巻号一覧
84 巻, 2 号
選択された号の論文の29件中1～29を表示しています
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I. Effects of pH and Salt Concentration on the Dissolution of the Fibrils1Toshihiko HAYASHI1978 年 84 巻 2 号 p. 245-249
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe time-dependent increase in stability, as measured in terms of the rate of dissolution, of collagen fibrils formed in vitro from pepsin-treated collagen was significantly affected only by temperature, and not by either ionic strength or pH. This is in contrast with collagen fibril formation, a process which is greatly affected by ionic strength and pH. Within the range of temperature 29-37°C, lower temperature caused slower fibril formation and faster fibril stabilization. These results suggest that the intermolecular interactions involved in stabilizing collagen fibrils are entirely different from those involved in fibril formation. Based on kinetic analysis of the dissolution and stabilization of the fibrils, it is proposed that collagen molecules first form unstable fibrils which become gradually stabilized on prolonged incubation, without necessarily introducing covalent cross-links.抄録全体を表示PDF形式でダウンロード (346K) -
Kimihiko GOTO, Shoji MIZUSHIMA1978 年 84 巻 2 号 p. 251-258
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーBovine serum albumin appreciably stimulated the initial rate and the steady-state level of proline uptake in membrane vesicles, while it had no effect on the oxidase activity for ascorbatephenazine methosulfate, on which the transport activity depends.
Bovine serum albumin showed the strongest stimulatory effect on the transport system among the proteins tested. Three other proteins did not show any effect, while β-lactoglobulin showed a weaker but appreciable effect on the transport activity.
The incubation of membrane vesicles with bovine serum albumin resulted in extensive removal of fatty acids, while none of the other membrane components, including proteins and phospholipids, was removed by this treatment. Fatty acids inhibited the proline transport activity, while the inhibited activity was appreciably restored by incubation with the albumin. An experiment with radioactive fatty acids showed that exogenously-added fatty acids bound firmly to the membrane vesicles and were removed by subsequent incubation with the albumin.
The incubation of membrane vesicles for several hours resulted in an increase of fatty acids with a concomitant loss of the transport activity. Subsequent incubation with the albumin resulted in the removal of fatty acids and the partial restoration of the transport activity.
Based on these results, it is concluded that bovine serum albumin specifically removed fatty acids from membrane vesicles, resulting in activation of the proline transport system.抄録全体を表示PDF形式でダウンロード (1642K) -
IV. Detection of Exposed Guanine Residues by Chemical Modification with Kethoxal1Kazuya NISHIKAWA, Shosuke TAKEMURA1978 年 84 巻 2 号 p. 259-266
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe reaction of Torulopsis (Candida) utilis2 5S ribosomal RNA with kethoxal (β-ethoxy-α-ketobutyraldehyde) was studied in an attempt to identify the exposed guanine residues. At most 7-8 out of 32 guanine residues in T. utilis 5S RNA were kethoxalated after reaction at 37°C for 4 h in the presence of magnesium ions. Localization of the kethoxalated guanine residues in T. utilis 5S RNA was achieved by sequence analyses of RNase T1 digests of the kethoxalated 5S RNA. These analyses showed that residues G37, G57, G91, and some of the three guanine residues G80, G82, and G85, are the most accessible sites. Residues G30, G41, and G49 also reacted with kethoxal though less strongly. These results are for the most part compatible with our secondary structure model for T. utilis 5S RNA (Nishikawa and Takemura (1974) J. Biochem. 76, 935-947). However, partial formation of some hydrogen bonds within the loop region of the model seems to be necessary to explain the inaccessibility of residue G101 to kethoxal. The results are also discussed in comparison with those of similar studies on E. coli 5S RNA.抄録全体を表示PDF形式でダウンロード (564K) -
Takuji SASAKI1978 年 84 巻 2 号 p. 267-276
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThree new protease inhibitors were isolated and purified about 200-fold from hemolymph of silkworm larvae, Bombyx mori, using ion-exchange and affinity chromatography. Two of the three inhibitors were basic proteins (SCI-I had pI 9.4 and SCI-11 had pI 9.6) and one was acidic (SCI-TII had pI 4.0). The molecular weight of each inhibitor was determined to be 7, 000 by the sedimentation equilibrium method. The amino acid compositions of the inhibitors were similar except for the contents of Asp, Glu, Ile, Len, and Lys. Val, His, and Trp were not present in the inhibitors and Met appeared only in SCI-III. The CD spectra of the inhibitors were all similar and indicated a low content of α-helical structure (10% at most). Each inhibitor could inhibit the protease and esterase activities of bovine α-chymotrypsin at a one-to-one molar ratio, and the dissociation constants were 3.1×10-9 M for SCI-I and II and 1.3×10-8 M for SCI-III. Only SCI-II showed a weak inhibitory activity against bovine trypsin. Subtilisin BPN' and papain were not inhibited by these inhibitors.抄録全体を表示PDF形式でダウンロード (1056K) -
I. Reconstruction of a Chemo-Mechanical System from Acto-HMM of Rabbit Skeletal Muscle1Masafumi YANO, Takenori YAMADA, Hiroshi SHIMIZU1978 年 84 巻 2 号 p. 277-283
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーSteady and uniform streamings (SUS) of HMM solutions were set up in the presence of Mg-ATP in a circular slit, on both side-walls of which a Millipore filter was fixed; F-actin filaments from rabbit skeletal muscle were bound onto the Millipore filter by cyanogen bromide in the flow. The direction of the SUS was specifically determined by that of the flow during the fixing of F-actin and was independent of the direction of the initial velocity applied externally to the HMM solutions. The SUS continued for about 90min with a velocity of about 20μm/s at 20°C. There was a strong correlation between the acto-HMM ATPase activity and the velocity of SUS when the salt concentration was varied. Moreover, this was also the case when the ATPase activity was controlled by Ca2+, when native tropomyosin was bound to F-actin in the circular slit. Careful examination led to the conclusions that F-actin filaments are fixed on the Millipore filter with a specific polarity and that a chemo-mechanical system had been successfully reconstituted in our “stream cells, ” in which chemical energy from ATP is converted to the mechanical energy of streaming.抄録全体を表示PDF形式でダウンロード (569K) -
Kikuko TAKEUCHI, Yuji TONOMURA1978 年 84 巻 2 号 p. 285-292
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーTransient and steady state kinetics were studied in the interactions of ATP with acto-Hmeromyosin reconstituted from bovine arterial heavy-meromyosin (HMM) and rabbit skeletal muscle F-actin. The results showed that the rate of dissociation of the hybrid acto-HMM induced by ATP was slower than the rate of the fluorescence enhancement of HMM, and that the rate of the P1 burst of HMM was unaffected by addition of skeletal muscle F-actin. The ATPase [EC 3. 6. 1. 3] activity of arterial HMM was activated only slightly even with addition of high concentrations of skeletal muscle F-actin. Furthermore, the rates of dissociation of the hybrid acto-HMM induced by ATP and reassociation of dissociated arterial HMM with skeletal muscle F-actin after decomposition of ATP were much lower than those of skeletal muscle acto-HMM.抄録全体を表示PDF形式でダウンロード (1112K) -
Toshihiko SUGANUMA, Ryuichi MATSUNO, Masatake OHNISHI1978 年 84 巻 2 号 p. 293-316
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe action pattern and mechanism of the Taka-amylase A-catalyzed reaction were studied quantitatively and kinetically by product analysis, using a series of maltooligosaccharides from maltotriose (G3) to maltoheptaose (G7) labeled at the reducing end with 14C-glucose. A marked concentration dependency of the product distribution from the end-labeled oligosaccharides was found, especially with G3 and G4 as substrates. The relative cleavage frequency at the first glycosidic bond counting from the nonreducing end of the substrate increases with increasing substrate concentration.
Further product analyses with unlabeled and end-labeled G3 as substrates yielded the following findings: 1) Maltose is produced in much greater yield than glucose from unlabeled G3 at high concentration (73mM). 2) Maltooligosaccharides higher than the starting substrate were found in the hydrolysate of labeled G3. 3) Nonreducing end-labeled maltose (G*-G), which is a specific product of condensation, was found to amount to only about 4% of the total labeled maltose. Based on these findings, it was concluded that transglycosylation plays a significant role in the reaction at high concentrations of G3, although the contribution of condensation cannot be ignored.
A new method for evaluating subsite affinities is proposed; it is based on the combination of the kinetic parameter (k0/Km) and the bond-cleavage distribution at a sufficiently low substrate concentration, where transglycosylation and condensation can be ignored. This method was applied to evaluate the subsite affinities of Taka-amylase A.
Based on a reaction scheme which involves hydrolysis, transglycosylation and condensation, the time courses of the formation of various products were simulated, using the Runge-Kutta-Gill method. Good agreement with the experimental results was obtained.抄録全体を表示PDF形式でダウンロード (1855K) -
Mariko MOMOI, Tamio YAMAKAWA1978 年 84 巻 2 号 p. 317-325
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーGlucosamine-containing sphingoglycolipids were isolated from sheep erythrocyte membranes, and the presence of glycolipids with long carbohydrate chains was demonstrated. The purification of highly polar glycolipids was achieved by high-performance liquid chromatography of acetylated samples followed by deacetylation with sodium methoxide. Their structures were elucidated by conventional methylation studies, oxidation with chromium trioxide and the direct measurement of permethylated glycolipids by GC-MS.
Forssman-active glycolipid2 was a major component of sheep erythrocytes and lacto-N-neotetraosylceramide (LcnOse4Cer) was found to be one of the components. The amount of tetraglycosylceramide was only 5% of that of Forssman-active glycolipid. Three highly polar glycolipid components with ten to twelve carbohydrate residues were also found in sheep erythrocytes.抄録全体を表示PDF形式でダウンロード (1853K) -
Purification and Comparison with Liver and Muscle Fructose-1, 6-BisphosphatasesHideo MIZUNUMA, Yohtalou TASHIMA1978 年 84 巻 2 号 p. 327-336
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe occurrence of specific fructose-1, 6-bisphosphatase [D-fructose-1, 6-bisphosphate 1-phosphohydrolase, EC 3. 1. 3. 11] (Fru-1, 6-P2ase) in the small intestine was confirmed. 1. Fru-1, 6-P2ase was isolated from mouse small intestine by a simple method. The isolated enzyme preparation was an electrophoretically homogeneous protein. 2. The molecular weight and subunit molecular weight were 140, 000 and 38, 000, respectively. 3. The intestinal enzyme was electrophoretically distinct from the liver enzyme. 4. The kinetic properties of the purified intestinal enzyme were compared with those of the mouse liver and muscle enzymes. 5. Mouse intestinal and muscle Fru-1, 6-P2ases hydrolyzed ribulose-1, 5-bisphosphate in addition to fructose-l, 6-bisphosphate and sedoheptulose-l, 7-bisphosphate.抄録全体を表示PDF形式でダウンロード (1331K) -
Hiroto AKIYOSHI, Setsuro FUJII1978 年 84 巻 2 号 p. 337-342
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーNucleosomes isolated from Yoshida sarcoma chromatin by micrococcal nuclease treatment were relatively inactive as templates for in vitro DNA synthesis. However, the template activity increased by trypsin digestion of nucleosomes or addition of heparin to the reaction mixture. This indicates that the nucleosomal template activity is masked. A crude extract of Yoshida sarcoma cells stimulated the nucleosomal template activity. The stimulatory factor was separated into three peaks by DEAE cellulose column chromatography. The same three peaks were observed in normal rat liver extract with much lower activities, but enhanced in regenerating liver. The factors seem to stimulate DNA synthesis by activating DNA template in nucleosomes without degrading histones or changing the primary structure of nucleosomal DNA.抄録全体を表示PDF形式でダウンロード (1006K) -
Oligo-L-Arginines of Various Chain Lengths and Herring Spern DNASeiichi KAWASHIMA, Toshio ANDO1978 年 84 巻 2 号 p. 343-350
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe interaction of DNA and oligo-L-arginines having definite chain lengths of 1-17 residues was studied by precipitate formation and thermal denaturation of the complexes in order to obtain a better understanding of the roles of nuclear basic proteins. The results can be summarized as follows.
1. Those oligo-L-arginines, (Arg)n, in which n≥4 can bind with DNA irreversibly to form precipitates of the complexes. Among them, oligomers larger than (Arg)5 precipitate DNA completely in Arg/P input ratios below 1. The Arg/P ratios in the precipitates are between 0.6-0.8.
2. The thermal stability of the complexes depends on the method of complex formation, and complexes formed by the dialysis method are more stable than those formed by the mixing method.
3. The binding of (Arg)n to DNA was found to be reversible and in an equilibrium for n≤6. In general, the longer the oligomer, the higher the stability of the complex at a definite Arg/P ratio.
4. For (Arg)7-10, three kinds of complexes with different stabilities are formed between DNA and oligopeptides.
5. For (Arg)14-17, only a restricted type of complexes can be formed between DNA and oligomers, as in the case with poly-L-arginine or protamines.
6. The interaction between basic nuclear proteins and DNA is discussed in the light of the basic region in protamine and histone molecules.抄録全体を表示PDF形式でダウンロード (521K) -
Toshikatsu NAKABAYASHI, Hiroh IKEZAWA1978 年 84 巻 2 号 p. 351-360
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe differentiation of rat liver lysosomal acid phosphatase, acid ATPase, acid phosphodiesterase, acid ribonuclease, and acid deoxyribonuclease was studied by isoelectric focusing. To prevent autolytic digestion, inhibitors of cathepsins and neuraminidase were used.
The proportion of acidic forms of acid phosphatase, acid ATPase and acid phospho-diesterase was increased by the use of extraction medium containing 0.05% Triton X-100. To investigate the identity of acid ATPase and acid phosphodiesterase, the relative activities among the multiple forms of these enzymes, the acid phosphodiesterase/acid ATPase ratio at each activity peak, and the degree of enzyme inhibition by p-chloromercuriphenyl sulfonic acid were estimated. The results suggest that acid ATPase is not identical with acid phosphodiesterase.
With extraction medium free of Triton X-100, acid ribonuclease appeared in two forms. However, in addition to these forms, a new form of this enzyme with a more acidic pI (4.22) emerged when extraction medium containing 0.05% Triton X-100 was used. The major peak of acid deoxyribonuclease with pI=8.40-9.39 was obtained regardless of the extracting method.抄録全体を表示PDF形式でダウンロード (724K) -
Kunio NAKASHIMA, Machiko OHTSUKI, Syozo TUBOI1978 年 84 巻 2 号 p. 361-367
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe cytosolic fumarase [EC 4.2.1.2] of rat liver was bound, after dialysis, to the microsomal membrane in vitro. Binding of the enzyme was dependent on pH, and was facilitated in the pH range below 7.5. The binding reaction was completely inhibited by 0.5mM fumarate, aurintricarboxylate or colchicine. The bound fumarase was released from the membrane by the substrates, isocitrate, citrate or 2, 3-diphosphoglycerate at low concentrations. Desorption of the enzyme by metabolites was also dependent on pH, and was more rapid in the alkaline pH range. The enzyme desorption curves were sigmoidal, and kinetic studies suggested a biphasic cooperative mechanism for the action of the metabolites. The apparent desorption constants (concentrations necessary for 50% desorption of the enzyme) estimated at pH 7.3 for isocitrate, 2, 3-diphosphoglycerate, L-malate, oxalacetate, fumarate, citrate, succinate, and KCl were 0.073, 0.074, 0.22, 0.32, 0.39, 0.56, 2.9, and 19mM, respectively. The bound fumarase showed little enzymatic activity, and its Km and Vmax values were fivefold and 31%, respectively, of those of the free enzyme.抄録全体を表示PDF形式でダウンロード (467K) -
Kosuke MORIKAWA, Toshio SAKAMAKI, Yoshifumi NISHIMURA, Yukio MITSU, Ka ...1978 年 84 巻 2 号 p. 369-375
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーCrystals as large as 0.5×0.3×0.2mm of purified arginine-transfer RNA from Escherichia coli have been prepared by a vapour diffusion method. X-ray diffraction photographs showed that the crystals gave reflections up to 3.7Å spacing. They have a trigonal space group P31 2 1 (or P32 2 1) and cell-dimensions a=97.2, b=97.2, c=94.8Å. Crystals of a mercury derivative of this transfer RNA have also been obtained, and an X-ray diffraction photograph of one of them is presented. Formylmethionine-transfer RNA from E. coli was crystallized in various forms, and the appearance of the polymorphs was found to depend upon the amount of spermine in the solution from which the crystallization took place. Crystals of tyrosinetransfer RNA and glycine-transfer RNA have also been obtained.抄録全体を表示PDF形式でダウンロード (1247K) -
Tetsuo MAITA, Morris GOODMAN, Genji MATSUDA1978 年 84 巻 2 号 p. 377-383
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーα and β chains from adult hemoglobin of the slender loris (Loris tardigradus) were isolated by Amberlite CG-50 column chromatography. After S-aminoethylation, both chains were digested with trypsin and the amino acid sequences of the tryptic peptides obtained were analyzed. Further, the order of these tryptic peptides in each chain was deduced from their homology with the primary structures of a and β chains of human adult hemoglobin. Comparing the primary structures of the α and β chains of adult hemoglobin of the slender loris thus obtained with those of adult hemoglobin of the slow loris, 4 amino acid substitutions in the α chains and 2 in the β chains were recognized.抄録全体を表示PDF形式でダウンロード (502K) -
I. Partial Purification and Characterization of Nuclear Ribonuclease HFumio TASHIRO, Yoshio UENO1978 年 84 巻 2 号 p. 385-393
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーA ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36, 000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cell.抄録全体を表示PDF形式でダウンロード (639K) -
II. Partial Purification and Characterization of Cytosol Ribonuclease HFumio TASHIRO, Yoshio UENO1978 年 84 巻 2 号 p. 395-402
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーWe have detected in rat liver cytosol three enzymes (termed C-1, C-2, and C-3) which cleaved the RNA moiety of RNA-DNA hybrid. These enzymes were separated from each other by DEAE-Sephadex and Sephadex G-200 chromatography. C-1 and C-2 specifically act on the RNA moiety of RNA-DNA hybrid, while C-3 degrades single-stranded RNA as well as the RNA of the hybrid. The molecular weights of C-1, C-2, and C-3 are about 110, 000, 35, 000 and 110, 000 daltons, respectively, and their activities are absolutely dependent on divalent cations such as Mg2+ and Mn2+. Cleavage by C-1 and C-2 is endonucleolytic, producing mostly oligonucleotides and a small amount of mononucleotides which possess 3'-hydroxyl termini. It seems likely that C-2 is originally present in the nucleus and is released into cytosol because of its loose binding to the nuclear components. As for biochemical properties, C-1 is very similar to the cytosol ribonuclease H initially reported by Roewekamp and Sekeris, and C-2 is very similar to the nuclear ribonuclease H reported by us in the preceding paper.抄録全体を表示PDF形式でダウンロード (462K) -
Hitoshi KONDO, Hiroshi NAKATANI, Keitaro HIROMI, Ryuichi MATSUNO1978 年 84 巻 2 号 p. 403-417
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリー1. A large fluorescence enhancement of 2 p-toluidinylnaphthalene-6-sulfonate (TNS) observed in the presence of amylose was utilized to monitor quantitatively the time course of porcine pancreatic α-amylase [EC 3. 2. 1. 1] (PPA)-catalyzed hydrolysis of amylose with a numberaverage degree of polymerization of 16.8.
2. The slope of the plot of decrease in the relative fluorescence intensity of the TNS-amylose system (termed as the fluorescence value) versus the number of linkages hydrolyzed (reducing value) (Kondo, H. et al. (1977) Agric. Biol. Chem. 41, 631-634) in the course of PPA-catalyzed hydrolysis was shown to be useful to describe the degree of “multiple attack”, which is defined by the number of reattacks on a long chain substrate molecule per one encounter of the enzyme and the substrate. A parameter γ was defined as the ratio of the reciprocal of the slopes obtained at each pH to that at pH 10.5, where the multiple attack is not operating.
3. The γ versus pH profile gave an apparent pK value of about 9, indicating that some ionizable groups participate in the multiple attack mechanism.
4. Based on a reaction scheme involving a “sliding” of the substrate molecule on the enzyme, which may contribute to the multiple attack mechanism, besides binding, dissociation, and cleavage steps of the substrate, and on the assumption of the steady state for the enzymesubstrate complex, rate equations were obtained to describe the time course of hydrolysis of a linear substrate. The product distribution with the progress of the reaction can be calculated theoretically, and is dependent on the number of multiple attack and the mode of sliding. The number of multiple attack can be estimated from this distribution, and the fluorescence value can be calculated theoretically by combining the product distribution with the relative efficiency of fluorescence intensity of each maltooligosaccharide (Nakatani, H. et al. (1977) Biopolymers 16, 2363-2370). By comparing the experimental data with the theoretical ones, it was suggested that the multiple attack occurs through the sliding by maltose unit of the retained fragment on the enzyme, which is one of the fragments produced by the initial cleavage of the substrate molecule.
5. It was found that anions (chloride, bromide, and nitrate ions) which critically affect the enzyme activity have no effect on the degree of multiple attack.抄録全体を表示PDF形式でダウンロード (1632K) -
Yu HOSOKAWA, Kenji YAMAGUCHI, Noriyuki KOHASHI, Yoshihiko KORI, Iwao U ...1978 年 84 巻 2 号 p. 419-424
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe hepatic cysteine dioxygenase activity of rats was markedly decreased by the intraperitoneal administration of glucagon. The enzyme activity was also decreased by either dibutyryl cyclic AMP or theophylline. The prior administration of actinomycin D completely blocked the glucagon-mediated decrease of enzyme activity, while administrations of this inhibitor of protein synthesis after glucagon injection did not block the decrease of enzyme activity. A single administration of actinomycin D resulted in a slight increase of cysteine dioxygenase activity in the rat liver. On the other hand, the injection of cycloheximide resulted in a rapid decrease of the hepatic cysteine dioxygenase with a half-life of 2.5h. The half-life of the enzyme in rat liver after glucagon administration was one hour. The administration of hydrocortisone or insulin had no effect on the glucagon-mediated decrease of cysteine dioxygenase of rat liver. The enzyme activity of alloxan diabetic rat liver was almost the same as that of the intact rat liver. The evidence obtained here suggests that enhancement of degradation or inactivation of cysteine dioxygenase is responsible for the glucagon-mediated decrease of the enzyme activity in rat liver.抄録全体を表示PDF形式でダウンロード (416K) -
Purification of the Heavy Enzyme Obtained from Some Mutants of Bacillus brevis1Kazuko HORI, Toshitsugu KUROTSU, Masayuki KANDA, Setsuko MIURA, Akiko ...1978 年 84 巻 2 号 p. 425-434
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe heavy enzyme of gramicidin S synthetase was purified to an almost homogeneous state by a combination of ammonium sulfate fractionation, ornithine-Sepharose 4B chromatography, DEAE-cellulose chromatography, and Ultrogel AcA 22 chromatography. The enzyme was proved to be essentially homogeneous by ultracentrifugation and polyacrylamide disc gel electrophoresis. The heavy enzymes of gramicidin S synthetase from various groups of mutant strains lacking the ability to form gramicidin S were also purified to a similar extent. The sedimentation rates of the purified enzymes from a wild strain and the mutant strains (BI-3, BII-3, BI-9) were studied by analytical centrifugation and sucrose density gradient centrifugation. The enzymes from the wild strain and these mutant strains were all found to have an S20, w value of 12.2 at a protein concentration of 2.5mg per ml.
These results strongly suggest that the failure of specific amino acid activation in the heavy enzyme of these gramicidin-lacking mutants might be due to some modification at the active center of the corresponding amino acid-activating enzyme rather than to a complete absence of the amino acid-activating enzyme protein in the heavy enzyme.抄録全体を表示PDF形式でダウンロード (1221K) -
Purification and Properties of the Light Enzyme Obtained from Some Mutants of Bacillus brevis1Masayuki KANDA, Kazuko HORI, Toshitsugu KUROTSU, Setsuko MIURA, Akiko ...1978 年 84 巻 2 号 p. 435-441
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe phenylalanine-activating and/or -racemizing enzyme, i.e., the light enzyme, of gramicidin S synthetase was purified to a homogenous state by n-phenylalanine-Sepharose 4B chromatography from a wild and some gramicidin S-lacking mutant strains of Bacillus brevis. The light enzyme obtained from a mutant strain E-1 could activate phenylalanine but not racemize it, and had no phenylalanine-dependent ATP-[14C]AMP exchange activity, whereas the same enzyme obtained from other mutants and the wild strain had all three activities. Furthermore, the light enzyme of the mutant E-1 could form only acid-labile enzyme-bound phenylalanine, while the same fraction of the wild strain carried half of the enzyme-bound phenylalanine as acid-labile adenylate and half as acid-stable thioester.
These results suggest that the thiol site of the light enzyme of mutant E-l might be damaged.抄録全体を表示PDF形式でダウンロード (1115K) -
Yoshinori KANBAYASHI, Tsutomu NAKAMURA, Koichi HOSODA, Katsumi NOGIMOR ...1978 年 84 巻 2 号 p. 443-451
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe subunit structure was studied of islets-activating protein (IAP), a new protein recently isolated from the culture media of Bordetella pertussis and possessing a unique action, i. e., potentiating insulin secretory responses of animals, IAP dissociated into three subunits, F-1, F-2, and F-3, when incubated in 8M urea. Three subunits isolated by chromatography on CM-Sepharose and DEAE-Sepharose columns showed different molecular weights (F-1: 44, 000, F-2: 20, 000, F-3: 11, 000) and different isoelectric points, but similar amino acid compositions. The F-1 subunit consisted of two polypeptide chains linked by S-S bonding(s), while the F-2 and F-3 subunits were single-chain peptides. These subunits, none of which was biologically active alone, associated upon incubation for 2h at 37°C and regained biological activities after association only when the F-3 subunit was present in the association product. Thus, the F-3 subunit was essential, and the F-1 and F-2 subunits were permissive, for the development of IAP activity in animals.抄録全体を表示PDF形式でダウンロード (1724K) -
Yoshinori KANBAYASHI, Tsutomu NAKAMURA, Koichi HOSODA, Katsumi NOGIMOR ...1978 年 84 巻 2 号 p. 453-460
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーBased on the finding reported in the preceding paper (Kanbayashi, et al.: J. Biochem.) that subunits of islets-activating protein (IAP), a new protein purified from the culture media of Bordetella pertussis, were inactive as such, but regained the original biological activities when recombined, the conditions required for recovery of the biological activities were studied. Essentially the same biological activities as the native IAP were recovered when the smallest subunit, F-3, was incubated with one of the other subunits, F-1 and F-2, at a pH of around 7, at temperatures below 30°C and for longer than 12h. During the incubation, association products were formed which were isolated by gel filtration as homogeneous proteins that consisted of two subunits probably in a molar ratio of 1:1. The native IAP (consisting of three kinds of subunits) and the two proteins prepared as described above (each consisting of two IAP subunits including F-3) were equipotent in enhancing insulin secretory responses, in inhibiting epinephrine-induced hyperglycemia, in inducing leukocytosis and in increasing histamine sensitivity in experimental animals.抄録全体を表示PDF形式でダウンロード (1141K) -
Study on Subunit Structure by Dimethyl SuberimidateTakashi MATSUMOTO, Jiro TOBARI1978 年 84 巻 2 号 p. 461-465
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーMethylamine dehydrogenase (MW: 105, 000) of Pseudomonas sp. J was treated with a bifunctional cross-linking reagent, dimethyl suberimidate. Cross-linked proteins having different molecular weights of 53, 000, 64, 000, 80, 000, 93, 000, and 103, 000 were found in addition to 13, 000 (light subunit) and 40, 000 (heavy subunit) by SDS polyacrylamide gel electrophoresis. Isolated light and heavy subunits were separately treated with the reagent. The product having a molecular weight of 80, 000 was found to be a major cross-linked protein for the heavy subunit but no product was found for the light subunit. A similar electrophoretic pattern was also obtained for the reconstituted enzyme from the subunits of Pseudomonas sp. J and for methylamine dehydrogenase of Pseudomonas AM1 These results suggest that methylamine dehydrogenases obtained from these two bacteria are both the α2β2-type subunit enzyme and have a geometrically analogous subunit structure.抄録全体を表示PDF形式でダウンロード (1177K) -
Daisuke TSURU, Kunio FUJIWARA, Kunio KADO1978 年 84 巻 2 号 p. 467-476
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーMicroorganisms capable of producing L-pyrrolidonecarboxylate peptidase [L-pyrrolidonyl peptidase, EC 3. 4. 11. 8] were screened and a strain of Bacillus amyloliquefaciens was chosen as one of the most potent producers of the enzyme. The enzyme was purified from lysozymelysate of the bacterial cells by salting out with ammonium sulfate, adsorption on DEAE-cellulose, covalent chromatography on PCMB-Sepharose and by gel filtration on Sephadex G-150. By these procedures, the enzyme was purified about 800-fold with an activity recovery of 9%, and the preparation was electrophoretically homogeneous. The enzyme was most active and stable at pH 7-8. The presence of 2-mercaptoethanol and EDTA was effective for stabilizing the enzyme. The molecular weight was estimated to be 72, 000 by the gel filtration method and to be 24, 000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a subunit oligomer, presumably trimer. The enzyme was inactivated by the addition of PCMB, sodium tetrathionate, Hg2+ and Cu2+, but the activity lost was restored by the addition of 2-mercaptoethanol and EDTA. The purified enzyme split amide and ester linkages in L-pyroglutamyl derivatives of L-alanine, β-naphthylamine, α-naphthol, and 4-methylumbelliferone, but was completely inert towards various peptides and esters used as substrates for usual amino- and carboxy-peptidases, and for endopeptidases such as trypsin, subtilisin and α-chymotrypsin.抄録全体を表示PDF形式でダウンロード (1118K) -
Keiko KOBAYASHI, Yukihiro SANADA, Nobuhiko KATUNUMA1978 年 84 巻 2 号 p. 477-481
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThe selective cleavage of peptide bonds by a serine protease from skeletal muscle (SK-protease) was examined using glucagon and neurotensin as substrates. Among the peptide bonds cleaved in these substrates, the most susceptible were Phe-Thr, Tyr-Ser, Tyr-Leu, Trp-Leu, and Tyr-Ile. These results indicate that the SK-protease hydrolyzed the carboxyl side of aromatic amino acid residues under the experimental conditions. When the amino acid on the carboxyl side of aromatic amino acid residues was serine, threonine or glutamic acid, these peptide bonds, such as Phe-Thr, Tyr-Ser, and Tyr-Glu, were not susceptible to another serine protease from small intestine (SI-protease) under the same experimental conditions. The peptide bond between the arginines of Pro-Arg-Arg-Pro in neurotensin was hydrolyzed by the SI-protease, but not by the SK-protease. Thus the specificity of the SK-protease differs from that of the SI-protease. These results suggest that the specificity of the hydrolytic action of the SK-protease is more like that of bovine chymotrypsin A than like that of porcine chymotrypsin C and of the SI-protease.抄録全体を表示PDF形式でダウンロード (332K) -
Seitaro MUTOH, Ikuo FUNAKOSHI, Ikuo YAMASHINA1978 年 84 巻 2 号 p. 483-489
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーA proteoglycan was isolated from plasma membranes prepared from AH 66 cells by the following procedure. The plasma membranes were isolated from cells according to the method devised by Funakoshi and Yamashina ((1976) J. Biochem. 80, 1185-1193), then the membranes were made lipid-free. The lipid-free membranes were solubilized with 5 mm sodium phosphate buffer, pH 7.0, containing 0.5% sodium dodecyl sulfate (SDS), then the solution was fractionated on a Sepharose CL 6B column. The proteoglycan eluted near the void volume fraction was further purified by repeated precipitation with cetylpyridinium chloride (CPC).
The proteoglycan isolated was homogeneous on electrophoresis on a cellulose acetate strip and was identified as proteoheparan sulfate. The preparation contained 10.6% protein, its amino acid composition being characterized by high contents of glutamic acid, aspartic acid, proline, glycine, threonine, and serine.抄録全体を表示PDF形式でダウンロード (1097K) -
Tsunehiro KITAGAWA, Tetsuhiro KANAMARU, Hideki WAKAMATSU, Hiromi KATO, ...1978 年 84 巻 2 号 p. 491-494
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーA new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugate raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4ng to 1μg. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin.抄録全体を表示PDF形式でダウンロード (274K) -
Kyoichi KOBASHI, Ikuo NISHIZAWA, Takehisa YAMADA, Jun'ichi HASE1978 年 84 巻 2 号 p. 495-497
発行日: 1978/08/01
公開日: 2008/11/18
ジャーナル フリーThrough the investigation of the bile acid-deconjugation activities of human intestinal anaerobes, a new enzyme was discovered in Peptostreptococcus intermedius which hydrolyzed specifically the taurine-conjugates, but not the glycine-conjugates of bile acids. However, the enzymes in Streptococcus faecalis and Lactobacillus brevis hydrolyzed chiefly the glycineconjugates.抄録全体を表示PDF形式でダウンロード (213K)
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