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Volume 96 (1984)
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Volume 96, Issue 5
Displaying 1-39 of 39 articles from this issue
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Nobuo IDA, Masanobu TOKUSHIGE1984 Volume 96 Issue 5 Pages 1315-1321
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAspartase purified from Escherichia coli W cells was inactivated by diethylpyrocarbonate following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with NH2OH, the enzyme activity was completely restored. The difference absorption spectrum of the modified vs. native enzyme preparations exhibited a prominent peak around 240 nm. The pH-dependence of the inactivation rate suggested that an amino acid residue having a pK value of 6.6 was involved in the inactivation. These results indicate that the inactivation was due to the modification of histidine residues. L-Aspartate and fumarate, substrates for the enzyme, and the Cl- ion, an inhibitor, protected the enzyme against the inactivation. Inspection of the spectral change at 240 nm associated with the inactivation in the presence and absence of the Cl- ion revealed that the number of histidine residues essential for the enzyme activity was less than two. Partial inactivation did not result in an appreciable change in the substrate saturation profiles. These results suggest that one or two histidine residues are located at the active site of aspartase and participate in an essential step in the catalytic reaction.View full abstractDownload PDF (534K) -
Yoshiji OHTA, Isao ISHIGURO, Junko NAITO, Rikio SHINOHARA1984 Volume 96 Issue 5 Pages 1323-1336
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have isolated a protein factor from rat liver which stimulates anthranilamide hydroxylation by the microsomes in the presence of NADPH and oxygen and showed this factor to contain Cu and Zn and to have superoxide dismutase activity [Biochim. Biophys. Acta 365, 148-157 (1974)]. In the present study, this protein factor was confirmed to be a superoxide dismutase (SOD) by comparison of the recovery of SOD activity with that of anthranilamide hydroxylation-stimulating activity at each step of its purification, by inhibition of SOD activity with NaCN and hydrogen peroxide (H2O2), and by recovery of the SOD activity of the protein factor after reconstitution with Cu2+ and/or Zn2+. At a given SOD activity level, there was no difference among the rat liver SOD, Cu, Zn-SOD from bovine erythrocytes, and Mn-SOD from Serratia marcescens in their ability to stimulate anthranilamide hydroxylation not only by rat liver microsomes, but also by the reconstituted cytochrome P-450-containing monooxygenase system. Rat liver SOD stimulated anthranilamide hydroxylation by the reconstituted system in proportion to its amount below a protein concentration of 1 μg/ml. In anthranilamide hydroxylation by the reconstituted system without SOD, only a slight hydroxylase activity was found at the initial stage of the reaction and a marked increase in the amounts of NADPH oxidized and H2O2 formed was observed after a lag time. In the presence of rat liver SOD, however, the hydroxylase activity was markedly and continuously increased almost proportionally to reaction time with a concomitant decrease in the amounts of NADPH oxidized and H2O2 formed. In addition, a trace of 3-OH anthranilamide, one of the products, not only stimulated NADPH-dependent H2O2 formation in the reconstituted system, but also inhibited the apparent reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-OH anthranilamide were diminished by rat liver SOD. When a trace of 3-OH anthranilamide were added to a system composed of NADPH-cytochrome c (P-450) reductase and NADPH, H2O2 formation and NADPH oxidation were markedly stimulated. However, on addition of 3-OH anthranilamide to the system containing rat liver SOD, no stimulation on either H2O2 formation or NADPH oxidation was found.
From these results, we propose the following mechanism for the stimulatory action of rat liver SOD on anthranilamide hydroxylation by the liver microsomal monooxygenase system: In anthranilamide hydroxylation by the monooxygenase, the SOD causes an increase in the amount of electrons flowing from NADPH to cytochrome P-450 by depressing an intensive NADPH-dependent H2O2 formation induced by 3-OH anthranilamide via NADPH-cytochrome c reductase, resulting in an enhancement of the efficiency of hydroxylation of the substrate by cytochrome P-450.View full abstractDownload PDF (1152K) -
Masaaki INABA, Takumi HATAYAMA, Masahisa WADA, Munehiko YUKIOKA1984 Volume 96 Issue 5 Pages 1337-1342
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen rat liver nuclei were digested with nuclease, we found that the chromatinbound RNA polymerase II was liberated as two distinct complexes, peak 1 and peak 2, which seemed to reflect different functional states in cell nuclei.
We further examined their occurrence in nuclear digests of various tissues of rats and the following results were obtained. (1) Upon digestion with micrococcal nuclease of nuclei from brain, spleen, testis and kidney, chromatin-bound RNA polymerase II was liberated as two distinct forms which sedimented differently in a sucrose density gradient. (2) The sedimentation rate of peak 1 varied depending on the tissue nuclei examined. After high salt or RNase treatment of the nuclear digests, peak 1 from liver, brain, spleen and testis nuclei showed the same sedimentation rate as did kidney peak 1, the rate for which remained unchanged by these treatments. The results suggested that peak 1 complexes from various tissue nuclei had basically the same structural organization, and we confirmed this by electrophoretic studies on RNase-treated liver and kidney nuclear digests. (3) Peak 2 from various tissue nuclei exhibited identical sedimentation rates. Thus, the chromatin-bound RNA polymerase II seems to exist commonly in two distinct states in cell nuclei of rats.View full abstractDownload PDF (457K) -
Ken-Ichi FURUKAWA, Hiroshi NAKAMURA1984 Volume 96 Issue 5 Pages 1343-1350
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA calmodulin inhibitor, trifluoperazine, suppresses ATP-dependent Ca2+ uptake into microsomes prepared from bovine aortic smooth muscle. From this microsomal preparation which we expected to contain calmodulin-dependent Ca2+-transport ATPase [EC 3. 6. 1. 3], we purified (Ca2+-Mg2+)ATPase by calmodulin affinity chromatography. The protein peak eluted by EDTA had calmodulindependent (Ca2+-Mg2+)ATPase activity. The major band (135, 000 daltons) obtained after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) accounted for about 80% of the total protein eluted. This major band was phosphorylated by [y-32P] ATP in a Ca2+-dependent manner. All the 32P incorporated into the major band was released by hydroxylaminolysis. The ATPase reconstituted in soybean phospholipid liposomes showed ATP, calmodulin-dependent Ca2+ uptake. The affinity of the ATPase for Ca2+, Km, was 7 μM and the maximum ATPase activity was 1.4 μmol/mg/min. These values were changed to 0.17 μM and 3.5 μmol/mg/min, respectively by the addition of calmodulin. The activity of the purified (Ce2+-Mg2+)ATPase was inhibited by orthovanadate, and the concentration required for half-maximal inhibition was about 1.8 μM which is close to that of plasma membrane ATPases. Judging from the effect of orthovanadate and the molecular weight, the purified (Ca2+-Mg2+)ATPase was considered to have originated from the plasma membrane not from the sarcoplasmic reticulum.View full abstractDownload PDF (1331K) -
Shigeo KOYASU1984 Volume 96 Issue 5 Pages 1351-1364
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDefective flagellar organella, stubs, and flagellin synthesis by L-[35S]methionine labeling of two non-motile mutants of C. crescentus CB15 were studied. CB15 wild type synthesized two flagellin species, flagellin A of molecular weight 26, 000 and flagellin B of molecular weight 28, 000. A DNA phage ø CbK-resistant nonmotile mutant, CB15 pdr-816 [Fukuda et al. (1981) J. Bacterial. 145, 559-572] did not synthesize flagellin B but synthesized large amounts of flagellin A and a novel flagellin of smaller molecular weight 22, 000 (22K flagellin), and formed stubs composed of both flagellin A and 22 K flagellin. The other mutant, CB 15 fla-801 synthesized only 22 K flagellin and formed very short stubs composed of 22 K flagellin. Motile revertants were isolated from these mutants. Full revertants were isolated from CB15 fla-801 at a frequency of ca. 10-7. However, only partial revertants swimming slowly were obtained from CB15 pdr-816 at a similar frequency. Such partial revertants synthesized only flagellin A and formed short flagella in spite of the fact that flagellin A was still synthesized at a higher level than in the wild type. Given the previous finding that flagellin B first polymerizes onto the hook followed by addition of flagellin A onto flagellin B filament [Koyasu et al. (1981) J. Mol. Biol. 153, 471-475], flagellin B seems very important in the formation of normal flagella in C. crescentus. A small amount of 22 K flagellin was detected even in wild type cell. Neither conversion nor alteration was observed in flagellin A or B or 22 K flagellin by short-pulse and pulse-chase experiments. It was thus suggested that 22 K flagellin was not a degradation product formed from other flagellins but was of different origin from flagellins A and B. C. crescentus flagellins consist of several subspecies of different isoelectric points. 22 K flagellin consisted of at least three subspecies, in two-dimensional gel, with more basic isoelectric points than other flagellins. Heterogeneity in isoelectric point of polymerized and nascent flagellins was similar, indicating that each subspecies of different isoelectric point can polymerize into filament, and that conversion between subspecies is not needed to form flagellar filament.View full abstractDownload PDF (5393K) -
Michihiro SUMIDA, Hiromichi OKUDA, Minoru HAMADA1984 Volume 96 Issue 5 Pages 1365-1374
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 μM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105, 000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 μm Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50 inhibition occurring at 180 μm. Fifty mm MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 μm to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15°C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16°C and 14.6 kcal/mol between 16 and 30°C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+, Mg2+-ATPase of the aortic microsomes.View full abstractDownload PDF (766K) -
Akira MURASUGI, Chiaki WADA NAKAGAWA, Yukimasa HAYASHI1984 Volume 96 Issue 5 Pages 1375-1379
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt has been reported that two kinds of Cd-binding peptide (Cd-BPI and Cd-BP2) are induced in fission yeast upon exposure to Cd, and that they consist of the same unit peptide (cadystin), but Cd-BP 1 binds 1.5 times more Cd atoms per cadystin than Cd-BP 2 (Murasugi, A., Wada, C., & Hayashi, Y. (1981) J. Biochem. 90, 1561-1564).
The relative amount of each allomorphic Cd-BP in the cell varied with time after induction and with the concentration of Cd in the induction medium. Further, the production of acid-labile sulfide in the cell increased greatly upon exposure to Cd and varied with time after Cd addition and with Cd concentration in the medium, as in the case of Cd-BP1. Since Cd-BP1 contains labile sulfide, the increase of labile sulfide production together with the increase of cellular Cd concentration may be the driving force to form Cd-BP1, resulting in the increase of the relative amount of Cd-BP1.View full abstractDownload PDF (370K) -
Hirosi ANZAI, Kazutosi NISIZAWA, Kazuo MATSUDA1984 Volume 96 Issue 5 Pages 1381-1390
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA highly purified cellulase [EC 3. 2. 1. 4] preparation (Bβ) was obtained from a crude extract of gastric teeth of Dolabella auricularia by successive chromatographies on Sephadex G-100, DEAE-Toyopearl and CM-Toyopearl. The purified cellulase showed a single protein band on disc electrophoresis, and its isoelectric point was at pH 8.6. It contained relatively large amounts of basic amino acids and its molecular weight was estimated to be approximately 44, 000 by sodium dodecyl sulfate (SDS) electrophoresis. The highest activity of this enzyme was attained at pH 6.3, but the enzyme was rather labile to heat. The activity of this enzyme was strongly inhibited by Hg2+, Mn2+, Zn2+, and Cu2+, whereas Ca2+ and Mg2+ showed no significant effect on the activity. The purified cellulase hydrolyzed sodium carboxymethyl cellulose (CMC) and phosphoric acid-swollen cellulose (swollen cellulose), as well as cellooligosaccharides and their reduction products, in an endowise fashion. It produced higher cellooligosaccharides effectively from swollen cellulose. Cellooligosaccharides with degrees of polymerization of 4-6 (G4-G6) were also hydrolyzed by the purified cellulase, but the modes of hydrolysis of these oligosaccharides were different from each other. The enzyme did not effectively attack cellooligosaccharides lower than G4. It produced G4 and G2 from G6, and G4 and glucose from G5. When these oligosaccharides were modified by reduction with sodium borotritide, the second linkage from the reducing end became, in each case, significantly susceptible to the enzyme and was preferentially cleaved.View full abstractDownload PDF (1179K) -
Munehiro YOSHIDA, Toshiaki FUKUNAGA, Kimikazu IWAMI, Kyoden YASUMOTO1984 Volume 96 Issue 5 Pages 1391-1397
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChicks were fed an amino acid mixture-based diet (basal diet) or one supplemented with selenium (Se, 0.2 μg/g as Na2SeO3) and/or vitamin E (100 μg/g as a-tocopherol). The group receiving the basal diet devoid of Se and vitamin E showed a tendency to grow slowly, but not significantly so, compared to the non-deficient control and manifested a symptom of exudative diathesis after the feeding period of 4 weeks. Supplementation of the basal diet with Se or vitamin E prevented the deficiency symptoms in the chicks. The hepatic GSH level and GSH synthesis activity were about three times as much in the Se- and vitamin E-deficient group as in the control. This was also the case for in vivo sulfur incorporation into hepatic GSH for 10 h post-injection with [35S]methionine. The increased level of GSH may partly compensate the hepatocytes for peroxidative damage.View full abstractDownload PDF (558K) -
Megumi NAKAMURA, Mitsushi INOMATA, Masami HAYASHI, Kazutomo IMAHORI, S ...1984 Volume 96 Issue 5 Pages 1399-1407
Published: 1984
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 under mild conditions, comprised high- and low-molecular-weight components. The latter (LMW-inhibitor; Mr= 50, 000) was purified to homogeneity by means of chromatography on DEAE-cellulose and phenyl-Sepharose CL-4B and chromatofocusing. The purified inhibitor is a protein composed of two polypeptide chains with molecular weights of 26, 000 and 24, 000 daltons. It contains large amounts of glutamic acid, alanine, and serine, and small amounts of aromatic amino acids. It was specific for CANPs having low (m-type) and high (μ-type) Ca2+-sensitivity, had no effect on any other protease examined (trypsin, α-chymotrypsin, bromelain, ficin, papain, thermolysin, etc.), and inhibited rabbit mCANP more effectively than rabbit μCANP or chicken mCANP. It was demonstrated that the inhibition is due to the formation of a stoichiometric complex between two molecules of rabbit mCANP and one inhibitor molecule.View full abstractDownload PDF (1827K) -
In Sook LEE, Sadao WAKABAYASHI, Kouichi MIYATA, Katsumi TOMODA, Masahi ...1984 Volume 96 Issue 5 Pages 1409-1418
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSReexamination of the molecular mass and the amino acid composition of Serratia protease revealed the presence of 1 mol of methionine per mol of protein (about 46 K daltons), and this was confirmed by BrCN cleavage followed by separation of the two fragments. The sole methionine residue was located near the middle region of the molecule. The amino(N)-terminal sequence was determined by Edman degradation of the protein and studies of several proteolytic peptides, establishing a sequence of 18 residues with a heterogeneous N-terminus. The carboxyl(C)-terminal sequence was determined by carboxypeptidase A digestion and tritiumlabeling of the citraconylated C-terminal half segment to be -Phe-Ile-Val. The sequences of a total of 53 residues containing the methionine residue and a total of 38 residues containing two histidine residues were established by the application of various conventional methods to a BrCN peptide and several proteolytic peptides. The segment containing the histidine residues was homologous with that containing the two histidine residues chelating the zinc atom of thermolysin. The 38-residue segment may be directly connected to the 53-residue segment.View full abstractDownload PDF (1371K) -
Hiroshi UEKI, Fumiaki HARAGUCHI, Aiichiro MOTOSHIMA, Takayuki FUNAKOSH ...1984 Volume 96 Issue 5 Pages 1419-1425
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the effects of elastase [EC 3. 4. 21. 11] on lipogenesis, antilipolysis, and pyruvate dehydrogenase activity in rat epididymal adipose tissue in comparison with those of insulin and trypsin [EC 3. 4. 21. 4]. The rate of conversion of [3-3H]-glucose into lipid in fat cells was stimulated by elastase, trypsin, and insulin. When fat pads were incubated with elastase, trypsin, or insulin in the presence of glucose, pyruvate dehydrogenase activity in the homogenate of the incubated fat pads was markedly increased. In the absence of glucose, elastase did not increase pyruvate dehydrogenase activity, though trypsin and insulin showed a slight but significant increase. Further, the increasing effect of elastase in the presence of glucose was inhibited by the addition of 3-O-methylglucose or phlorizin to the incubation mixture of the fat pads. Trypsin and insulin still showed a significant increase under similar conditions. When the homogenate of intact fat pads was incubated with elastase, the pyruvate dehydrogenase activity was progressively decreased with increase in the concentration of elastase. Concanavalin A showed an additive effect on the pyruvate dehydrogenase activity increase caused by elastase, whereas such an effect was not observed with insulin or H2O2. The stimulation of lipolysis by epinephrine in the fat cells was not suppressed by elastase, in contrast to trypsin and insulin. These results suggest that elastase reacts with the cell surface, facilitates glucose transport into the fat cells, and consequently affects glucose and lipid metabolism by somewhat different mechanisms from those of insulin and trypsin.View full abstractDownload PDF (499K) -
Kiyoshi IKEDA, Shin-ichiro SAND, Keizo TESHIMA, Yuji SAMEJIMA1984 Volume 96 Issue 5 Pages 1427-1436
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe phospholipase A2 from the venom of A. halys blomhoffii was titrated with micellar n-hexadecylphosphorylcholine (an analog of lysolecithin) by following the tryptophyl fluorescence change at 25°C and ionic strength 0.1. The data were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N=10.0 and 6.7 for the apoenzyme and its Ca2+, complex, respectively. The binding constant of the enzyme to the substrate micelle was found to be enhanced by Ca2+ binding to the enzyme.
The pH dependence of the binding constant of the apoenzyme to the micelle was well interpreted in terms of pK shifts of two ionizable groups from 5.16 to 5.67 and from 6.45 to 6.6. The pH-dependence curve for the enzyme-Ca2+ complex, which lacked the former transition, was interpreted in terms of the pK shift of a single ionizable group from 5.55 to 5.76. The former ionizable group was assigned as Asp 49, to which Ca2+÷ ion can coordinate, and the latter as His 48 in the active
site. No participation of the α-amino group with a pK value of 7.30 was observed. The binding constant of the enzyme to the substrate micelle, Kmic=0.45-2.3×106 M-1, was found to be far greater than that to the monomeric substrate, Kmon=0.2-1.0×104M-1. This was interpreted in terms of the presence of an additional weak substrate-binding site in the enzyme molecule.View full abstractDownload PDF (720K) -
Eiki KOMINAMI, Yoshiaki BANDO, Nobuaki WAKAMATSU, Nobuhiko KATUNUMA1984 Volume 96 Issue 5 Pages 1437-1442
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe concentrations of two types of endogenous inhibitors of thiol proteinases were determined in soluble extracts of various rat tissues by means of a sensitive enzyme immunoassay method, which consisted of solid-phase immobilized anti-rat liver inhibitor or anti-rat epidermal inhibitor and antibodies labeled with horseradish peroxidase. The minimum detectable amounts of inhibitors from liver and epidermis were 30 pg and 3 pg/assay, respectively. The tissue distributions of the epidermal and liver-type inhibitors were found to differ. The liver-type inhibitor was found to be widely distributed in various tissues at levels of 76-420 ng/mg protein, whereas the epidermal-type inhibitor was found at high levels in the skin, tongue, esophagus, stomach, intestine, and vagina, but at quite low levels in other tissues tested.View full abstractDownload PDF (1864K) -
Shuji TANAKA, Yoshito TAKAHASHI, Noriko MOHRI, Hiroshi KIHARA, Motonor ...1984 Volume 96 Issue 5 Pages 1443-1448
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOxidation of reduced T. flavoviridis phospholipase A2 under suitable conditions resulted in recovery of its active, native structure. The oxidized product was eluted at the same positions as native phospholipase A2 on reserved phase column and DEAE-Toyopearl 650M column chromatographies. The native and regenerated proteins were also identical in mobility on polyacrylamide gel electrophoresis and in circular dichroism spectra. Des-octapeptide(1-8)-phospholipase A2 (L-fragment), which shows only greatly reduced activity, was reduced and oxidized but no effective reformation of the native structure was found, indicating that the entire sequence is required for efficient reorganization.View full abstractDownload PDF (910K) -
Hiroaki HAYASHI, Minoru NOMOTO, Koichi IWAI1984 Volume 96 Issue 5 Pages 1449-1456
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe H4 histone of the protozoan Tetrahymena pyriformis, as obtained previously (Nomoto, M. & Iwai, K. (1982) J. Biochem. 91, 719-723), was completely sequenced; the total sequence reported preliminarily (Hayashi, H., Nomoto, M., & Iwai, K. (1980) Proc. Jpn. Acad. 56 B, 579-584) is one of the two variant sequences determined here. The intact H4 was directly sequenced by automated Edman degradation from the N-terminal through residue 92. Sequence determination was further performed with tryptic peptides and peptic peptides, both covering the whole sequences. Thus, the complete sequences of two variants were determined; both consist of a total of 102 amino acid residues, have identical compositions, have the same molecular weights of 11, 228 in the unmodified form, and are partially acetylated at four lysine residues from the N-terminal. The sequences differ in two positions from each other (-Lys-Arg-/-Arg-Lys- at residues 19 and 20, 7:3 mol/mol), and in 22 or 20 positions from the human spleen H4 sequence. The implications of these results for the structure-function relationship of this histone species and also for the phylogeny of protozoa are discussed.View full abstractDownload PDF (510K) -
Hiroyasu INOUE, Tamio NOGUCHI, Takehiko TANAKA1984 Volume 96 Issue 5 Pages 1457-1462
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of fructose on the induction of L-type pyruvate kinase mRNA in diabetic rat liver was studied by using a cloned cDNA probe. Fructose feeding resulted in a 5- to 6-fold increase in the L-type enzyme mRNA level after 1 to 3 days. These changes were approximately proportional to the changes in the level of translatable mRNA of this enzyme. A significant increase in total cellular L-type enzyme mRNA level was observed within 2 h after fructose feeding and the level reached a maximum after 8 h. Dietary glycerol also markedly increased the L-type mRNA level. These alterations were essentially due to the changes in the cytosolic mRNA. Northern blot analysis of total cellular RNA revealed that two L-type enzyme mRNA species with molecular sizes of 2.1 and 3.6 kilobases were proportionally increased during the fructose induction. The two mRNA forms were found in immunopurified L-type enzyme mRNA and directed synthesis of the L-type subunit in vitro; they are therefore functional mature forms. In contrast, analysis of nuclear RNA showed five putative precursor RNA species for the enzyme, up to 9.4 kilobases in length, in the liver of fructose-fed rats, while no band of the RNA species was found in the nuclei of control liver. The changes in the number of bands of these RNA species and their intensities after fructose feeding preceded the changes in the level of total cellular L-type enzyme mRNA sequences. These results indicate that dietary fructose causes a rapid increase in the level of L-type pyruvate kinase mRNA sequences by acting at the nuclear level.View full abstractDownload PDF (1753K) -
Chiharu KIMURA, Akira KONDO, Noriko KOEDA, Hiroshi YAMANAKA, Michinao ...1984 Volume 96 Issue 5 Pages 1463-1469
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPeroxisomal 2, 4-dienoyl-CoA reductase was purified from rat liver to homogeneity. The subunit molecular weight of 33, 000 was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The native molecular weight close to 120, 000 was estimated by gel filtration on Sephacryl S-300 Superfine. trans-2, trans-4-Decadienoyl-CoA was the most active substrate among the dienoyl-CoA's of various chain lengths. The total activity of peroxisomal 2, 4-dienoyl-CoA reductase exceeded that of the mitochondrial one even in the livers of rats fed with a standard diet. Furthermore both reductases were remarkably and coordinately induced in the livers of clofibrate-treated rats.View full abstractDownload PDF (501K) -
Hideki NAKAKUMA, Yutaka SANAI, Kazuko SHIROKI, Yoshitaka NAGAI1984 Volume 96 Issue 5 Pages 1471-1480
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMalignant transformation is known to be associated with characteristic changes of cell surface glycolipids, but the genetic background and underlying molecular mechanism of these changes are still unknown. Incorporation of oncogenic elements into cultured cells should provide a useful model for studies on this problem. We analyzed gangliosides of a clonal and fibroblastic rat cell line, 3Y1, and its transformed counterparts, E1Y, HY1, and CY1 cell lines. E1Y cells were established by transfection of 3Y1 cells with the transforming gene E1, minimum DNA fragment of human adenovirus type 12 (Ad 12) required for complete transformation of cells. HY1 cell line is the cells established with E1A which is a transcriptional subunit of the E1 and can transform incompletely cells. CY1 was established by co-transfection with parts of non-transforming gene E3 and E4 of Ad 12 DNA in addition to the E1. GD3 ganglioside (NeuAcα2-8NeuAcα2-3Galβ1-4Glcβ1-1Cer) appeared in all these transfection-transformed cells but was undetectable in untransformed control cells. The appearance of GD3 was undoubtedly due to expression of E1A gene in association with oncogenic cell transformation. The expression of other gangliosides, particularly GM2 and GM1, were also influenced by transfection, but their expressions depended on the kind of oncogenic DNA fragments used. These results all suggest that ganglioside metabolism is under unexpectedly complicated gene control.View full abstractDownload PDF (2421K) -
Yasufumi MINAMI, Sachiko ENDO, Hikoichi SAKAI1984 Volume 96 Issue 5 Pages 1481-1490
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have already reported that neurofilaments are capable of stimulating microtubule assembly and causing gelation. After separation of each of the triplet proteins of neurofilaments it was demonstrated that only the 200 K subunit shows the activity to promote tubulin polymerization (Minami, Y. & Sakai, H. (1983) J. Biochem. 94, 2023-2033). The separation of each subunit protein led us to attempt the reconstitution of filaments from the 200 K and 70 K subunits or from the 150 K and 70 K subunits. It was found that both the 200 K and 150 K subunits independently contribute to the formation of intermediate-sized filaments, provided that each subunit was combined with the 70 K subunit before removing urea by dialysis for reconstitution. On the other hand, the 200 K subunit alone formed a very short thread-like structure after removal of urea, and the 150 K subunit formed a filamentous structure, both incapable of being incorporated into filaments made of the 70 K subunit alone. These observations suggest that the 200 K and 150 K subunits are not peripherally attached to a filament core made of 70 K protein, but they take part in the formation of the core. Moreover, both proteins can co-polymerize with the 70 K protein at a weight ratio of about 1:1 at least, which is in excess of that of the intact neurofilament. We investigated whether or not the 200 K subunit incorporated with the 70 K subunit into filaments could also stimulate tubulin polymerization. Low-shear viscometry measurements suggested that the 200 K subunit retains the activity to initiate tubulin polymerization. This was confirmed by measuring viscosity changes with an Ostwald-type viscometer. In contrast, filaments reconstituted from the 70 K and 150 K proteins were incapable of increasing low-shear viscosity when mixed with tubulin. These observations suggest that the domain of the 200 K protein embedded in the core of intermediatesized filament is separate from the site responsible for promotion of tubulin polymerization.View full abstractDownload PDF (2985K) -
Tsuneo OMURA, Hiroyuki SADANO, Tohru HASEGAWA, Yuzo YOSHIDA, Shiro KOM ...1984 Volume 96 Issue 5 Pages 1491-1500
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHemoprotein H-450 was purified from rat liver cytosol to homogeneity by an improved procedure. The purified H-450 showed a subunit molecular weight of 64, 000 daltons and contained 0.7-0.9 mol of protoheme per mol subunit. Among rat tissues examined, liver and kidney contained significant amounts of H-450 in the cytosol.
Oxidized H-450 showed a Soret peak at 428 nm and a broad β band at around 550 nm. Reduced H-450 was found to exist in two interconvertible forms, alkaline and acid forms. The alkaline form showed Soret, β, and α peaks at 448, 540, and 571 nm, whereas the acid form showed Soret, β, and α peaks at 425, 530, and 558 nm. The spectral properties of both oxidized and reduced H-450 in alkaline medium resemble those of cytochrome P-450 having a nitrogenous ligand at the 6 th coordination position of the heme. Upon addition of low concentrations of HgCl2. H-450 was converted to a denatured form both in the oxidized and the reduced states and lost its unique spectral characteristics. Addition of carbon monoxide to reduced H-450 produced a new spectral species which resembled that of the reduced carbon monoxide complex of P-420, a denatured form of cytochrome P-450. Comparison of the EPR signal of oxidized H-450 with those of a cytochrome P-450, P-450 (PB-1), and several model compounds indicated the presence of a thiolate anion at the 5 th coordination position of the heme of H-450. Judging from EPR data, oxidized H-450 also converts between acid and alkaline forms, whose signals were observed at g=1.867, 2.31, and 2.507 and at g=1.910, 2.28, and 2.424, respectively.
These lines of evidence indicate that the 5 th and 6 th coordination positions of the heme of H-450 are a thiolate and a nitrogenous group, respectively. With respect to the heme environments, H-450 is a member of the cytochrome P-450 family, and has a nitrogenous ligand at the 6 th coordination position of the heme.View full abstractDownload PDF (1447K) -
Chikao NAKAYAMA, Mineo SANEYOSHI1984 Volume 96 Issue 5 Pages 1501-1509
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVarious 5-substituted UTPs (methyl, ethyl, n-propyl, n-butyl, fluoro, chloro, bromo, and iodo) and sulfur-containing UTP analogues (4-thio-, 2-thio-, 5-methyl-2-thio-, and 5-methyl-4-thio-) were synthesized chemically and their utilization by DNA-dependent-RNA polymerases I and II of the cherrry salmon (Oncorhynchus masou) were studied in substitution experiments under the condition of limited RNA synthesis in vitro. RNA polymerase I utilized the 5-methyl-, chloro, bromo, and iodo derivatives of UTP more efficiently than unmodified UTP, but RNA polymerase II utilized UTP most efficiently. 5-Methyl-4-thiouridine 5'-triphosphate (4-thio TTP) was utilized more efficiently than UTP by RNA polymerase I. On the other hand, it was found that 4-thio TTP was a selective substrate for RNA polymerase I and that its incorporation by RNA polymerase II was very slow. Thus recognition of UTP analogues as substrates by RNA polymerase I and II was different. These observation were attributed from kinetic analyses to differences in catalytic activity (Vmax).View full abstractDownload PDF (519K) -
Shuzo YAMAGATA1984 Volume 96 Issue 5 Pages 1511-1523
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA crude extract of Schizosaccharomyces pombe cells catalyzed sulfhydrylation of both O-acetyl-L-serine and O-acetyl-L-homoserine with H2S, but did not synthesize cystathionine from O-acetyl-L-homoserine and L-cysteine. The O-acetylhomoserine sulfhydrylase [EC 4. 2. 99. 10] was very unstable; however, it could be stabilized by the addition of 25% (w/w) sucrose or glycerol. The optimal pH for activity was 8.0 and that for stability was 7.0.
The enzyme was purified approximately 300-fold from an ammonium sulfateprecipitated fraction. L-Methionine was the most effective inhibitor among the amino acids examined. It inhibited the enzyme competitively with respect to OAH with a K1 value of 2.6mM. Sulfhydrylase activity was inhibited to various extents by some carbonyl reagents, but sulfhydryl reagents such as p-chloromercuribenzoic acid, 5, 5'-dithio-bis (2-nitrobenzoic acid), and monoiodoacetic acid had no inhibitory effect.
The enzyme also reacted with O-succinylhomoserine and L-homoserine to synthesize homocysteine directly, but could not utilize cysteine as a co-substrate in place of H2S. In the sulfhydrylation reactions, Km values for the three substrates ranged from 10.4-12.5mM. The enzyme was resolved to the apoenzyme by incubation with phenylhydrazine and reactivated by the addition of pyridoxal 5'-phosphate, whose Km value was 0.083μM. The molecular weight of the enzyme was estimated to be approximately 186, 000 by gel filtration and 170, 000 by ultracentrifugation in sucrose density gradients. The isoelectric point of the protein was pH 4.1. The characteristics of this enzyme are compared with those of physiologically functional sulfhydrylases reported for other organisms, and the possibility of the enzyme functioning as a homocysteine synthase is discussed.View full abstractDownload PDF (1699K) -
Senya MATSUFUJI, Kazunobu FUJITA, Takaaki KAMEJI, Ryuhei KANAMOTO, Yas ...1984 Volume 96 Issue 5 Pages 1525-1530
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA monoclonal antibody was obtained against rat liver ornithine decarboxylase by using hybridoma technology with a small amount of partially purified enzyme. The antibody, IgG1 of κ-type, was affinity-purified to homogeneity from culture supernatants of hybridoma cells. While the antibody had no inhibitory effect on ornithine decarboxylase activity when tested alone, it precipitated up to 87 units (60 ng) of the enzyme per microgram in the presence of formalin-fixed Staphylococcus aureus Cowan I bacteria. Immunoadsorption on a column of the mono-clonal antibody-Sepharose 4B was shown to be useful for the removal of ornithine decarboxylase from antizyme inhibitor preparations, an essential procedure for the accurate assay of either ornithine decarboxylase-antizyme complex or antizyme inhibitor. It was also shown that antizyme could be affinity-purified by using a column of the monoclonal antibody-Affi-Gel 10 to which ornithine decarboxylase had been bound.View full abstractDownload PDF (1101K) -
Noboru YUMOTO, Takanobu KIKUCHI, Takashi SASAKI, Takashi MURACHI1984 Volume 96 Issue 5 Pages 1531-1537
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo different forms of Ca2+-dependent cysteine proteinase, low-Ca2+-requiring calpain I and high-Ca2+-requiring calpain II, are known to be heterodimers, each composed of one heavy (called 80 K) and one light (called 30 K) subunit. The most probable identity of the 30 K and the substantial difference between the 80 K subunits of porcine calpains I and II were clearly demonstrated by comparing the tryptic peptide maps obtained upon running a high performance liquid chromatography which permitted parallel detection of tryptophan-containing peptides by fluorometry. Comparison of the amino acid compositions of the two 30 K and 80 K subunits also confirmed this conclusion. The same chromatographical analysis also revealed close structural similarity of the human calpain I 30 K subunit, and even some similarity existing between the calpain I 80 K subunits of human and porcine origins.View full abstractDownload PDF (475K) -
Michiko IIJIMA, Takashi TOBE, Terufumi SAKAMOTO, Motowo TOMITA1984 Volume 96 Issue 5 Pages 1539-1546
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSC3-translational product, which was synthesized with rabbit liver mRNA in a reticulocyte lysate protein-synthesizing system, did not react with [14C] methylamine, indicating the lack of an internal thioester bond. Instead, the C3-translational product reacted with iodo [1-14C] acetamide, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate after immunoprecipitation of the product, indicating the presence of a reactive thiol group. When the C3-translational product was treated with rabbit liver homogenate, the product acquired reactivity with [14C] methylamine and lost the reactivity with iodo[1-14C]acetamide. Thus, the liver homogenate seemed to contain a factor (or factors) required for the formation of an internal thioester bond. The factor was partially purified from the liver homogenate by ammonium sulfate precipitation and ion-exchange chromatography on DEAE-cellulose.View full abstractDownload PDF (581K) -
Yasutaka OHTA, Sachiko ENDO, Eisuke NISHIDA, Hiromu MUROFUSHI, Hikoich ...1984 Volume 96 Issue 5 Pages 1547-1558
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeveral actin binding proteins were isolated from ascites hepatoma cells AH 7974 by DNase I affinity chromatography. Among them, a protein having a molecular weight of 18, 000 was further purified by DEAE cellulose and hydroxyapatite column chromatographies and gel filtration on a Sephadex G-75 column. The 18 K protein not only inhibits actin polymerization but also depolymerizes actin filaments. This conclusion was supported by viscosity and fluorescence intensity measurements and the DNase I inhibition assay. A chemical cross-linking experiment suggested that the 18 K protein binds to monomeric actin and forms an 18 K-actin 1:1 complex. The net depolymerization rate by the 18 K protein measured by the DNase I inhibition assay was slower than the rapid reduction of the fluorescence intensity of pyrene-labeled F-actin upon addition of the 18 K protein. This result suggests that the 18 K protein not only binds to monomeric actin but also binds to actin filaments directly. The sedimentation assay showed that a part of the 18 K protein was cosedimented with actin filaments. Electron microscopic observations demonstrated that the 18 K protein decreased the amount of actin filaments and the remaining filaments appeared to be decorated and distorted by the 18 K protein. The 18 K protein had no Ca2+ ion sensitivity and exhibited the same effect on both this tumor actin and muscle actin.View full abstractDownload PDF (5273K) -
Kazuhiko WAKABAYASHI, Yasuji FURUTANI1984 Volume 96 Issue 5 Pages 1559-1564
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondrial DNA of an erythromycin-resistant petite mutant of yeast, E734, showed physical maps of inversion, which occurred between two cross-over sites in the fragments Hae A and Hae B. The pair of cross-over sites was inferred to be accommodated within a repeat unit of E734 mtDNA during petite mutation by joining two fragments excised from non-adjacent region of wild type mtDNA.View full abstractDownload PDF (1026K) -
Fusahiro OGATA, Satoru MAKISUMI1984 Volume 96 Issue 5 Pages 1565-1574
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree trypsin inhibitors were isolated from tubers of taro (Colocasia antiquorum var. nymphaifolia?) and named taro trypsin inhibitors (TTI)-I, -II, and -III. The final preparations were homogeneous by polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The three inhibitors showed strong and stoichiometric inhibition against bovine trypsin and the inhibitor constants (Ki) were estimated to be of the order of 10-9 to 10-10M. In contrast, they had only a weak capacity to inhibit bovine α-chymotrypsin and did not inhibit subtilisins (BPN' and Carlsberg), porcine pepsin, or papain. Each inhibitor appeared to be a protein with a molecular weight of 40, 000 which could be dissociated into two subunits, both of which had a molecular weight of 20, 000. The inhibitors formed complexes with trypsin at molar ratios of 1:2, suggesting that each subunit of these inhibitors can react with the enzyme in a 1:1 molar ratio. The three inhibitors had similar amino acid compositions and none of them contained carbohydrate or free sulfhydryl group. The antitryptic activity of all three inhibitors was suppressed by treatment with 1, 2-cyclohexanedione (CHD) but not with 2, 4, 6-trinitrobenzenesulfonate (TNBS), thus demonstrating each of the inhibitors to contain an arginyl residue at the reactive site.View full abstractDownload PDF (1729K) -
Shigeo NAKAJO, Katsumi SHINKAWA, Takashi SHIMIZU, Kazuyasu NAKAYA, Yas ...1984 Volume 96 Issue 5 Pages 1575-1585
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCasein kinase 1 (CK 1) and casein kinase 2 (CK 2) were purified from the cytosol fraction of AH-66 cells to electrophoretic homogeneity by a simple procedure based on our finding that CK 1 and CK 2 are chromatographically distinct on phosvitin-Sepharose. The amino acid composition of CK 2 resembles those of cyclic AMP-dependent and cyclic GMP-dependent protein kinases but is considerably different from that of CK 1. Both CK 1 and CK 2 were markedly stimulated by low concentrations of spermine and spermidine but were practically unaffected by putrescine. When CK 1 and CK 2 were added back to AH-66 cytosol, they promoted the phosphorylation of the same cytosolic proteins that were phosphorylated endogenously. Although most of the cytosolic proteins phosphorylated by CK 1 and CK 2 were common, some proteins were preferentially phosphorylated by either CK 1 or CK 2. Interestingly, CK 1 was able to phosphorylate the plasma membrane proteins of AH-66 cells. In contrast, enhancement of the phosphorylation of the membrane proteins by CK 2 was practically undetectable.View full abstractDownload PDF (2878K) -
Susumu OGUSHI, Makoto ANDO, Daisuke TSURU1984 Volume 96 Issue 5 Pages 1587-1591
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe NAD -dependent formaldehyde dehydrogenase from Pseudomonas putida C-83 was found to contain 4 gram atoms of zinc per mol, corresponding to 2 gram atoms of zinc per subunit monomer. Treatment of the enzyme with o-phenanthroline resulted in removal of 1 gram atom of zinc per subunit and caused a complete inactivation of the enzyme. The activity lost was restored by the addition of zinc ions, by which the zinc content was also reversed to almost the same level as that of the native enzyme. Another zinc atom that was resistant to metal chelator-treatment was liberated from the enzyme only after the irreversible denaturation of the enzyme. These results indicate that the formaldehyde dehydrogenase of P. putida is a zinc metallcenzyme and one of two zinc atoms per subunit participates in the catalytic activity of the enzyme, another zinc being presumably involved in maintaining the native conformation of the enzyme. Treatment of the enzyme with bipyridine also caused a reversible inactivation of the enzyme, but the zinc content remained unchanged. The spectrophotometric analysis indicated that the formation of a enzyme-Zn-bipyridine complex took place. Incubation of the enzyme with p-chloromercuribenzoate also resulted in a complete loss of the activity. These results suggest that an intrinsic zinc and sulfhydryl group together with NAD+ participate in the dehydrogenation reaction of substrate by the enzyme.View full abstractDownload PDF (351K) -
Tomofusa TSUCHIYA, Setsuo SAITO1984 Volume 96 Issue 5 Pages 1593-1597
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSn-Octyl-β-D-thioglucopyranoside (octylthioglucoside), a new nonionic detergent, was synthesized. Properties of this detergent and its applicability to membrane biochemistry were investigated. The critical micelle concentration of this detergent was determined to be 9mM. Membrane proteins of Escherichia coli were effectively solubilized with octylthioglucoside at 25-35mM. The solubilizing power was equivalent to that of n-octyl-β-D-glucopyranoside (octylglucoside). Reconstitution of the melibiose carrier into liposomes was performed by the detergent dilution procedure. It was found that the concentration range of octylthioglucoside for successful reconstitution (45-70mM) was considerably wider than that of octyiglucoside (43-46mM), thus giving more reproducible results. Octylthioglucoside is more stable than octyiglucoside. Furthermore, the former is synthesized with high yield at low cost whereas the latter is very expensive. Thus, we conclude that octylthioglucose is superior to octylglucoside, and is very useful in membrane biochemistry.View full abstractDownload PDF (389K) -
Miki HARA-YOKOYAMA, Shigeyuki YOKOYAMA, Tatsuo MIYAZAWA1984 Volume 96 Issue 5 Pages 1599-1607
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlutamyl-tRNA synthetase has been isolated from an extreme thermophile, Thermus thermophilus HB 8. The enzyme has been purified to homogeneity by successive chromatography on columns of DEAE-cellulose, DEAE-Sephacel, phosphocellulose and hydroxyapatite. 11.7mg of purified enzyme has been obtained from 2kg of T. thermophilus cells, with a purification factor of 600 with an 11% yield. From gel permeation chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme is found to be a monomer protein with a molecular weight of 50, 000. The optimum temperature for the aminoacylation of T. thermophilus tRNAGlu is 65°C, and the optimum pH range is 8.0-9.0, in the presence of 5mM Mg2+. The Km values for ATP, L-glutamate, and T. thermophilus tRNAGlu are 230 μM, 70 μM, and 0.65 μM, respectively, in the presence of 50mM KCl and 10mM MgCl2 at pH 8.0 at 65°C. Escherichia coli tRNA2Glu is also a good substrate with a Km value of 0.60 μM at 65°C. The mole fractions of Arg and Leu residues are higher and that of Asx residues is lower than those of E. coli glutamyl-tRNA synthetase. Glutamyl-tRNA synthetase from T. thermophilus is remarkably thermostable; even after incubation for 9 h at 65deg;C, 70% of the enzyme activity is retained in the absence of any protecting factors. Such an extremely thermostable enzyme with a low molecular weight will be useful for detailed physicochemical analyses on the molecular mechanism of strict recognition by aminoacyl-tRNA synthetases.View full abstractDownload PDF (1265K) -
Hiroshi NAKADA, Takaya SAWAMURA, Yutaka TASHIRO1984 Volume 96 Issue 5 Pages 1609-1617
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycosylation of a rat receptor specific for asialoglycoproteins was investigated in vivo by using monospecific antibody. After intravenous injection of [3H]mannose, the receptor protein was immunoprecipitated from various subcellular fractions and the glycopeptide and oligosaccharide chains of the protein were prepared by treatment with pronase and endo-β-N-acetylglucosaminidase H. The glycopeptides thus prepared from the rough and smooth microsomes and Golgi heavy fraction (GF3) were all sensitive to endo H and most of the endo H-sensitive oligosaccharides were eluted at the position considered to correspond to Man8GIcNAc on high-resolution Bio-Gel chromatography. Endo H-resistant forms were first detected in the Golgi intermediate fraction (GF2) and significantly in the light Golgi fraction (GF1), suggesting the formation of the complex-type oligosaccharide chains in the latter fraction. This view was also supported by the almost exclusive addition of the terminal sugars such as N-acetylglucosamine, galactose, and sialic acid in GF1.
These results suggest that the major form of the oligosaccharide chains from the endoplasmic reticulum to the Golgi apparatus is Man8GlcNAc2, and that the processing of these large mannosyloligosaccharide chains and subsequent addition of terminal sugars to them are performed successively in the trans-Golgi region.View full abstractDownload PDF (1428K) -
Yohtalou TASHIMA, Hideo MIZUNUMA, Hiroshi SHIMA, Ryoji KOBAYASHI, Yosh ...1984 Volume 96 Issue 5 Pages 1619-1624
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlucocorticoids significantly affected the developmental appearance of fructose-1, 6-bisphosphatase [EC 3. 1. 3. 11] and glucose-6-phosphatase [EC 3. 1. 3. 9] in fetal mouse liver. In fragments of 15- or 16-day-old fetal livers maintained in organ culture in the absence of serum, induction of the bisphosphatase by dibutyryl cyclic AMP was repressed completely when the tissue was treated with 10-7M dexamethasone for 24 h during the second day of culture. The induction of the glucose phosphatase was greatly stimulated after a lag of 1 to 2 days. The glucocorticoid action continued over a period of 2 days even though the steroid had been washed out. The dose response curve of hydrocortisone with the half-maximally effective concentration of roughly 2×10-8M is in the physiological range. The corticoid action was specific for glucocorticoids, and aldosterone or progesterone was ineffective. When the tissue was cultured for 4 days before addition of dexamethasone, the bisphosphatase induction became insensitive to the steroid. Glucose-6-phosphatase induction, however, remained sensitive, but the long latent period required for the appearance of the hormone action disappeared. These results indicate the involvement of glucocorticoids in the developmental appearance of glucose-6-phosphatase in fetal liver.View full abstractDownload PDF (440K) -
Kimitsuna WATANABE, Hyouta HIMENO, Takahisa OHTA1984 Volume 96 Issue 5 Pages 1625-1632
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn extreme thermophile, Thermus thermophilus HB 8, contains two types of tRNAs, T- and s2T-containing tRNAs. Their relative content changes depend on the growth temperature of the bacterial cells (1-3). To elucidate the reason why the extreme thermophile possesses the two types of tRNAs, an attempt was made to clarify how these tRNAs are utilized in in vivo protein synthetic systems of the bacteria cultured at different temperatures.
First, a method was developed to isolate active polysomes from the thermophile cells cultured at 55°C, 65°C, and 77°C. Then, tRNAs were separated from the polysomes and the T- and s2T-contents of the tRNAs were determined by HPLC. The relative content of s2T-tRNAs in the polysomes from 77°C cells was much higher than that in bulk tRNAs from whole cells cultured at the same temperature, but the situation was reversed in 50°C cells.
These results clearly show that the protein synthetic systems of the thermophile have some selection mechanism to utilize either T- or s2T-containing tRNAs preferentially depending on the environmental temperature.View full abstractDownload PDF (633K) -
Hitoshi SHICHI, Robert L. SOMERS1984 Volume 96 Issue 5 Pages 1633-1636
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rod GTP-binding protein in the bovine disk membrane seems to exist as oligomers in the dark. Gαand Gβ do not interact strongly, and Gγ may be required for Gα•GDP to dimerize.View full abstractDownload PDF (306K) -
1984 Volume 96 Issue 5 Pages 1637a
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (39K) -
1984 Volume 96 Issue 5 Pages 1637b
Published: 1984
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (39K)
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