巻号一覧
75 巻, 2 号
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Ryuji HOSONO, Sigeru KUNO1974 年 75 巻 2 号 p. 215-220
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリー1) The growth of Escherichia coli K12 W3110 was inhibited by the addition of adenine to the medium. The syntheses of DNA, RNA, and protein also were coordinately inhibited by adenine.
2) The growth inhibition caused by adenine was overcome by uridine, but was enhanced in the presence of adenosine, inosine, or guanosine.
3) In the presence of adenine, the pool size of ATP increased two-fold while those of CTP and UTP were markedly reduced. The pyrimidine nucleotide pools were restored to normal levels when uridine was added together with adenine.
4) The incorporation of uracil or orotic acid into nucleic acid was inhibited by adenine, while that of uridine was not inhibited.
5) These results suggest that the growth inhibition provoked by adenine is attributable to inhibition of the de novo synthesis of pyrimidine nucleotides, probably at the step from orotic acid to UMP.抄録全体を表示PDF形式でダウンロード (445K) -
Yoshio YOSHINO, Yoshiko HIRAMATSU1974 年 75 巻 2 号 p. 221-228
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーThe subcellular distribution of 59Fe was analyzed by Sephadex G-200 gel filtration of the supernatant fraction of mucosal cells and extract of insoluble particles of duodenal mucosa with SDS Ihr after administration of 59Fe-iron.
Two fifths of the 59Fe in the supernatant fraction was identified as ferritin by gel filtration and the immunoprecipitin reaction (S-l peak). The remainder seemed to consist of free ionic iron or large molecular iron hydroxide. Three fifths of the 59Fe in the SDS extract was incorporated in a protein containing iron (SDS peak), the elution pattern of which did not correspond to that of rat liver ferritin. The incorporation of 59Fe into ferritin fraction increased with time from 1/2 to 3 hr after iron administration, but that into the SDS peak reached a maximum after 1 hr and then gradually decreased. Cycloheximide inhibited 59Fe incorporation into mucosal cells, but did not inhibit the iron-binding capacities of the two iron binding proteins preferentially.抄録全体を表示PDF形式でダウンロード (494K) -
VIII. Properties and Regulation of the Active Transport Reaction of Proline in Escherichia coliAkinobu MORIKAWA, Haruo SUZUKI, Yasuhiro ANRAKU1974 年 75 巻 2 号 p. 229-241
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーStudies were made on the kinetic properties of the active transport reaction of proline in cells of Escherichia coli. The kinetics of the entry of substrate gave a nonlinear, hyperbolic curve when the initial rates of uptake were plotted by the Lineweaver-Burk method. The two Km values thus obtained with cells grown in a synthetic medium were 0.44 and 40μM, respectively. When the cells were grown in a semi-defined nutrient medium, the activity of proline uptake increased without alteration in its Km values.
Formation of an intracellular pool of proline was determined as a function of the concentration of substrate. A 1000-fold intra- to extracellular concentration gradient was formed at substrate concentrations below 3μM, followed by rapid overshooting of the proline pool.
The proline uptake reaction was greatly affected by potassium and sodium ions and their effects in stimulating and inhibiting the uptake reaction were studied as function of their concentrations.
The effects of various drugs inhibiting energy metabolism on the uptake reaction were analyzed. 2, 4-Dinitrophenol and potassium cyanide mainly affected the uptake reaction, reducing the Vmax values, while iodoacetate and iodoacetamide affected it greatly, increasing the Km values. The effects of the drugs on the exchange reaction of proline transport were studied in detail. The exchange-entry reaction of substrate was facilitated by, and dependent on the concentration of substrate in the cells. The rate of exchange-entry was inhibited by 2, 4-dinitrophenol and iodoacetate.
A single, homogeneous transport carrier model with multi-binding sites for substrate was proposed to explain the active transport system of bacteria which gives curvilinear Lineweaver-Burk plots, and shows negative cooperativity.抄録全体を表示PDF形式でダウンロード (950K) -
IX. Studies on the Active Transport Reaction in Sodium Azide- and 2, 4-Dinitrophenol-sensitive Mutants of Escherichia coliFumiko GOTO, Yasuhiro ANRAKU1974 年 75 巻 2 号 p. 243-251
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーThe effects of sodium azide and 2, 4-dinitrophenol on the active transport reaction of proline in Escherichia coli cells were studied using various mutants with altered sensitivities to these drugs. The mutants used were KY2671 (azide-sensitive), KY131 (azide-resistant), DS-1 (2, 4-dinitrophenol-sensitive), and DR-51 (2, 4-dinitrophenol-resis-tant). The former two strains were isolated by T. Yura and C. Wada (1) and the latter two were isolated in this laboratory.
The respiratory activities of the mutants were examined by measuring the rates of oxygen uptake with various carbon sources as substrate, and the effects of the drugs in enhancing or inhibiting the rates of oxygen consumption were studied as functions of the concentrations of the drugs.
The active transport reaction of proline was inhibited by sodium azide and 2, 4-dinitrophenol. Uptakes by mutants which are resistant to sodium azide and 2, 4-dinitrophenol were also found to be resistant to the inhibitory effects of the drugs while those of mutants which are sensitive were affected greatly by the drugs.
The correspondence of the drug sensitivities of the mutants to the drugs on their active transport was abolished when the uptake reaction was carried out in the presence of a carbon source.
The inhibitory effects of 3, 5-di-tert-butyl-4-hydroxybenzylidenemalononitrile and diphenylphosphoryl azide on the proline uptake were described.抄録全体を表示PDF形式でダウンロード (606K) -
Hiroyuki SUNI, Setsuro FUJII1974 年 75 巻 2 号 p. 253-260
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーThe activation of the enzymes hydrolyzing ethyl Na-acetyl-L-tyrosinate (ATEE) and methyl Na-tosyl-L-arginate (TAME) and the changes in various inhibitors of human serum due to the egg albumin antigen-antibody reaction were studied.
The first component of complement (Cl) was adsorbed on “antigen-antibody, ” while the inhibitor of Cl was not.
Dissociation of these components resulted in the activation of Cl to Cl esterase, which hydrolyzes ATEE.
The enzyme hydrolyzing TAME was also activated by the antigen-antibody reaction and was identified as plasmin [EC 3. 4. 4. 14].
“Antigen-antibody” did not directly activate plasminogen but the results suggested some participation of complements in the activation.抄録全体を表示PDF形式でダウンロード (664K) -
Daisuke TSURU, Kunio FUJIWARA, Reiko WATANABE, Tadashi YOSHIMOTO, Shin ...1974 年 75 巻 2 号 p. 261-268
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーAn acid protease from Rhizopus chinensis [EC class 3. 4. 4] was inactivated by incubation with carbobenzoxy-L-phenylalanyldiazomethane (ZPDM) as well as with diazoacetyl-DL-norleucine methyl ester (DAN) at pH 5 and 25°C for 10min in the presence of cupric ions. The presence of 5mM L-glutamyl-glycyl-L-phenylalanine (L-Glu-Gly-L-Phe) protected the enzyme almost completely from inactivation by these reagents. Incorporation of norleucine into the enzyme caused by incubation with DAN was also prevented by the presence of L-Glu-Gly-L-Phe or ZPDM. An aromatic diazonium compound, p-nitrobenzenediazonium fluoroborate (PNBD), inactivated the Rhizopus acid protease on incubation at 25°C and pH 7 for 10min, and this inactivation was also prevented by preincubating the enzyme with 5mM L-Glu-Gly-L-Phe. Spectrophotometric and amino acid analyses of the modified enzyme indicated that azocoupling had occurred at tyrosyl residues of the enzyme. From these results, it was concluded that one carboxyl group and tyrosyl residue(s) of the Rhizopus chinensis acid protease are closely rolated to the enzyme activity.抄録全体を表示PDF形式でダウンロード (629K) -
Frederick VARRICCHIO, Takeshi SENO1974 年 75 巻 2 号 p. 269-275
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーTransfer RNA's isolated from Bacillus subtilis, Bacillus stearothermophilus, and chicken liver have been separated by polyacrylamide gel electrophoresis at pH 5.8 in the presence of 7M urea. The tRNA's from each source separated into unique patterns of RNA bands. The B. subtilis tRNA's separated into 5 bands, as did the B. stearothermophilus tRNA's. The chicken liver tRNA's separated into 4 or 5 bands. In each case the location of several specific tRNA's was determined by aminoacylating the bulk tRNA with one [14C]amino acid according to the method described previously (1). When the relative migration rates of tRNA's from these three sources and from Escherichia coli (1) were compared, it was noted that the tRNA's specific for each amino acid generally migrated similarly, relative to each other. A possible basis for the separation on polyacrylamide gels of RNA species of similar chain length is discussed.抄録全体を表示PDF形式でダウンロード (2147K) -
II. Effect of Metal Ions on the Electrophoretic Behavior of Myosin SubunitsStephen L. GAFFIN, Avraham OPLATKA1974 年 75 巻 2 号 p. 277-281
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーThe myosin light chain of intermediate molecular weight, LMP-II, undergoes a marked reduction in its electrophoretic migration rate in response to Ca2+, compared to the other light chains run under the same conditions. Other divalent, but not monovalent, cations had a similar effect. These results support earlier tryptophan fluorescence measurements showing that LMP-II binds Ca2+ions. The possible role played by these light chains-which are not required for the ATPase [EC 3. 6. 1. 3] activity of myosin, and which have therefore been considered as nonessential-is discussed.抄録全体を表示PDF形式でダウンロード (1562K) -
X. Direct Evidence for Ca2+ Translocation Coupled with Formation of a Phosphorylated IntermediateMichihiro SUMIDA, Yuji TONONURA1974 年 75 巻 2 号 p. 283-297
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーThe time course of Ca2+ uptake by the SR was compared with those of formation and decomposition of a phosphorylated intermediate, EP, in the Ca2+, Mg2+-dependent ATPase reaction [EC 3. 6. 1. 3]. Ca2+ uptake was measured by the Millipore filtration method, using 45CaCl2 as a substrate. The SR was allowed to react with 10μM ATP at 0°C in the presence of 20μM CaCl2 without adding MgCl2. Under the conditions used, the formation and decomposition of EP could be measured separately, since the rate of EP formation was much higher than that of its decomposition. The following results were obtained.
1. When EP formation and Ca2+ uptake by the SR were terminated by adding 1mM EGTA+2.5mM MgCl2, EP was rapidly decomposed into E+Pi. Ca2+ uptake consisted of two phases: a rapid, initial one and a slow, steady one, which was almost linear with time. The time course of the former corresponded to that of EP formation, and that of the latter to Pi liberation before the addition of EGTA+MgCl2. During both phases, the molar ratio of Ca2+ uptake/ATP hydrolysis was about 1/1.
2. When the reaction was stopped by adding 1mM EGTA+0.25mM ADP, ATP was formed immediately from EP and ADP. The time course of Ca2+ uptake showed only a slow, steady phase, corresponding to that of Pi liberation before the addition of EGTA+ADP. The molar ration of Ca2+ uptake/ATP hydrolysis was smaller than 1/1.
3. After Ca2+ uptake had been stopped by adding 1Mm EGTA+10μM ADP, the time course of release of Ca2+ ions from the SR was measured by adding 20mM MgCl2 after various intervals, since the release was stopped by adding MgCl2. The time course observed corresponded to that of disappearance of EP.抄録全体を表示PDF形式でダウンロード (1075K) -
Tsuneyoshi HORIGOME, Hisataka KASAI, Tsuneo OKUYAMA1974 年 75 巻 2 号 p. 299-307
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーTaka-amylase A [EC 3. 2. 1. 1] was immobilized by covalent bonding on Sephadex G-25, Sephadex G-200, Sepharose 6B and Sepharose 2B, which were activated with cyanogen bromide by the method of Porath. The properties of the bound enzymes were compared with one another and with those of the soluble enzyme.
1. Among the bound amylases, T2B (bound on Sepharose 2B) showed the highest activity towards soluble starch (about 7% that of the soluble amylase), and T6B (bound on Sepharose 6B) showed the highest activity towards phenyl maltoside (about 76% that of the soluble amylase).
2. From the results of staining of the immobilized enzymes, it is suggested that there are two species; the surface-bound type and the entrapped type.
3. The effect of pH, heating, EDTA or urea on the stability of soluble and immobilized amylases was estimated. The series of T6B, T2B≈TG200, and TG25≈soluble amylase represents the order of decreasing stability.
4. From these facts, it was suggested that there should be a most effective matrix size to stabilize the enzyme.
5. The soluble and immobilized amylase were inactivated by the addition of EDTA. Inactivated entrapped-type amylase, however, showed partially restored amylase activity on the addition of calcium ions, whereas the soluble and surface-bound amylases did not recover any activity under the same conditions.抄録全体を表示PDF形式でダウンロード (1766K) -
Keishiro SHIRAHAMA, Kaoru TSUJII, Toshio TAKAGI1974 年 75 巻 2 号 p. 309-319
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーFree-boundary electrophoresis was carried out with complexes formed between sodium dodecyl sulfate and polypeptides derived from various proteins by cleavage of their disulfide linkages. It was found that the electrophoretic mobilities of the complexes were independent of the moleculat weights of the polypeptides in the range 10, 000to 70, 000 daltons. It is clear that the apparent electrophoretic mobilities of the polypeptides in SDS-polyacrylamide gel electrophoresis become uniquely dependent on the molecular weights of the polypeptides chiefly due to the molecular sieve effect of the gel matrix. The mobilities of complexes formed between sodium dodecyl sulfate and polyvinylpyrrolidone were also found to be independent of the molecular weight of the polymer. The electrophoretic mobility observed was about -2.6×10-4cm2•sec-1•volt-1 at 25°C, whether the polymer component of the complex was a protein polypeptide or polyvinylpyrrolidone. A prolate ellipsoid model has been proposed for the complexes formed between sodium dodecyl sulfate and protein polypeptides by Reynolds and Tanford (J. Biol. Chem., 245, 5161 (1973)). An alternative model that is applicable to either of the two kinds of complexes studied was propsed based on the present findings. In the model, the polymer chain is considered to be mostly flexible, and sodium dodecyl sulfate clusters are arranged along the polymer chain.抄録全体を表示PDF形式でダウンロード (906K) -
II. Role of Sulfhydryl Groups and Subunit StructureMiyoko ONO-KAMIMOTO, Shiro SENOH1974 年 75 巻 2 号 p. 321-331
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーCrystalline 3, 4-dihydroxyphenylacetate 2, 3-dioxygenase from Pseudomonas ovalis has a molecular weight of 140, 000 and contains 4-5g atoms of non-heme iron (ferrous state) and 16 half-cystine residues.
The enzyme was strongly inhibited by anionic detergents, such as sodium dodecyl sulfate, but on inhibition was observed by cationic or nonionic detergents. Apparently, the inhibition was due to dissociation of the enzyme into homologous subunits, the molecular weight of which was estimated to be 35, 000 by SDS-polyacrylamide gel electrophoresis and by gel filtration on Sephadex G-100.
By selective tritium labeling and by carboxypeptidase digestion, the carboxylterminal amino acid was determined to be leucine, The minimum molecular weight per carboxyl-terminal amino acid was calculated to be approximately 35, 000. These observations shwed that the holo-enzyme consisted of 4 identical subunits of molecular weight 35, 000.
From the titration of SH groups, a total of 16 half-cystine residues were found to exist in the form of SH groups, not as disulfide linkages. These 16 Sh groups could be classified into three types according to their reactivity toward various SH reagents. In the first group, 4 SH groups in the native enzyme were radily titrated with Ag+, Hg2+, 5, 5'-dithiobis-(2-nitrobenzoate) (DTNB) or p-chlormercuriphenyl sulfate (PCMS) without loss of enzymic activity. In the second group, however, 4 Sh groups of the native enzyme were specially reactive with PCMS but not with the other reagents used. This modification caused the enzyme to dissociate into subunits having molecular weights of 35, 000 each and resulted in complete loss of enzymic activity. In the third group, the remaining 8 SH groups were titrated only in the subunit state with Ag+, hg2+, DTNB of PCMS. These results indicated that the 4 SH groups belonging to the second group participated in the association of subunits.抄録全体を表示PDF形式でダウンロード (2388K) -
IV. Purification and Some Properties of D-Erythrulose Reductase from Beef LiverKihachiro UEHARA, Tsuyoshi TANIMOTO, Hisashi SATO1974 年 75 巻 2 号 p. 333-345
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーA procedure for the purification of D-erythrulose reductase from beef liver is described. D-Erythrulose reductase was purified approximately 1, 700-fold from acetone powder extracts of beef liver. The purified enzyme was proved to be homogeneous by ultracentrifugation and polyacrylamide gel electrophoresis. D-Erythrulose reductase was active specifically with D-erythrulose as a substrate in the presence of NADH as well as NADPH as a coenzyme, and catalyzed the reduction of D-erythrulose, yielding erythritol as the product. Attempts to demonstrate reversibility of the reaction were unsuccessful. NADH was less effective than NADPH and the Ka value for NADH (2.2×l0-4M) was much larger than that for NADPH (6.8×10-6M). The molecular weight of D-erythrulose reductase was estimated to be 90, 000±1, 000 by Sephadex G-200 gel filtration, sedimentation equilibrium analysis and sucrose density gradient centrifugation, and that of its subunit was estimated to be 22, 000±500 by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was determined to be pH 6.75 by Ampholine isoelectric focusing. D-Erythrulose reductase was inactivated by exposure to low temperatures. This
cold inactivation was at least partially reversible by simply rewarming, and protection against cold inactivation was afforded by NADP+ at low concentrations. NAD+. ATP and other compounds with structures related to that of NADP+ did not exert protection.抄録全体を表示PDF形式でダウンロード (1571K) -
Multiplicity and Enzyme Activities for Ceramide Trihexoside and Some Aryl αGalactosidesItsu KANO, Tamio YAMAKAWA1974 年 75 巻 2 号 p. 347-354
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリー1. Human kidney α-galactosidase [EC 3. 2. 1. 22], assayed with p-nitrophenyl α-galactoside as a substrate, was separated into two major components, Fr. I and Fr. II, by isoelectric focusing and Cellogel electrophoresis. The isoelectric points were 4.5 and 4.3 for Fr. I and Fr. II, respectively.
2. Thermal inactivation experiments at 55°C revealed that Fr. I was thermolabile and Fr. II thermostable.
3. The Michaelis constant of Fr. I for pNPG at the optimal pH (pH 4.6) was 6.O×10-3M and that of Fr. II was 13.3×lO-3M.
4. p-Nitrophenyl α-galactoside hydrolase of Fr. I was inhibited 50% by 400mM myoinositol. However, Fr. II was insensitive to this inhibitor.
5. Ceramide trihexosidase activity was found in both fractions, and the Michaelis constants were 5.7×lO-4M and 4.7×lO-4M for Fr. I and Fr. II, respectively.抄録全体を表示PDF形式でダウンロード (1024K) -
II. Modification by Lysosomal Cathepsin B1 from the Same CellKunio NAKASHIMA, Kuniko OGINO1974 年 75 巻 2 号 p. 355-365
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーIn the modification of rabbit liver D-fructose-1, 6-diphosphate 1-phosphohydrolyase [fructose-1, 6-diphosphatase, EC 3. 1. 3. 11], cathepsin B1 [EC 3. 4. 4. -] from the lysosomal particles was considered to play an important role. The modifying reaction required a sulfhydryl compound and was inhibited by N- α-p-tosyl-L-lysine chloromethyl ketone (TLCK) and L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK). The optimum pH for the modification reaction was 5.0. the affinity of the modifying enzyme for fructose-1, 6-diphsphatse was very high with and apparent Km of 5×10-8. The modified fructose-1, 6-diphosphatase showed an alkaline pH optimum, and the activity at pH 8.6 was increased by approximately four-fold, wheras the untreated enzyme showed a neutral pH optimum. The modification was considered to be a result of partial proteolysis of fructose-1, 6diphosphatase subunis by the modifying enzyme.抄録全体を表示PDF形式でダウンロード (1987K) -
Tomoh MASAKI, Osashi TAKAITI1974 年 75 巻 2 号 p. 367-380
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーM-Protein was contained in an extract of muscle mince with Hasselbach-Schneider's solution. The crude M-protein fraction isolated from this extract by the method previously reported could be separated into two components, component I, true M-protein, and component II. M-Protein thus prepared was immunochemically identical with the protein which constitutes the M-line of myofibrils. The molecular weight of the purified M-protein was estimated to be 165, 000 by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The binding activity of the M-protein fraction to myosin was shown to be solely due to true M-protein, not to component II. The maximum ratio of bound M-protein to myosin by weight was about 0.26. Tryptic digestion of M-protein did not decrease this binding activity, but rather increased it.
M-Protein was digested by a relatively small amount of trypsin [EC 3. 4. 4. 4] and degraded into smaller fragments including one with a molecular weight of 109, 000. This fragment still reacted with anti-M-protein antibody and was resistant to further tryptic digestion.抄録全体を表示PDF形式でダウンロード (4682K) -
Tsuneo KAMEYAMA, Shizuko HAYAKAWA, Takamitsu SEKINE1974 年 75 巻 2 号 p. 381-387
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーThe enzymatic properties of heavy meromyosin and subfragment 1 were studied with special reference to the response to various effectors and to chemical modification, in order to investigate the interaction, if any, between the two subunits which make up the head of myosin A.
1. EDTA activated the ATPase [EC 3. 6. 1. 3] activity not requiring additional cofactors (K+-ATPase) of heavy meromyosin and of subfragment 1 in the same manner.
2. Pentachlorophenol, as well as 2, 4-dinitrophenol, a well-known effector, was found to activate Ca2+-ATPase and to inhibit EDTA-ATPase of both heavy meromyosin and subfragment 1. p-Nitrophenol inhibited only the EDTA-ATPase. The degree of effectiveness was in the order pentachlorophenol>2, 4-dinitrophenol>p-nitrophenol.
3. Michaelis constants of heavy meromyosin ATPase in the presence of phenol derivatives were not affected by tryptic digestion to subfragment 1. The values of the constants varied slightly in the presence of different phenols.
4. The S1 blocking of subfragment 1 proceeded much faster than that of heavy meromyosin.
5. ATP-induced conformational changes around the active site of myosin A and heavy meromyosin, which were assumed to occur on the basis of the change in the rate of chemical modification of a specific sulfhydryl group (S2) by N-ethylmaleimide, were preserved in subfragment 1.
From these results, it was suggested that the effect of phenol on myosin A
ATPase and that of ATP on the conformation around S2 could not be interpreted in terms of subunit-subunit interactions.抄録全体を表示PDF形式でダウンロード (493K) -
Kenzoh NAGAMI, Siro SENOH1974 年 75 巻 2 号 p. 389-398
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーBreuibacterium pyrocatechase. [EC 1. 13. 1. 1] contains a bound ferric iron essential for its enzymic activity. This bound ferric iron plays a role in the enzymic reaction at both the catalytic and substrate binding sites.
A sulfhydryl group of apopyrocatechase (iron-free) reacts with 5, 5'-dithiobis(2-nitro-benzoic acid) (DTNB) resulting in the formation of the TNB-bound apoenzyme (TNB-apoenzyme) which has no ability to bind iron. By reacting this with cysteine, to reduce the disulfide bond, the reconstitution of holopyrocatechase (iron-containing) is achieved and full enzymic activity restored. Both native and reconstituted holoenzyme have no DTNB-detectable sulfhydryl group. The holoenzyme as well as the apoenzyme reacts with one molar equivalent of p-chloromercuribenzoate (PCMB) and completely loses enzymic activity. These observations indicate the existence of an iron-sulfur linkage in pyrocatechase.
By chemical modification using N-bromosuccinimide (NBS) together with fluorometric studies, the participation of a tryptophanyl residue in the binding of iron to pyrocatechase was strongly suggested. On treatment of a single tryptophanyl residue in apopyrocatechase with small amount of NBS, the ability to recombine an iron atom is lost. In the holoenzyme, this tryptophanyl residue is protected from NBS reaction. Both the holo- and apoenzyme showed a fluorescence emission maximum at 342nm depending on tryptophanyl residue in the protein moiety after excitation at 280nm. The emission intensity at 342nm was 3-fold higher for the apoenzyme. The decrease in fluorescence intensity of the apoenzyme accompanied by the addition of iron was in proportion to the binding of iron to the apoenzyme and vice versa.
The structure of the active site of pyrocatechase is discussed on the basis of the above results.抄録全体を表示PDF形式でダウンロード (739K) -
V. Possible role of Free Sulfhydryl Group in Apoprotein of Glucose Oxidase and 6-Amino Group in Adenine Moiety of FADHaruhito TSUGE, Hisateru MITSUDA1974 年 75 巻 2 号 p. 399-406
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリー1. Reconstitution of FAD and the apoenzyme of glucose oxidase [EC 1. 1. 3. 4] was strongly inhibited by p-chloromercuribenzoate (PCMB), while the enzyme actvity per se was not inhibited by this compound. This inhibition was reversed by the addition of dithiothreitol.
2. It was observed that loss of sigmoidicity in the FAD “concentration-activity” curve occurred in the reconstitution reaction of the enzyme when the apoenzyme was incubated with FAD in the presence of PCMB.
3. Adenosine and its analogues in which the amino group in the C-6 position of the purine base was replaced by a mercaptogroup showed strong inhibition of FAD incorporation in the reconstitution reaction.
4. It was found that in the reconstitution reaction initiated by flavin-hypoxanthine dinucleotide, prolonged incubation and a high concentration of this compound were necessary to regenerate the activity because of the lack of an initial binding site.抄録全体を表示PDF形式でダウンロード (597K) -
Toshio NIIMURA, Toshie TOKIEDA, Tsutomu YAMAHA1974 年 75 巻 2 号 p. 407-417
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーAn enzyme was partially purified 620-fold from extracts of Pseudomonas sp.; it catalyzes the following reaction:
cyclohexylsulfamate+H2O→ cyclohexylamine+SO42-+H+.
The pH optimum of the reaction was between 6.5 and 6.7. The Km value of the enzyme for cyclamate was 5×lO-3M. The enzyme was comparatively heatstable, and the enzyme could be stored at -20°C in Tris-HCl containing 0.1% gelatin for at least 3 months with only 10% loss of activity.
The enzyme was found to be specific for the hydrolysis of sulfamates, and it hydrolyzed aliphatic sulfamates with three to eight carbons preferentially. The sulfamates of secondary amines such di-n-propylamine and N-methylcyclohexylamine were scarcely hydrolyzed. Sulfanilamide, sulfamate (NH2SO3-), and cyclohexyl sulfate were not hydrolyzed.
EDTA caused complete loss of activity, which could be partially restored by the addition of various metal ions. No metal ion specificity was observed as regards the reversal of EDTA inhibition.
Hg2+, Cd2+, and iodoacetic acid showed strong inhibitory effects, but PCMB did not show as strong an inhibition as expected. The effects of various other cations and anions on the enzyme activity are also described.抄録全体を表示PDF形式でダウンロード (771K) -
Tadazumi KOMIYAMA, Masachika IRIE1974 年 75 巻 2 号 p. 419-428
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリー1) A ribonuclease [EC 2. 7. 7. 17] from Rhizopus sp. (RNase Rh) was found to be inactivated by various halo-fatty acids and iodoacetamide. The inactivation of RNase Rh by iodoacetamide and bromoacetate was most marked at pH 8-9 and 3-4, respectively, on the basis of the present results.
2) Determination of the amino acid composition of RNase Rh inactivated by iodoacetamide at pH 7.0 indicated the formation of two residues of 3-carboxamidomethylhistidine with the loss of enzymatic activity. Similar experiments with bromoacetate at pH 4.0 and 7.0 showed the formation of about 1.6 and 2.0 residues of 3-carboxymethylhistidine, respectively.
3) Further studies on the identification of the carboxamidomethylated amino acid residues in the modified RNase Rh indicated that carboxamidomethyl groups were introduced on histidine, lysine, and carboxylic groups of RNase Rh. The formation of 3-carboxamidomethylhistidine was markedly inhibited by the presence of 2', (3')-AMP, but those of 1-carboxamidomethylhistidine, ε-carboxamidomethyllysine and carboxamidomethyl esters of acidic amino acids were not depressed -by the presence of the inhibitor.
4) Since, in the case of carboxamidomethylation of RNase Rh, the formation of 3-carboxamidomethylhistidine was in linear correlation with the loss of enzymatic activity, and formation is inhibited by the presence of inhibitor, it was concluded that one or two residues of histidine was involved in the active site of RNase Rh.
5) The gross conformation of carboxamidomethyl RNase Rh, as investigated by fluorescence emission and spectrophotometric titration of phenolic hydroxyl groups, was very similar to that of RNase Rh.抄録全体を表示PDF形式でダウンロード (734K) -
Keiichi SHIMAZAKI, Shunrokuro ARIMA1974 年 75 巻 2 号 p. 429-432
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーPDF形式でダウンロード (292K) -
Keitaro HIROMI, Masatake OHNISHI, Yoshiaki KAMA1974 年 75 巻 2 号 p. 433-435
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーPDF形式でダウンロード (1137K) -
Toshisuke KAWASAKI, Kazuyuki SUGAHARA, Tadayoshi OKUMURA, Ikuo YAMASHI ...1974 年 75 巻 2 号 p. 437-439
発行日: 1974/02/25
公開日: 2008/11/18
ジャーナル フリーPDF形式でダウンロード (210K)
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