radish (Raphanus sativus L. var. acantiformis cultivar Miyashige) and two isoproteins (L-Fd A and L-Fd B) from leaves. The amino acid sequences of three of them (L-Fd A, R-Fd B-1, and R-Fd B-2) were determined and compared with one another and with those of other higher plant ferredoxins so far studied. L-Fd A and R-Fd B-1 had heterogeneities at four and two amino acid sites, respectively. Two isoprotein (R-Fd B-1 and R-Fd B-2) were deduced to be expressed only in root tissue on the basis of sequence studies and amino acid compositions of all isoferredoxins isolated from the radish plant. The root ferredoxins sequenced in this study were similar to each other, but quite different from other higher plant ferredoxins, all of which were isolated from leaf tissue. The coupling activities of these ferredoxin isoproteins were measured in the NADP+- photoreduction system of radish chloroplasts and glutamate synthase [EC 1.4.7.1] systems isolated from radish leaf and root tissues. No distinctive physiological characteristics were observed among these isoferredoxins.View full abstractDownload PDF (864K) -
Xuemin Xu, Shigenori Kanaya, Noriyuki Koyama, Takeshi Sekiguchi, Yoshi ...1989 Volume 105 Issue 4 Pages 626-632
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe alkalophile NADH dehydrogenase (NADH: 2, 6-dichlorophenolindophenol oxido-reductase) [EC 1. 6. 99. 3] consists of two identical subunits of 65 kDa, and each subunit contains the catalytic and liposome-binding regions. On treatment with trypsin, the polypeptide exhibiting the liposome-binding property in one of the subunits was digested to form an enzymatically active hetero-dimer (40 and 65 kDa), and then the polypeptide in the other subunit was digested to form an active homo-dimer (40 and 40 kDa). The hetero-dimer bound to liposomes, but the homo-dimer did not. Kinetic analysis showed that removal of one or two of the polypeptides in the enzyme slightly affects its kinetic parameters. For all the enzyme species, NAD inhibited competitively with respect to NADH and non-competitively with respect to 2, 6-dichlorophenolindophenol. The partially determined amino acid sequence of this alkalophile enzyme suggested that (i) a long randomcoiled peptide (58 amino acid residues) or a portion of the peptide is located between the polypeptides with liposome-binding and catalytic properties, (ii) the polypeptide exhibiting liposome-binding property is in the amino terminal region of the enzyme, (iii) the amino acid sequences around the subtilisin and trypsin cleavage sites of the peptide are hydrophilic and on the surface of the protein molecule and therefore are susceptible to digestion, and (iv) the FAD-binding site is located near the amino terminal region of the catalytic region.View full abstractDownload PDF (1797K) -
Kazunobu Matsushita, Emiko Shinagawa, Osao Adachi, Minoru Ameyama1989 Volume 105 Issue 4 Pages 633-637
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSD-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent oxidoreductase linked to the respiratory chain of a wide variety of bacteria. There is a controversy as to whether the glucose dehydrogenase is linked to the respiratory chain via ubiquinone or cytochrome b. In this study, it was shown that the glucose dehydrogenase of Gluconobacter suboxydans has the ability to react directly with ubiquinone. The enzyme purified from the membranes of G. suboxydans was able to react with ubiquinone homologues such as ubiquinone-1, -2, or -6 in detergent solution. Furthermore, in order to demonstrate the reactivity of the enzyme with native ubiquinone, ubiquinone-10, in the native membranous environment, the dehydrogenase was reconstituted together with cytochrome o, the terminal oxidase of the respiratory chain, into a phospholipid bilayer containing ubiquinone-10. The proteoliposomes thus reconstituted exhibited a reasonable glucose oxidase activity, the electron transfer reaction of which was able to generate a membrane potential and a pH gradient. Thus, D-glucose dehydrogenase of G. suboxydans has been demonstrated to donate electrons directly to ubiquinone in the respiratory chain.View full abstractDownload PDF (585K) -
Hideo Higuchi, Shigeru Takemori1989 Volume 105 Issue 4 Pages 638-643
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of 2, 3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20°C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20°C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.View full abstractDownload PDF (647K) -
Mitsukuni Yasui, Masato Ohe, Akihiko Kajita, Toshiaki Arata, Akio Inou ...1989 Volume 105 Issue 4 Pages 644-647
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]P1, species produced by the hydrolysis of [γ-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two P1, species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20°C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KC1. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KC1 at 20°C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissocia-tion of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.View full abstractDownload PDF (513K) -
Kinuko Kimura, Hazumi Suzuki, Yoshio Nakano1989 Volume 105 Issue 4 Pages 648-652
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlutamine synthetase (L-glutamate: ammonia ligase [ADP forming]) [EC 6. 3. 1. 2] has been purified from a Gram-positive, acid-fast bacterium, Mycobacterium phlei, by simple procedures with 57% recovery. The enzyme resembled that from Mycobacterium smeg-matis in the subunit size (56, 000), molecular weight (670, 000), amino acid composition, the amino acid sequence of the NH2-terminal, and the secondary structure. The enzyme activity was regulated by adenylylation of each subunit in the dodecameric molecule. M. phlei glutamine synthetase possesses two useful characteristics: high thermostability and resistance to protease digestion. The enzyme was not inactivated on exposure to 60°C for 2 h or 37° for 72 h, or after incubation with 1% trypsin or chymotrypsin at 37°C for 12 h, pH 7.8. With saturating substrate levels, the Arrhenius plot was nonlinear and concave downward with an intersection point at 45°C, and the activation energies were calculated to be 3.2 and 9.6 cal/mol from the slopes. The specific activity of the highly adenylylated enzyme (E10.7) was remarkably lower than that of the slightly adenylylated enzyme (E2.5); however, both enzymes show similar profiles of the Arrhenius plot. These results indicate that the adenylylation of the enzyme does not affect its activation energies.View full abstractDownload PDF (1724K) -
Masayuki Kanda, Kazuko Hiori, Toshitsugu Kurotsu, Setsuko Miura, Yoshi ...1989 Volume 105 Issue 4 Pages 653-659
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have demonstrated that gramicidin S synthetase 1 (GS 1), phenylalanine racemase [EC 5. 1. 1. 11], of Bacillus brevis catalyzes the exchange between a proton in the medium and α-hydrogen of phenylalanine in the course of the racemase reaction by using tritiated water or L-phenyl [2, 3-3H] alanine. GS 1 from some gramicidin S non-producing mutants of B. brevis lacking phenylalanine racemase activity did not catalyze the tritium exchange reaction. The proton exchange between phenylalanine bound as thioester on the GS 1-phenylalanine complex and water in the medium was detected, but 5, 5'-dithiobis(2-nitrobenzoic acid)-modified complex lacked both the proton exchange and phenylalanine racemase activity. It is suggested that a base group, probably a sulfhydryl group, on the enzyme functions as proton donor and acceptor during the phenylalanine racemase reaction.View full abstractDownload PDF (918K) -
Shoji Odani, Takehiko Koide, Teruo Ono, Yoshiaki Takahashi, Jun-ichi S ...1989 Volume 105 Issue 4 Pages 660-663
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA low-molecular-mass protein, tentatively named meleagrin, was isolated from a commer-cial preparation of turkey (Meleagris gallopavo) ovomucoid. This 40-amino acid protein contains 3 disulfide bonds and high concentrations of aromatic residues (2 tryptophans and 3 tyrosines). It lacks threonine, methionine, phenylalanine, and arginine residues. The complete amino acid sequence was determined to be the following: <Glu-Val-Len-Lys-Tyr-Cys-Pro-Lys-Ile-Gly-Tyr-Cys-Ser-Ser-Lys-Cys-Ser-Lys- Ala-Glu-Val-Trp-Ala-Tyr-Ser-Pro-Asp-Cys-Lys-Val-His-Cys-Cys-Val-Pro-Ala-Asn-Gln-Lys-Trp. One of the three disulfide bonds exists between Cys12 and Cys28, and the two others links Cys32-Cys33 with Cys6 and Cys16. The amino acid sequence of meleagrin shows a strong homology to a similar basic protein, cygnin (Simpson, G. R. & Morgan, F. J. [1983] Int. J. Pep. Protein Res. 22, 476-481), of a rather distantly related ayes, black swan (Cygnus atratus), suggesting some vital role of this protein in avian eggs. Similarity to a part (exon 9) of transferrins was also recognized.View full abstractDownload PDF (481K) -
Heinz Beitinger, Florian Schifferer, Mutsumi Suffita, Shigeko Araki, M ...1989 Volume 105 Issue 4 Pages 664-669
Published: April 01, 1989
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe surface properties of four negatively charged glycosphingolipids from vertebrates, the sialo-glycosphingolipids (=gangliosides) GM1, GD1a, GT1b and a sulfo-glycosphingolipid (=sulfatide), and of the two negatively charged glycosphingolipids from lower inverte-brates, the glucurono-glycosphingolipid Lipid IV and the aminophosphono-glycosphingo-lipid SGL-II were investigated in monolayers at the air/water interface. The molecular peculiarities under investigation were surface pressure (π) and surface potential (ΔV) which are described for Lipid IV and SGL-II for the first time. The surface pressure/area isotherms of all glycosphingolipids were typical of a liquid-expanded monolayer and, with the exception of SGL-II, exhibited a phase transition to a liquid-condensed state at surface pressures above 20 mN/m. The surface potential/molecular area data found for gan-gliosides in the closely packed state at π =30 mN/m (GM1: ΔV=-17 mV; GD1a: ΔV=-35mV; GTlb: ΔV =-39 mV) showed only a slight influence of the additional number of negatively charged residues. For Lipid IV, the surface behavior was very similar to GM1, both possessing one negative group per molecule, whereas in SGL-II also the surface potential data (ΔV=+173 mV) were different compared with GDla both possessing two negative groups per molecule. The addition of Ca2+ condensed the monolayers of all glycolipids and increased the potential in the direction to more positive values, but these findings were less effective in SGL-II films. On the basis of monolayer results presented here, in biological membranes of invertebrates especially Lipid IV might play a similar role as the ganglioside GM1 in vertebrate cells.View full abstractDownload PDF (614K)
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