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Volume 91 (1982)
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Volume 91, Issue 2
Displaying 1-36 of 36 articles from this issue
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Terutoshi MORI, Yoshimi TAKAI, Binzu YU, Juro TAKAHASHI, Yasutomi NISH ...1982 Volume 91 Issue 2 Pages 427-431
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe specificity of the fatty acyl moieties of diacylglycerol for the activation of Ca2+-activated, phospholipid-dependent protein kinase was investigated. Diacylglycerol has been previously shown to activate this enzyme by increasing the affinity for Ca2+ and phospholipid, both of which are indispensable for the enzyme activation. Diacylglycerols containing at least one unsaturated fatty acid at either position 1 or 2 are fully active in this capacity, irrespective of the chain length of the other fatty acyl moiety in the range tested, C2 to C18. Diacylglycerols containing two saturated fatty acids such as dipalmitin and distearin are far less effective. Mono- and triacylglycerols and free fatty acids are totally inactive, indicating that the diacylglycerol structure is essential.View full abstractDownload PDF (280K) -
Tohru YUTAKA, Arvan L. FLUHARTY, Richard L. STEVENS, Hayato KIHARA1982 Volume 91 Issue 2 Pages 433-441
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIduronate sulfatase was purified from human liver for an investigation of the degradative pathway of dermatan sulfate. An overall 80-fold purification was achieved and, more importantly, the preparation was free of α-L-iduronidase, β-glucuronidase, N-acetylgalactosamine 4-sulfate sulfatase (arylsulfatase B) and highly enriched in β-N-acetylhexosaminidase. The liver enzyme appeared to be composed of several molecular species. The enzyme activity was optimal at pH 4.0 and its Km was 10-20 μM with sulfoiduronyl sulfoanhydromannitol. Chloride was inhibitory at high concentration and among divalent metal ions, only copper was inhibitory. Nitrocatechol sulfate was not a substrate, but did show competitive inhibition. Its Ki for iduronate sulfatase was similar to its Km for arylsulfatase, suggesting a similarity in the substrate binding sites of iduronate sulfatase and arylsulfatases.View full abstractDownload PDF (495K) -
Seiji HORI1982 Volume 91 Issue 2 Pages 443-448
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen mice were exposed to 100% oxygen at a pressure of 6 atm. absolute, the animals suffered from severe convulsions. The content of γ-aminobutyric acid (GABA) in synaptosomes was lower in the exposed animals than in unexposed ones. The exposure to high pressure oxygen produced a considerable reduction in GABA formation rate in synaptosomes, due to the inhibition of glutamic acid decarboxylase activity. Exposure to air at the same high pressure produced no reduction in the GABA levels in synaptosomes. The results support the view that low GABA levels in synaptosomes were involved in the etiology of the seizures.View full abstractDownload PDF (372K) -
Yoichi NAKAMURA, Yuji TONOMURA1982 Volume 91 Issue 2 Pages 449-461
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amount of Ca2+ bound to the Ca2+, Mg2+-dependent ATPase of deoxycholic acid-treated sarcoplasmic reticulum was measured during ATP hydrolysis by the double-membrane filtration method [Yamaguchi, M. & Tonomura, Y. (1979), J. Biochem. 86, 509-523]. The maximal amount of phosphorylated intermediate (EP) was adopted as the amount of active site of the ATPase.
In the absence of ATP, 2 mol of Ca2+ bound cooperatively to 1 mol of active site with high affinity and were removed rapidly by addition of EGTA. AMPPNP did not affect the Ca2+ binding to the ATPase in the presence of MgCl2.
Under the conditions where most EP was ADP sensitive at steady state (58 μM Ca2+, 50 μM EGTA, and 20mM MgCl2 at pH 7.0 and 0°C), bound Ca2+ increased by 0.6-0.7 mol per mol active site upon addition of ATP. The time course of decrease in the amount of bound 45Ca2+ on addition of unlabeled Ca2++EGTA was biphasic, and 70% of bound 45Ca2+ was slowly displaced with a rate constant similar to that of EP decomposition. Similar results were obtained for the enzyme treated with N-ethylmaleimide, which inhibits the step of conversion of ADP-sensitive EP to the ADP-insensitive one.
Under the conditions where most EP was ADP insensitive at steady state (58 μM Ca2+, 30 μM EGTA, and 20mM MgCl2 at pH 8.8 and 0°C), the amount of bound Ca2+ increased slightly, then decreased slowly by 1 mol per mol of EP formed after addition of ATP. Under the conditions where about a half of EP was ADP sensitive (58 μM Ca2+, 25 μM EGTA, and 1mM MgCl2, at pH 8.8 and 0°C), the amount of bound Ca2+ did not change upon addition of ATP.
These findings suggest that the Ca2+ bound to the enzyme becomes unremovable by EGTA upon formation of ADP-sensitive EP and is released upon its conversion to ADP-insensitive EP.View full abstractDownload PDF (797K) -
Hiroko HASEGAWA-SASAKI, Terukatsu SASAKI1982 Volume 91 Issue 2 Pages 463-468
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConcanavalin A stimulated the [32P] phosphate incorporation into phospholipids of rat lymph-node cells in a dose-dependent manner up to 200 μg of the lectin per ml of the lymphocyte culture. The initial step in the concanavalin A-stimulated phospholipid metabolism was examined by use of rat lymphocytes prelabeled with radioactive arachidonic acid. Concanavalin A induced a small but significant decrease, up to 10%, in the phosphatidylinositol radiolabel at 1 to 5min after the lectin addition. A small increase, about 10%, in the diacylglycerol radiolabel and a twofold increase in the phosphatidate radiolabel were observed concomitant with the breakdown of phosphatidylinositol. The increase in the phosphatidic acid radioactivity amounted to about one-fifth of the decrease in the phosphatidylinositol radioactivity. No significant change in radiolabel was found in the other major phospholipids or in triacylglycerol. The results suggest that the breakdown of pre-existing phosphatidylinositol is the initial step in the lymphocyte phosphatidylinositol response.View full abstractDownload PDF (394K) -
Thermodynamics of Association of 8-Substituted Riboflavins with Egg White Riboflavin Binding ProteinKunio MATSUI, Kyoko SUGIMOTO, Sabu KASAI1982 Volume 91 Issue 2 Pages 469-475
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAssociation constants of 14 species of riboflavin derivatives with hen egg-white riboflavin binding protein were measured by a fluorometric titration method using flavin and protein fluorescence in a temperature range of 10-42°C. The derivatives were riboflavins whose 8-CH3 group was substituted with alkylamino, alkyloxy, halogen or hydrogen. The flavin derivatives were classified into four groups, i.e., RR'N, HRN, RO, and halogen, on the basis of the linear relation between enthalpy and entropy changes (linear free energy relationship), and from the chemical structures of the 8-substituents. The relation between the entropy change and the bulkiness of 8-substituents suggested burying of the substituents in a cavity of the protein, and provided an indication of the size of the substituent-accepting cavity. The relation also suggested an electric repulsion between halogen substituents and the binding site. A very good correlation was found between the constant term of the linear free energy relation and the wavelength of the visible absorption peak of flavins, and this correlation suggested the electronic nature of the flavin-protein interaction.View full abstractDownload PDF (443K) -
Taibo YAMAMOTO, Yuji TONOMURA1982 Volume 91 Issue 2 Pages 477-486
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle were solubilized with a high concentration of dodecyl octaethyleneglycol monoether (C12E8) and the kinetic properties of the Ca2+, Mg2+-dependent ATPase [EC 3. 6. 1. 3] were studied. The following results were obtained:
1. SR ATPase solubilized in C12E8 retains high ability to form phosphoenzyme ([EP]=4-5 mol/106g protein) for at least two days in the presence of 5mM Ca2+, 0.5M KCl, and 20% glycerol at pH 7.55.
2. The ATPase activity was dependent on both Mg2+ and Ca2+. However, the rate of E32P decay after the addition of unlabeled ATP was independent of Mg2+.
3. Most of the EP formed in the absence of Mg2+ was capable of reacting with ADP to form ATP in the backward reaction. However, in the presence of 5mM Mg2+, the amount of ATP formed was markedly reduced without loss of the reactivity of the EP with ADP.
4. The removal of C12E8 from the ATPase by the use of Bio-Beads resulted in the full restoration of the Mg2+ dependency of the EP decomposition.
5. These results strongly suggest that in the case of SR solubilized with a high concentration of C12E8 the decomposition of phosphoenzyme is Mg2+ independent and ATP is mainly hydrolyzed through Mg2+-dependent decomposition of an enzyme-ATP complex, which is in equilibrium with phosphoenzyme and ADP.View full abstractDownload PDF (586K) -
Hisaya IIDA, Yoshinori NOZAWA, Tetsutaro IIZUKA, Yuzuru ISHIMURA, Toku ...1982 Volume 91 Issue 2 Pages 487-496
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochromes in Tetrahymena pyriformis cultured under various growth conditions were analyzed by the aid of a low temperature spectroscopic technique which allowed us to quantitate the cytochrome contents in the whole cell. When the protozoa were grown in a nutrient-rich medium at 39.5°C, the presence was clearly demonstrated of two cytochromes with α-bands at 553 and 558 nm, respectively. By subcellular fractionation, the former cytochrome was shown to be located in the mitochondria, which also contained another cytochrome with its a-band at 548 nm, whereas the latter was from the microsomes.
When the cells were grown either in glucose-deficient medium or at 15°C, the total amounts of cytochromes were depressed significantly compared with those grown in the enriched medium, although the number of cell divisions and final cell densities were not markedly affected. Glucose-supplementation of the deficient medium after 36 h of incubation did not restore the cytochrome level. On the other hand, both the cytochrome contents and growth rate were significantly decreased in an iron-deficient medium. The addition of iron to the deficient medium resulted in an increase in the level of a cytochrome with α-band at 553 nm, but did not affect the level of other cytochromes or the growth rate.
On starvation of the cells in a minimum medium containing only phosphate and a few minerals, the cytochrome contents were remarkably diminished. Electron microscopic examination revealed that the starvation process was accompanied by decreases in cell size, the number of mitochondria, and the amounts of endoplasmic reticulum in the cell. On transferring the starved cells to the enriched medium, the levels of the mitochondrial 553-nm peak cytochrome increased concomitantly with an increase in mitochondrial number, then the microsomal cytochromes began to accumulate as a well-developed endoplasmic reticulum was restored.
Based on these findings, the regulation mechanism for the biosynthesis of subcellular organelles and their components is discussed. The mitochondrial function appears to be prerequisite for microsomal hemoprotein accumulation in this protozoon.View full abstractDownload PDF (1586K) -
Masaru KANASHIRO, Tadashi TANABE, Retsu MIURA, Toshio YAMANO, Yoshihir ...1982 Volume 91 Issue 2 Pages 497-506
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSelective labeling with N-(1-oxyl-2, 2, 6, 6-tetramethyl-4-piperidinyl)-maleimide of human serum LDL has been performed. The spin-labeled LDL exhibited an ESR spectrum containing signals of a strongly immobilized component only. The signals were completely reversible between 4°C and 37°C and fairly stable at each temperature. The spin-labeled LDL which was prepared by the usual method exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components (5, 6). The latter was unstable above 25°C and changed irreversibly. The strongly binding site showed higher affinity for the nitroxide radical than the weakly binding site, and two kinds of the strongly binding site were demonstrated kinetically. The rate of binding of the nitroxide radical to the two kinds of strongly binding site were estimated to be 4.7×104M-1•day-1 and 0.16×104M-•day-1 at pH 7.4 and 4°C, respectively. Both the strongly immobilized and weakly immobilized radicals were reduced with ascorbate at the same rate. It was also shown on gel filtration of the SDS-treated LDL derivatives that the strongly immobilized component was on the apoprotein B moiety, whereas either noncovalent binding to LDL or binding to some small molecular species other than protein was suggested for the weakly immobilized component.View full abstractDownload PDF (666K) -
Akio SUGIHARA, Mieko IWAI, Yoshio TSUJISAKA1982 Volume 91 Issue 2 Pages 507-513
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLipase from Geotrichum (Geo.) candidum was rapidly inactivated by incubation with water-soluble carbodiimide, 1-ethyl-3-dimethylaminopropyl carbodiimide (EDC), or 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl) carbodiimide metho-p-toluenesulfonate (CMC), at pH 4.8. The pH dependence of the rate of inactivation was consistent with the modification of carboxyl groups in the lipase. Reaction of the lipase with EDC in the presence of the nucleophile taurine showed that about 9 carboxyl groups per molecule of enzyme were modified with concomitant total loss of activity. This number was reduced to 4 when CMC was used as a carbodiimide instead of EDC. The modification had no effect on the CD spectrum in the ultraviolet region. Kinetic analysis of the effect of CMC on the lipase indicated that at least 1 CMC molecule bound to the enzyme during inactivation.View full abstractDownload PDF (406K) -
Makoto SHIMIZU, Kunio YAMAUCHI1982 Volume 91 Issue 2 Pages 515-524
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMilk fat globule membrane (MFGM) enclosing fat droplets in human milk was found to contain a high molecular weight glycoprotein which did not migrate in 10% acrylamide gel on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This glycoprotein (termed PAS-0) was isolated by Sepharose CL-4 B chromatography. Isolated PAS-0 gave one band on SDS-PAGE using 5% acrylamide gel (acrylamide : bisacrylamide=4:1, w/w) and gave one peak on analytical ultracentrifugation, indicating its homogeneity.
PAS-0 was rich in serine, threonine, proline, glycine, and alanine. In contrast, contents of sulfur-containing amino acids were very low. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid were detected as constituent sugars of PAS-0 and the total carbohydrate content was about 50%. Alkaliborohydride treatment suggested that the carbohydrate moiety was linked to the polypeptide core with O-glycosidic bond (s). These results suggested that PAS-0 was a mucin-like glycoprotein. PAS-0 was shown to be resistant to pepsin, trypsin and chymotrypsin digestion, but susceptible to Pronase and Subtilisin BPN'. Extraction of intact milk fat globules (cream) with MgCl2 and guanidine hydrochloride solutions suggested that PAS-0 was an intrinsic component of MFGM. Digestion of cream with Subtilisin BPN' demonstrated that PAS-0 was located on the external surface of fat globules and was accessible to molecules outside the globules. By agglutination-inhibition tests using eight lectins, PAS-0 was suggested to act as surface receptors for Ricinus communis agglutinin, wheat germ agglutinin and peanut agglutinin.View full abstractDownload PDF (2602K) -
Tsuneo MIYAHARA, Tatsuya SAMEJIMA1982 Volume 91 Issue 2 Pages 525-535
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inactivating effects of halide ions on porcine kidney catalase were investigated. It was found that the inactivation of catalase was dependent on incubation time with halides and on their concentrations: the addition of 2M NaCl reduced the activity to 18% of the original level, whereas the same extent of inactivation was obtained in the presence of 0.1M NaF. Removal of excess halide ions by dialysis resulted in good recovery of enzyme activity for all halides examined except for KI. Additions of halide ions to catalase solution caused subtle but evident alterations in the absorption and CD spectra. We could detect clear difference spectra between the salttreated and native catalase solutions. These difference spectra showed halide concentration dependence with an evident isosbestic point. From these changes and also changes in CD spectra, we deduced that fluoride, chloride, and bromide ions can bind with heme iron of the catalase molecule as ligands to form stable catalasehalide complexes, but iodide ions showed a different reactivity with catalase from other halides and may cause gross alteration in the structure or conformation of catalase. Dissociation constants (Kd) were estimated to be 2.5, 0.23, and 26M for chloride, fluoride, and bromide complexes with catalase, respectively, and there is no heme-heme interaction during formation of the catalase-halide complexes as estimated from the Hill plot.View full abstractDownload PDF (669K) -
Yoshiaki NAKAMARU, Chusuke SATO1982 Volume 91 Issue 2 Pages 537-551
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of dimeric binding of bromocresol purple (BCP) anions to Mg2++Ca2+-ATPase of the sarcoplasmic reticulum (SR) and the resulting partial inhibition of the ATPase activity were studied.
BCP anions in three states, free monomer, bound monomer, and bound dimer, were spectrophotometrically calculated by solving simultaneous equations, ΔA_??_1-_??_2=∑Δαi(ε1λ1-ε1λ1), and concentration changes of these states were analyzed. The addition of ATP caused an increase in the bound dimer and a decrease in the free monomer, but the change of the bound monomer was slight. The decrease in ΔA (decrease phase) on the addition of ATP on dual-wavelength spectrophotometry at 585-610 nm was related to an increase in the amount of dimer bound to the SR membranes. The magnitude of the decrease phase increased with an increase in Mg2+ concentration and decreased with an increase in the concentration of Ca2+.
BCP anions at the probe concentration partially inhibited the ATPase activity, and brought about a decrease in the ADP-sensitive E-P (E1P) and an increase in the ADP-insensitive E-P (E2P), though BCP anions did not affect the amount of total E-P. On elimination of Mg2+ at the steady-state E-P level both E2P and E2P•(BCP)2 were decomposed, suggesting that the enzyme form binding the BCP dimer was Mg•E-P. An increase in Mg2+ concentration increased E2P but an increase in Ca2+ concentration decreased E2P. Decomposition of E2P to P1 was inhibited by BCP anions. The following simple scheme was suggested to explain the partial inhibition of the ATPase activity,
_??_
Application of BCP anions was discussed for use as a probe for Mg•E-P in the steady-state ATP hydrolysis.View full abstractDownload PDF (857K) -
Sadayuki MATUDA, Takeyori SAHEKI1982 Volume 91 Issue 2 Pages 553-561
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLipoamide dehydrogenase (LADase) was purified to homogeneity from rat liver mitochondria, and the intracellular distribution and biosynthesis of the LADase were investigated with antibody prepared against the purified enzyme. 1) LADase activity was mostly found in mitochondria; the activity in cytosol was about onetenth of that in mitochondria. 2) LADase in the crude mitochondrial and cytosolic extracts and the purified LADase were immunologically identical as judged from the Ouchterlony double diffusion test. These LADases were indistinguishable from each other on immunochemical titration; i.e., the amount of LADase precipitated by a fixed amount of the anti-LADase antibody was the same for all the preparations. However, cytosolic LADase activity was inhibited by the antibody more strongly than mitochondrial LADase activity. 3) Two min after intravenous injection of [35S] methionine, more radioactivity was incorporated into cytosolic LADase than into the mitochondrial enzyme in the liver. This result suggests that localization of LADase in the cytosolic fraction is not an artifact due to leakage from mitochondria during homogenization of rat liver. 4) LADase was synthesized predominantly on free ribosomes, which indicates that LADase is synthesized on cytoplasmic ribosomes and translocated into mitochondria just as other mitochondrial proteins are. 5) After cell-free protein synthesis with post-mitochondrial supernatant, radioactivity immunoprecipitated with anti-LADase antibody was detected as a major peak with the same molecular weight as the purified LADase.View full abstractDownload PDF (2258K) -
Tetsuo MORITA, Masataka MORI, Masamiti TATIBANA1982 Volume 91 Issue 2 Pages 563-569
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of dietary conditions on the degradation of hepatic N-acetyl-L-glutamate, an essential activator of carbamoyl phosphate synthase I [EC 6. 3. 4. 16], were studied by injecting mice with [14C] glutamate and by following the fate of labeled N-acetyl-L-glutamate. In fasted mice, the radioactivity in N-acetyl-L-glutamate reached a maximum 10min after injection and decreased rapidly with an apparent half-life of about 14min. In mice fed on a 60% casein diet, the radioactivity in N-acetyl-L-glutamate increased up to 30min and then decreased more slowly with an apparent half-life of about 60min. The remarkably prolonged retention of the radioactivity in N-acetyl-L-glutamate in fed animals was not due to continued entry of the label into the compound but principally to an increase in the half-life of the compound. A protein-free diet failed to induce a similar response. Glucagon induced a retention of the radioactivity, though the extent was much less. When mitochondria from fasted rats were incubated at 25°C, about 40% of N-acetyl-L-glutamate passed from the mitochondria to the medium in 60min. On the other hand, the rate of efflux from the mitochondria of fed rats was comparable to that of fasted animals in spite of a 6-fold higher concentration of the compound in the mitochondria. The observations, together with previous results, indicate that N-acetyl-L-glutamate degradation is positively regulated by a modification of its efflux through mitochondrial membranes.View full abstractDownload PDF (467K) -
Kiyoshi KONDO, Hajime FUJIO, Tsunehisa AMANO1982 Volume 91 Issue 2 Pages 571-587
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree lysozymes (DLs-1, -2, and -3) were purified from Peking-duck egg white by adsorption on CM-Sephadex C-25 resin, followed by CM-Sephadex C-25 and Sephadex G-50 column chromatographies. The three enzymes each moved as a single band, but showed different electrophoretic mobilities, on disc-polyacrylamide gel electrophoresis at pH 4.1. Final yields of DL-1, DL-2, and DL-3 were 18.2%, 22.0%, and 6.0%, respectively, from the crude material adsorbed on CM-Sephadex resin. The enzymatic activities of DL-1, DL-2, and DL-3 were 1.53, 1.52, and 1.34 times that of hen egg white lysozyme, respectively, using Micrococcus lysodeikticus cell wall as a substrate at pH 6.2 and 37°C.
All the DLs lacked histidine and their amino acid compositions differed from each other by a few amino acid exchanges. The amino acid sequences of DL-2 and DL-3 differed from that of DL-1 by two displacements (Ser-37 to Gly and Gly-71 to Arg) and three displacements (Pro-79 to Arg in addition to the same substitutions), respectively. In comparison with Duck II and Duck III lysozymes from Kaki-duck (Hermann and Jollès (1970) Biochim. Biophys. Acta 200, 178-179; Hermann et al. (1971) Eur. J. Biochem. 24, 12-17) as regards amino acid sequences, Duck II is identical to DL-1 except for one displacement of Gln-57 in DL-1 to Gin in Duck II, while Duck III is rather different from our three lysozymes.
All DLs gave single precipitin lines with any rabbit antisera against DLs and each precipitin line fused completely with that of any of the DLs in Ouchterlony double diffusion tests. However, the radiobinding inhibition assay showed that some anti-DL antisera clearly discriminated fine differences among the three DLs. The displacement at residue 79 (Pro↔Arg) gave the clearest immunological difference among the three kinds of displacements found in DLs.View full abstractDownload PDF (1578K) -
Mitsuo TAGAYA, Shoji SHIMOMURA, Kenichi NAKANO, Toshio FUKUI1982 Volume 91 Issue 2 Pages 589-597
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe process of reconstitution of potato apophosphorylase with pyridoxal 5'-phosphate (PLP) has been investigated to elucidate the structure-function relationship in phosphorylase [EC 2. 4. 1. 1]. In time-course studies, the recovery of enzyme activity was found to be delayed, especially at low temperatures, compared with the binding of PLP to protein. On polyacrylamide gel electrophoresis, an intermediary enzyme species was detected which bound PLP in the same binding mode as the native holoenzyme does, but which had neither enzyme activity nor affinity for the substrate amylopectin. The intermediate is monomeric and is converted to the active holoenzyme with concomitant dimerization. NaBH4-treatment of the monomeric intermediate produced the reduced monomeric enzyme, which could be converted into the reduced dimeric enzyme with considerable enzyme activity. The findings support the view that the catalytic activity of phosphorylase requires the dimeric structure of protein. As in the animal enzyme, the aldimine bond between the PLP and the ε-amino group of the lysyl residue is also not essential for enzyme activity in plant phosphorylase.View full abstractDownload PDF (586K) -
Mitsuo TAGAYA, Kenichi NAKANO, Shoji SHIMOMURA, Toshio FUKUI1982 Volume 91 Issue 2 Pages 599-606
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSP1, P2-bis (5'-pyridoxal) diphosphate crosslinks between the original cofactor (pyridoxal 5'-phosphate) linking residue and Lys-573 in rabbit muscle phosphorylase (Shimomura, S., Nakano, K., & Fukui, T. (1978) Biochem. Biophys. Res. Commun. 82, 462-468). We have applied the same technique to potato phosphorylase to compare the structures of the active-site regions of the two enzymes, which have different regulatory properties. The reagent was bound to the potato enzyme in the same binding mode as to the rabbit muscle enzyme. A sequence study on the potato enzyme labeled with this reagent revealed that it crosslinks between the original cofactor-linking lysyl residue and another lysyl residue, respectively corresponding to Lys-679 and Lys-573 in the rabbit muscle enzyme, and that the sequence Lys-573 to Leu-577 in the rabbit muscle enzyme is conserved in the potato enzyme. These findings indicate structural similarities in the active-site region between the phosphorylases, and suggest the importance of a lysyl residue in the catalytic mechanism of the phosphorylase reaction.View full abstractDownload PDF (490K) -
Tatsuo YAGURA, Tomoko KOZU, Takeshi SENO1982 Volume 91 Issue 2 Pages 607-618
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mouse DNA polymerase accompanied by a novel RNA polymerase activity and its specific protein factor (stimulating factor) were purified from Ehrlich ascites tumor cells and partially characterized. The DNA polymerase was thought to be a subspecies of DNA polymerase α, and to be accompanied by or copurified with RNA polymerase activity capable of synthesizing RNA, which was probably utilized as a primer for subsequent DNA polymerization on a template of poly (dT) or poly (dC). This coupled reaction by RNA and DNA polymerase activities required the stimulating factor in addition to ribo- and deoxyribonucleotide substrates, although the degree of requirement depended on the kind of template and ribonucleotide substrate: the activity to incorporate dATP with poly (dT) plus ATP depended greatly on the stimulating factor, while the activity to incorporate dGTP with poly (dC) did not when GTP was added at high concentrations. GDP could be substituted for GTP, but the activity with poly (dC) plus GDP depended largely on the stimulating factor. Involvement of known RNA polymerases in the activity with poly (dT) was excluded, because addition of purified mouse RNA polymerases I and II had no effect on the incorporation of dATP, and α-amanitin (100 μg/ml) did not inhibit the incorporations of dATP and ATP. Analysis of the inhibition by the nucleotide analog 2', 3'-dideoxynucleoside 5'-triphosphate (ddNTP) further supported the involvement of new RNA polymerase; ddNTPs inhibited the activities with poly (dT) and poly (dC) significantly more than RNA polymerases I and II or DNA polymerase α activity with poly (dT)•oligo (rA) and poly (dC)•oligo (dG) as template. Lineweaver-Burk analysis of the inhibitions showed that ddATP inhibited competitively with respect to ATP, and ddGTP inhibited competitively with respect to GDP but noncompetitively with respect to GTP.View full abstractDownload PDF (827K) -
Tamo FUKAMIZO, Katsuya HAYASHI1982 Volume 91 Issue 2 Pages 619-626
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the course of a study on the lysozyme-catalyzed reaction of chitooligosaccharides, it was found that each chitooligosaccharide gave two completely separated peaks on high-performance liquid chromatography with a partition column.
Synthetic 2-acetamido-2-deoxy-β-D-glucopyranose gave [α]D14=-18.1° (c=0.51, H2O) and a large second peak with a minor first peak on high-performance liquid chromatography. When an aqueous solution of the β-anomer was allowed to stand, the area of the first peak on high-performance liquid chromatography increased, together with a decrease in the area of the second peak and an increase in [α]D value. It was concluded that the two peaks of each chitooligosaccharide on high-performance liquid chromatography were due to the separation of α- and β-anomers.
The mutarotation of 2-acetamido-2-deoxy-β-D-glucopyranose was followed by monitoring the [α]D value and the peak area of the two peaks on high-performance liquid chromatography. It was found that the ratios of α- and β-anomers of chitooligosaccharides produced by the lysozyme-catalyzed reaction of chitopentaose were different from those of the corresponding authentic chitooligosaccharides which were allowed to stand in the absence of the enzyme under the conditions used for the enzymatic reaction.View full abstractDownload PDF (434K) -
Yuhachiro YODA, Akira MAKITA, Shinsei GASA1982 Volume 91 Issue 2 Pages 627-632
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree glucosamine-containing sphingoglycolipids were isolated from human lung tissue of a blood group-A subject. They were hexaglycosyl, pentaglycosyl, and tetraglycosyl ceramides. The hexaglycosyl ceramide exhibited blood group-A antigenicity, and the following chemical structure was proposed on the basis of sequential glycosidase treatment and methylation analysis.
GalNAcα1→3 _??_alβ1→4 GlcNAcβ1→3 Galβ1→4 Glc→ceramide
α1Fuc
The pentaglycosyl ceramide from human lung was a blood group H (O)-active glycolipid having a carbohydrate composition of Fuc:GlcNAc:Gal:Glc (1:1:2:1). The tetraglycosyl ceramide is probably paragloboside (lactoneotetraosyl ceramide).View full abstractDownload PDF (348K) -
Miyoko OKUDA, Kikuo ARAKAWA1982 Volume 91 Issue 2 Pages 633-641
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTrypsin was found to generate depressor substances from human plasma protein fraction IV-4 at all pH values from 2.0-10.0. The depressor substances generated at three representative pH values, i.e., 3.2, 5.0, and 8.0 were purified and isolated. The procedures used for the isolation were: (a) gel-filtration through Sephadex G-25; (b) desalting by ion retardation chromatography on AG 11 A 8; (c) equilibrium chromatography on SP-Sephadex C-25; (d) desalting by AG 11 A 8 again, and (e) partition chromatography on a high pressure liquid chromatograph. Upon high pressure liquid chromatography, the depressor substances formed at each of the three pH values were separated into two fractions. In thin layer electrophoresis, the two depressor substances moved with mobilities identical with those of synthetic bradykinin and synthetic [des-Pro3]-bradykinin but not those of methionyl-lysyl- or lysyl-bradykinin. The amino acid analysis of the hydrolysate of the depressor substance 1 generated at pH 3.2 showed the following proportional composition: Arg, 2; Pro, 2; Gly, 1; Phe, 2; Ser, 1. The product thus lacked 1 mol of proline residue as compared with bradykinin, and the retention time of this depressor substance 1 was identical with that of synthetic [des-Pro3]-bradykinin on high pressure liquid chromatography. The depressor substance 2 showed Arg, 2; Pro, 3; Gly, 1; Phe, 2; Ser, 1. This is identical with the composition of bradykinin. Finally, substances 1 and 2 showed oxytocic activities identical to those of synthetic [des-Pro3]-bradykinin and synthetic bradykinin, respectively. Substance 1 showed a bradykinin-potentiating activity similar to that of synthetic [des-Pro3]-bradykinin.View full abstractDownload PDF (499K) -
Kaname MOGAMI, Shinobu C. FUJITA, Yoshiki HOTTA1982 Volume 91 Issue 2 Pages 643-650
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen proteins of whole Drosophila thorax were analyzed by two-dimensional gel electrophoresis, 186 spots were detected by protein staining with Coomassie brilliant blue R-250. Two methods were developed to identify proteins which exist in indirect flight muscle (IFM) and its myofibrils. 1) A whole fly was freeze-dried in a dry ice-acetone mixture, and indirect flight muscle fibers were cleanly dissected out from the thorax. The muscle cells and the rest of the thorax were analyzed separately. The muscle contained 146 polypeptides, of which 12 were not detected elsewhere. 2) Flies were frozen in liquid nitrogen and shaken vigorously so that their thoraces broke off from heads and abdomens. The thoraces were separated from the rest by sieving and centrifugation. After homogenization of the thorax, myofibrils were prepared by centrifugation in a discontinuous sucrose density gradient. The myofibril fraction contained at least 20 proteins. There were two types of actin (II and III), myosin heavy chain, tropomyosin and paramyosin. Nine of the other myofibrillar proteins were specific to this muscle.View full abstractDownload PDF (2124K) -
Takato MAYAHARA, Kunihiko SUZUKI, Hisayuki OHATA, Tetsuya KAMIMURA1982 Volume 91 Issue 2 Pages 651-655
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin B was extracted from rabbit fast skeletal muscle. Caffeine (2-70mM), theophylline (2-30mM), and theobromine (2mM) were examined for their effects on the rate and extent of myosin B superprecipitation at a low ionic strength (50mM KCl) and a low concentration of ATP (40 μm) by the turbidimetric method. The rate of the superprecipitation was significantly (p<0.05 and p<0.01) reduced by 30-70mM caffeine and 2-30mM theophylline, while the extent was significantly (p<0.01) increased by 10-30mM theophylline. The onset of the superprecipitation was also delayed and a clearing phase was even induced by 50-70mM caffeine. Theobromine had no significant effect on the rate or extent of the superprecipitation at the concentration used. These results indicate that caffeine and theophylline are retardants of the myosin B superprecipitation reaction in the concentration ranges investigated.View full abstractDownload PDF (323K) -
Kazuhiko IKEDA, Shigeru KUNUGI, Norio ISE1982 Volume 91 Issue 2 Pages 657-663
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding and catalytic activities of dimeric α-chymotrypsin for specific amino acid derivatives were investigated with special reference to the equilibrium between active and inactive monomeric forms of this enzyme occurring in a low pH range. The low catalytic activity of the dimeric enzyme towards these specific substrates was revealed to be due to the difficulty in binding of the dimer. However the free tryptophanates and N-acetyl-L-tryptophan, which are slightly smaller (in molecular size) than the above substrates and comparable to the nonspecific phenyl acetate substates (towards which the dimeric α-chymotrypsin showed an distinct catalytic activity in our previous study [J. Biochem. 87, 871-880 (1980)]), were bound to the dimer more strongly than to the monomeric enzyme. Hence they enhanced dimer formation when added at low concentrations.View full abstractDownload PDF (376K) -
Takao TAKI, Ryoei KAMADA, Makoto MATSUMOTO1982 Volume 91 Issue 2 Pages 665-674
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycolipid biosynthesis in rat bone marrow cells has been studied with reference to four kinds of glycosyltransferases catalyzing the transfer of N-acetylgalactosamine, galactose, N-acetylneuraminic acid, and fucose to each glycolipid acceptor. It was demonstrated that glycosyltransferase activities which synthesize galactosylglucosylceramide (CDH) from glucosylceramide (CMH), N-acetylgalactosaminylgalactosylglucosylceramide (GA2) from CDH, galactosyl-N-acetylgalactosaminylgalactosylglucosylceramide (GA1) from GA2 and N-acetylneuraminylgalactosyl-N-acetylgalactosaminylgalactosylglucosylceramide (GM1b) from GA1 were all present in rat bone marrow cell homogenate. Fucosyltransferase activity catalyzing the transfer of fucose from GDP-fucose to GA1 was also recognized in the cell homogenate. Neutral glycolipid extracted from rat bone marrow cells was analyzed by thin layer chromatography and glycosidase treatments. The presence of glycolipids corresponding to GA2, GA1, and fucolipid was demonstrated. From these results, it was concluded that the biosynthesis of glycolipid through asialogangliosides is a major biosynthetic route in rat bone marrow cells.View full abstractDownload PDF (1899K) -
Yoshimasa KYOGOKU, Mayumi WATANABE, Masatsune KAINOSHO, Tairo OSHIMA1982 Volume 91 Issue 2 Pages 675-679
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRibonuclease T1 is highly specific for the guanylic acid residue in polyribonucleotides. To clarify the origin of the substrate specificity, the interaction sites of guanylic acid with ribonuclease T1 were investigated by the use of 15N-NMR. 95% 15N-enriched guanosine-3'-phosphate was prepared and mixed with purified ribonuclease T1. 15N-NMR spectra of the mixtures at different concentrations were obtained and compared with that of the 15N-enriched substrate alone. Upon complex formation, a 15N signal assigned to the amino group nitrogen at position 2 of guanine shifted and was significantly broadened, suggesting a strong interaction with the enzyme through the amino group. This observation is consistent with the results of studies on the substrate specificity by chemical modification. Nuclear Overhauser effects of signals assigned to N-7 and N-3 were also changed, but no shift was observed. The observations do not support the occurrence of protonation at N-7 upon complex formation, which was previously proposed.View full abstractDownload PDF (298K) -
Toshikazu NAKAMURA, Katsuhiko YOSHIMOTO, Kazushi AOYAMA, Akira ICHIHAR ...1982 Volume 91 Issue 2 Pages 681-693
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn primary cultured monolayer hepatocytes of adult rats, insulin (1×10-8M) induced glucose-6-phosphate dehydrogenase [EC 1. 1. 1. 49, G 6 PDH] several fold in 48 h. It also induced lipogenesis, measured as [1-14C] acetate incorporation in 2 h, in these cells. Of the various lipids, triglycerides and phospholipids were induced markedly, while cholesterol and its esters were not induced. The increase of G 6 PDH and lipogenesis were parallel. Glucagon, dibutyryl cyclic AMP, triiodothyronine, dexamethasone, epinephrine, isoproterenol, and dibutyryl cyclic GMP were also tested under similar conditions, but none of them caused significant induction of G 6 PDH or lipogenesis.
Use of anti-G 6 PDH serum showed that induction of G 6 PDH by insulin was due to increase in the amount of enzyme protein. Insulin was found to increase the rate of synthesis of G 6 PDH about 3-fold. SDS-polyacrylamide gel electrophoresis of the immunoprecipitable protein revealed that besides G 6 PDH another radioactive fraction (Mr 37, 000) was increased by insulin. This suggests that complete synthesis of G 6 PDH protein is slowed down in primary cultured hepatocytes and that an apparent nascent peptide of the enzyme accumulates.
Although on long-term treatment (48 h), glucagon and dibutyryl cyclic AMP had no effect on lipogenesis, when added with [14C] acetate for 2 h they strongly inhibited lipogenesis. Significant inhibition of lipogenesis by short-term treatment with glucagon was seen even in cells with a high capacity for lipogenesis induced by long-term treatment with insulin. Insulin again stimulated lipogenesis in short-term treatment, but its effect was slight.
It is concluded from these results that insulin exerts long-term stimulation of lipogenesis by inducing enzymes related to lipogenesis including G 6 PDH as well as causing slight stimulation by enhancing supply of substrate for lipogenesis. Glucagon seems to play a minor role in long-term control, but it causes short-term inhibition of lipogenesis.View full abstractDownload PDF (1475K) -
Keiko TAKEDA, Midori HIRANO, Hiroshi KANAZAWA, Noriko NUKIWA, Yasuo KA ...1982 Volume 91 Issue 2 Pages 695-701
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSATPase complexes were reconstituted from homologous and heterologous combinations of α, β, and γ subunits of coupling factor ATPase TF1 of thermophilic bacterium PS 3 and EF1 of Escherichia coli. TF1 and the αβγ complex reconstituted from TF1 subunits were thermostable and activated by methanol, sodium dodecyl sulfate and anions and they were halophilic, whereas EF1 and its three-subunit complex did not show these properties. The hybrid ATPase αTβTγE (complex of the α and β subunits of TF1 and the γ subunit of EF1) showed closely similar properties to TF1 except for thermostability, while αEβEγT (α and β from EF1 and γ from TF1) had similar properties to EF1. These results suggest that α and/or β is required for the properties of F1. The complex αEβTγE showed similar properties to EF1 except for its optimum pH: this complex had a broad pH optimum at about pH 7, whereas EF1 had an optimum at pH 8.5. No hybrid complexes were thermostable, suggesting that all three subunits of TF1 are required for heat stability. These hybrids showed higher halophilicity than EF1, although they were less halophilic than TF1. The hybrid enzymes studied here are the first thermophilic-mesophilic hybrid enzymes obtained.View full abstractDownload PDF (434K) -
Shoii SHIMOMURA, Masami NAGAI, Toshio FUKUI1982 Volume 91 Issue 2 Pages 703-717
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo types of α-glucan phosphorylase [EC 2. 4. 1. 1] from spinach leaves have been separately purified to near homogeneity. Type I enzyme shows a subunit molecular weight of 92, 000 and Km values for amylopectin, glycogen and amylose much smaller than that for maltopentaose. Cyclodextrin is a normal competitive inhibitor with respect to maltopentaose, while it is a multi-site competitive type inhibitor with respect to amylopectin, glycogen or amylose. Type II enzyme shows a subunit molecular weight of 108, 000, and utilizes amylopectin, amylose and maltopentaose well, but glycogen very poorly. On affinity electrophoresis, Type II enzyme shows no affinity for glycogen and the dissociation constant for amylopectin is more than a thousand-fold greater than that of Type I enzyme. Type II enzyme has similar characteristics (subunit size, glucan specificity, and mode of cyclodextrin inhibition) to potato phosphorylase, for which cyclodextrin is a normal competitive inhibitor with respect to either maltopentaose or amylopectin.
Enzyme-glucan binding models have been proposed to explain these different kinetic properties. In spinach Type I phosphorylase, multiple glucan binding sites are located on the same face of the enzyme molecule to enable a single large substrate to be bound by riding on all the site; in spinach Type II and potato phosphorylases, two glucan binding sites are three-dimensionally arranged in a manner that excludes the possibility of the binding of amylopectin by riding on the two sites.View full abstractDownload PDF (1530K) -
Minoru NOMOTO, Koichi IWAI1982 Volume 91 Issue 2 Pages 719-723
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe histones of the ciliated protozoan Tetrahymena were isolated and fractionated on a large scale by revised procedures, and the H 2 B and H 4 histones were highly purified for sequence determination. The nuclei of an amicronucleate strain, GL, of Tetrahymena pyriformis were isolated, without cell harvesting, from cultures reaching the stationary state, which were continuously centrifuged through 1.0M sucrose, pH 7.5, containing Nonidet P-40. From the nuclei further purified and washed with 0.15M NaCl, pH 7.5, histones were extracted with 0.25M HCl. Thus 1, 620 liters of cultures yielded 5.20g of histone extract. The histones were first separated into a 0.5M HClO4 soluble fraction (H 1) and an insoluble fraction (H 2 A, H 2 B, H 3, and H 4). The insoluble fraction was chromatographed on a large column of Bio-Gel P-60 with 20mM HCl containing 250mM NaCI at 15°C, yielding H 2 A+H 3, H 2 B, and H 4 fractions. The H 4 fraction was highly pure; the H 2 B fraction was further purified by the same Bio-Gel P-60 chromatography without NaCl, yielding pure H 2 B.View full abstractDownload PDF (248K) -
Koukichi MARUYAMA1982 Volume 91 Issue 2 Pages 725-730
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo short-chain fatty acyl-CoA synthetases were extracted from the photosynthetic bacterium, Rhodopseudontonas sphaeroides, and partially purified by column chromatography on Sephacryl S-200 and DEAE-cellulose. One enzyme activated propionate, valerate, acrylate, butyrate, and acetate, and was designated as propionyl-CoA synthetase, since the highest activity and lowest Km value (0.6mM) were observed with propionate. The other enzyme activated acetate, propionate and acrylate. It showed the highest activity and lowest Km value (0.37mM) for acetate, and was identified as acetyl-CoA synthetase [EC 6. 2. 1. 1].View full abstractDownload PDF (364K) -
Mamoru ISEMURA, Zensaku YOSIZAWA, Takehiko KOIDE, Teruo ONO1982 Volume 91 Issue 2 Pages 731-734
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of porcine plasma fibronectin and its proteolytic fragments with hyaluronic acid was investigated by affinity chromatography using hyaluronic acidlinked Sepharose 4 B. Hyaluronic acid was found to interact with all major fragments including the gelatin-binding polypeptide which has no affinity for heparin. A difference between hyaluronic acid and heparin was also noted in the relative interaction with two larger fragments. This type of multiple interaction may account for the affinity for hyaluronic acid strong enough to prevent the elution of fibronectin from the hyaluronate-column with a buffer containing 2M NaCl.View full abstractDownload PDF (1320K) -
Identification of the Trimannosyl-Chitobiose Structure in Sugar Moieties of Japanese Quail OvomucoidSumihiro HASE, Kazunobu OKAWA, Tokuji IKENAKA1982 Volume 91 Issue 2 Pages 735-737
Published: February 01, 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOvomucoid isolated from egg white of japanese quail contained mannose, galactose, glucosamine, and sialic acid (8.6:1.3:6.6:0.5 mol/mol) as sugar components. Sugar chains were liberated from the polypeptide portion by hydrazinolysis, N-acetylated and fractionated on Bio-Gel P-2. The reducing ends of the thus obtained sugar moieties were pyridylaminated. Chemical and exoglycosidase digestion studies of the fluorescent pyridylamino derivative of a major sugar fraction indicated the following structure for one of the major sugar chains of japanese quail ovomucoid; Manα1→6 (Manα1→3) Manβ1→4 GlcNAcβ1→4 GlcNAc.View full abstractDownload PDF (184K) -
1982 Volume 91 Issue 2 Pages 739a
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (20K) -
1982 Volume 91 Issue 2 Pages 739b
Published: 1982
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (20K)
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