巻号一覧
89 巻, 3 号
選択された号の論文の33件中1～33を表示しています
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Koscak MARUYAMA, Sumiko KIMURA, Kazuyo OHASHI, Yuh KUWANO1981 年 89 巻 3 号 p. 701-709
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーWhen whole muscle fibers or myofibrils of rabbit and chicken skeletal muscles are directly solubilized in hot SDS solution, a very high molecular protein called titin can be isolated by gel filtration (Wang et al. 1979). Connectin, an elastic protein of muscle (Maruyama et al. 1977), can be isolated by a similar method from thoroughly extracted muscle residues. Studies of electrophoretic mobility on 2-3% polyacrylamide gel electrophoresis, amino acid composition, and localization in myofibrils determined by the indirect immunofluorescence technique showed that titin and connectin are identical. Connectin was found to be unstable in SDS solution on storage for a few days at room temperature; the doublet band of connectin on SDS gel electrophoresis became diffuse and eventually disappeared.
Connectin was concentrated around the A-I junction region of a myofibril, although it was present in an entire sarcomere except for the Z lines. On removal of myosin, the A-I junction was still fluorescent, when treated with fluorescent antibody against connectin. In the KI-extracted myofibril, materials accumulated on both sides of the Z lines were strongly stained, and there were fluorescent filaments between the neighboring Z lines, but the Z lines were not stained at all.抄録全体を表示PDF形式でダウンロード (1707K) -
Koscak MARUYAMA, Masanori KIMURA, Sumiko KIMURA, Kazuyo OHASHI, Koichi ...1981 年 89 巻 3 号 p. 711-715
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThe effects of various proteolytic enzymes on the high molecular weight protein (connectin) present in a direct sodium dodecyl sulfate extract of myofibrils from chicken breast muscle were studied in detail. To keep the high molecular weight proteins intact, myofibrils had to be prepared in the presence of EGTA. Trypsin, chymotrypsin, papain, and nagarse readily hydrolyzed connectin (doublet band of titin) and the band 3 protein (N2-line protein). Pepsin did not attack connectin, but digested the band 3 protein and myosin. Calcium-activated neutral proteinase hydrolyzed the band 3 protein, leaving connectin intact. On the other hand, serine protease digested connectin but not the band 3 protein.抄録全体を表示PDF形式でダウンロード (2287K) -
Hiroyuki KOMIYA, Makoto KAWAKAMI, Shosuke TAKEMURA1981 年 89 巻 3 号 p. 717-722
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーA low molecular weight RNA fraction was obtained from the posterior silk glands of Bombyx mori. 5 S rRNA was purified from this fraction by gel filtration on a column of Sephadex G-100 and electrophoresis on a discontinuous polyacrylamide gel devised for large scale preparation. The nucleotide sequence of this RNA was determined mainly by a chemical sequencing method on polyacrylamide gels using RNA labeled with [32P]pCp at the 3' terminus (1). An enzymatic sequencing method on gels using 32P-labeled RNA at the 5' end (2) and conventional sequence analyses of the complete digests of the unlabeled RNA with RNase T1 [EC 3. 1. 27. 3] and RNase A [EC 3. 1. 27. 5] were used for complete determination of the total sequence. 16 base substitutions were observed between B. mori and Drosophila melanogaster 5 S rRNAs.抄録全体を表示PDF形式でダウンロード (1002K) -
Masanao KOBAYASHI, Takayuki OZAWA1981 年 89 巻 3 号 p. 723-730
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーWhen a plasma membrane preparation isolated from rat liver was incubated with [r-32P]ATP and Mg2+, protein-bound 32P increased rapidly, followed by a gradual decrease. The time course suggested the existence of membrane-bound kinase (s) and phosphatase (s) phosphorylating and dephosphorylating endogenous proteins. The extent of phosphorylation was not affected by inclusion of cyclic AMP in the reaction mixture. The extent of the maximum phosphorylation was dependent on membrane concentration, owing to rapid hydrolysis of ATP by the membrane-bound ATPase activity. Thus, phosphorylation proceeded further on repeated addition of ATP. Both phosphorylation and dephosphorylation were stimulated by Mg2+, an effective rate of phosphorylation being obtained at 15mM. P1 up to 20mM stimulated phosphorylation with little effect on the rate of dephosphorylation. At higher phosphate concentrations, the maximum 32P-incorporation decreased again, and at 100mM, dephosphorylation was prevented significantly. Autoradiography after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea revealed six main phosphorylated bands, two of which (Band 3 and 5) were partly extractable with 1M NaCl. In the presence of 100mM P1, very strong phosphorylation of Band 5 (about 23, 000 daltons) was noted, and a new strongly labeled band (Band P, about 20, 000 daltons) was observed. It was concluded that the phosphoproteins in the membrane may be turned over at different rates and high concentrations of P1 may affect the turnover rate of some phosphoproteins, probably through interference with the phosphatase.抄録全体を表示PDF形式でダウンロード (1277K) -
Masanao KOBAYASHI, Takayuki OZAWA1981 年 89 巻 3 号 p. 731-740
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーPlasma membrane isolated from rat liver contained activities of phosphoprotein phosphatase dephosphorylating [32P] phosphorylase a or [32P] phosphohistone. The properties of the membrane-bound phosphatase were examined using these exogenous substrates. The optimal reaction rate was at pH near neutrality At concentrations as low as 0.1-1.0mM, Mg2+ or Mn2+ slightly stimulated the activity for phosphorylase a or phosphohistone, respectively; at higher concentrations, they were inhibitory with both substrates. Co2+ was inhibitory with both substrates, while Ca2+ had no significant effect. The phosphatase activities were inhibited by ATP, ADP, or AMP; the extents of inhibition were in opposite order with the two substrates. Phosphorylase phosphatase activity was strongly inhibited by KF or P1. Phosphorylase phosphatase activity could be completely solubilized by incubating the membrane with 0.5M NaCl or trypsin, and this was associated with several-fold activation. While Vmax values were increased, Km values for phosphorylase a were not much affected by these treatments. Unlike the soluble phosphatase, freezing in the presence of mercaptoethanol or by precipitation with ethanol failed to activate or to solubilize the membrane-bound phosphatase. The molecular weights of the NaCl- and the trypsin-solubilized phosphatase were estimated on gel filtration to be about 42, 000 and 32, 000, respectively. The present results indicate that the phosphoprotein phosphatase associated with liver plasma membrane shares several properties in common with phosphatases from other sources reported, and that, like those in the soluble fraction, it may be bound to some inhibitory proteins.抄録全体を表示PDF形式でダウンロード (690K) -
Tetsuro YONESAKI, Ichiro HARUNA1981 年 89 巻 3 号 p. 741-750
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーAn RNA replicase of GA phage, one of the Group II RNA phages, was isolated and purified to a homogeneous state. By SDS polyacrylamide gel analysis, the purified GA replicase was found to contain four different subunits, numbered I, II, III, and IV, the molecular weights of which were 74, 000, 60, 000, 47, 000, and 36, 000, respectively. Three of them, I, III, and IV, proved to be host-coded proteins, ribosomal protein SI (I), and elongation factors Tu (III) and Ts (IV) of protein biosynthesis, respectively. On a phosphocellulose column, the RNA replicase was separated into two components: One composed of subunits I and II, and the other composed of subunits III and IV. Each component alone had no replicase activity. However, when the two components were combined at 0°C, 60% of the replicase activity was restored within 10min.
The purified GA replicase catalyzed the GA phage RNA-directed synthesis of template-size RNA. However, the maximum level of product RNA synthesized was less than 20% of the amount of template RNA added. RNA-RNA hybridization experiments indicated that the product RNA included only the RNA strand complementary to the template RNA, and not the viral strand.抄録全体を表示PDF形式でダウンロード (764K) -
Tetsuro YONESAKI, Akira AOYAMA1981 年 89 巻 3 号 p. 751-757
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーA certain factor (s) derived from Escherichia coli was found to extensively stimulate RNA synthesis by the RNA replicase of phage GA. This factor (s), named GA-HF (host factor (s) for GA RNA replication), was partially purified from an uninfected cell extract and characterized. In the presence of GA-HF, GA replicase synthesized 50-100 times more RNA than was synthesized in its absence, and was capable of synthesizing both the viral strand as well as its complementary strand. This factor (s) could not be replaced by HFI, which is necessary for the replication of Qβ RNA by Qβ replicase.
In the presence of GA-HF, the GA RNA replication system has a characteristic template specificity. Group I and II phage RNAs, but none of the Group III and IV phage RNAs, showed template activity.抄録全体を表示PDF形式でダウンロード (520K) -
Kunihiro KUWAJIMA, Yuji OGAWA, Shintaro SUGAI1981 年 89 巻 3 号 p. 759-770
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーPrevious studies of the reversible unfolding of α-lactalbumin in the acid to neutral pH region have shown that the unfolding transition with guanidine hydrochloride involves a stable intermediate which is similar to a partially unfolded state produced by acid transition. In order to clarify how the interaction of ionizable groups takes part in stabilization of the native structure during the folding of the protein, the transitions were further investigated in the alkaline region in the presence of the denaturant by means of circular dichroism, difference spectra and pH jump measurements, and the effects of pH on the equilibrium and kinetics of the unfolding in the whole pH region are discussed. The alkaline state is indistinguishable from the acid state in equilibrium and kinetic properties. Thus, as a first approximation, the total unfolding in the whole pH region can be expressed as a three-state mechanism involving the native (N), the intermediate (A), and the fully unfolded (D) states. The strong pH dependence of the N_??_A transition above pH 10 is almost entirely ascribable to the abnormal tyrosines in the N state previously detected by the pH-jump titration method, while the dependence between pH 7 and 10 also suggests the presence of an abnormal α-amino group. The normalization of most of the alkaline and the acidic abnormally ionizable groups in the N state occurs simultaneously in the first step of the unfolding pathway, i.e., the forward activation of the N_??_A transition, and the final step of the folding may be associated with the interaction of the ionizable groups. Among the abnormally ionizable groups detected, the tyrosyl and carboxyl groups are most important in view of the large changes in their pK values, suggesting the presence of some interactions, even if only indirect, between these groups. Alignment data of amino acid residues also suggest that at least one such abnormal tyrosyl residue (Tyr 50) and its neighbors are conserved throughout in the α-lactalbumin-lysozyme group of proteins. Possible mechanisms of the interaction between the tyrosyl and carboxyl groups are discussed.抄録全体を表示PDF形式でダウンロード (880K) -
Kiyoshi SUGAWARA, Fumitaka OYAMA1981 年 89 巻 3 号 p. 771-774
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThe fluorogenic reaction of ammonia with o-phthalaldehyde (OPT) and dithiothreitol (DTT) is reported. Optimal detection wavelengths are λex=413nm and λem=476nm. This fluorescence is specific for ammonia: other amino compounds, such as amino acids, amines and proteins, do not show emission at 476nm when activated at 413nm. This reaction permits specific microdetermination of ammonia in biological materials down to the nanomole range.抄録全体を表示PDF形式でダウンロード (254K) -
Shigeki TSUCHIDA, Fusako IMAI, Kiyomi SATO1981 年 89 巻 3 号 p. 775-782
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThe immunological properties of β-glutamyltransferases (β-GTs) from human serum, liver and tonsil were studied by using a monospecific antibody to human kidney β-GT for the purpose of elucidating their isozymic relationships. β-GTs partially purified from liver and tonsil were indistinguishable in this respect from kidney β-GT. β-GT in sera from patients with hepato-biliary diseases, on the other hand, was heterogeneous in molecular size as revealed by sucrose density gradient centrifugation and Sephadex G-150 gel filtration, and was inhibited and precipitated by the above antibody relatively poorly as compared with the kidney enzyme. When these sera were treated with bromelain, however, the molecular size of β-GT was reduced and the enzyme now reacted with the antibody as strongly as kidney β-GT. β-GT from bromelain-treated sera also exhibited a single immunoprecipitin line smoothly fusible with that from kidney β-GT; the enzyme-antibody complex still exhibited β-GT activity. The major form of β-GT partially purified from papain-treated sera, even though indistinguishable from kidney β-GT immunologically and in molecular size, exhibited a mobility on polyacrylamide gel electrophoresis which was higher than that of kidney β-GT but similar to that of liver β-GT. It is suggested that β-GT in human sera is heterogeneous in molecular size and electric charge but is composed of common peptide chains, probably identical to those of kidney β-GT.抄録全体を表示PDF形式でダウンロード (1805K) -
Toshiaki ODA, Nuran NABI, Tsuneo OMURA1981 年 89 巻 3 号 p. 783-790
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーPuromycin-mediated in vitro release of nascent peptides from rat liver ribosomes was significantly stimulated by the presence of low concentrations of a detergent, and the stimulation was much more marked with bound ribosomes than with free ribosomes. The release of nascent peptides from ribosomes could be carried out in two steps, first with puromycin in the absence of a detergent and then with a detergent, to give two separate nascent peptide fractions S 1 and S 2, respectively. Although S 1 and S 2 fractions were not significantly different in hydrophobicity and in the size of the puromycin-conjugated peptides when examined by alkyl-Sepharose column chromatography and SDS-polyacrylamide gel electrophoresis, the fractionation of the released peptides by immunoprecipitation showed significant difference in the distribution of the nascent peptides of two specific proteins, serum albumin and NADPH-cytochrome c reductase, between these two fractions. The nascent peptides of serum albumin were found mainly in fraction S1 obtained from bound ribosomes. On the other hand, a larger portion of the nascent peptides of NADPH-cytochrome c reductase was detected in fraction S2 from free ribosomes than in other fractions. The presence of a detergent is indispensable for efficient in vitro release of nascent peptides from ribosomes by puromycin and this finding may be important in studying the synthesis of specific proteins in mammalian cells.抄録全体を表示PDF形式でダウンロード (631K) -
Hiroshi YOSHIDA, Katsutoshi ISHIMARU, Osamu TSUCHIKURA1981 年 89 巻 3 号 p. 791-794
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThe products derived from the degradation of the sixteen possible diribonucleoside monophosphates (NpN') by Fusarium phosphodiesterase-phosphomonoesterase were analyzed by means of thin layer chromatography. The analysis showed that NpN' was first cleaved into nucleoside N and 5'-nucleotide pN', which was then dephosphorylated to yield nucleoside N'. The dephosphorylation was fast when N' was adenosine or cytidine but slow when N' was guanosine or uridine. The cleavage reaction was followed by measuring the increase of absorbance due to hyperchromicity, and the kinetic constants, Km and kcat, were determined for the sixteen dinucleoside phosphates. The Km value was higher, for a given N, when N' was a pyrimidine nucleoside than when N' was a purine nucleoside. For a given N', uridine as N gave the highest Km value and adenosine gave the lowest one. The kcat value was the highest, for a given N, when N' was cytidine. For a given N', uridine as N gave by far the lowest kcat value. These results can be interpreted in terms of two binding sites on the enzyme with different base preferences. Comparison of kcat/Km values suggested that the base of nucleoside N plays an important role in determining whether a dinucleoside phosphate is a good substrate of the enzyme. The dinucleoside phosphates with uridine as N were found to be particularly poor substrates of the enzyme.抄録全体を表示PDF形式でダウンロード (285K) -
Masako TANIGUCHI, Makoto AIKAWA, Toshio SAKAGAMI1981 年 89 巻 3 号 p. 795-800
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーA very rapid hemolysis was found to be caused by active oxygen species produced by a hypoxanthine-xanthine oxidase system with very low concentrations of hypoxanthine. The addition of superoxide dismutase or catalase inhibited the hemolysis, indicating that O2- and H2O2 participate in this system.
The extent of erythrocyte hemolysis was found to depend on the sex of the human donor. The change in phospholipid composition before and after hemolysis in human erythrocytes from donors of each sex was compared by thin layer chromatography. A significant decrease in phosphatidylethanolamine content and a concomitant increase in altered phospholipid fraction were observed in erythrocytes from male donors, suggesting that these erythrocytes were easily attacked by active oxygen species to produce modified phosphatidylethanolamine.抄録全体を表示PDF形式でダウンロード (1116K) -
Takemi ENOMOTO, Sei-ichi TANUMA, Masa-atsu YAMADA1981 年 89 巻 3 号 p. 801-807
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThe ATP requirement for two steps of DNA replication, the synthesis and subsequent joining of Okazaki fragments, was investigated by using isolated HeLa cell nuclei. Among adenine nucleotides tested, high levels of dATP and ADP stimulated DNA synthesis. In the presence of high levels of ATP, the addition of high levels of dATP or ADP resulted in about 70% inhibition of DNA synthesis. The optimal concentration of ATP for the stimulation of DNA synthesis varied depending on the magnesium ion concentration. When the molar ratio of magnesium ion to ATP was approximately 1, maximal stimulation was attained. Product analysis by sedimentation in an alkaline sucrose gradient revealed that both Okazaki fragments and high molecular weight DNA were synthesized in the presence of high levels of ATP, whereas in the case of dATP and ADP, little high molecular weight DNA was synthesized. The ability to synthesize high molecular weight DNA was restored to nuclei by adding low levels of ATP in the presence of high levels of ADP but not of dATP.抄録全体を表示PDF形式でダウンロード (542K) -
Shin-ichi YAMAMOTO, Shinya KOYAMA, Takashi KAWASAKI1981 年 89 巻 3 号 p. 809-816
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThiamine transport in Ehrlich ascites tumor cells was investigated. The rate of [35S] thiamine transport in the presence of an Na+ gradient was dependent on temperature but not on the presence of glucose, although the steady-state level of the uptake was reduced in the absence of glucose. Approximately 65% of thiamine taken up by cells for 5min at 37°C in the presence of glucose was present in unaltered form, and the distribution ratio of intra-/extracellular thiamine concentration was 1.8. This ratio increased to 2.3 either in the absence of glucose or in the presence of 2, 4-dinitrophenol and KCN without glucose, which indicated a concentrative uptake of thiamine after incubation for 5min under any conditions employed. When the incubation was prolonged to 60min in the presence of glucose, thiamine taken up was metabolized to thiamine pyrophosphate; 10.7% of the total uptake was thiamine and its distribution ratio was 1.0.
The initial rate of transport as a function of thiamine concentrations showed saturation kinetics with a Km value of 43.5 nM and a Vmax of 0.71 pmol/mg of protein/min. Pyrithiamine and chloroethylthiamine, antimetabolites of thiamine, competitively inhibited thiamine uptake with K1 values of 21.0 and 10.1nM, respectively, but oxythiamine was not inhibitory. These results indicate the presence of a specific thiamine carrier of high affinity to the substrate.
When the effect of Na+ concentration on the kinetics of thiamine transport was determined, it was found that a decrease in Na+ concentration increased the Km value for thiamine without changing the Vmax value. Treatment of the cells with gramicidin reduced the rate of thiamine uptake to a half of that in untreated cells. The uptake rate in the presence of an Na+ gradient (out>in) was also independent of cellular ATP level in the range from 0.1 to 2.4mM. These results indicate that thiamine is actively transported by coupling to the Na+ gradient as a direct driving force.抄録全体を表示PDF形式でダウンロード (598K) -
Kazuo AISAKA, Osamu TERADA1981 年 89 巻 3 号 p. 817-822
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーLipase [EC 3. 1. 1. 3] was purified from the culture supernatant of Rhizopus japonicus KY 521 by ethanol precipitation, chromatography on Column-lite, affinity chromatography on heparin-Sepharose 4 B, and separation into two lipases, I and II, by isoelectric focusing. The purified lipases I and II were found to be homogeneous by disc electrophoresis, and showed isoelectric points at pH 7.4 and pH 7.9, respectively. They both had an apparent molecular weight of about 42, 000, hydrolyzed tricaprin very rapidly, and exhibited a pH optimum around pH 7.0-8.5. These lipases were inhibited by the addition of serum to the reaction mixtures. These lipases were enhanced slightly in the absence of serum by high concentrations of NaCl and protamine, but were inhibited strongly by these compounds in the presence of serum.抄録全体を表示PDF形式でダウンロード (881K) -
Mitsuo TORII, Setsuko TANAKA, Toshiyuki URYU, Kei MATSUZAKI1981 年 89 巻 3 号 p. 823-829
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーAlpha (1-6) specific anti-dextran antibody was raised in rabbits by injecting N 4 dextran-concanavalin A conjugate, and the interactions of five synthetic linear dextrans (α(1→6)-D-glucopyranans) with rabbit anti-N4 dextran were studied. The ability of glucans to precipitate antibody depended on their average molecular weight, samples with higher molecular weight precipitating more antibody nitrogen under the same conditions. This phenomenon was shown to be due to solubility of the antigen-antibody complex. Oligosaccharide inhibition assay indicated that the maximum size of the α(1→6)-specific antibody combining site corresponded to isomaltopentaose. The precipitated antibody class was shown to be IgG immunoglobulin, and it was mostly directed to linear non-terminal glucosidic linkages. Determination of antibody nitrogen and glucan in the precipitates indicated that the ratio of antibody molecule to numbers of glucose residues was 1:16 in the extreme antibody excess region.抄録全体を表示PDF形式でダウンロード (1589K) -
Takayuki MIYANISHI, Yuji TONOMURA1981 年 89 巻 3 号 p. 831-839
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーWe previously reported ((1979) J. Biochem. 85, 747-753) that both of the burst and nonburst heads contain one reactive lysine residue per head, and that only the reactive lysine residue in the burst head is trinitrophenylated in the presence of PPi whereas both reactive lysine residues are modified in the absence of PPi. In the present study, subfragment one (S-1) prepared from trinitrophenyl (TNP) myosin was subjected to a limited tryptic digestion, a BrCN cleavage, and a thorough tryptic and α-chymotryptic digestion, and the TNP peptides thus obtained were analyzed by SDS-gel electrophoresis and by gel filtration. In the limited digestion, it was found that the reactive lysine residues are both located in the tryptic peptide of 25-27 K daltons containing the N-terminus of the heavy chain and not in the 19-21 K peptide containing reactive thiols. In the BrCN cleavage, it was found that the reactive lysine residues are not located in the 17 K peptide containing the essential arginine residues (Morkin, E., et al. (1979) J. Biol. Chem. 254, 12647-12652). In the thorough digestion, S-1 prepared from myosin modified in the absence of PPi gave two equimolar fractions of TNP peptides in Sephadex G-25 column chromatography, whereas that of S-1 prepared from myosin modified in the presence of PPi gave a single fraction of TNP peptide which corresponds in size to one of the two fractions of TNP peptides obtained above. These findings strongly suggest that the chemical structure around the reactive lysine residue in the burst head is different from that in the nonburst head.抄録全体を表示PDF形式でダウンロード (1066K) -
Takashi MANABE, Kiyotsugu KOJIMA, Setsuko JITZUKAWA, Tadao HOSHINO, Ts ...1981 年 89 巻 3 号 p. 841-853
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーA “normalized map” of human plasma proteins, which illustrated the standard distribution pattern of the proteins, was prepared by comparing eight two-dimensional electrophoretic gels of normal human plasma. Phenotypes of several plasma proteins were detected. The pI and molecular weight of each spot in the normalized map were calculated and 84 spots out of 128 were identified or tentatively identified as known plasma proteins by several methods including micro amino acid analysis of the proteins extracted from the stained gel pieces. Human cerebrospinal fluid and urine protein patterns were compared with the normalized map and the positions of proteins characteristic of each body fluid sample were determined.抄録全体を表示PDF形式でダウンロード (3499K) -
Kiyoshi TATSUMI, Takayoshi DOI, Nobuyuki KOGA, Hidetoshi YOSHIMURA, Hi ...1981 年 89 巻 3 号 p. 855-859
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーOxygen-insensitive nitrofuran reductase in Escherichia coli B/r was clearly resolved by DEAE-cellulose column chromatography into two components, one NADPH-linked, and the other both NADPH- and NADH-linked. It is known that the strain acquires resistance to nitrofurazone in two mutational steps. In such a case, the first step mutants had no NADPH-linked component and the second step ones had neither this component nor the NAD(P)H-linked one. The NADPH- and NADH-linked activities of the latter component were similarly inactivated by heat or urea treatment. In addition, it was found that these activities were significantly inhibited by dicoumarol, an NAD(P)H dehydrogenase inhibitor, to similar extents. These results suggest that the activities of the NAD(P)H-linked component originate from a single enzyme. On the other hand, the NADPH-linked component was less sensitive to heat, urea and dicoumarol.抄録全体を表示PDF形式でダウンロード (327K) -
Hideo INOUE, Yotaro KONISHI, Yoshiro TAKEDA, Alois CIHÁK1981 年 89 巻 3 号 p. 861-869
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThe relationship between polyamine metabolism and DNA synthesis in regenerating liver was investigated using normal and 5-azacytidine-treated rats.
The increase of DNA synthesis in regenerating rat liver was completely inhibited by administration of the analogue 1h after partial hepatectomy. On the contrary, early and enhanced increase (modulation) of DNA synthesis was observed when the analogue was injected 24h before partial hepatectomy.
Changes in ornithine decarboxylase [EC 4. 1. 1. 17] activity and the tissue levels of polyamines in liver remnants showed similar patterns in normal and 5-azacytidine-pretreated rats, except that the putrescine level in removed liver and the spermidine level in the S-phase were significantly higher in the drug-pretreated rats. On the other hand, a single injection of the analogue into intact rats evoked marked increase in hepatic ornithine decarboxylase activity followed by increase in the tissue level of putrescine, but not of spermidine or spermine. No similar increase in the enzyme activity was detected after injection of 5-aza-2'-deoxycytidine, which did not cause modulation of DNA synthesis.
No modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine was observed when partial hepatectomy was carried out before increase of the hepatic level of putrescine. In addition, a positive and highly significant correlation was observed between the level of putrescine in the liver removed at partial hepatectomy and the extent of modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine.
These results suggest that putrescine plays an important role in modulation of DNA synthesis and support our previous proposal that putrescine may have at least two roles in DNA synthesis.抄録全体を表示PDF形式でダウンロード (643K) -
Hiroshi SUZUKI, Sawako SUZUKI, Shizuo WATANABE1981 年 89 巻 3 号 p. 871-878
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーIt has long been known that very high concentrations of magnesium are required for contraction of vertebrate smooth muscle (1-6). We thought of three possible reasons to account for the requirement of high concentrations of magnesium; 1) A high concentration of magnesium may be required for activation of myosin lightchain kinase. 2) It may be required for actin-activation of myosin-ATPase. 3) It may be required for formation of thick filaments.
The three possible reasons were examined by studying superprecipitation of chicken gizzard actomyosin and its ATPase reaction. The following results were thus obtained. a) A MgCl2 concentration higher than 1mM (in the presence of 1mM ATP) was required for activity of myosin light-chain kinase. b) However, high concentrations of magnesium (>ca. 3mM MgCl2 in the presence of 1mM ATP) were also required for superprecipitation of acto-phosphorylated myosin. c) In the presence of 1mM MgCl2 (and 1mM ATP), superprecipitation of acto-phosphorylated myosin did not occur but actin-activation of phosphorylated myosin-ATPase did occur to a full extent. d) The ATPase activity of unphosphorylated myosin was about 1 nmol P1/min/mg of myosin at all the concentrations of magnesium we tested (1-20mM MgCl2), but that of phosphorylated myosin increased from 3 to 9 nmol P1/min/mg of myosin when magnesium concentration increased to higher than approximately 3mM. e) Judging from the turbidity of the myosin suspensions, a magnesium concentration higher than approximately 3 mM (in the presence of 1mM ATP) was also required for formation of thick filaments even when myosin was phosphorylated. It is therefore suggested that formation of thick filaments is responsible for the increase in the ATPase activity of phosphorylated myosin from 3 to 9 nmol P1/min/mg of myosin.抄録全体を表示PDF形式でダウンロード (1264K) -
Mitsuyo OKAZAKI, Yoshimi OHNO, Ichiro HARA1981 年 89 巻 3 号 p. 879-887
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーA simple and rapid method for the quantitation of cholesterol in human serum lipoproteins (VLDL, LDL, HDL2, and HDL3) was developed (1). The content of cholesterol in each lipoprotein fraction was determined by means of a commercial enzymatic reaction kit after separation by high performance liquid chromatography with gel permeation columns. The quantitation of cholesterol could be performed with only 10-20 μl of serum in less than 50min by measuring A550, after passing the mixed eluate and enzyme solution through an on-line reactor system of a high-speed chemical derivatization liquid chromatograph.
The precision, reproducibility and sensitivity of the quantitation of cholesterol with this method were acceptable, and the values of HDL-cholesterol determined by this method correlated well with those found by the heparin-manganese chloride precipitation method (r=0.958, n=93).抄録全体を表示PDF形式でダウンロード (623K) -
Hiroko OHBA, Tomoyuki HARANO, Tsuneo OMURA1981 年 89 巻 3 号 p. 889-900
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThe intracellular distribution of a membrane-bound protein disulfide isomerase (PDI) in rat liver was studied by quantitative immunoprecipitation, and its microsomal localization was confirmed. The content of the enzyme was 1 to 2% of total microsomal protein, and it was almost equally distributed between rough and smooth microsomes. The enzyme was not solubilized from microsomes by high concentrations of KCl, but was readily solubilized by detergents.
Since PDI in microsomes was susceptible to digestion by trypsin, at least some parts of the enzyme molecule are exposed on the outside surface of microsomal vesicles. However, the binding of antibodies to microsomal PDI and the modification of the glutamine residues of PDI molecules by transglutaminase suggested that the molecules are not extensively exposed on the surface. Solubilized PDI was unable to rebind to microsomes or to become incorporated into reconstituted membrane of detergent-solubilized microsomes, showing that the association of this enzyme with the membrane is not simply mediated by hydrophobic interaction.抄録全体を表示PDF形式でダウンロード (1540K) -
Hiroko OHBA, Tomoyuki HARANO, Tsuneo OMURA1981 年 89 巻 3 号 p. 901-907
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThe biosynthesis and turnover of one type of microsomal protein disulfide isomerase (PDI) in rat liver were studied. The enzyme is predominantly synthesized by the membrane-bound ribosomes of rough endoplasmic reticulum, as judged from the results of immunological analyses of puromycin-released nascent peptides and in vitro translation products of isolated free and bound polyribosomes. Nascent peptides of PDI were released from rough microsomes by treatment with puromycin in the presence of 0.5M KCl, which indicates that the nascent peptides of this enzyme are released on the outer surface of microsomal vesicles. This enzyme accounts for about 0.1 to 0.2% of the total protein synthesis by membrane-bound ribosomes. The half-life of PDI was about 7 days, which was significantly longer than the average half-life of microsomal proteins.抄録全体を表示PDF形式でダウンロード (563K) -
Ken HASHIMOTO, Mitsuo NISHIMURA1981 年 89 巻 3 号 p. 909-918
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーIt was demonstrated that one of the main parameters regulating the cyclic electron transfer during and shortly after a steady state under illumination (monitored by following the re-reduction of cytochrome c-555 after 10-s illumination) was the H+ concentration in the surface region on the periplasmic side of the photosynthetic membrane (outer surface of the photosynthetic membrane in cells or spheroplasts, or inner surface of the chromatophore membrane) under aerobic conditions in a purple sulfur bacterium, Chromatium vinosum. Membrane surface potential and membrane surface pH on the periplasmic side of the photosynthetic membrane were changed in cells, spheroplasts and chromatophores by changing pH and salt concentration in the suspending medium.
Salt addition (NaCl, KCl, Na2SO4, choline chloride, CaCl2, or MgCl2) did not affect the rate of re-reduction of cytochrome c-555 in chromatophores. Only when chromatophores were washed with gramicidin D in low-salt buffer and measurements were carried out in the presence of gramicidin D did salt addition (NaCl, KCl, Na2SO4, CaCl2, MgCl2) affect the rate of re-reduction. Even in this case, choline chloride had no effect on the re-reduction rate. Salts of divalent cations were effective at much lower concentrations than monovalent salts. Among the salts of cations of the same valence, there was no ionic species specificity. Similar concentration and valence dependences were also observed in cells and spheroplasts without gramicidin D. The membrane surface pH on the photosynthetic membranes was calculated from the Boltzmann distribution of H+ and the membrane surface potential determined from the ionic concentrations and surface charge density by using the Gouy-Chapman relationship. From the pH-dependences of the dark reduction rate at low and high salt concentrations, the membrane surface pH on the periplasmic side of the photosynthetic membrane was found to be the major factor in determining the rate of re-reduction in cells, spheroplasts and chromatophores. It was also suggested that the electrochemical potential difference of H+ across the membrane was another factor determining the rate of re-reduction.抄録全体を表示PDF形式でダウンロード (744K) -
Tsuneo MIYAHARA, Tatsuya SAMEJIMA1981 年 89 巻 3 号 p. 919-928
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーSubcellular distribution of catalase for porcine kidney was analyzed by differential centrifugation of kidney homogenate. The specific activity of catalase was the highest in light mitochondrial fraction, followed by mitochondrial, cytosolic, nuclear, and microsomal fractions. However, about a half of the total activity was found in supernatant (cytosol) fraction and the other half was mainly associated with both mitochondrial and light mitochondrial fractions. Osmotic shock using hypotonic solution, 50mM sodium phosphate buffer (pH 7.0) was found to be most effective for solubilization of particulate-bound catalase. Both particulate and cytosol catalases from porcine kidney were purified by ammonium sulfate fractionation followed by DEAE-cellulose, CM-cellulose, and Sephadex G-100 column chromatographies. The purified enzymes showed two distinct bands, one major and the other minor, on disc gel electrophoresis. Both particulate and cytosol enzymes showed identical molecular weights estimated from disc gel electrophoresis; that of the major component was 219, 000 corresponding to native molecule and that of the minor one 421, 000. A similar value, 210, 000, was also obtained for the major component by gel filtration on a Bio Gel A-1.5m column. It was inferred that the minor component was formed by dimerization of native molecule caused by formation of disulfide cross-links due to oxidation of SH groups in protein moiety. The particulate and cytosol catalases showed essentially identical molecular characteristics, although a slight difference was detected in stability in guanidine-HCl solution. The effect of NaCl on enzyme activity and optical properties of catalase were also measured.抄録全体を表示PDF形式でダウンロード (1240K) -
Eiko YAJIMA, Tamitaro MIZUNOYA1981 年 89 巻 3 号 p. 929-936
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThe kinetic relation between the photoinactivation and photooxidation of mitomycin C in the presence of riboflavin was investigated. The photoinactivation was tested for λ-phage induction in Escherichia coli K-12 (λ) cells and colony formation of E. coli Bs-1, cells. Mitomycin C lost its phage-inducing and antibiotic activities when the antibiotic was irradiated in vitro with visible light in the presence of riboflavin. The loss of phage-inducing activity followed a Stern-Volmer type equation with respect to the dose of irradiation, and the inactivation constant was evaluated to be 0.96×10-4m2/J. The initial rate of decay of mitomycin C by photooxidation in the presence of riboflavin obeyed first order kinetics, and its cross section was estimated to be 2.51×10-6m2/J independent of the intensity of incident light. The cross section for photooxidation was found to be proportional to the inactivation constant. These results suggest that the photoinactivation of mitomycin C is caused by its photooxidation. In order to rationalize this conclusion, a mechanism of photooxidation was proposed and reactions in vivo of the photoproduct were discussed in relation to the inactivation.抄録全体を表示PDF形式でダウンロード (458K) -
Yuji SHIMADA, Mieko IWAI, Yoshio TSUJISAKA1981 年 89 巻 3 号 p. 937-942
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーRhizopus (Rh.) delemar (ATCC 34612) lipase is modified by its binding with phosphatidylcholine (PC); such binding enhances the lipoprotein lipase (LPL) activity, shifts the isoelectric point (pI) to the acidic side and decreases its α-helical content ((1980) J. Biochem. 88, 533-538).
The results of density gradient ultracentrifugation proved that PC binding to lipase molecule was depleted by the treatment of PC-bound lipase with 0.3% Triton X-100 and 0.1M NaCl. By this treatment, LPL activity was decreased almost to the original activity. At the same time, α-helical content recovered to that of the original lipase and the isoelectric point recovered from pI 6.5 to nearly the pI of the original lipase. These data indicate that the modification of Rh. lipase by PC is reversible. Furthermore, the results of an experiment with 2-[1-14C]oleoyl PC showed that lipase having high LPL activity contained about 5 mol of PC per mol of lipase.抄録全体を表示PDF形式でダウンロード (437K) -
Inhibition of the Anion Permeability of Sarcoplasmic Reticulum Vesicles by Some Stilbene DerivativesMichiki KASAI1981 年 89 巻 3 号 p. 943-953
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーThis paper concerns a study of the inhibition of sulfate permeability of sarcoplasmic reticulum vesicles by stilbene derivatives, such as 4-acetoamido-4'-isothiocyano-2, 2'-stilbene-disulfonic acid (SITS), 4, 4'-diisothiocyano-stilbene-2, 2'-disulfonic acid (DIDS), and diisothiocyano-1, 2-diphenyl-ethane-2, 2'-disulfonic acid (H2DIDS). The level of sulfate permeability was measured by using a radioactive tracer. The sulfate efflux curves comprise two phases. This is explained by the existence of two types of vesicles with different permeability. The permeability of the rapidly permeated vesicles was at least 100 times higher than that of the slowly permeated vesicles. The permeability of both types of vesicles was inhibited by the above inhibitors. Apparent dissociation constants for the inhibitors of sulfate permeability were 5 μM, 6 μM, and 40 μM for DIDS, H2DIDS, and SITS, respectively.
The relation between sulfate permeability and the amount of the bound inhibitors was studied. To effect complete inhibition of permeation by sulfate in both vesicles, the binding of 5 μmol of inhibitors/g protein was required. However, kinetic analysis of the sulfate efflux of the rapidly permeated vesicles suggested that the amount of the anion transport system is much smaller than 5 μmol/g protein.
Permeability for Na+ and choline was not affected by the same concentration of inhibitors. However, Ca2+ permeability was increased by the inhibitors, and at the same time Ca2+ uptake was reduced. This inhibition of Ca2+ uptake was explained by the increase of Ca2+ permeability.抄録全体を表示PDF形式でダウンロード (783K) -
Mitsuhiro OKAMOTO, Retsu MIURA, Toshio YAMANO, Motoji FUJIOKA1981 年 89 巻 3 号 p. 955-962
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーRat liver microsomes labeled with spin-labeled phosphatidylcholine release the label into the aqueous phase during the aerobic incubation with NADPH (Biochem. Biophys. Res. Commun. (1979) 87, 300-307). To establish the chemical nature of the released moiety, microsomes were labeled with [14C] phosphatidylcholine. When the 14C-labeled microsomes were incubated with NADPH under aerobic conditions, a few percent of the radioactivity was liberated into the aqueous phase within 60min. Thin-layer chromatographic analysis of the radioactive substance liberated showed the presence of hydroxylated fatty acids derived from the 2-position of glycerol moiety. About one-third of the fatty acids formed from [14C] phosphatidylcholine during the incubation were converted into hydroxy-derivatives. Gas chromatography/mass spectrometry analysis further confirmed an NADPH-dependent formation of 16-hydroxypalmitic acid, 15-hydroxypalmitic acid, and hydroxy-derivatives of other fatty acids from the phospholipids of the microsomal membrane. Evidence was also obtained indicating the formation of ketopalmitic acid.抄録全体を表示PDF形式でダウンロード (1242K) -
Atsushi HIRAYAMA, Hajime FUJIO, Yoshitane DOHI, Yutaka TAKAGAKI, Kiyos ...1981 年 89 巻 3 号 p. 963-974
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーTwo derivatives of hen egg-white lysozyme (lysozyme) with single substitutions of a 2, 4-dinitrophenyl (DNP) residue were prepared. The reaction of lysozyme with a 10-fold molar excess of 2, 4-dinitrobenzene sulfonic acid provided one mono-DNP substituted lysozyme (DNP1-33lysozyme), which was purified by ion-exchange chromatographies. The other one (DNP1-96lysozyme) was prepared as follows. After maleylation of lysozyme in the presence of a 7-fold molar excess of maleic anhydride, the derivative with one free amino group was purified on DE-52. This material was dinitrophenylated with 2, 4-dinitrobenzene sulfonic acid and the mono-DNP substituted derivative was purified on DE-52. DNP1-96lysozyme was finally purified on SE-Sephadex C-25, after demaleylation at pH 3.5, at 37°C, for 5 days. DNP1-33lysozyme and DNP1-96lysozyme each migrated as a single band with slower mobility than that of native lysozyme. On reduction, carboxymethylation and chymotrypsin digestion, both mono-DNP substituted lysozymes yielded a single yellow peptide. The amino acid compositions or partial sequence of these peptides indicated that lysine-33 and lysine-96 were the only dinitrophenylated residues in DNP1-33lysozyme and DNP1-96lysozyme, respectively. DNP1-33lysozyme and DNP1-96lysozyme showed antigenic reactivities equal to that of native lysozyme with antisera to lysozyme. The DNP residues on the protein were accessible to anti-DNP antibodies, but the affinities of DNP1-33lysozyme to anti-DNP antibodies were lower than those of DNP1-96lysozyme. This result is discussed with respect to the local environments of the DNP residues in these proteins.抄録全体を表示PDF形式でダウンロード (2239K) -
Yutaka KIRINO, Ken-ichiro HIGASHI, Mikiko MATSUI, Hiroshi SHIMIZU1981 年 89 巻 3 号 p. 975-978
発行日: 1981年
公開日: 2008/11/18
ジャーナル フリーWhether or not the thermotropic change at about 18°C in the physical state of Ca2+-ATPase protein molecules of sarcoplasmic reticulum membranes could be transmitted to lipids through protein-lipid interactions was investigated using a spin-label technique. Fatty acid spin labels were used to probe the bulk membrane lipids while long-chain spin labels attached at one end to the Ca2+-ATPase molecules through a covalent bond were used to monitor the boundary lipids. The results on the temperature-dependence of alkyl-chain flexibility of lipid molecules indicate that the change in the state of the protein molecules is accompanied by one of the boundary lipids, but not of the bulk lipids.抄録全体を表示PDF形式でダウンロード (272K)
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