The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
80 巻 , 3 号
選択された号の論文の30件中1~30を表示しています
  • Seiki KURAMITSU, Yongsok YANG, Kiyoshi IKEDA, Kozo HAMAGUCHI
    1976 年 80 巻 3 号 p. 417-423
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The interactions of deoxy derivatives of GlcNAc, 6-deoxy-GlcNAc, and 3-deoxy-GlcNAc with hen egg-white lysozyme [EC 3.2.1. 17] were studied at various pH's by meas-uring the changes in the circular dichroic (CD) band at 295 nm. It was shown that 6-deoxy-GIcNAc and 3-deoxy-G1cNAc bind at subsite C of lysozyme and compete with GIcNAc. The pH dependence of the binding constant of 6-deoxy-G1cNAc was the same as that of GlcNAc. On the other hand, the binding constants of 3-deoxy-GlcNAc were 3-10 times smaller than those of GlcNAc in the pH range from 3 to 9. X-ray crystallographic studies show that O(6) and O(3) of G1cNAc at subsite C are hydrogen-bonded to the indole NH's of Trp 62 and Trp 63, respectively, but the above results indicate that Trp 63, not Trp 62, is important for the interaction of GlcNAc with lysozyme.
  • Yongsok YANG, Seiki KURAMITSU, Yoshiko NAKAE, Kiyoshi IKEDA, Kozo HAMA ...
    1976 年 80 巻 3 号 p. 425-434
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The binding constants of α- and β-GlcNAc to hen and turkey lysozymes [EC 3.2.1.17] were determined at various pH's using the method proposed by Ikeda and Hama-guchi ((1975) J. Biochem. 77, 1-16). The pH dependence of the binding of β-GlcNAc to hen lysozyme was essentially the same as that for turkey lysozyme. The pH dependence curves of the binding constants of β-GlcNAc to hen and turkey lysozymes were interpreted in terms of the participation of Glu 35 (pK 6.0), Asp 52 (pK 3.5), Asp 48 (pK 4.5), and Asp 66 (pK 1.5). The binding constants of α-GlcNAc to hen and turkey lysozymes were the same below pH 3.5 but were different above this pH. The main participant residues in the binding of α-GlcNAc were Glu 35, Asp 48, and Asp 66 for hen lysozyme and Glu 35 and Asp 66 for turkey lysozyme. The results obtained here were well explained by the following assumptions: (1) above about pH 4, α-GlcNAc binds to hen lysozyme in both α- and β-modes, which correspond to the binding orientation of α-GlcNAc and that of β-GlcNAc, respectively, as deter-mined by X-ray crystallographic studies, but it binds predominantly in the β-mode below about pH 4, (2) β-GlcNAc binds to hen and turkey lysozymes predominantly in the β-mode above about pH 4 and in both α- and β-modes below pH 4, and (3) α-GlCNAc binds to turkey lysozyme predominantly in the β-mode over the whole pH rangse studied.
  • Yoshiko NAKAE, Kiyoshi IKEDA, Kozo HAMAGUCHI
    1976 年 80 巻 3 号 p. 435-447
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The interactions of the substrate analogs β-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which an ester linkage had been formed between Glu 35 and Trp 108 (108 ester lysozyme), were studied by the circular dichroic and fluorescence techniques, and were compared with those for intact lyso-zyme. The binding constants of β-methyl-GlcNAc and (GlcNAc)2 to 108 ester lyso-zyme were essentially the same as those for intact lysozyme in the pH range of 1 to 5. Above pH 5, the binding constants of these saccharides to 108 ester lyso-zyme did not change with pH, while the binding constants to intact lysozyme de-creased. This indicates that Glu 35 (pK 6.0 in intact lysozyme) participates in the binding of these saccharides. The extent and direction of the pK shifts of Asp 52 (pK 3.5), Asp 48 (pK 4.4), and Asp 66 (pK 1.3) observed when β-methyl-GlcNAc is bound to 108 ester lysozyme were the same as those for intact lysozyme. The par-ticipation of Asp 101 and Asp 66 in the binding of (GlcNAc)2 to 108 ester lysozyme was also the same as that for intact lysozyme. These findings indicate that the conformations of subsites B and C are not changed by the formation of the ester linkage. On the other hand, the binding constants of (GlcNAc)3 to 108 ester lyso-zyme were higher than those for intact lysozyme at all pH values studied. This result is interpreted in terms of an increase in the affinity for a GlcNAc residue of subsite D, which is situated near the esterified Glu 35.
  • Chiaki YOSHIDA, Mitsuyoshi YOSHIKAWA, Toshio TAKAGI
    1976 年 80 巻 3 号 p. 449-454
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The trypsin inhibitor of adzuki (Phaseolus angularis) beans shows a CD spectrum with a negative extremum at 280nm and a positive shoulder around 245nm. Since the inhibitor lacks tryptophan and tyrosine, the observed CD spectrum can be attrib-uted to the six disulfide groups in the molecule. The CD features completely disap-peared on reduction of the disulfide groups, and converged into a single negative extremum at 270nm when the groups were modified to form mixed disulfides with glutathione. These observations of the CD properties of the inhibitor strongly suggest the presence of disulfide groups constrained with respect to their dihedral angles.
  • Circular Dichroism and Fluorescence Studies
    Nobuo OKABE, Noriko MANABE, Ryozi TOKUOKA, Ken-ichi TOMITA
    1976 年 80 巻 3 号 p. 455-461
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    As a model study to investigate the binding mechanism between thyroid hormones and carrier protein, the interaction of diiodo-L-tyrosine (DIT) and triiodophenol (I3φ) with bovine serum albumin (BSA) was investigated by circular dichroism (CD) and fluorescence methods.
    In both the DIT-BSA system and the I3φ-BSA system, induced Cotton effect was observed in the wavelength region near 320nm. This induced Cotton effect was measured at various molar ratios of ligands to BSA (L/P). The value of the ellipticity at 319 nm, [θ]319, in the I3φ-BSA system was remarkably large compared with that of the DIT-BSA system, and [θ]319 at an L/P ratio of one was -1.96X 104 (degree cm2 decimole-1) for the I3φ-BSA system and -0.1×104 for the DIT-BSA system.
    The binding constants for the combination of BSA with a single molecule of ligand, calculated by measuring the quenching of the fluorescence of the protein, were 1.33×105M-1 at 15° for the DIT-BSA system and 1.6×109M-1 at 28° for the I3φ-BSA system.
    These results suggest that the binding of I3φ to BSA is stronger than that of DIT and a cleft may exist more congruent with the molecular dimensions of I3φ than with those of DIT.
  • VI. Binding of Cations to Transfer RNA and Its Role in Aminoacyl Transfer RNA Formation
    Yoshifumi TAKEDA, Takeo OHNISHI, Yuko OGISO
    1976 年 80 巻 3 号 p. 463-469
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The role of cations (polyamines and M2+) in isoleucyl-tRNA formation catalyzed by purified isoleucyl-tRNA synthetase [EC 6.1.1.5] from Escherichia coli was studied. It was found that spermine, spermidine, and Mg2+ bind to tRNA and that when bound to these cations, tRNA acts as substrate of aminoacylation without requiring further cations. These findings suggest that the primary function of cations in aminoacyl-tRNA formation is to bind to tRNA to stablize its structure, not to bind to the enzyme to activate it.
  • VII. Lack of Correlation between Aminoacylation and PPi-ATP Exchange Catalyzed by Isoleucyl-tRNA Synthetase of Escherichia coli in the Presence of Various Divalent Cations
    Yoshifumi TAKEDA, Takeo OHNISHI, Yuko OGISO
    1976 年 80 巻 3 号 p. 471-475
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Isoleucyl-tRNA formation and isoleucine-dependent PP1-ATP exchange catalyzed by purified isoleucyl-tRNA synthetase [EC 6.1.1.5] of Escherichia coli were studied in the presence of various amounts of either Mg2+, Ca2+, Fe2+, Ni2+, or Cu2+. In the presence of Mg2+, isoleucine-dependent PP1-ATP exchange was observed in parallel with isoleucyl-tRNA formation, while in the presence of Ca2+, isoleucyl-tRNA for-mation was observed without isoleucine-dependent PP, -ATP exchange. Moreover, isoleucine-dependent PPi-ATP exchange was much more in the presence of Fe2+ than in the presence of Mg2+, while little isoleucyl-tRNA was formed in the presence of Fe2+. In the presence of Ni2+ or Cu2+, neither reaction was observed.
    These data, indicating that formation of an isoleucyl-AMP-enzyme complex is not a necessary step in isoleucyl-tRNA formation, support the existence of a concerted mechanism of isoleucyl-tRNA formation in E. coli.
  • Genichiro OSHIMA, Kinzo NAGASAWA, Jyoji KATO
    1976 年 80 巻 3 号 p. 477-483
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Angiotensin I-converting enzyme [EC 3.4.15.1] was rapidly and highly purified from a particulate fraction of hog kidney cortex with 13% yield. The procedure, which was rapid, included fractionation on DEAE-cellulose and calcium phosphate gel, chromatographies on DEAE-Sephadex A-50 and hydroxylapatite columns, and gel filtration on a Sephadex G-200 column. The purified enzyme preparation gave two protein bands on standard disc gel electrophoresis, but showed a single protein component on the gel after treatment with neuraminidase [EC 3.2.1.18]. The data strongly suggest that the purified enzyme preparation was a mixture of sialo- and asialo-enzyme. Sialic acid residues apparently do not contribute to the catalytic activity of the enzyme.
    The enzyme was activated more by chloride ions than by other halide ions tested, using Bz-Gly-Gly-Gly as a substrate. The dissociation constant for chloride ions was determined to be 2.2mM. Chloride did not protect the enzyme against heat or low pH. The enzyme was resistant to inactivation by trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1].
  • Namiko MURAMATSU, Yoshiaki NOSOH
    1976 年 80 巻 3 号 p. 485-490
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Threonine deaminase [EC 4.2.1.16] was highly purified from Bacillus stearothermophilus. The enzyme exhibited maximum activity at 65° and at pH 9.2-9.6. It was inactivated on dilution and on storage at 4°, but was protected by egg albumin. The enzyme was labile at 65°, but became stable in the presence of egg albumin and isoleucine at pH 7.0. The substrate saturation curve for the enzyme reaction at 40 or 65° was hyperbolic, but in the presence of isoleucine, the curve became sigmoidal (n=2). The enzyme was more sensitive to isoleucine at 40° than at 65°, while valine slightly inhibited the enzyme at both 40 and 65°. Inhibition of the enzyme by isoleucine was antagonized by valine at 40 and 65°. These properties were essentially similar to those of the enzymes from mesophilic and thermophilic bacteria. The enzyme existed in two forms with different molecular sizes, 1.5-5×106 and 2×105 daltons, at pH 7.0 and at temperatures below 40°. The larger component disaggregated into the small one at pH 8.5 or above, at temperatures above 50° or in the presence of isoleucine and valine.
  • Kazumitsu HANAI
    1976 年 80 巻 3 号 p. 491-495
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The interaction of chymotrypsinogen A with benzeneboronic acid (BBA), a transition state analog inhibitor of serine proteases, was investigated by the temperature-jump method using pH indicators. It was found that 1/τ is dependent on BBA concen-tration, in contrast to the case of the a-chymotrypsin [EC 3.4.21.1]-BBA system in which 1/τ is independent of BBA concentration. By examination of the pH depend-ences of the kinetic parameters, the acid dissociation behavior of His 57 in chymotry-psinogen, chymotrypsinogen-trigonal BBA complex and chymotrypsinogen-tetrahedral BBA complex was analyzed. The kinetic deuterium isotope effect was also examined and found to occur principally on the acid dissociation constants. The state of the catalytic residues in the zymogen molecule is discussed based on these results.
  • V. Comparative Studies on the Specific Inhibition of Acid Proteases by Diazoacetyl-DL-norleucine Methyl Ester, 1, 2-Epoxy-3-(p-nitrophenoxy) propane and Pepstatin
    Kenji TAKAHASHI, Wen-Jong CHANG
    1976 年 80 巻 3 号 p. 497-506
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Comparative studies have been made on the effects of diazoacetyl-DL-norleucine methyl ester (DAN), 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and pepstatin on acid proteases, including those from Acrocylindrium sp., Aspergillus niger, Aspergillus saitoi, Mucor pusillus, Paecilomyces varioti, Rhizopus chinensis, and Trametes sanguinea, and also porcine pepsin [EC 3.4.23. 1] and calf rennin [EC 3.4.23.4] for comparative purposes.
    These enzymes were rapidly inactivated at similar rates and in 1:1 stoichiometry by reaction with DAN in the presence of cupric ions. The pH profiles of inactivation of these enzymes were similar and had optima at pH 5.5 to 6. They were also inactivated at similar rates by reaction with EPNP, with concomitant incorporation of nearly 2 EPNP molecules per molecule of enzyme. The pH profiles of inactivation were again similar and maximal inactivation was observed at around pH 3 to 4. Some of the EPNP-inactivated enzymes were treated with DAN and shown still to retain reactivity toward DAN. All these enzymes were inhibited strongly by pepstatin, and the reactions of DAN and EPNP with them were also markedly inhibited by prior treatment with pepstatin.
    These results indicate that the active sites of these enzymes are quite similar and that they presumably have at least two essential carboxyl groups at the active site in common, one reactive with DAN in the presence of cupric ions and the other reactive with EPNP, as has already been demonstrated for porcine pepsin and calf rennin. Pepstatin appears to bind at least part of the active site of each enzyme in a simmilar manner.
  • Yoshiyuki TAMURA, Setsuro FUJII
    1976 年 80 巻 3 号 p. 507-511
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    L-Homoarginine benzylester was coupled with Sepharose 4B and used for affinity chromatography of human plasma kallikrein [EC 3.4.21.8] and urokinase [EC 3.4.99.26]. The euglobulin fraction of human plasma was treated with disodium ethylenediamine-tetraacetate (EDTA) and activated with acetone, and then kallikrein was purified 16-fold by affinity chromatography. The yield of kallikrein activity on affinity chromatog-raphy was 53%. The purified preparation of human plasma kallikrein appeared to be homogenous on gel electrophoresis. Commercial urokinase was also purified 4.8-fold with 64% yield by affinity chromatography.
  • Isolation and Characterization
    Sadako INOUE, Mariko IWASAKI
    1976 年 80 巻 3 号 p. 513-524
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Dermatan sulfate-chondroitin sulfate copolymers have been isolated from human umbilical cord as a major galactosaminoglycan component of this tissue. The galactos-aminoglycan fraction was obtained from this tissue by papain [EC 3.4.22.2] digestion followed by precipitation with cetylpyridinium chloride in a yield of 700 mg per 100 g of dry tissue. Ethanol fractionation resolved 4-5 subfractions differing in relative content of L-iduronic acid and D-glucuronic acid. No galactosaminoglycan containing either solely L-iduronic acid or D-glucuronic acid was obtained. The copolymeric structure of the material in each subfraction was demonstrated by analysis of oligosaccharide fragments obtained by chondroitinase-AC [EC 4.2.2.5] digestion. All the polymers contained repeating disaccharide units, D-glucuronosyl-N-acetylgal-actosamine, D-glucuronosyl-N-acetylgalactosamine 4-sulfate, D-glucuronosyl-N-acetyl-galactosamine 6-sulfate, and L-iduronosyl-N-acetylgalactosamine 4-sulfate, of which D-glucuronosyl-N-acetylgalactosamine 6-sulfate and L-iduronosyl-N-acetylgalactosamine 4-sulfate were predominant. Both iduronic acid- and glucuronic acid-containing units were arranged in clusters. The presence of a considerable amount of nonsulfated disaccharide units was noted. The copolymers show extensive polydispersity in elec-trophoresis on cellulose acetate and gel chromatography on Sephadex G-200.
  • Takeshi KOMURA, Setsu WADA, Hideo NAGAYAMA
    1976 年 80 巻 3 号 p. 525-529
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    1. Retinol exerted a remarkable stimulating effect (approx. 260% increase), essentially similar to that (300%0) of phytol, on the so-called esterase activity displayed by crude pancreatic lipase [EC 3.1.1.3] toward true solutions of esters, but none on the typical lipase activity toward emulsions of water-insoluble esters.
    2. Comparison of the stimulatory effects of retinol derivatives on the esterase activity revealed that retinyl acetate was the most active, being substantially similar in effect to retinol; retinal was fairly active, while retinoic acid, retinyl palmitate, andβ-ionone were far less active.
    3. With various isoprenoid compounds, the efficiency of stimulation increased with the carbon chain length, attaining a maximum at 15 to 20 carbon atoms. Above this chain length the efficiency decreased rapidly.
    4. Comparison of the effects of retinol and phytol on the esterase activity of various other lipolytic enzymes indicated that this kind of activator may be relatively specific to porcine pancreatic esterase activity.
  • Shin HASEGAWA, Masato TAMARI, Masao KAMETAKA
    1976 年 80 巻 3 号 p. 531-535
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Diacylglyceryi-2-aminoethyiphosphonate was isolated from bovine liver by a combination of silicic acid column and silica gel thin-layer chromatographic techniques, and was identified from the results of elementary analysis, the infrared spectrum, chemical properties, and chromatographic behavior. This is the first isolation of a lipid-bound form of ciliatine in mammals.
  • Michio OGURA, Yoshikazu AYAKI, Midori GOTO
    1976 年 80 巻 3 号 p. 537-545
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    In vivo and in vitro experiments with rats were carried out to investigate the cholesterol pool in subcellular organelles of the liver that acts as the immediate precursor for the biosynthesis of cholic acid.
    When rats with a bile-fistula were given a mixture of [2-14C]mevalonate and [1, 2-3H]cholesterol intravenously, the 14C:3H ratio in cholic acid in both whole homogenate and cytosol prepared from their livers was higher than that in free cholesterol in any subcellular fraction of the livers. When [2-14C] mevalonate was administered intravenously to bile-fistula rats, the specific radioactivity of free cho-lesterol in the hepatic microsomal fraction exceeded that in any other fraction, and the specific radioactivity of biliary cholic acid was remarkably high, exceeding that of microsomal free cholesterol. In similar experiments with [4-14C]cholesterol, the specific radioactivity of free cholesterol in the hepatic microsomal fraction exceeded that in any other subcellular fraction and the specific radioactivity of biliary cholic acid was lower than that of free cholesterol in any hepatic subcellular fraction. Tissue suspensions of rat livers in Krebs-Ringer bicarbonate (pH 7.4)-5.5mm glucose were incubated with [2-14C]mevalonate in O2-CO2 (95:5, v/v) at 37°. The specific radioactivity of free cholesterol in the microsomal fraction prepared from the incu-bated tissue exceeded the specific radioactivities of free cholesterol in the other subcellular fractions. The estimated specific radioactivity of taurocholate formed during the incubation was far higher than that of microsomal free cholesterol.
    These data indicate that hepatic microsomal free cholesterol which was newly synthesized in situ was preferentially incorporated into cholic acid.
  • Mitsuaki SAKODA, Keitaro HIROMI
    1976 年 80 巻 3 号 p. 547-555
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The best-fit values of the Michaelis constant (Km) and the maximum velocity (V) in the Michaelis-Menten equation can be obtained by the method of least squares with the Taylor expansion for the sum of squares of the absolute residual, i.e, the difference between the observed velocity and the corresponding velocity by calculation. This method makes it possible to determine the values of Km and V not in a trial-and-error manner but in a deductive and unique manner after some iterative procedures starting from arbitrary approximate values of Km and V. These values can be said to be uniquely determined for a set of data as the finally converged values are no longer dependent upon the initial approximate values of Km and V.
    It is also very important to obtain initial approximate values of parameters for the application of the method described above. A simple method is proposed to estimate the approximate values of parameters involved in fractional functions. The method of rearrangement after canceling the denominator of a fractional function can be utilized to obtain approximate values, not only for cases of two unknown parameters such as the Michaelis-Menten equation, but also for cases with more than two unknowns.
  • I. Effects of Cellular Growth, Hormones, and Actinomycin D
    Yasuhide YAMASAKI, Akira ICHIHARA
    1976 年 80 巻 3 号 p. 557-562
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The activity of ornithine decarboxylase [EC 4.1.1.17] (ODC) in mouse L cells in the confluent state was induced within 4 hr by cyclic AMP (cAMP) or by insulin. During growth of L cells the concentration of cAMP increased first, then induction of ODC occurred and finally the cell number increased: the levels of cAMP and ODC increased only transitorily and returned to the basal levels when the cells became confluent. In growing cultures, however, the presence of cAMP reduced induction of ODC and cell growth. These results suggest that cAMP is involved in induction of ODC and that its concentration may be important for enzyme induction as well as for cell growth.
    Actinomycin D with or without these inducers stimulated induction of ODC in L cells, whereas cycloheximide inhibited it, suggesting that these hormones affect the translational level of ODC synthesis. The effect of actinomycin D on induction of ODC was much greater in non-growing cells than in growing cells.
    It was also found that the half life of ODC was 81 min in non-growing cells and 112 min in growing cells. This suggests that turnover of the enzyme is more rapid in the non-growing than in the growing state and that there may be an RNA fraction which controls its turnover and which also has a very short half life.
  • II. Effects of Fasting and Refeeding
    Masayuki SAITO, Eiko MURAKAMI, Teruo NISHIDA, Yoshiki FUJISAWA, Masami ...
    1976 年 80 巻 3 号 p. 563-568
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The effects of fasting were examined on the rhythmic changes in the activities of maltase [EC 3.2.1.20] and leucine aminopeptidase [EC 3.4. 11. 1] in the small intestine of rats which has been kept under scheduled feeding conditions. Irrespective of whether the rats had been kept on a daytime or nighttime feeding schedule, the rhythms of maltase and leucine aminopeptidase persisted when the animals were starved. However, the amplitude of the leucine aminopeptidase rhythm began to decrease from the first day of fasting, while that of maltase did not. Conspicuous rhythms persisted for at least 2 days during fasting, but they gradually became vague and disappeared after 5 days. When rats were refed after fasting, the leucine aminopeptidase activity increased within a few hours, but the maltase activity did not. It is suggested that the rhythms of the digestive enzymes in the small intestine of rats are not a direct consequence of food intake, but are triggered off by the anticipatory mechanism which operates when rats expect to be fed. The rhythmic change of leucine aminopeptidase seemed to be intensifed by food intake.
  • Tsuyako INOMATA, Masataka HIGUCHI
    1976 年 80 巻 3 号 p. 569-578
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    When Rhodopseudomonas spheroides cells grown aerobically in the dark were incubated in medium containing tritiated water (THO), incorporation of T into the bacterial cell materials occurred under growth and no-growth conditions. The overall T incorporation under no-growth conditions was stimulated by vigorous aeration and was suppressed strongly in the presence of either 10-3M KCN or 0.3% HgCl2, indicating that the bulk of the incorporation might depend upon bacterial cell metabolism or respiration. 10 μg/ml chloramphenicol and 20 μg/ml rifampicin slightly suppressed the T incorporation. The extent of T incorporation was proportional to the concentration of T in the medium. Accordingly, regardless of differences in the concentration of T in the medium, the maximum ratio of T content per hydrogen atom in the cell materials to that of THO in the medium was approximately 0.2 in non-growing cells and 0.5 in growing cells, whereas the value was 0.02-0.03 in cells incubated in medium containing KCN or HgCl2. The non-growing cells aerated in THO medium were lyophilized and fractionated by the modified method of Schneider. More than 40% of the total T incorporated into the cell materials was recovered in the cold PCA-soluble fraction, whereas the distribution of T into fractions soluble in ether-ethanol, hot PCA and alkali was 10 to 20% each. More than 75% of the T extracted in the cold PCA-soluble fraction was volatile. While the amounts of RNA and protein in the non-growing cells decreased on adding chloramphenicol or rifampicin, the distribution of T in these fractions did not change much. Our results on T incorporation into non-growing cells indicate that the major T incorporation into bacterial cell materials is independent of biosynthetic reactions using labeled precursors produced by the assimilation of T into metabolites, but presumably depends on energy-linked conformational changes of macromolecules.
  • Shigeo HORIE, Tomino WATANABE, Satoshi NAKAMURA
    1976 年 80 巻 3 号 p. 579-593
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    A green iron-chlorin protein was purified 160-fold from a lyophilized extract of Aspergillus niger by ion-exchange chromatography and gel filtration with a yield of 25%. The purified preparation appeared nearly homogeneous on sedimentation analysis and the sedimentation coefficient of the protein at infinite dilution was 13.4 S. Its molecular weight was calculated to be 2.8-3.2×105 from sedimentation and gel filtration data.
    The ferric form of the protein had absorption maxima at 587.5 and 708 nm in the visible region and a Soret band at 404 nm. High-spin ESR signals of a rhom-bically distorted ferric iron-chlorin complex were observed at g=6.5 and 5.3 together with unidentified, weaker signals at g=4.3 and 2.0. The ferric form reacted readily with cyanide to give a complex showing absorption peaks at 422 and 632 nm and a shoulder at about 595 nm. When the protein combined with cyanide its high-spin ESR signals disappeared and low-spin signals at g=1.88, 2.29, and 2.42 appeared. The ferric high-spin form was slowly converted by dithionite to a ferrous form having absorption maxima at 622 and about 410 nm. The rate of reduction by dithionite was slightly reduced by the presence of either nitrite or sulfite, and greatly accelerated by the presence of hydroxylamine. The reduced spectrum obtained in the presence of hydroxylamine had maxima at 620 and about 420 nm. The ferric cyanide complex did not show any spectral change on addition of dithionite.
    The green prosthetic group could be extracted with acidified acetone and the absorption maxima of the pyridine ferrihemochrome were at 413 and 599nm. On removal of metal from the prosthetic group the characteristic spectrum of a chlorin was obtained. The absorption maxima of a solution of the chlorin in benzene were at 403, 501, 537, 576, 595, and 655nm, the 655nm band being strongest of those in the visible region. No significant amount of flavin was detected in the purified preparation. The iron-chlorin protein catalyzed methyl viologen-linked reduction of hydroxylamine and also that of nitrite at a slower rate under the same conditions, but no evidence that it reduced sulfite was obtained in the present study. The purified preparation also had high catalase [EC 1.11.1.6] activity.
    Crystalline material was obtained by gradual concentration of the purified pre-paration at about pH 6.
  • Reversible Oxidation of a Sulfhydryl Group and Structural Change
    Shin'ichi ISHIWATA
    1976 年 80 巻 3 号 p. 595-609
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    It was found that the essential change in actin (whether G- or F-actin) on freeze- thawing was the specific oxidation of one of five sulfhydryl (SH) groups, i.e. the SH group of Cys 373 in the amino acid sequence. Oxidized SH groups formed an inter-molecular disulfide (SS) bond to yield an actin dimer. F-actin, subjected to freeze- thawing (or F-actin obtained by the transformation of once frozen G-actin which is essentially a dimer), has anomalous physico-chemical properties and a different con-formation from normal F-actin, as determined by optical and electron microscopic observations, as well as high steady ATP-splitting activity in the presence of Mg2+ However, it was found that those peculiarities disappeared and normal actin was reformed on reducing the oxidized SH group with dithiothreitol (DTT). It was also found that the normal characteristics of actin were preserved for more than four months on freezing in the presence of a sufficient amount of DTT.
  • Purification and Enzymatic Properties
    Umeji MURAKAMI, Koki UCHIDA, Toshiaki HIRATSUKA
    1976 年 80 巻 3 号 p. 611-619
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    A method is described for the preparation of high purity myosin from the left ventricle of pig heart. The purified myosin was free from nucleic acid, actin, tropo-myosin, troponin, the 150, 000 molecular weight protein and other contaminants. Analyses of subunits in the purified myosin were carried out on 3.5% acrylamide gel with 0.1% SDS. Of the total protein present in myosin, 11.3% was in the light chains; light chain 1 (LC1), 5.9% and light chain 2 (LC2), 5.4%. Urea gel elctro-phoresis of the purified myosin showed three closely spaced bands corresponding to the 20, 000 dalton, the charge-modified 20, 000 dalton and the phosphorylated 20, 000 dalton components. The properties of the Ca2+-activated and K+-activated ATPases [EC 3.6.1.3] of the purified myosin were also studied. The Km values were 27 and 55μM and the Vmax values were 0.263 and 0.317 μmole P1/mg/min for the Ca2+ activated and K+-activated ATPases, respectively. The pH-activity profiles and the effects of SH modification were of the skeletal myosin type except that the activities were lower.
  • Masami TAKAHASHI
    1976 年 80 巻 3 号 p. 621-624
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    1. The smallest light chain of myosin, g3, was not transferred from adult HMM to fetal myosin in alkali (pH 10.5) under conditions when the light chains dissociated from myosin.
    2. The g3 isolated from adult myosin did not bind to fetal myosin at either pH 7.8 or 10.5.
  • Cyclic Synthesis of Histones
    Kazutaka KANO, Yoshitake MANO
    1976 年 80 巻 3 号 p. 625-632
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Synthesis of the acid-soluble proteins in the early cleavage stage of the sea urchin, Hemicentrotus pulcherrimus, was investigated.
    As detected by the incorporation of lysine, the acid-soluble proteins were synthe-sized periodically even before the first cleavage, differing from the pattern of incor-poration of tryptophan into the fraction. Cyclic synthesis occurred almost in parallel with DNA synthesis. However, the phase and periodicity of cyclic synthesis of the acid-soluble protein fraction were quite different from those found in the hot TCA-insoluble (acid-insoluble) protein fraction. The acid-soluble proteins were adsorbed on cation exchange resin, Amberlite CG-50, and gave an elution profile similar to that found for calf thymus histones. The migration pattern of these proteins on acrylamide gel also resembled that of histones.
  • Etsuo YAMAMOTO, Takao MINAMIKAWA
    1976 年 80 巻 3 号 p. 633-635
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Dehydroquinate synthase, an enzyme catalyzing the conversion of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) to 3-dehydroquinate, was detected in cell-free extracts of etiolated Phaseolus mungo seedlings. The reaction product, 3-dehydro-quinate, formed from [1-14C]DAHP was identified by paper-radiochromatography. The enzyme required NAD+ and Co2+ for activity.
  • Mutsumi SUGITA, Taro HORI
    1976 年 80 巻 3 号 p. 637-640
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Three gangliosides were isolated from the starfish by Folch's partition, dialysis, silicic acid column chromatography, Iatrobead column chromatography and preparative thin-layer chromatography. Partial acid hydrolysis of the three lipids in 0.1 N HCl at 80° for 90min liberated all the lactosyl ceramide and water-soluble degradation products, but no sialic acid. Gas-liquid chromatography of the trimethylsilylated methyl glycosides of the water-soluble products demonstrated the presence of arabinose in addition to the usual sugar units of gangliosides.
  • Shoji ODANI, Tokuji IKENAKA
    1976 年 80 巻 3 号 p. 641-643
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    Two proteinase inhibitors, C-II and D-II, were isolated from soybeans. C-II was shown to be an inhibitor of bovine trypsin [EC 3.4.21.4], bovine α-chymotrypsin [EC 3.4.21. 1], and porcine elastase [EC 3.4.21. 11], whereas D-II inhibited only trypsin. The complete amino acid sequences of the two inhibitors established by conventional methods showed that C-II and D-II were so-called double-headed inhibitors. On the basis of the specificities of the inhibitors and their homologies with other double- headed inhibitors, the reactive sites of C-II seem to be alanine-22 for elastase and arginine-49 for trypsin (and probably also for chymotrypsin).
    D-II was quite unique because its both reactive sites are arginine residues and it only inhibits trypsin. It is suggested that D-II might be a primitive form of double-headed inhibitor and that the prototype single-headed inhibitor was a trypsin inhibitor with an arginine residue as the reactive site.
  • Toshihiko SUGANUMA, Masatake OHNISHI, Ryuichi MATSUNO, Keitaro HIROMI
    1976 年 80 巻 3 号 p. 645-648
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    The action of Taka-amylase A from Asp. oryzae was studied quantitatively by the product analysis method using unlabeled maltotriose and maltotriose labeled at the reducing end as substrates. It was found that the ratio of the unlabeled products, maltose (G2) and glucose (G1) exceeded unity, and that the ratio of the labeled products, G2*/Gl* was strongly dependent on the initial substrate concentration. The results can only be explained by a transglycosylation or condensation mechanism or both. Analysis of maltose labeled at the nonreducing or reducing end revealed that the ratio of the transglycosylation to the condensation mechanism was about 20:1 with about 80mM maltotriose. A computer simulation was made on a reaction scheme involving the termolecular-shifted complex, transglycosylation and conden-sation besides hydrolysis, by using reported subsite affinities as modified by the authors. The simulation reproduced the experimental results satisfactorily.
  • Structural Change of Its Polypeptide Chain during Gel Formation
    Shin NAKAMURA, Takashi TAKAGI, Sadaaki IWANAGA, Makoto NIWA, Kenji TAK ...
    1976 年 80 巻 3 号 p. 649-652
    発行日: 1976/09/25
    公開日: 2008/11/18
    ジャーナル フリー
    A clottable protein (coagulogen) isolated from a hemocyte lysate of the Japanese horseshoe crab (Tachypleus tridentatus) was incubated with an endotoxin-activated clotting enzyme(s) partially purified from the same lysate, and its structural change during gel formation was examined. The results indicated that the enzymatic formation of gel involved limited proteolysis of the 46Arg-47Gly and 18Arg-19Thr linkages located in the N-terminal portion of the coagulogen, liberating peptide C.
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