巻号一覧
81 巻, 2 号
選択された号の論文の31件中1～31を表示しています
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Takayoshi IIO, Koshin MIHASHI, Hiroshi KONDO1977 年 81 巻 2 号 p. 277-283
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe kinetics of the conformational change of troponin-C induced by binding or removal of protons was studied by a stopped-flow pH-jump spectrofluorometric method. In the pH-down experiment (to investigate the kinetics of conformational change from the deprotonated state to the protonated state), a single first-order reaction with a rate constant and amplitude of 1.75-2.4 sec-1 and around 10%, respectively, was observed. On the other hand, two firstorder reactions with rate constants of 0.84-1.6 sec-1 and 0.08-0.4 sec-1 were observed in the pH-up experiment, the total amplitudes of these reactions being around 10-20%. The pH dependences of the rate constants of these reactions were analyzed in terms of a three-species mechanism.抄録全体を表示PDF形式でダウンロード (476K) -
Effects of Hydrogen Ion, Potassium Chloride, and Protein ConcentrationsMichinori MIYAHARA, Haruhiko NODA1977 年 81 巻 2 号 p. 285-295
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe in vitro assembly of rabbit skeletal myosin was studied by flow birefringence. Filaments were obtained from a solution of myosin in 0.5M KCl by rapid dilution to lower ionic strength. In most cases, the filament length as determined from extinction angle measurements increased or decreased gradually for about 1h after dilution, depending on pH, KCl concentration and the previous history. The filament length (l) immediately after dilution also showed a marked dependence on pH, KCl concentration and protein concentration (c) at the moment of assembly. The general characteristics obtained from our limited study (0.04-6.0mg/ml) show three distinctive modes of effect of the protein concentration on the filament length: d logl/ d log c is positive (0.1-1) at small c, negative (from -1 to -0.2) at intermediate c, and zero or slightly positive (0.0-0.3) at large c. Lowering of the KCl concentration (75-250mM) as well as increase of the hydrogen ion concentration (pH 6-8) influenced the filament length in qualitatively the same manner as increase of the protein concentration. A model of the assembly reaction of myosin in which the polarity of filaments is crucial was constructed and shown to give qualitatively the experimental dependence of the filament length on the protein concentration.抄録全体を表示PDF形式でダウンロード (860K) -
Fumitsugu ISHIGAMI, Fumi MORITA1977 年 81 巻 2 号 p. 297-303
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe pH-activity curve of heavy meromyosin ATPase [EC 3. 6. 1. 3] was measured at various temperatures. The pH-activity curve at higher temperatures showed a maximum at low pH and a minimum at pH 7 to 8 as has been already reported. At lower temperatures it was sigmoidal in shape, similar to a simple dissociation curve of pKa 6 to 7. The pH-activity curve at intermediate temperatures appeared to be inbetween the two extreme shapes. These changes in pH-activity curve with temperature were found to be common in the presence of divalent cations such as Mg2+, Mn2+, and Ca2+. The ATPase mechanism may be identical in the presence of any divalent cation, and the rate determining step revealing the steady state rate alters by changing the temperature. The transition temperatures estimated at pH 8 were 10°, 8°, and about 5° in the presence of MnCl2, CaCl2, and MgCl2, respectively. The difference in the temperature coefficients above and below the transition temperature was most distinct in the presence of MnCl2, and vague in the presence of CaCl2. A similar change of pH-activity curve with temperature was found with heavy meromyosin ITPase in the presence of MgCl2.抄録全体を表示PDF形式でダウンロード (362K) -
Fumi MORITA, Fumitsugu ISHIGAMI1977 年 81 巻 2 号 p. 305-312
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe UV absorption difference spectrum of heavy meromyosin induced by ATP was measured at various temperatures. At higher temperatures, the difference spectrum formed rapidly after adding ATP and continued steadily during the steady state which we have called the ATP-form of difference spectrum. At lower temperatures, the ATP-form of difference spectrum decayed into the other form before the steady state was attained. This was identical to the difference spectrum obtained by adding ADP and has been called the ADP-form of difference spectrum. At intermediate temperatures, biphasic decay was observed. The results indicate that the dominant intermediate at the steady state is altered from the one showing the ATP-form of difference spectrum at higher temperatures to that showing the ADP-form at lower temperatures. The population of the two intermediates depends on the temperature between the two extremes. This temperature-induced transition was observed in the presence of any divalent cation such as Mg2+, Mn2+, or Ca2+. A similar transition was observed with the difference spectrum induced by ITP in the presence of MgCl2. The pH dependence of the single early decay of the ATP-induced difference spectrum was measured in the presence of MnCl2 at 1°. The apparent rate constant of the early decay showed a biphasic pH dependence, having the same shape as the pH activity curve of ATPase [EC 3. 6. 1. 3] observed at higher temperatures. The rate determining step for the steady state ATPase at higher temperatures is thought to be the step of changing from the intermediate complex showing the ATP-form of difference spectrum to that showing the ADP-form. This is inconsistent with our previous mechanism (Yazawa, M. et al. (1973) J. Biochem. 74, 1107-1117). The rate determining step at lower temperatures was assigned as a step of ADP dissociation.抄録全体を表示PDF形式でダウンロード (546K) -
Fumi MORITA1977 年 81 巻 2 号 p. 313-320
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe UV absorption difference spectrum of heavy meromyosin induced by adenylyl imidodi-phosphate (AMP-PNP) was found to be changed by temperature. At higher temperatures, the shape of the difference spectrum resembled the ATP-form of difference spectrum induced by ATP. At lower temperatures, a different shape was observed, resembling that induced by ADP. This temperature transition was found in the presence of both MgCl2 and MnCl2. The transition temperatures, were 21° and 9° in the presence of MnCl2 and MgCl2, respectively. A similar temperature dependence was observed with the difference spectrum induced by ATP at the steady state. The transition temperatures in this case were 11° and 4.5° in the presence of MnCl2 and MgCl2, respectively. The similarity of the effects of the two kinds of divalent cation on both transitions indicates that the temperature induced transition between two species of heavy meromyosin-AMP-PNP complex mimics the step in ATPase [EC 3. 6. 1. 3] reaction in which the intermediate complex showing the ATP-form of difference spectrum changes to that showing the ADP-form. The equilibrium constant of the decay step of the ATP-form of difference spectrum to the ADP-form in ATPase is, therefore, thought to be highly temperature dependent. Thermodynamic parameters were calculated for the transition between the two species of heavy meromyosin AMP-PNP complex. Large decreases in enthalpy and entropy were observed, while the standard free energy change was small. The results suggest that the intermediate showing the ATP-form of difference spectrum hardly changes to the forward direction in the ATPase reaction at higher temperature. The complex appears to be so stable in the steady state that almost all the myosin is present as this complex. The decay step in ATPase of the difference spectrum from the ATP-form to the ADP-form may be coupled to muscular contraction. The temperature induced transition of heavy meromyosin AMP-PNP complex may, therefore, provide information concerning the state of myosin in active muscles.抄録全体を表示PDF形式でダウンロード (573K) -
Yoshiaki NAKAMARU, Keizo NOMURA1977 年 81 巻 2 号 p. 321-328
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe interaction of Pi with sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was studied using bromocresol purple (BCP) as a probe and a dual-wavelength spectrophotometer. Two kinds of absorption-intensity changes controlled by a low concentration of Ca2+(>10-6M) were observed after addition of Pi; an increase phase (in the presence of Ca2+), and a decrease phase (in the presence of EGTA).
The increase phase was rapid, Ca2+-dependent, Mg2+-enhanced (depressed by high Mg2+ concentration) and not inhibited by PCMB and was suggested to reflect the formation of an SR. Pi complex.
The decrease phase was slower than the increase phase, and was strongly inhibited by the low concentration of Ca2+. It required Mg2+, and was completely inhibited by p-chloromercuribenzoate or deoxycholate. It was suggested to reflect the formation of SR-Pi (phosphorylated protein). ATP inhibited this phase by converting it completely to an SR•MgATP phase. PPi was effective for inducing the decrease phase but PPPi was not.
From measurements of these phases, the association constants of the SR•Pi complex and SR-Pi at pH 8.8 in the reaction scheme, SR+Pi_??_SR•Pi_??_SR-Pi, were calculated as 5.4×10 M-1 and 1.8×103M-1, respectively.
From the completely different responses of SR•Pi and SR-Pi observed with BCP a marked difference in the conformations of these enzyme states was suggested.抄録全体を表示PDF形式でダウンロード (555K) -
Tetsu HOZUMI1977 年 81 巻 2 号 p. 329-332
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーSuperprecipitation of an actomyosin suspension was measured at various temperatures (2.5°-20°) using Mg-ITP as substrate. Superprecipitation was induced by the addition of Mg-ITP at all temperatures, but decreased in extent with decrease in temperature. The predominant intermediate in the Mg-ITP hydrolysis of myosin depends on the temperature; at 20° it is the myosin*-IDP-Pi complex, while below 8° it is the myoisn-ITP complex (Hozumi, T. (1976) Eur. J. Biochem. 63, 241). Therefore, the occurrence of superprecipitation below 8° is not compatible with muscle models in which formation of a myosin*-product complex is the rate-limiting intermediate.抄録全体を表示PDF形式でダウンロード (248K) -
Yoshihiko URATANI, Makoto KAGEYAMA1977 年 81 巻 2 号 p. 333-341
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーAddition of pyocin R1, a bacteriocin of Pseudomonas aeruginosa, to sensitive cells caused a fluorescence increase of 8-anilino-1-naphthalenesulfonate (ANS) in the cell suspension. The reaction was rapid, starting with a short time lag after adsorption of pyocin onto the cells and finishing within several minutes. The fluorescence response was attributed to the interaction of the cell body and ANS, not to that of the medium outside the cells and ANS. The maximal amplitude of fluorescence after pyocin addition was dependent on temperature, and the relation appeared to be biphasic. Similarly, Arrhenius plots of the initial rate of fluorescence change were biphasic. The transition of slopes in both cases occurred in the temperature range between 18 and 19°. These results suggest that ANS interacts with lipids in the cell envelope and that pyocin causes a structural change of the cell envelope leading to increased fluorescence of ANS.抄録全体を表示PDF形式でダウンロード (715K) -
Eisuke NISHIDA, Takaaki KOBAYASHI1977 年 81 巻 2 号 p. 343-347
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーGuanine nucleotides bound to both the non-exchangeable sites (N sites) and exchangeable sites (E sites) of tubulin were completely released after 7 moles of SH groups per tubulin subunit (55, 000 molecular weight) had reacted with PCMPS. The blockage of 2 moles of SH groups in the glycerol-reassembly buffer or 1 mole of SH groups in glycerol-free reassembly buffer resulted in complete loss of tubulin polymerizability. However, under both sets of experimental conditions, the amount of guanine nucleotides released from the E sites was less than 8Y. and the loss of total guanine nucleotides was only 5%. Addition of GSH did not induce reassociation of released guanine nucleotides, although it restored tubulin polymerizability. These results indicate that the loss of tubulin polymerizability on blockage of the SH groups was not due to dissociation of bound guanine nucleotides and that the binding sites of the nucleotides were independent of the SH groups in tubulin required for polymerization. Fur-thermore, blockage of SH groups did not change the ratio of GTP to GDP bound to tubulin.抄録全体を表示PDF形式でダウンロード (358K) -
Yukio IKEHARA, Kimimitsu ODA, Keitaro KATO1977 年 81 巻 2 号 p. 349-354
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe acceptor activities of subcellular membrane preparations for the terminal sugars, galactose and sialic acid, were compared using a Golgi fraction purified from rat liver as an exogenous emzyme source for sugar transfer. Data are presented which strongly suggest that completion of carbohydrate chains of membrane glycoproteins and glycolipids occurs in the Golgi apparatus. Significant differences of acceptability of galactose and sialic acid were found between plasma membranes of rat liver and hepatoma cells (AH-130), indicating “incompleteness” of sugar chains in the latter.抄録全体を表示PDF形式でダウンロード (448K) -
Hiroyuki KONISHI, Teruo NAKAJIMA, Isamu SANO1977 年 81 巻 2 号 p. 355-360
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe metabolism of putrescine in rat brain was studied systematically by the intraventricular injection of radioactive diamine.
Putrescine injected into the brain was metabolized mainly to polyamines, spermidine, and spermine. A small portion of the radioactivity of putrescine was incorporated into γ-gluta-mylputrescine, putreanine, γ-aminobutyric acid, and homocarnosine.
Comparison of the specific radioactivities of γ-aminobutyric acid and homocarnosine after the injection of radioactive putrescine with those after the injection of radioactive glutamic acid indicated that there may be a metabolic pool of γ-aminobutyric acid (putrescine-γ-amino-butyric acid system) which is different from the glutamic acid-γ-aminobutyric acid system and which is effectively used for synthesis of the dipeptide.抄録全体を表示PDF形式でダウンロード (447K) -
Shigeru KUROOKA, Setsuko OKAMOTO, Masahisa HASHIMOTO1977 年 81 巻 2 号 p. 361-369
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーA new and simple colorimetric method for human serum lipase [EC 3. 1. 1. 3] assay has been developed, using 2, 3-dimercaptopropan-l-ol tributyroate as a substrate, 5, 5'-dithiobis(2-nitro-benzoic acid) as a chromogenic reagent, phenylmethylsulfonyl fluoride as an inhibitor of serum esterases, and sodium dodecylsulfate as a lipase activator. The method requires only 50 μl×2 of serum sample and a reaction time of less than 30min. The method is reproducible and sensitive enough to measure low levels of lipase activity in normal and abnormal sera. The gel filtration of serum samples on a Sephadex G-200 column gave one peak of lipase activity, when measured by the present method, and the molecular weight of the enzyme was identical with that of lipase of human pancreatic origin, confirming the specificity of this new method for the serum lipase.抄録全体を表示PDF形式でダウンロード (666K) -
Kunitada SHIMOTOHNO, Kin-ichiro MIURA1977 年 81 巻 2 号 p. 371-379
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーNucleoside triphosphate phosphohydrolase [EC 3. 6. 1. 15] activity was found to be included in silkworm cytoplasmic polyhedrosis (CP) virus, which synthesizes mRNA carrying the 5'-terminal modification. This enzyme releases orthophosphate from the γ-position in a nucleoside triphosphate, leaving nucleoside diphosphate. The rate of hydrolysis of ATP is faster than that of any other ribonucleoside triphosphate. Deoxy ATP is hydrolyzed rather faster than ATP. However, polynucleotides carrying triphosphate at the 5'-terminus, that is, 4S RNA which was synthesized by E. coli RNA polymerase [EC 2. 7. 7. 6] using calf thymus DNA as a template, and the phage Qβ RNA (30S), are not effective substrates for this enzyme. Although the CP virion loses the viral genome and one kind of protein component on proteolytic treatment with pronase, the partially degraded virion still retains phosphohydrolase activity. The phosphohydrolase must therefore be associated firmly with the virion. This enzyme does not require the presence of nucleic acid for its function. Phosphohydrolysis of ATP by this enzyme activity represents a first step in the synthesis of the 5'-terminal modified mRNA of CP virus.抄録全体を表示PDF形式でダウンロード (1811K) -
Hiroshi KUMAGAI, Kazuei IGARASHI, Masaru YOSHIKAWA, Seiyu HIROSE1977 年 81 巻 2 号 p. 381-388
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by E. coli ribonuclease I [ribonucleate 3'-oligonucleotidohydrolase, EC 3. 1. 4. 23] and ribonuclease II [EC 3. 1. 4. 1] have been studied. The degradation of poly(C) by RNase II was stimulated by spermine and spermidine, while that of poly(A) by RNase II was not affected by polyamines. Under our standard experimental conditions, the breakdown of poly(U) by RNase II was inhibited slightly by polyamines. The stimulatory effect of spermine and spermidine on the breakdown of poly(C) occurred in the absence of monovalent cations but not in the absence of divalent cations. When polyamines were used as a stimulant of RNase II, the ratio of poly(C) degradation to poly(U) degradation was greater in the presence of inhibitors such as poly(G) than in their absence.
Although the breakdown of all synthetic polynucleotides by RNase I was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)>poly(A)_??_poly(U). However, the difference in degree of stimulation among polynucleotides decreased as monovalent cation concentration was increased.抄録全体を表示PDF形式でダウンロード (498K) -
Kazuei IGARASHI, Hiroshi KUMAGAI, Hirofumi OGUCHI, Seiyu HIROSE1977 年 81 巻 2 号 p. 389-394
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U) ] by polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2. 7. 7. 8] from Micrococcus luteus have been studied. Although the breakdown of all the synthetic polynucleotides tested was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)>poly(A)>poly(U) at pH 7.5. However, the difference in degree of stimulation among polynucleotides decreased as the pH or monovalent cation concentration was increased. In the presence of heparin, an inhibitor of polynucleotide phosphorylase hydrolysis of polynucleotides, spermidine clearly stimulated the breakdown of poly(C) and poly(A), while the breakdown of poly(U) was stimulated only slightly by the addition of spermidine. Although binding of [14C]spermine to polynucleotide phosphorylase was observed by gel filtration, the amount of spermine bound to the enzyme was much less than that to RNA.抄録全体を表示PDF形式でダウンロード (416K) -
Kenji TAKAHASHI1977 年 81 巻 2 号 p. 395-402
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリー1. The reaction of phenylglyoxal (PGO), glyoxal (GO), and methylglyoxal (MGO) with amino acids were investigated at mild pH values at 25°. These aldehydes reacted most rapidly with arginine and the rate of reaction increased with increasing pH values. Histidine, cystine, glycine, tryptophan, asparagine, glutamine, and lysine reacted with these aldehydes at significant but various rates, depending on the pH and the kind of the reagent used. The reactions with these amino acids seemed to involve both the α-amino groups and the side chain groups, and no significant reaction appeared to occur with the side chain alone except with those of arginine, lysine, and cysteine. These reagents were similarly reactive with the guanidinium group of arginine, but PGO appeared to be much less reactive with the ε-amino group of lysine than MGO and GO. The other ordinary amino acids were very much less reactive or did not react at all with these reagents, with the exception of cysteine.
2. Di-PGO-L-arginine was prepared from Nα-benzyloxycarbonyl-L-arginine, and di-PGO-methylguanidine from methylguanidine, and the stoichiometry of the reaction of two PGO molecules with one guanidino group was confirmed. A glyoxal derivative of L-arginine (GO-arginine) was prepared by reaction of glyoxal with arginine. GO-arginine was fairly unstable, especially at higher pH values. A similar derivative (MGO-arginine) was also found to be formed by reaction of MGO with L-arginine, and was similarly unstable. These derivatives, however, did not regenerate arginine upon acid hydrolysis.抄録全体を表示PDF形式でダウンロード (595K) -
Kenji TAKAHASHI1977 年 81 巻 2 号 p. 403-414
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリー1. The reactivities of phenylglyoxal (PGO), glyoxal (GO), and/or methylglyoxal (MGO) with several proteins, including ribonuclease A [EC 3. 1. 4. 22] and its derivatives, α-chymotrypsin [EC 3. 4. 21. 1], trypsin [EC 3. 4. 21. 4], lysozyme [EC 3. 2. 1. 17], pepsin [EC 3. 4. 23. 1], rennin [EC 3. 4. 23. 4], thermolysin, and insulin and its B chain, have been examined. From analyses of the reaction products, PGO was shown to be the most specific for arginine residues. GO and MGO also reacted rapidly with arginine residues, but they also reacted with lysine residues to a significant extent. A side reaction with N-terminal α-amino groups was observed with each of these reagents.
2. Two arginine residues out of four in ribonuclease A, two out of three in α-chymotrypsin, one out of two in trypsin, one out of two in pepsin, and one out of five in rennin appeared to react with PGO fairly rapidly, indicating a difference in the relative accessibility of these residues by the reagent. Extensive modification of the arginine residues by PGO occurred with RCM-derivatives of ribonuclease A and insulin B chain. The N-terminal isoleucine residues of α-chymotrypsin and trypsin appeared to be unreactive with PGO because of salt bridge formation with an aspartyl residue. The activity of α-chymotrypsin toward N-benzoyl-L-tyrosine ethyl ester and the lytic activity of lysozyme were lost rapidly on treatment with PGO, as in the case of ribonuclease A. Pepsin and rennin were only partially inactivated by reaction with PGO.抄録全体を表示PDF形式でダウンロード (851K) -
XXII. Tryptic Cleavages of the Single Lysyl and Arginyl Bonds in Ribonuclease T1Kenji TAKAHASHI, Nobuo INOUE1977 年 81 巻 2 号 p. 415-421
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリー1. When ribonuclease T1 [EC 3. 1. 4. 8] was treated with trypsin [EC 3. 4. 21. 4] at pH 7.5 and 37°, activity was lost fairly slowly. At higher temperatures, however, the rate of inactivation was markedly accelerated. The half life of the activity was about 2.5 h at 50° and I h at 60°. 3'-GMP and guanosine protected the enzyme significantly from tryptic inactivation.
2. Upon tryptic digestion at 50°, the Lys-Tyr (41-42) and Arg-Val (77-78) bonds were cleaved fairly specifically, yielding two peptide fragments. One was a 36 residue peptide comprizing residues 42 to 77. The other was a 68 residue peptide composed of two peptide chains crosslinked by a disulfide bond between half-cystines -6 and -103, comprizing residues 1 to 41 and 78 to 104.
3. When the trinitrophenylated enzyme, in which the α-amino group of alanine-1 and the ε-amino group of lysine 41 were selectively modified, was treated with trypsin at 37°, the activity was lost fairly rapidly with a half life of about 4 h. In this case, tryptic hydrolysis occurred fairly selectively at the single Arg-Val bond. Thus the enzyme could be inactivated by cleavage of a single peptide bond in the molecule, an indication of the importance of the peptide region involving the single arginine residue at position 77 in the activity of ribonuclease T1.抄録全体を表示PDF形式でダウンロード (479K) -
VII. Distribution and Some Properties of Acid Proteases in Monkey TissuesKazuhiro SOGAWA, Kenji TAKAHASHI1977 年 81 巻 2 号 p. 423-429
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリー1. The distribution of acid protease activity in various tissues of Japanese monkey (Macaca fuscata fiascata) was investigated with hemoglobin as a substrate at pH 3.0. The activity per protein weight in crude extracts was highest in spleen and lung, and decreased in the order: spleen, lung>kidney, testis>brain>liver, placenta>thyroid gland, muscle. The activity in crude muscle extract was about one-tenth those of spleen and lung. The activity per wet tissue weight was in roughly the same order except for a lower activity per wet weight of brain.
2. Upon chromatography of each crude extract on a Sephadex G-100 column, one major activ-ity peak was eluted at a position corresponding to a molecular weight of about 41, 000. This enzyme activity is attributed to cathepsin D [EC 3. 4. 23. 5]. In addition, a minor activity peak was eluted in the case of spleen, lung and kidney at the break-through position, corresponding to a molecular weight of more than 100, 000. This activity peak is presumably due to cathepsin E. These acid protease activities were, in most cases, strongly inhibited by pepstatin, an acid protease-specific peptide inhibitor.
3. The distribution of acid protease activity was investigated in the brain of crab-eating monkey (Macaca fascicularis). The activity was fairly evenly distributed among several regions of the brain, and its distribution was similar to those of other acid hydrolases, especially N-acetyl-β-D-glucosaminidase [EC 3. 2. 1. 30] and acid phosphatase [EC 3. 1. 3. 2], which are marker enzymes of lysosomes.抄録全体を表示PDF形式でダウンロード (515K) -
I. Mass Spectrometric Method for Determination of the pKa of the β-146 Histidine Residue in Human HemoglobinMasato OHE, Akihiko KAJITA1977 年 81 巻 2 号 p. 431-434
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーA mass spectrometric method was developed to determine pH-dependent hydrogen-deuterium exchange at the C-2 position of the imidazole ring of histidine, after converting the amino acid to the methylthiohydantoin derivative. The amount of deuterium exchange in N-acetyl-histidine estimated by the present method was confirmed to be in good agreement with that determined by NMR spectrometry.
N-Acetylhistidine was deuterated at various pH's. From the amount of deuterium exchange, a pseudo-first order rate constant (Kφ, ) was calculated. A pKa value of 7.2 for the amino acid was obtained from the relation between Kφ and pH.
This method was applied to estimate the pKa, value of β-146 histidine in human hemoglobin. Human hemoglobin deuterated at various pH's was digested with carboxypeptidase A [EC 3. 4. 12. 2] to release the β-146 histidine. The amount of deuterium exchange in the isolated histidine was determined to obtain Kφ. From these measurements pKa values of 7.0 for the histidine in oxyhemoglobin and of 8.2 for that in deoxyhemoglobin were found at 36.5°, respectively.抄録全体を表示PDF形式でダウンロード (328K) -
Yukio KAWAMURA, Teruyoshi MATOBA, Tadao HATA, Etsushiro DOI1977 年 81 巻 2 号 p. 435-441
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe substrate specificities of two different molecular sizes of cathepsin A, A, L (large form) and A, S (small form), for synthetic substrates were examined kinetically. Both enzymes showed a similar broad substrate specificity against various acyl dipeptides, amino acid esters, and amino acid amides. Z-Phe-Ala and Ac-Phe-OEt were good substrates. Peptides containing hydrophobic amino acids were hydrolyzed rapidly. The presence of hydrophobic amino acid residues, not only at the C-terminal position but also at the second position and probably the third position from the C-terminal, resulted in an increase in the rate of hydrolysis. Peptides containing glycine and proline were hydrolyzed slowly.
Inhibition studies with Z-D-Phe-D-Ala and Z-Phe suggested that the peptidase and esterase activities of the enzymes are both catalyzed by the same site of the enzyme molecule, but it remains to be elucidated whether or not the binding sites for peptides and esters are the same.抄録全体を表示PDF形式でダウンロード (445K) -
Jun-ichi KURISAKI, Kunio YAMAUCHI1977 年 81 巻 2 号 p. 443-449
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe selective solubilization of apo-very low density lipoprotein (apoVLDL) of hen's egg yolk was achieved from intact VLDL with guanidine hydrochloride (GuHCl) or urea. The amount of extracted apoVLDL increased with increase of the reagent concentration. GuHCl was more effective than urea and more than 60%, of apoVLDL was solubilized with 6M GuHCl.
Previously we reported the presence of five major apoVLDL components, GPI, ApoA, GPII, ApoB, and ApoC in order of size, and found that GPI and GPII were periodic acid-Schiff staining positive, while ApoA, ApoB, and ApoC were negative. With GuHCl or urea, GPI and GPII were easily solubilized, while ApoA and ApoB could not be extracted. The solubilized apoVLDL was rich in carbohydrates, especially sialic acid, compared with the residual apoVLDL. However, only slight differences in amino acid composition were found between the soluble and the residual apoVLDL.
After the partial removal of apoVLDL with GuHCl or urea, VLDL retained its particulate nature, and no destruction of the lipid core was observed.
These results were interpreted as indicating that the release of apoVLDL with GuHCl or urea occurred from the surface of the VLDL particle and that the selectively solubilized apo-VLDL fractions, such as GPI and GPII, were weakly bound to lipids on the surface of VLDL, while ApoA and ApoB were tightly associated with the VLDL particle.抄録全体を表示PDF形式でダウンロード (2930K) -
Yoko HAYASHI, Ryo KOMINAMI, Masami MURAMATSU1977 年 81 巻 2 号 p. 451-459
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーThe synthesis and incorporation into ribosomes of 5S RNA were investigated under conditions of protein synthesis inhibition by cycloheximide. Within 1 h after addition of cycloheximide to the culture medium, the synthesis of 5S RNA in HeLa cells, as measured by the pulse labeling of total 5S RNA, was inhibited by 25-30 per cent, but the degree of inhibition did not further increase for several hours. The synthesis of 4S RNA was much less markedly inhibited, especially at earlier time periods.
Although the appearance of labeled 5S RNA in cytoplasmic ribosomes after chase was inhibited by approximately 60% or more, this presumably reflects the inhibition of 60S ribo-somal subunit formation by cycloheximide. Some utilization of preformed 5S RNA for new ribosomes was suggested after resumption of protein synthesis.
In confirmation of the results of Kumar and Wu (1), a low dose of actinomycin D inhibited the appearance of 28S RNA in the cytoplasm rather specifically, whereas cycloheximide showed an apparently opposite differential effect.抄録全体を表示PDF形式でダウンロード (1199K) -
II Effects of Additions of Amino Acids and SerumYasuhide YAMASAKI, Akira ICHIHARA1977 年 81 巻 2 号 p. 461-465
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーInduction of ornithine decarboxylase [EC 4. 1. 1. 17] (ODC) in mouse L cells by components of the culture medium was investigated. It was found that further addition of amino acid mixture, but not of calf serum, to confluent cells of 5 day culture induced ODC and that this induction was accelerated by actinomycin D. In salt solution, addition of either amino acids or serum alone did not cause full induction, but addition of both together did. This induction, in contrast, was inhibited by actinomycin D. Induction by insulin, but not by cyclic AMP, was enhanced by a higher concentration of amino acids.
These results can be explained by supposing that in non-growing cells there is stable RNA which is involved in ODC induction, possibly mRNA of ODC, and that the observed induction is caused by inhibition of enzyme degradation and accelerated translation, while in growing cells this RNA is unstable and ODC induction is controlled at the level of transcription.抄録全体を表示PDF形式でダウンロード (367K) -
I. Purification and PropertiesKinuko KIMURA, Atsuo MIYAKAWA, Takeo IMAI, Taiji SASAKAWA1977 年 81 巻 2 号 p. 467-476
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーBacillus subtilis PCI 219 has a single glutamate dehydrogenase (GDH) [EC 1. 4. 1. 3] with dual coenzyme specificity [for NAD(H) and NADP(H)]. The enzyme was purified 800-fold from crude extracts of B. subtilis from the post-exponential phase of growth and showed one significant protein band on gel electrophoresis. This band was determined, by activity staining, to have all the GDH nucleotide specificities. Its molecular weight was estimated to be 250, 000±20, 000 by gel filtration, and 270, 000±30, 000 by zone centrifugation in a sucrose density gradient. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that GDH has a subunit size of about 57, 000. The pI of GDH was found to be pH 3.7 by isoelectric focusing. GDH exhibited nonlinear kinetics in the reduction of NAD+, and in the reverse direction, the substrate, NH4+, was strongly inhibitory at high concentrations. Purine nucleotides did not affect the activity. The oxidative deamination of glutamate was significantly inhibited by the metabolites oxaloacetate and citrate, which acted as allosteric effectors of this enzyme, inhibiting the reaction in one direction. The pH optimum of each of the activities of GDH and the stability of GDH are also reported.抄録全体を表示PDF形式でダウンロード (1244K) -
Noriko TOMINAGA, Takeshi MORI1977 年 81 巻 2 号 p. 477-483
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーAn inorganic pyrophosphatase [EC 3. 6. 1. 1] was isolated from Thiobacillus thiooxidans and purified 975-fold to a state of apparent homogeneity. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate and no activity was found with a variety of other phosphate esters.
The cation Mg2+ was required for maximum activity; Co2+ and Mn2+ supported 25 per cent and 10.6 per cent of the activity with Mg2+, respectively. The pH optimum was 8.8.
The molecular weight was estimated to be 88, 000 by gel filtration and SDS gel electrophoresis, and the enzyme consisted of four identical subunits. The isoelectric point was found to be 5.05. The enzyme was exceptionally heat-stable in the presence of 0.01M Mg2+.抄録全体を表示PDF形式でダウンロード (473K) -
Masaaki YAMADA, Eiichi HASEGAWA, Masao KANAMORI1977 年 81 巻 2 号 p. 485-494
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーHyaluronidase [EC 3. 2. 1. 35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7×104 based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30°, but was almost completely inactivated at 60° within 30min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18×10-1 mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skin of a rabbit.抄録全体を表示PDF形式でダウンロード (1195K) -
Yutaka ORII, Noriaki WASHIO1977 年 81 巻 2 号 p. 495-503
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーChlorocruorin was purified from Potamilla leptochaeta and the spectral properties of its derivatives were investigated. Ferri- or ferrochlorocruorin did not exhibit a ferrihemochrome or ferrohemochrome spectrum, respectively. Oxy- and carbonmonoxy-ferrochlorocruorin did show ferrohemochrome-type spectra. Ferrihemochromes were formed, however, when oxyor ferrichlorocruorin was treated with 0.02-0.05% SDS, and they were transformed to ferrohemochromes by reduction with sodium dithionite. Ferrihemochrome formation was also brought about by increasing the pH of a ferrichlorocruorin solution to 9, or by liganding of extrinsic imidazole or cyanide to the ferric pigment. Therefore, it is apparent that at least one of the coordination positions on the heme iron in ferri- and ferrochlorocruorin is vacant or occupied by a weak-field ligand.
Titration studies of ferrichlorocruorin with imidazole indicated that this supposedly vacant coordination position was occupied first by the imidazole, and that the intrinsic ligand of protein origin was replaced finally at higher concentrations. The extrinsic ligands in the cyanide and imidazole complexes of ferrichlorocruorin were excluded from their coordination positions as the protein moiety assumed conformations inherent to the reduced pigment. Spectral analyses indicated that the intrinsic ligand is an imidazole moiety of a histidyl residue.
When chlorocruorin was intact, carbonyl reagents such as cyanide and sodium bisulfite did not add to the formyl group of chlorocruoroheme. When the protein conformation was perturbed by SDS, addition to ferrichlorocruorin occurred appreciably. This addition was accelerated if the heme iron coordination position had been occupied by strong field ligands, and was reversed to some extent as the chlorocruorin complexes were reduced.抄録全体を表示PDF形式でダウンロード (673K) -
Yutaka ORII, Masao MANABE, Machiko YONEDA1977 年 81 巻 2 号 p. 505-517
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーIn dimeric cytochrome oxidase [EC 1. 9. 3. 1], one of the two heme a molecules of one monomeric unit has been proposed to be converted by the other unit, thus becoming latent in terms of catalytic functions (1). As the dimer was split into two monomers by treatment with alkali or sodium dodecyl sulfate (SDS), it was shown that the intensity of circular dichroism (CD) in the Soret region due to heme a decreased, probably reflecting release of the strain on the latent heme. On the other hand, the profile of magnetic circular dichroism (MCD) was nearly unchanged during this conversion, except for a weakening of the signal due to deprotonation of the heme during the alkali treatment.
When the monomer was further dissociated into constituent subunits in strong alkali or at high concentrations of SDS, the CD spectrum disappeared almost completely, indicating loss of the asymmetric interactions of the chromophoric heme a with its immediate environments, consisting of the subunit assembly. The MCD pattern also suffered a small change as the dissociation proceeded, and a specific pattern appeared as the Schiff base was finally formed.
The Schiff base formation of cytochrome oxidase in strong alkali proceeded in two steps whether the heme iron was in the oxidized or reduced state. As a consequence of the initial rapid reaction, the enzyme was suggested to have been disintegrated into constituent subunits with heme a being attached nonspecifically to either one, and structural characteristics dependent on the redox state were completely lost. The Arrehenius plot for this rapid change showed a break, indicating a transition in the structure of the cytochrome oxidase assembly, although no such phenomenon was observed during the slow reaction. Activation parameters in the rapid and slow reactions for the oxidized and reduced oxidase are given.
Based on these findings, as well as other considerations, a molecular architecture of this enzyme is proposed; the role of heme a in anchoring four 14, 000-dalton polypeptides into the minimal functional unit catalyzing the aerobic oxidation of ferrocytochrome c is emphasized.抄録全体を表示PDF形式でダウンロード (945K) -
Nobuhito SONE, Masasuke YOSHIDA, Hajime HIRATA, Yasuo KAGAWA1977 年 81 巻 2 号 p. 519-528
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリー1. A stable ATPase [EC 3. 6. 1. 3] complex (TF0•F1) from the thermophilic bacterium PS3 was reconstituted into vesicles capable of energy transformation, measured as ATP-dependent enhancement of fluorescence of 8-anilinonaphthalene-l-sulfonate.
2. The factors necessary for obtaining highly active vesicles were investigated. Cholate and deoxycholate were both required for solubilization of TF0•F1 and P-lipids, and removal of the detergents by dialysis resulted in vesicle formation. Medium of around pH 8 and low ionic strength containing 2.5mM MgSO4 was found suitable for dialysis. The optimal temperature for reconstitution was 30° with soybean P-lipids and 45° with PS3 P-lipids. The optimal ratio of protein to lipid was about 1/50.
3. The vesicles obtained under these conditions were mainly 100-200nm in diameter, covered with 9.5nm spheres, and had a bouyant density of 1.06 in sucrose and an internal volume of about 0.5 μl per mg of P-lipids.抄録全体を表示PDF形式でダウンロード (1312K) -
Keizo OGAWA, Tomitake TSUKIHARA, Hiromasa TAHARA, Yukiteru KATSUBE, Yo ...1977 年 81 巻 2 号 p. 529-531
発行日: 1977/02/25
公開日: 2008/11/18
ジャーナル フリーA chloroplast-type ferredoxin containing two non-heme iron and two labile sulfur atoms per molecule was prepared from Spirulina platensis. The protein crystallized in the orthorhombic system with cell dimension a=62.32, b=28.51, and c=108.09A. The space group is C2221 and one asymmetric unit contains one molecule.
The electron density maps at 5A and 3.5A resolutions were synthesized utilizing the best phase angles calculated by the single isomorphous method coupled with the anomalous dispersion method. The difference Fourier synthesis with the anomalous scattering difference of the native data showed the location of the iron atoms clearly. Comparing the location of the iron atoms with the best phase angle electron density map, it was concluded that the active center of the present molecule is close to the surface of the molecule.抄録全体を表示PDF形式でダウンロード (185K)
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