巻号一覧
72 巻, 6 号
選択された号の論文の28件中1～28を表示しています
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Kinji TSUKADA, Nobuyuki ITO1972 年 72 巻 6 号 p. 1299-1305
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーPolynucleotide ligase, which catalyzes the covalent joining of two segments of an interrupted strand in a DNA duplex, was studied in normal rat kidney and kidney tumors induced by N-nitrosodimethylamine, The activities of this enzyme in the fraction, precipitated at pH 5 with acetic acid, and in the nuclei fraction of kidney tumors were about 5 and 3 times higher than in the respective fractions of normal rat kidney. The activity of polynucleotide ligase in kidney tumors is correlated with increased DNA synthesis.抄録全体を表示PDF形式でダウンロード (482K) -
Etsuro SUGIMOTO, Yasuo KITAGAWA, Kazuo NAKANISHI, Hideo CHIBA1972 年 72 巻 6 号 p. 1307-1315
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーAn improved procedure for purification of D-glycerate dehydrogenase [EC 1. 1. 1. 29] from beef liver is described. The purified enzyme was proved to be homogeneousby ultracentrifugation and zone electrophoresis. Its molecular weight was determinedto be 72, 000. The Vlichaelis constants of the enzyme for DL-glycerate and hydroxy-pyruvate were 1.4mM and 4μM, respectively. The enzyme activity was inhibitedby hydroxypyruvate, a substrate, with an inhibition constant of O.08mM. Among the many metabolites tested, glycolytic intermediates such as pyruvate, α-D-fructose1, 6-diphosphate, 3-phospho-D-glycerate, 2, 3-diphospho-D-glycerate, and 2-phospho-D-glycerate as well as ATP strongly inhibited the enzyme activity in the concentration range from O.1 to 1mM. The inhibitory action of pyruvate was affected by the concentrations of the substrate and sodium chloride. Both NAD+ (NADH) andNADP+ (NADPH) could act as coenzymes for the enzyme, though NADP+ (NADPH) was less effective than NAD+ (NADH). In the presence of 1mM pyruvate, however, NADP+ showed a higher coenzyme activity than NAD+ (NADH). Based on thesefindings, the possibility is discussed that the nonphosphorylated pathway for L-serinesynthesis (including D-glycerate dehydrogenase) is actually responsible for cataboliccoversion of L-serine to glycolytic intermediates and the latter act as feedback effectors for this catabolic pathway.抄録全体を表示PDF形式でダウンロード (1306K) -
Etsuro SUGIMOTO, Yasuo KITAGAWA, Masaaki HIROSE, Hideo CHIBA1972 年 72 巻 6 号 p. 1317-1325
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーFluorescence titration of beef liver D-glycerate dehydrogenase [EC 1. 1. 1. 29] provided evidence for the presence of two substrate binding sites (S and S' sites) on the enzyme protein. Kinetic studies indicated that inhibition of the enzyme by inorganic anions resulted from competition of the anions with the substrate at the S site. It was also shown that the binding of hydroxypyruvate to the S' site was responsible for the substrate inhibition, and that inorganic anions, when bound to the S' site, activated the enzyme. These fiindings explained the release by addition of inorganic anions of the substrate inhibition in the presence of high concentrations of hydroxypyruvate. When the activity was plotted against the anion concentration, a sigmoid curve was obtained. An analysis of this sigmoid curve demonstrated that the enzyme possessed at least three anion binding sites, one of which being the S' site. Based on these findings, a hypothetical model is presented for the mechanisms of inhibition and activation of the enzyme by inorganic anions.抄録全体を表示PDF形式でダウンロード (613K) -
Taibo YAMAMOTO, Yoshitaka KURIKI, Yuji TONOMURA1972 年 72 巻 6 号 p. 1327-1337
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーKinetic studies were made on uncoupled GTPase in the steady state and in the initial phase of the reaction using ribosomes and G-factor purified from E. coli Q13. The reaction was started by adding GTP, and the amount of Pi liberated was measured after stopping the reaction with TCA. The following results were obtained.
1. At low concentrations of GTP (1-200μM) and at pH 7.0 and 25°C, the dependence of the rate in the steady state, v0, on the concentrations of GTP and G-factor was given by
_??_
where φ1 and φ2 are constants which are independent of the concentrations of GTP, [S], and G-factor, [G]. V0 is v0 at an infinite concentration of G-factor. The values of φ1 and φ2, were 50-110μM and 0.6mg/ml, respectively.
2. In the steady state GTPase was competitively inhibited by GDP. The apparent inhibition constant, KI', for GDP was about 1/10 of the apparent Michaelis constant, φ1'. The value of KI' decreased with increase in [G].
3. A Lineweaver-Burk plot of the rate of the GTPase reaction in the steady state gave two straight lines intersecting at about 0.2mM GTP. With 1mM GTP, v0 increased linearly with increase in the concentration of G-factor, and reached the maximum velocity, V0, when the molar concentration of G-factor was equal to that of ribosomes. The value of V0 in the high concentration range of GTP was 1.5 fold that in the low concentration range of GTP.
4. A lag phase before Pi-liberation was observed immediately after addition of GTP. The lag phase was about 0.15sec, and was independent of the concentrations of both GTP and G-factor. After the lag phase, a definite burst of Pi-liberation occurred, which was followed by the steady state within 1sec. Both the amount of Pi liberated in the burst and v0 increased with increase in the concentrations of GTP and G-factor, while the ratio of v0 to the amount of the burst was independent of these concentrations, and was 1.5sec-1.
5. These results were explained by the following reaction mechanism:
_??_
where R, S, and G are ribosomes, GTP, and G-factor, respectively. X is a secondd ternary complex which does not liberate Pi even in the presence of TCA and contains bound GTP and G-factor. Y is a terminal intermediate which liberates Pi, at least in the presence of TCA, and contains G-factor. The values of the dissociation constants and the rate constants given in the above scheme are those estimated from the results at pH 7.0 and 25°C and in the presence of low concentrations of GTP. High concentrations of GTP increase the rate constant of step 3 to well above 1.2sec-1. Parallels between the reaction mechanisms of G-factor-dependent GTPase of ribosomes and myosin-ATPase are discussed.抄録全体を表示PDF形式でダウンロード (763K) -
Shuichi HASHIMOTO, Masaharu KAWATA, Shosuke TAKEMURA1972 年 72 巻 6 号 p. 1339-1349
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーTyrosine transfer ribonucleic acid from Torulopsis utilis was split into 3'- and 5'-half molecules at the G-Ψ bond of the anticodon GΨ A with limited RNase T1 [EC 2. 7. 7. 26] digestion. When the isolated two half molecules were combined and annealed in the presence of Mg2+, about 40% of tyrosine acceptor activity was restored. After chemical removal of the 3'-terminal residue Gp of the 5'-half molecule and enzymatic removal of several residues from the 5'-terminal of the 3'-half molecule, the two products were mixed and annealed. Tyrosine acceptor activity of the reconstituted complex was about 25%. No other acceptor activity was detected. Since the complex did not contain any anticodon nucleotides, the anticodon of T. utilis tyrosine tRNA is not essential for tyrosine specific acceptance.抄録全体を表示PDF形式でダウンロード (882K) -
Masachika IRIE, Masatomi HARADA, Fumio SAWADA1972 年 72 巻 6 号 p. 1351-1359
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリー1) In order to investigate the roles of tryptophan residues in the active site of ribonuclease from Aspergillus saitoi [RNase M, EC 2. 7. 7. 17] several lines of experiments were carried out.
2) Chemical modifications of RNase M by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide indicated that tryptophan residues were rather resistant toward these reagents at pH's between 3.5 and 6.0.
3) The reactivity of these tryptophan residues increased with the denaturation of RNase M by acid or 6M guanidine-HCl.
4) N-Bromosuccinimide oxidation of RNase M at pH 2.0 indicated that 4 residues of tryptophan out of seven are reactive, and 1 residue of them is particularly reactive with simultaneous loss of enzymatic activity.
5) H2O2-dioxane oxidation of RNase M indicated that about 5 residues of tryptophan are reactive at pH 8.6, and the rest of 2 residues are rather resistant.
6) The fluoresence emission spectrum of RNase M excited by 295mμ light was found to have a maximum at 330mμ and the maximum was shifted to 350mμ in 6M guanidine-HCl. From these results, it was suggested that most of tryptophan residues in RNase M were buried inside of molecule and the conversion to accessible states took place in parallel with the shift of the fluorescence band.
7) Quenching of RNase M fluorescence caused by addition of 2'-AMP was studied and the dissociation constant of RNase M-2'-AMP complex was determined.
8) New fluorescence peak at 360mμ was induced by the interaction of RNase M with 6-thioguanosine 2' (3'), 5'-diphosphate. The results indicated that base moiety of this nucleotide exist in a hydrophobic region of RNase M molecule.
9) The possible roles of tryptophan residues for the RNase M activity were discussed.抄録全体を表示PDF形式でダウンロード (637K) -
Koichiro TAKESHIGE, Akio ITO, Shigeki MINAKAMI1972 年 72 巻 6 号 p. 1361-1367
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリー1. The content of cytochrome P-450 as well as the activities of NADPH-cytochrome c reductase, NADPH-cytochrome P-450 reductase, aniline hydroxylase [EC 1. 14. 1. 1] and aminopyrine N-demethylase in liver microsomes from rats treated or untreated with phenobarbital or 3-methylcholanthrene were examined in the presence and absence of 1 % Triton X-100.
2. Approximately 50% of the total cytochrome P-450 in liver microsomes was rapidly destroyed on exposure to Triton X-100, but the remainin portion of the cytochrome was resistant to the detergent even when the detergent concentration was increased and incubation time was prolonged.
3. Aniline hydroxylase activity of microsomes from rats treated with 3-methyl-cholanthrene was reduced to 25% on exposure to Triton X-100, whereas the activity from control and phenobarbital-treated rats was decreased to only about 50% by the detergent.
4. Aminopyrine N-demethylase activity of microsomes from control as well as pheno-barbital- or 3-methylcholanthrene-treated rats was all reduced to about 50% upon addition of Triton X-100.
5. The intensity of aminopyrine-induced difference spectrum of liver microsomes was increased after phenobarbital induction. The shape of this spectrum was anomalous in microsomes from 3-methylcholanthrene-induced rats. The aminopyrine-induced difference spectrum disappeared completely on addition of Triton X-100, though half of the aminopyrine N-demethylase activity was unaffected by this treatment.
6. NADPH-cytochrome P-450 reductase activity was reduced to about one tenth in the presence of Triton X-100. Under similar experimental conditions, the overall hydroxylation rates were decreased to only half the original values, suggesting that the reduction of cytochrome P-450 is not the main rate-limiting step.
7. Based on these findings, the possible existence of multiple types of microsomal drug hydroxylase system and their dependence on the administered inducer drugs are discussed.抄録全体を表示PDF形式でダウンロード (513K) -
Kunio YAGI, Morimitsu NISHIKIMI, Nobuko OHISHI1972 年 72 巻 6 号 p. 1369-1377
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリー1. The time sequence of the appearance of the purple intermediate in the anaerobic reaction of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1. 4. 3. 3] with the substrate, revealed the existence of two forms in the oxidized enzyme. The major portion of the enzyme molecules rapidly formed the purple intermediate and the minor portion of the molecules reacted slowly. The aerobic binding of the enzyme with o-aminobenzoate, an inhibitor, gave the same conclusion.
2. Ultracentrifugal analysis indicated that the rapidly reacting and slowly reacting forms corresponded to the dimer and monomer of the oxidized enzyme, respectively. This view was further supported by the fact that the slowly reacting form increased upon lowering the enzyme concentration.
3. The spectrum obtained during the rapid phase of the anaerobic reaction of the enzyme with the substrate was a sum of the spectrum of the purple intermediate produced by the dimer and the spectrum of the unreacted oxidized enzyme of monomeric form. These results led us to the conclusion that the purple intermediate produced by the dimer is identical with the purple complex crystallized earlier in this laboratory.抄録全体を表示PDF形式でダウンロード (3568K) -
Takashi MATSUBARA, Toshiaki HASEGAWA, Yoshihiro TOCHINO, Akira TANAKA, ...1972 年 72 巻 6 号 p. 1379-1390
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーThe effects of hypophysectomy on the oxidative functions and morphology of rat liver mitochondria were examined. The respiratory rate of mitochondria both in states 3 and 4 decreased with time after the operation. However, the respiratory control index and P:O ratio did not change appreciably. After hypophysectomy the respiratory cytochrome contents of isolated mitochondria decreased in parallel with decrease in respiratory activities, but the respiratory cytochrome contents of whole liver homogenates, though first decreasing markedly were later restored to almost normal levels. The microsomal cytochrome content also decreased but then gradually increased again from 9 days after hypophysectomy. Electron microscopic observations on hepatocytes revealed that the mitochondrial number and volume per unit of cytoplasm increased after the operation, and the cytoplasm became almost completely filled with mitochondria. The rough surfaced endoplasmic reticulum appeared to surround each mitochondrion. Subsequent administration of growth hormone increased the cytochrome contents of both the mitochondria and microsomes.抄録全体を表示PDF形式でダウンロード (5102K) -
IV. Acceptor Specificity and Quantitative Representation of the Transglycosylation ReactionTomohiro MEGA, Tokuji IKENAKA, Yoshio MATSUSHIMA1972 年 72 巻 6 号 p. 1391-1396
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーIt was found that several sugars accepted the N-acetylglucosaminyl residue in the transglycosylation reaction catalyzed by N-acetyl-β-D-glucosaminidase [EC 3. 2. 1. 30]. To examine the acceptor site in the enzyme protein, the efficiencies of the various acceptors were examined. It was found that their efficiencies decreased in the following order: N-acetylglucosamine>N-formylglucosamine>glucose (galactose)>2-deoxyglucose>mannose. Among the alcohols tested as acceptors, the order was: primary alcohols>the corresponding secondary alcohols (tertiary alcohols had no, acceptor ability); 1-pentanol>1-butanol>1-propanol>ethanol>methanol, glycerol>ethylene glycol>erythritol>xylitol>glucitol>N-acetylglucosaminitol. These results suggested that the structure of the acceptor hexose at carbon 2 is important in the interaction of the acceptor and the acceptor site of the enzyme, that an alkyl chain easily enters the acceptor site and that the acceptor ability of polyalcohols decreases with increase in chain length.
The three step mechanism shown in the following equation was postulated for the transglycosylation reaction:
_??_
The ratio kt/kh, defined as the transfer ratio (Rt), was determined for several alcohols.抄録全体を表示PDF形式でダウンロード (405K) -
Noboru YAMAMOTO, Setsuko YOSHIMURA, Tomihiko HIGUTI, Katsuzo NISHIKAWA ...1972 年 72 巻 6 号 p. 1397-1406
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリー1) Chromatophores from Rhodospirillum rubrum cells were bound with 0.14 nmole of ADP and 1-4 nmoles of Pi per A880nm unit (7.1 nmoles of bacteriochlorophyll). These values correspond to approximately 16 molecules and 110-440 molecules in each chromatophore of the average size, respectively. The binding of ADP was tight, but that of Pi was loose; the exchange of the bound ADP for added ADP did not occur, whereas the exchange between the bound and added Pi proceeded at a slow rate.
2) All the bound ADP was phosphorylated into ATP in the light (approximately 1, 000 foot-candles); the ATP thus formed was also at the bound state. The phosphorylation was completed within 0.2 sec at 0°C. The phosphorylation of added ADP occurred after completion of the phosphorylation of the bound ADP.
3) The phosphorylation of the bound ADP was not inhibited either by oligomycin or by Triton X-100, both of which were potent inhibitors for the phosphorylation of added ADP.
4) Besides the bound ADP, approximately 0.022 nmole/A880nm of added ADP was weakly adsorbed by chromatophores. This value corresponds to approximately 3 molecules in each chromatophore of the average size. The adsorption was prevented in the presence of oligomycin.
5) Possibly, the phosphorylation of the bound ADP is the primary step of the phosphorylation of added ADP, and the phosphoryl transfer from the resulting bound ATP to added ADP is inhibited by the oligomycin such bound on the chromatophore membrane as to prevent the access of added ADP to the bound ATP.抄録全体を表示PDF形式でダウンロード (783K) -
Masahiko NEGISHI, Tsuneo OMURA1972 年 72 巻 6 号 p. 1407-1417
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーThe early stage of labeling of microsomal membrane proteins in rat liver by radioactive amino acids was examined by using 1-14C-L, 1-14C-D-, and 1-14C-DL-leucines and guanidino-14C-L-arginine. Radioactivity was incorporated into microsomal proteins from the radioactive D-leucine as well as from the corresponding L-isomer, but the rate of incorporation in the former case was much more slower than in the latter. The time course of radioactivity incorporation into microsomal proteins from 1-14C-DL-leucine could be explained by assuming additive contributions of the L- and D-isomers in the labeling. Almost all radioactivity incorporated from 1-14C-D-leucine into protein was recovered in the leucine fraction when the labeled protein was hydrolyzed and examined.
The experiments with 1-14C-L-leucine and guanidino-14C-L-arginine showed that more than half of the newly incorporated radioactivity was lost from microsomal proteins during the initial few hours after the labeling. Since this initial loss of the once-incorporated radioactivity from microsomal proteins was equally or even more pronounced for purified NADPH-cytochrome c reductase than for total microsomal protein, we regarded this observation as signifying the actual behavior of newly synthesized microsomal membrane proteins in their early stage of binding to the membrane. Thus, we concluded that the turnover of microsomal NADPH-cyto-chrome c reductase in rat liver was biphasic consisting of a rapid reaction and a slow one, and the rapid phase with a half life of about 1 hr has been overlooked in previous turnover studies. The injection of phenobarbital, an inducer of microsomal NADPH-cytochrome c reductase, to the rats caused the disappearance of this rapid turnover phase of the reductase.抄録全体を表示PDF形式でダウンロード (845K) -
XVI. The Complete Amino Acid Sequence of Clupeine YIIKoichi SUZUKI, Toshio ANDO1972 年 72 巻 6 号 p. 1419-1432
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーClupeine from Pacific herring was fractionated into Z and Y fractions by chromatography on CM-cellulose. After trinitrophenylation of the clupeine Y fraction with 2, 4, 6-trinitrobenzenesulfonic acid it was further fractionated into 2 homogeneous fractions, trinitrophenyl-clupeine YI and free clupeine YII. Clupeine YII was subjected to sequence analyses. It was composed of 30 amino acids (Arg20, Thr1, Ser2, Pro3, Ala2, and Val2) and the molecular weight was 4777 as hydrochloride. Clupeine YII was fragmented by the two complemental methods, tryptic digestion and selective chemical cleavage at amino sides of hydroxyl amino acids. The peptides thus obtained were purified by column chromatography and the sequences of the purified peptides were determined. From the sequences of the individual peptides and the overlaps between them, the total amino acid sequence was deduced as follows: H-Pro-Arg3-Thr-Arg2-Ala-Ser-Arg-Pro-Val-Arg4-Pro-Arg2-Val-Ser-Arg4-Ala-Arg4-OH.抄録全体を表示PDF形式でダウンロード (1001K) -
XVII. The Complete Amino Acid Sequence of Clupeine YIKoichi SUZUKI, Toshio ANDO1972 年 72 巻 6 号 p. 1433-1446
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーClupeine extracted from sperm nuclei of Pacific herring is composed of three main molecular species, YI, YII, and Z. The complete amino acid sequences of the two components Z and YII have already been determined. The third component, clupeine YI, was isolated homogeneously as its TNP-derivative by CM-cellulose column chromatography and submitted to structural analyses.
The amino acid composition of clupeine YI is Arg 20, Thr 2, Ser 3, Pro 2, Gly 1, Ala 2, and Ile 1, and the molecular weight is 4841 as hydrochloride. The complete amino acid sequence was established by the use of two complemental degradation procedures, hydrolysis by trypsin [EC 3. 4. 4. 4] and thermolysin. Individual peptides were separated and the sequences were determined. Mainly from the results of two sets of peptides the total sequence was derived: H-Ala-Arg-Arg-Arg-Arg-Ser-Ser-Ser-Arg-Pro-Ile-Arg-Arg-Arg-Arg-Pro-Arg-Arg-Arg-Thr-Thr-Arg-Arg-Arg-Arg-Ala-Gly-Arg-Arg-Arg-Arg-OH.抄録全体を表示PDF形式でダウンロード (992K) -
I. Regulation of Anthranilate SynthetaseIsamu SHIIO, Ryuichi MIYAJIMA, Michiko NAKAGAWA1972 年 72 巻 6 号 p. 1447-1455
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーAnthranilate synthetase was purified about 20-fold from sonic extracts of Brevibacterium flavum No. 2247. This enzyme required chorismate, glutamine, and Mg2+ for its activity. Double reciprocal plots of the reaction rate against one substrate concentration at several fixed levels of another substrate gave straight lines parallel to each other, indicating that the reaction mechanism is of ping pong type. Km values for chorismate and glutamine were 0.04 and 1.9mM, respectively. The molecular weight was estimated as approximately 1.8×105 by Sephadex G-200 gel filtration. The enzyme was strongly and specifically inhibited by tryptophan, the metabolic end product. Thus, 50 percent inhibition took place at about 1.5μM. The inhibition by tryptophan was competitive with respect to chorismate and uncompetitive with, glutamine. In the presence of tryptophan, homotropic cooperativity of chorismate was observed. D-Tryptophan, phenylalanine, tyrosine, histidine, and indole scarecely affected the enzyme activity. Among structural analogues of tryptophan tested, 4-methyltryptophan (4MT), * 5-methyltryptophan (5MT), and 6-fluorotryptophan (6FT), gave a strong inhibitory effect but 6-methyltryptophan (6MT), 7-azatryptophan (7AT), and 5-hydroxytryptophan (5HT) did not. The inhibition type by 5MT was similar to that by tryptophan. Anthranilate synthetase of strain No. 555, a mutant derived from strain No. 2247, which is resistant to 5MT and accumulates L-tryptophan, was found to be fairly desensitized to the feedback inhibition by tryptophan. Thus, tryp tophan concentration required for, 50 percent inhibition was 45μM, 30 times that in the parent enzyme. These results indicate that tryptophan biosynthesis in B. flavum may be regulated through the feedback inhibition of anthranilate synthetase by tryptophan.抄録全体を表示PDF形式でダウンロード (672K) -
Takachika AZUMA, Kozo HAMAGUCHI, Shunsuke MIGITA1972 年 72 巻 6 号 p. 1457-1467
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーThe denaturation by guanidine hydrochloride (GuHCl) of two type κ and three type λ Bence Jones proteins was studied by means of optical rotation and circular dichroic (CD) techniques. All the Bence Jones proteins began to be denatured at a concentration of GuHCl as low as 0.9M. The transitions were not of a two-state process and consisted of two stages. The results suggested that the constant half of the Bence Jones protein molecule was less stable than the variable half against GuHCl. Comparison of the CD spectra of λ proteins in the native, intermediate, and denatured states made it possible to draw CD spectra of the constant and variable halves separately. All the CD spectra of the constant halves of the three λ proteins were very similar to each other and had a positive maximum at 300 mμ and negative maxima at 293 and 218mμ. The CD spectra of the variable halves were different from each other. These facts demonstrate that the constant and variable halves of Bence Jones proteins are folded independently.抄録全体を表示PDF形式でダウンロード (752K) -
XVIII. Gel Filtration Studies on the Interaction of Ribonuclease T1 with Substrate AnalogsKenji TAKAHASHI1972 年 72 巻 6 号 p. 1469-1481
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリー1. The interaction of ribonuclease T1 [EC 2. 7. 7. 26] with its substrate analogs has been investigated by a gel filtration method. The enzyme bound maximally one molecule of 3'-GMP per molecule of protein, indicating the presence of one specific binding site in the enzyme molecule.
2. At pH 5.5 and 25°C, the apparent binding strengths of the substrate analogs were in the order: 2'-GMP>3'-GMP>3', 5'-GDP>9-(2'-hydroxyethyl)guanine 2'-phosphate>5'-GMP>guanosine>8-bromoguanosine>3'-AMP> deoxyguanosine>5'-1-MeGMP>3'-CMP, 3'-UMP, guanine and the other eight nucleosides examined. The results show the importance of the integrity of the guanine, ribose, and phosphate portions for the binding. The importance of the N-1 and N-7 positions and the 2-amino and 6-oxo (or hydroxy) groups in the guanine portion for the specific binding was also suggested.
3. The optimum pH for the binding of 3'-GMP and guanosine to the enzyme was around pH 5. The pH dependence of the binding of these analogs to the enzyme indicated the implication in the binding of at least two groups in the enzyme with pKa values of about 3.5 and 7, respectively, presumably a carboxyl and an imidazole groups. The significant loss of the binding ability toward 3'-GMP at temperatrues above 40°C or in the presence of urea demonstrates the importance of the native conformation of the enzyme for the binding. The effects of some added substances on the binding were also investigated.
4. The Glu-58-carboxynmethylated, inactive enzyme retained a considerable binding ability toward 3'-GMP and moreover it possessed almost the same binding ability toward guanosine over a wide pH range as that of the native enzyme. These rueults appear to indicate that Glu-58 is part of the catalytic site rather than part of the binding site, although the latter possibility cannot be excluded. The binding ability toward 3'-GMP was lowered significantly by the modification of Arg-77 with phenylglyoxal and also by the cleavage of the disulfide bonds with β-mercaptoehanol. Arginine-77 may be the phosphate binding site or may be situated near the active site.抄録全体を表示PDF形式でダウンロード (1032K) -
Its Purification and Biological PropertiesGenichiro OSHIMA, Tamotsu OMORI-SATOH, Sadaaki IWANAGA, Tomoji SUZUKI1972 年 72 巻 6 号 p. 1483-1494
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーOne of two hemorrhagic factors, hemorrhagic factor I (HR-I), in the venom of Agkistrodon halys blomhoffii (Mamushi) was highly purified by column chromatography on DEAE-cellulose followed by vertical zone electrophoresis and gel chromatography on a Sephadex G-100 column. Sedimentation, gel chromatography, polyacrylamide gel electrophoresis, and immunoelectrophoresis indicated a high degree of homogeneity of the purified material. It possessed no detectable activities of enzymes present in the crude venom; activities tested are those of proteinases, phospholipase A's [EC 3. 1. 1. 4], nonspecific phosphatases [EC 3. 1. 3. 1 or 3. 1. 3. 2], arginine ester hydrolases, bradykinin releasing, and clotting enzynmes, L-amino-acid oxidase [EC1. 4. 3. 2], and hyaluronidase [EC 3. 2. 1. 35].
The purfied HR-I preparation had a sedimentation coefficient of 6.08S and diffusion coefficient of 5.40×10-7cm2•sec-1. Using the observed balues of s20, w, D20, w, and_??_(assumed as 0.7) in the Svedberg equation, the molecular weight was calculated as 91, 000. The molecular weight determined by Archibald's method was 80, 000. The apparent isoelectric point was pH4.70 and the mobility was estimated to be -3.55×10-5cm2•sec-1•volt-1 at pH8.5. The amino acid composition of the HR-I preparation was examined. High contents of aspartic and glutamic acids, arginine, tyrosin, and half-cystine residues were found. Among its neutral amino acids the content of tyrosine was examined, High contents of aspartic and glutamic acids, arginine, tyrosine, and half-cystine residues were found. Among its neutral amino acids the content of tyrosine was highest followed by that of half-cystine. HR-I contained about 12 percent of neutral sugars. Thus, HR-I was concluded to be an acidic glycoprotein.
The hemorrhagic activity of the purfied material was completely neutralized by Mamushi antivenin, and minimum hemorrhagic dose of HR-I was 0.0012μg. The purified material also had a potnent lethal effect on mice (L. D.50=0.36(0.32-0.45)mg/kg). Thus, the HR-I principle seems to be a major toxin in this venom.抄録全体を表示PDF形式でダウンロード (1701K) -
Shin-ichiro OHKAWA, Ryuichiro HATA, Yutaka NAGAI, Masaya SUGIURA1972 年 72 巻 6 号 p. 1495-1501
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーThe excretion of acidic glycosaminoglycans in urines of 30 aged subjects was investigated in relation to age and d'seaEe. The patl ern of glycosaminoglycans excreted in urines of the aged subjects was compared with each other and with those of healthy young adults (26-31yrs., male), using two dimensional electrophoresis on cellulose acetate strips.
1. The average amount of acidic glycosaminoglycans excreted in urine of the healthy aged was 1.74±0.51mg/day as uronic acid, indicating that the aged shows a tendency of less excretion of urinary acidic glycosaminoglycans, compared with healthy young adults.
2. Among the aged with individual diseases examined, only patients with diabetes mellitus showed a higher excretion of acidic glycosaminoglycans in their urines, compared with the healthy aged.
3. The analysis of urinary acidic glycosaminoglycans from the aged by two dimensional electrophoresis demonstrated that among 30 aged examined 8 cases excreted low sulfated chondroitin sulfate as a main component and other 7 less but higher than that of young adults. In the remaining 15 cases, rather young adult type of patterns were obtained.
4. Physiological meaning of the excretion of low sulfated chondroitin sulfate in urine is discussed in relation to renal function and aging process.抄録全体を表示PDF形式でダウンロード (2115K) -
Tadashi YOSHIDA, Goro KIKUCHI1972 年 72 巻 6 号 p. 1503-1516
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーThe metabolism of glycine and serine in the liver of various species of vertebrates including mammals, birds, reptiles, amphibians, and fishes was studied using mitochondria and cell homogenates. It was shown that the most significant pathway of glycine catabolism in the liver of these animals is the direct cleavage of glycine to form methylene-tetrahydrofolate (methylene-THF), CO2, and ammonia in mitochondria. Methylene-THF derived from the α-carbon of glycine can be further oxidized to CO2. Liver homogenates as well as liver mitochondria from all the animals tested also catalyzed 14CO2 formation from either serine-1-14C or -3-14C. Moreover, with ureotelic and ammonotelic animals, the soluble liver fraction alone could also oxidize the β-carbon of serine to C02, indicating the possible occurrence of 10-formyl-THF: NADP+ oxidoreductase in both mitochondrial and soluble liver fractions. However, the soluble liver fraction of uricotelic animals such as a bird and a snake was apparently devoid of 10-formyl-THF: NADP+ oxidoreductase. With all the animals tested, both the mitochondrial and soluble liver fractions contained considerable activities of serine hydroxymethyltransferase [EC 2. 1. 2. 1], methylene-THF dehydrogenase [EC 1. 5. 1. 5], and methylene-THF cyclohydrolase [EC 3. 5. 4. 9], whereas the activity of serine dehydratase [EC 4. 2. 1. 13] was relatively low and in some animal species it was hardly detectable. It was assumed that under physiological conditions serine catabolism in vertebrate livers would proceed mainly by way of the cleavage of serine to form methylene-THF and glycine. In ureotelic and ammonotelic animal livers, methylene-THF derived from the β-carbon of serine as well as the α-carbon of glycine is further oxidized to CO2 in either the soluble fraction or mitochondria, but in uricotelic animal livers methylene-THF would be utilized mainly for purine synthesis.抄録全体を表示PDF形式でダウンロード (1128K) -
Toshisuke KAWASAKI, Ikuo YAMASHINA1972 年 72 巻 6 号 p. 1517-1525
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーIsolated nuclei from rat liver contained carbohydrates consisting mainly of glucosamine and mannose which are normally components of glycoproteins. About 60% of the glucosamine and all of the mannose were localized in the nuclear membranes with nearly equal distribution between outer and inner membranes.
Glycopeptides were prepared by pronase digestion from isolated membrane, then fractionated to produce neutral and acidic glycopeptide fractions. The neutral one was major, comprising 74% of the total glucosamine, and its carbohydrate moiety consisted mainly of glucosamine and mannose with a molar ratio of about 1:3. This composition is very similar to that of the corresponding glycopeptide fraction from hepatic microsomes.
Metabolic studies using 14C-glucosamine suggested that carbohydrate moieties of the nuclear membrane are synthesized in nuclei or their vicinity.抄録全体を表示PDF形式でダウンロード (771K) -
Hideharu TAIRA, Shin-ichiro EJIRI, Kensuke SHIMURA1972 年 72 巻 6 号 p. 1527-1535
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーA procedure for the purification of G-factor from the silk gland of silkworm is described. The purified G-factor was homogeneous on disc gel electrophoresis. A molecular weight of 80, 000 was estimated from a calibrated Sephadex G-200 column. The purified G-factor exhibited the uncoupled ribosome-dependent GTPase activity and was inhibited by fusidic acid, SH inhibitors, and diphtheria toxin plus NAD. G-factor from E. coli and that from silk gland were not interchangeable in the GTPase reaction.抄録全体を表示PDF形式でダウンロード (1252K) -
VIII. Molecular Mechanism of the Conversion of Osmotic Energy to Chemical Energy in the Sarcoplasmic ReticulumSinpei YAMADA, Michihiro SUMIDA, Yuji TONOMURA1972 年 72 巻 6 号 p. 1537-1548
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーThe SR was loaded with Ca2+ ions by simply pre-incubating it in solutions containing various concentrations of CaCl2, 0.1M KCl, and 50mM Tris-maleate at pH7.0 and 0°C for several hour. When the Ca2+-loaded reticulum was put into a solution contazning 10mM EGTA, 20mM MgCl2, 5mM 32Pi, 90mM KCl, and 50mM Tris-maleate at pH 7.0 and 20°C, P was rapidly incorporation into the SR. The maximum amount of P-incorporation reached 4moles/106g SR protein.
The time-course of P-incorporationn showed a lag phase, and a remarkably large dependence on temperature.
The dependence of the amount of P incorporated ([EP]) in the steady state on the Ca2+-gradient across the membrane and the concentrations of Mg2+ ions and Pi, in the phosphorylation medium was given by _??_, where ε is the total concentration of phosphorylation site, and [Cai] and [CaO] are the electrochemical activities of CO2+ ions inside and outside the membrane, respectively. When [EP]/ε was 0.5 in the presence of 20mM MgCl2 and 5mM Pi, the free energy obtained by transporting 2 moles of Ca2+ ions from inside to outside the membrane was estimated to be about 12kcal.
The phosphorylation of the Ca2+-loaded SR with Pi was competitively inhibited by ATP. At various pH values the stability of the acid-denatured phosphorylated intermediate formed by the reaction with Pi was equal to that of the intermediate formed by the reaction with ATP. Decomposition of the acid-denatured phosphorylated intermediate produced by the reaction with Pi was accelerated by adding hydroxylamine in the same way as that of the intermediate produced by the reaction with ATP. Furthermore, the SDS-gel electrophretogram of the phosphorylated intermediate showed that P was incorporated in the protein moiety of the ATPase [EC 3. 6. 1. 3.].
When ADP was added to the Ca2+-loaded SR 30 sec after starting the phosphorylation experiment, the amount of P incorporated decreased markedly and rapily, and ATP was formed. The amount of ATP synthesized was exactly half the amount of Ca2+ ions loaded into the SR during the pre-incubation.抄録全体を表示PDF形式でダウンロード (841K) -
IV. Isolation of a Disaccharide, 6-O-(Strontium 2-Acetamido-2-deoxy-β-D-mannopyranosyluronate)-D-glucoseSumihiro HASE, Yoshie TSUJI, Yoshio MATSUSHIMA1972 年 72 巻 6 号 p. 1549-1555
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーAn acidic polysaccharide from the cell walls of Micrococcus lysodeikticus was subjected to controlled acid hydrolysis and the products were chromatographed on a Dowex 1×4 column (acetate form). Four peaks were obtained and the main one was purified further on a Bio Gel P2 column. To determine its structure, the material in this fraction was subjected to peracetylation and borohydride reduction, then its carboxyl group was esterified with diazomethane and the product was reduced with borohydride. The resulting material was found to contain nearly equimolar amounts of N-acetyl-mannosamine and glucitol. It yielded gentiobiitol on deacetylation with anhydrous hydrazine and deamination with nitrous acid. Thus there was a β-l, 6 linkage between mannosaminuronic acid and glucose in the isolated disaccharide. After N-acetylation of purified material from a partial hydrolysate of the polysaccharide, a microcrystalline disaccharide was obtained as a strontium salt.抄録全体を表示PDF形式でダウンロード (1936K) -
Akria TAKETO, Sigeru KUNO1972 年 72 巻 6 号 p. 1557-1563
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーWhen subjected to osmotic shock, cells of E. coli release various enzymes participating in nucleoside catabolism. These enzymes include uridine phosphorylase [EC 2. 4. 2. 3], (deoxy) cytidine deaminase [EC 3. 5. 4. 5], and adenosine deaminase [EC 3. 5. 4. 4]. Release of these enzymes was inhibited remarkably by an addition of Mg2+. Treatment in situ of E. coli cells with diazo-7-amino-1, 3-naphthalene-disulfonic acid failed to inactivate these enzymes while RNase I [EC 2. 7. 7. 16] activity was destroyed almost completely. In contrast to conventional periplasmic proteins, enzymes of nucleoside catabolism are not released upon conversion into spheroplasts by lysozyme [EC 3. 2. 1. 17] and EDTA. When spheroplasts were lyzed and fractionated in a medium containing Mg2+, more than half of these activities were detected in the membrane fraction. These enzymes were, however, quantitatively released into the cytoplasmic supernatant by omission of Mg2+ or by addition of Brij 58. Based on these results cellular localization of these enzymes in E. coli is discussed.抄録全体を表示PDF形式でダウンロード (570K) -
Michiko MASAKA, Hiromichi OKUDA, Setsuro FUJII1972 年 72 巻 6 号 p. 1565-1566
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーPDF形式でダウンロード (114K) -
Hiroko ONO, Fumimaro TAKAKU1972 年 72 巻 6 号 p. 1567-1569
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーPDF形式でダウンロード (182K) -
Tsuneo BABA, Hiroshi SUGIYAMA, Shuichi SETO1972 年 72 巻 6 号 p. 1571-1573
発行日: 1972/12/25
公開日: 2008/11/18
ジャーナル フリーPDF形式でダウンロード (161K)
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