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Volume 86 (1979)
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- Issue 4 Pages 833-
- Issue 3 Pages 597-
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Volume 86, Issue 3
Displaying 1-30 of 30 articles from this issue
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I. Effects of Preincubation of Poly(U) with 10 S Subunits on the Interactions of 40 S Subunit Proteins with Aurintricarboxylic Acid and with N, N'-p-PhenylenedimaleimideKazuo TERAO, Kikuo OGATA1979 Volume 86 Issue 3 Pages 597-603
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe proteins of rat liver 40 S ribosomal subunits that interact with poly(U) were studied by the use of ATA and PDM, which are known to interact and cross-link, respectively, with ribosomal proteins.
(1) When rat liver 40 S ribosomal subunits were incubated with ATA at concentrations higher than 10-5M, their poly(U)-binding activity decreased, the decrease depending on the concentration of ATA. When 40 S subunits were incubated with various concentrations of ATA from 10-5 M to 10-3 M, two-dimensional electrophoresis of the ribosomal proteins showed that stained spots of several ribosomal roteins, S1, S6, S7, S9, S10, S23, and S24, became smaller and fainter, and finally disappeared, as the ATA concentration increased. It was found that preincubation of 40 S subunits with poly(U) almost completely prevented the disappearance of S7 and S10 proteins on treatment with 10-4 M ATA.
(2) When 40 S subunits were incubated with PDM at concentrations higher than 0.05mM, the poly(U)-binding activity showed a PDM concentration-dependent decrease of up to about 50% of the control in the case of 0.4mM PDM. When 40 S subunits were incubated with 0.4mM PDM, the protein spots S7, S9, S10, S22, S23, and S24 became smaller and fainter than the corresponding spots of non-treated 40 S subunits. On preincubation of 40 S sub-units with poly(U), distinct spots of S7 and S22 were recovered on the two-dimensional gel even after incubation with 0.4mM PDM.
Based on these results it seems probable that S7 protein is the poly(U)-interacting protein.View full abstractDownload PDF (2032K) -
II. Cross-links between Poly(U) and Ribosomal Proteins in 40 S Subunits Induced by UV IrradiationKazuo TERAO, Kikuo OGATA1979 Volume 86 Issue 3 Pages 605-617
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS(1) When rat liver 40 S ribosomal proteins in 6 M urea were mixed with poly(U) at an appro-priate ratio, a precipitate was formed which was also insoluble in the sample solution for two-dimensional acrylamide gel electrophoresis. Analyses by two-dimensional acrylamide gel electrophoresis showed that S7 and SIO proteins (according to our numbering system) had disappeared selectively from the fraction soluble in 6 M urea. These two proteins were present in the fraction insoluble in 6 M urea, and became soluble in the sample solution after treating it with RNase. The results suggest that S7 and SIO proteins have strong affinities for poly(U). When rat liver 40 S subunits were incubated with poly(U), similar results were obtained.
(2) After incubation of 40 S subunits with [3H]poly(U) and then with unlabeled poly(U), UV irradiation cross-linked poly(U) to the protein moiety of the 40 S subunit. When the protein fraction insoluble in the sample solution for two-dimensional electrophoresis was prepared from 40 S subunits cross-linked to poly(U) and then subjected to two-dimensional acrylamide gel electrophoresis after RNase treatment, S7 and S10 proteins were detected on the gel. In addition to the S7 protein spot, a triangular area spreading from the spot to the origin contained radioactivity. The results suggest that poly(U) is cross-linked to S7 protein and oligo(U) fragments bound to S7 protein affect its electrophoretic mobility.
(3) Ribosomal proteins were prepared from 40S subunits cross-linked to carrier-free [3H]poly(U) and analyzed by three-dimensional acrylamide gel electrophoresis (Terao, K. & Ogata, K. (1975) Biochim. Biophys. Acta 402, 214-229) after RNase treatment. It was found that S7, S6, and S15 proteins are cross-linked to poly(U). From the results of the present and preceding experiments it is concluded that S7 is the poly(U)-binding protein. The possibility that other proteins in 40 S ribosomal subunits interact with poly(U) is discussed.View full abstractDownload PDF (3166K) -
Sachio GOTO, George P. HESS1979 Volume 86 Issue 3 Pages 619-625
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteractions between trypsin and each of five dansyl-arginine derivatives, dansyl-L-arginine methyl ester (L-DAME), dansyl-D-arginine methyl ester (D-DA ME), dansyl-L-arginine amide (L-DAA), dansyl-L-arginine (L-DA), and dansyl-D-arginine (D-DA), are accompanied by a fluorescence intensity change which can be followed by the stopped-flow method. These compounds are substrates or products in trypsin-catalyzed hydrolysis reactions. All of these compounds, except L-DAA, show a considerable fluorescence intensity increase in the reaction with trypsin. The observed rate constant, T-1 obisd, for the initial fluorescence intensity enhancement in the reaction between trypsin and D-DAME yields a typical hyperbolic curve when the rate is plotted as a function of the ligand concentration. This result is consistent with a two-step mechanism (1) in which a fast bimolecular association process is followed by a slower unimolecular isomerization process. The isomerization process may be con-sidered to be associated with a conformational change of the enzyme molecule, induced by the formation of the enzyme-substrate complex (1). The rate of the isomerization process depends on pH. The rates obtained for L-DAME and D-DAME increase linearly with decrease of the hydrogen ion concentration in the pH range below neutral.View full abstractDownload PDF (1662K) -
Kouji MATSUMOTO, Tadahiko ANDO, Hiuga SAITO, Yonosuke IKEDA1979 Volume 86 Issue 3 Pages 627-637
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus subtilis Marburg TI (thy, trpC2) has at least four endonuclease activities as assayed by measuring the conversion of single-stranded circular fl DNA to the linear form by agarose gel electrophoresis. One of them, which is specific for single-stranded DNA (named endo-nuclease MU), was purified about 320 times by two chromatographic steps and gel filtration, thereby eliminating exonuclease and phosphomonoesterase activities. This activity requires divalent cations but does not require ATP. The molecular weight estimated by gel filtration was about 57, 000 daltons. The cleavage products have 5'-phosphoryl termini. At low con-centrations, double-stranded DNA is not split to any detectable extent. At high concen-trations, however, double-stranded superhelical DNA is attacked to yield open-circular and linear DNA's. The activity of the enzyme towards single-stranded circular DNA relative to that towards double-stranded linear DNA was calculated to be approximately 5, 000: 1 by comparing the initial rates of introducing single-strand breaks into the DNA's.View full abstractDownload PDF (2060K) -
Takeyoshi IMAMURA, Kazue KONISHI, Masao YOKOYAMA, Katsutoshi KONISHI1979 Volume 86 Issue 3 Pages 639-642
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe separation of polypeptides treated with SDS was studied using G3000SW packing prepared from silica for high-speed gel filtration. The peaks of ovalbumin, chymotrypsinogen A, cytochrome c, aprotinin, and insulin B chain were completely separated in the presence of 0.1% SDS and 0.05 NI sodium phosphate buffer (pH 7.0). A plot of the logarithm of molecular weight of polypeptides versus Kd was linear over a molecular weight range of 3, 000 to 50, 000 at the above concentrations of SDS and sodium phosphate. The slopes of the plots of log molecular weight versus Kd depend to a significant extent on the concentration of the sodium phosphate buffer (pH 7.0).View full abstractDownload PDF (268K) -
Izumi OHKAWA, Sawako SHIGA, Makoto KAGEYAMA1979 Volume 86 Issue 3 Pages 643-656
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new esterase activity which hydrolyzes palmitoyl-CoA was found in the membrane fraction of Pseudomonas aeruginosa. All the 11 strains of P. aeruginosa tested possessed this esterase activity. The esterase was constitutive and was fully active on the intact cell bodies toward substrates in the medium. It was located on the outer membrane of the cell envelope, and was not released into the culture medium. This activity was designated as OM (outer membrane) esterase.
OM esterase was solubilized from the cell envelope with EDTA-Triton X-100 and purified 690-fold. It was a minor component of the outer membrane. Its molecular weight was approximately 55, 000. The activity was rather stable to heat, a wide range of pH, and treatment with detergents and organic solvents. No cofactors were required. The pH optimum of the reaction was 8.5.
Among various acyl-CoAs, only long chain (C12-C18) thioesters were hydrolyzed. OM esterase also hydrolyzed some kinds of oxy-esters such as p-nitrophenyl acyl esters, monoacyl esters of sucrose and Tween 80 (polyoxyethylene sorbitan monooleate). On the other hand, triglycerides, phospholipids, or hydrophobic monoesters were not hydrolyzed at all. Thus, this enzyme seems to have specificity for long chain acyl esters with hydrophilic groups, whether thio- or oxy-ester.
Mutants deficient in this esterase activity were isolated. These mutants were unable to grow on Tween 80 as a sole carbon source. This suggests a possible role of OM esterase in the utilization of acyl esters as carbon sources.View full abstractDownload PDF (1066K) -
Alois CIHÁK, Hideo INOUE1979 Volume 86 Issue 3 Pages 657-662
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCadmium affects the induction of thymidine and thymidylate kinases in regenerating rat liver. EDTA administered simultaneously with cadmium reverses its inhibitory action on enzyme synthesis, and prevents the depression of thymidine incorporation into DNA observed in cadmium-treated animals. Zinc does not abolish the inhibitory action of cadmium on the synthesis of DNA in regenerating liver, and the incorporation of thymidine into DNA in the testes was inhibited more by intraperitoneal injection of cadmium plus zinc than by injection of cadmium alone. Inhibition of thymidine incorporation into DNA in the liver and testes was proportional to the amount of cadmium administered up to about 2mg CdCl2/kg body weight, but surprisingly, higher doses of cadmium caused less inhibition.View full abstractDownload PDF (394K) -
Kiyoyoshi NISHITA, Takao OJIMA, Shizuo WATANABE1979 Volume 86 Issue 3 Pages 663-673
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin was isolated from striated adductor muscle of Akazara shell-fish, and purified on DEAE-Sephadex A50. The sedimentation constant (s020, w) and the intrinsic viscosity, [η] of Akazara myosin thus purified were estimated to be 6.6S and 2.10dl/g, respectively. In many respects, Akazara myosin was similar to scallop myosin. (1) Only one size of light-chain component (17, 000 daltons) was detectable in SDS-gel electrophoresis of Akazara myosin, but two types of light-chain component were seen in urea-gel electrophoresis; these were equivalent to EDTA-light chain and SH-light chain of scallop myosin. The molar ratio of heavy chain (206, 000 daltons), EDTA-light chain, and SH-light chain in Akazara myosin was estimated, from the staining densities of gel-electrophoretic bands, to be approximately 1:1:1. (2) The EDTA-washing procedure removed EDTA-light chain only, causing desensitization of Akazara myosin. EDTA-light chain isolated from Akazara myofibrils was able to resensitize EDTA-washed Akazara myosin.
Akazara myosin, however, was found to be different from scallop myosin in two important properties: (1) complete removal of EDTA-light chains was required to achieve a complete loss of calcium sensitivity, and full resensitization was attained on recombination of EDTA-light chains with desensitized myosin prepared essentially free from EDTA-light chains. (2) EDTA-light chains isolated from Akazara myofibrils show a calcium-induced UV absorption difference spectrum.View full abstractDownload PDF (4145K) -
Ichiro MATSUOKA, Takao NAKANURA1979 Volume 86 Issue 3 Pages 675-681
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Oleic acid at low concentrations (0-70 nmol/mg protein) stimulated mitochondrial state 4 respiration 4-fold, increased the apparent enthalpy change of the respiration per gram atom of oxygen consumed from -112 to -208kJ/O and completely inhibited ATP synthesis without significant effect on the Mg-ATPase activity of mitochondria.
2. Similar effects on mitochondrial respiratory activities were observed with other fatty acids.
3. Bovine serum albumin (BSA) protected mitochondria from the effects of oleic acid irrespective of the order of addition of oleic acid and BSA to mitochondria. The capacity of BSA to bind oleic acid was calculated to be 3.6-7.1 (mean, 4.9) mol of oleic acid/mol of BSA.
4. The response time of mitochondrial respiration to added oleic acid or BSA was 20-25s.View full abstractDownload PDF (499K) -
Yoshiko NARAHASHI, Kaoru YODA1979 Volume 86 Issue 3 Pages 683-694
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new carboxypeptidase was isolated from a mixture of proteolytic enzymes of St. griseus K-1 (pronase) and purified about 30-fold by a procedure including CM-cellulose chromatography, D-Arg-CH-Sepharose chromatography, Sephadex G-100 gel filtration, and ST-Sepharose chromatography with a yield of 55 %. In these purification procedures, two kinds of substrates, Cbz-Gly-Leu and Bz-Gly-Arg, which are representative substrates of mammalian pancreatic carboxypeptidase A [EC 3.4.12.2] and B [EC 3.4.12.31], respectively, were used to detect the activity. The activities towards the two substrates were eluted at the same position, and the ratio of the activity with Cbz-Gly-Leu to that with Bz-Gly-Arg was always constant.
On isoelectric focusing, the purified enzyme protein appeared as a single symmetrical peak at pH 5.2, and the two activities overlapped completely. Metal analysis of the enzyme showed 0.88 zinc atom per mol of the enzyme; its molecular weight determined by the sedimentation equilibrium method was 34, 000. In studies on the enzymic properties, such as pH optimum, pH stability, stability at various temperatures, effect of buffer composition, and optimum activity temperature, no difference between the two activities could be observed. Reversible inhibitions of the activities towards Bz-Gly-Leu and Bz-Gly-Lys by reaction products were studied. Lys or Leu showed competitive-type inhibition and the IC, values for the two activities were parallel. The carboxypeptidase activity was tested with reduced and carboxymethylated pancreatic ribonuclease. The enzyme liberated amino acids sequentially, except for Pro.
These observations led us to conclude that the carboxypeptidase of St. griseus K-1 has a new substrate specificity which includes the specificities of mammalian carboxypeptidases A and B.View full abstractDownload PDF (1342K) -
Sumiko HAMANAKA(née YASUE), Shizuo HANDA, Jun INOUE, Atsuhiko H ...1979 Volume 86 Issue 3 Pages 695-698
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe glycolipids of erythrocytes from individual cats were examined. The main glycolipid
of cat erythrocytes was generally NeuGc-NeuGc-Gal-Glc-ceramide (NeuGc-GD3), but among 41 cats of 5 breeds and 2 mongrels examined, 2 Persian cats were found to have NeuAc-NeuAc-Gal-Glc-ceramide (NeuAc-GD3). This is the first report of the occurrence of NeuAc-GD3 in cat erythrocyte membranes.View full abstractDownload PDF (693K) -
Osao ADACHI, Kazunobu MATSUSHITA, Emiko SHINAGAWA, Minoru AMEYAMA1979 Volume 86 Issue 3 Pages 699-709
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOld yellow enzyme system has been found in the cytosol fraction of Gluconobacter suboxydans. This is the first time that the enzyme has been found in organisms other than yeast cells. Old yellow enzyme [EC 1.6.99.1], D-glucose-6-phosphate dehydrogenase [EC 1.1.1.49], and catalase were isolated and crystallized separately from the organism. The old yellow enzyme from G. suboxydans showed catalytic and physicochemical properties almost identical with those of the enzyme from yeast cells. NADPH was specifically oxidized by the old yellow enzyme and the reduced enzyme was spontaneously reoxidized by atmospheric oxygen. The old yellow enzyme from G. suboxydans also contained FMN as a prosthetic group, and two mol of FMN were found per mol of enzyme (molecular weight, 88, 000 as determined by gel filtration). In the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconate, cyclic regeneration of NADP occurred smoothly in the presence of D-glucose-6-phosphate dehydrogenase and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.View full abstractDownload PDF (3523K) -
Kazuo SHISHIDO1979 Volume 86 Issue 3 Pages 711-717
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCovalently closed-circular, superhelical DNAs, including viral DNAs, bacterial plasmid DNAs, and bacteriophage replicative-form DNA, were treated with a small amount of Haemophilus gallinarum DNA-relaxing enzyme to generate incompletely relaxed DNA molecules. Each sample consisted of a set of closed-circular DNA molecules differing by one turn in their number of superhelical turns. The DNA samples were analyzed by agarose gel electrophoresis under conditions such that the electrophoretic mobility was a function of the number of turns. The numbers of superhelical turns (at 37°C in 20mM Tris-HCl (pH 7.5)-5 mM MgCl2) in the DNAs of pSC101 (5.8megadaltons), Colicin E1 (4.2megadaltons), pMR4 (4.0megadaltons; recombinant between pBR322 and λDNA fragment), φX174 replicative-form (RF) I, Simian virus 40 (SV40), and polyoma virus (3.4-3.6 megadaltons each), and λdv021 (2.05 megadaltons) were estimated to be 36, 27, 23-24, 20-21, 20-21, 20-21, and 11-13, respectively. It appears that the number of superhelical turns is mainly a function of the molecular weight of the DNA, at least in the substrates tested here.View full abstractDownload PDF (1963K) -
Naohito FUJIMOTO, Ikuo YASUMASU1979 Volume 86 Issue 3 Pages 719-724
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activity of fructose 1, 6-bisphosphatase [EC 3.1.3.11] in sea urchin eggs decreased following fertilization. During the first 30min after fertilization, the activity was considerably lower than that in unfertilized eggs, but by 30min the activity was similar to that in unfertilized eggs. The enzyme activity in fertilized eggs, estimated in the presence of EGTA, was similar to that in unfertilized eggs. The activity in unfertilized eggs was reduced by Ca2+ at concentrations between 1×10-5M and 5×10-3M. Immediately after fertilization, the enzyme was insensitive to concentrations of Ca2+ lower than 2×10-4M, but the Ca2+ sensitivity of the enzyme recovered 30min after fertilization. In the presence of Ca2+ at concentrations higher than 2×10-4M, the enzyme activity in unfertilized eggs was similar to that in fertilized eggs. Mg2+ restored the Ca2+-induced inhibition of fructose 1, 6-bisphosphatase. 3-Phos-phoglycerate and citrate hardly affected the enzyme activity, and AMP at concentrations above 10mM inhibited it.View full abstractDownload PDF (429K) -
Evidence for Differences in Structure around the Active Sites of Cardiac, Smooth, and Skeletal Muscle Myosin ATPaseSudhir K. SRIVASTAVA, Yuji TONOMURA, Akio INOUE1979 Volume 86 Issue 3 Pages 725-731
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosins purified from cardiac (porcine heart) and smooth (chicken gizzard) muscles were modified with 2, 4, 6-trinitrobenzenesulfonate (TNBS) and the effects on the kinetic properties of myosin ATPase [EC 3.6.1.3] were studied. The following results were obtained.
1. About 0.5mol of TNBS per mol of myosin head was incorporated rapidly, irrespective of the presence of PP1 (2mM), into both types of myosin studied.
2. The size of the initial burst of P1 liberation for both myosins was found to be 0.5-0.6 mol/mol head.
3. The rapid incorporation of TNBS into cardiac muscle myosin was accompanied by a rapid decrease in the size of the initial P1 burst, and it was completely lost after modification for 20 min. However, smooth muscle myosin retained its P1 burst.
4. The EDTA (K+)-ATPase activity of both myosins modified in the presence or absence of PP1 decreased sharply with incorporation of TNBS.
5. Superprecipitation and ATPase activity of reconstituted actomyosin from cardiac myosin and skeletal F-actin decreased only after 10min of modification with TNBS in the absence of PP1.
6. The spectra of TNP bound to myosins from cardiac and smooth muscles were unchanged by the addition of PP1.
The above findings are compared with those previously obtained for skeletal muscle myosin [Miyanishi, T., Inoue, A., & Tonomura, Y. (1979) J. Biochem. 85, 747-753], and the structural and functional differences among the myosins derived from skeletal, cardiac, and smooth muscles are discussed.View full abstractDownload PDF (575K) -
I. A Potentiometric Titration StudyShozo IIDA1979 Volume 86 Issue 3 Pages 733-743
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStructural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.View full abstractDownload PDF (764K) -
Takeyori SAHEKI, Yuki SATO, Shigeo TAKADA1979 Volume 86 Issue 3 Pages 745-750
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of inhibition of urea synthesis by L-norvaline was investigated in vitro with perfused rat liver and purified enzymes of the urea cycle, and in vivo. L-Norvaline caused potent inhibition of urea synthesis from ammonium chloride in the perfused liver and increases in citrulline and arginine in the liver. The increased ratio of the concentration of arginine to ornithine suggested that the main inhibition step of the urea cycle with L-norvaline is arginase. Kinetic studies with purified enzymes showed that L-norvaline inhibits arginase as well as ornithine transcarbamylase and argininosuccinate synthetase competitively with respect to their amino acid substrates, arginine, ornithine, and citrulline, respectively. The inhibition of arginase by L-norvaline was much stronger at pH 7.5 than at pH 9.7. An in vivo experiment was performed to determine whether the same inhibition mechanism operates in the rat. Judging from a decrease in urea and an increase in ammonia in the liver, intraperitoneal injection of L-norvaline caused inhibition of urea synthesis in vivo. The same conclusion, that L-norvaline inhibits urea synthesis mainly through the inhibition of arginase, was also obtained in the in vivo experiment showing an increased ratio of the concentration of arginine to ornithine. The concentration of acetylglutamate was increased by the addition of L-norvaline both in the perfused liver and in the liver in vivo, probably as a result of secondary effects of the increase in arginine.View full abstractDownload PDF (447K) -
Takao NAKAMURA1979 Volume 86 Issue 3 Pages 751-755
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe catalytic hydrolysis of p-nitrophenyl acetate by α-chymotrypsin was studied by stoppedflow calorimetry and spectrophotometry at pH 8.0 and 25°C. The initial burst and sub-
sequent steady state of the reaction were observed by rapid calorimetry and spectrophotometry. Based on the three-step mechanism established for the enzymatic reaction,
E +S_??_ES→acetyl-E+P1 (p-nitrophenol)→E+P1+acetic acid,
the enthalpy change of formation of the acetyl enzyme from ES complex was estimated to be -29 kJ•mol-1.View full abstractDownload PDF (327K) -
Yasuko SAWAI, Mieko YANOKURA, Kinji TSUKADA1979 Volume 86 Issue 3 Pages 757-764
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree forms (termed I, II, and III) of ribonuclease H (RNase H) [EC 3.1.4.34] activity are present in rat liver cytosol. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at 0 M, 0.25 M, and 0.5 M KCl in phosphocellulose chromatography, and were further purified by using blue Sepharose. They are further distinguished from one another by their ionic requirements, optimal pH, molecular weights, sedimentation coefficients, and sensitivity to the -SH reagent, p-chloromercuribenzoate, although I and III have similar characteristics. They liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini.View full abstractDownload PDF (601K) -
III. Isolation and Structural Studies of Two Novel Gangliosides Containing Internal Sialic Acid ResiduesMutsumi SUGITA1979 Volume 86 Issue 3 Pages 765-772
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo gangliosides, provisionally named Gangliosides 1 and 2 in previous studies, were isolated from starfish, Asterina pectinifera by silicic acid, DEAE-Sephadex, and Iatrobeads column chromatography procedures, and preparative thin-layer chromatography, and their structures were established. On the basis of the results of partial acid hydrolysis, methylation and oxidation with chromium trioxide, Gangliosides 1 and 2 were proposed to be Arapβ(1 →6)-Galpβ(1→4)8-0-MeNeuGc(2→, 3)Galpβ(1→4)G1cpβ(1→1)-ceramide and Arapβ(1→ 6)- Galpβ(1 4)NeuGc(2 →)3)Galpβ(1 →4)Glcpβ(1 →1)-ceramide, respectively.
The ceramide moieties of both gangliosides had similar phytosphingosine and 2-hydroxy fatty acid compositions, and both gangliosides were structurally related to the previously described Ganglioside 3.View full abstractDownload PDF (1890K) -
Takashi MIURA, Shizuo HANDA, Tamio YAMAKAWA1979 Volume 86 Issue 3 Pages 773-776
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of glycolipids on the interaction of the MIF (migration inhibition factor) with rat macrophages were examined using a migration inhibition assay system. MIF activity was specifically blocked by fucosylated Glycolipid RM [Galαl-3Gal(2-1αFuc)β1-3GaINAcβ1-3Ga1β1-4Glcβ1-lceramide, (1978) J. Biochem. 83, 85-90], but not by Cytolipin R, hematoside, or blood group B active glycolipid [Galα1-3Gal(2-1αFuc)β1-4GlcNAcβ1-3Galβ1-4G1cβ1-lceramide]. Inhibition of MIF activity was proportional to the concentration of Glycolipid RM. These findings suggest that Glycolipid RM acts as a receptor for MIF.View full abstractDownload PDF (328K) -
Toshihiro TSUDZUKI1979 Volume 86 Issue 3 Pages 777-782
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+ uptake by synaptic vesicle fractions isolated from bovine caudatolenticular nuclei and from rat brain was studied. The purified vesicle fractions from both materials took up very little Ca2+ even in the presence of ATP and Mg2+, but the crude fractions took up Ca2+ actively, showing the maximum uptake around pH 7.0. Since the crude fractions were contaminated by microsomes, which are known to accumulate Ca2+ actively (Yoshida, H., Kadota, K., & Fujisawa, H. (1966) Nature 212, 291-292; Otsuka, M., Ohtsuki, I., & Ebashi, S. (1965) J. Biochem. 58, 188-190), the active uptake of Ca2+ appeared to be largely, if not wholly, due to microsomal contamination.View full abstractDownload PDF (1033K) -
Yasuhito NAKAGAWA, Hideo UTSUMI, Keizo INOUE, Shoshichi NOJIMA1979 Volume 86 Issue 3 Pages 783-787
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relation between the molecular motion of a steroid in lipid membranes and the transfer rate between membranes was examined using radioactive cholestane spin label.
Order parameters of the molecule were determined in bilayers composed of dipalmitoyl-glycerophosphocholine or egg yolk phosphatidylcholine at various temperatures. The line widths of the ESR signal of the cholestane spin label in membranes, which depend upon the rate of molecular axial rotation in the membranes, were also measured. The temperature dependences of these two parameters and of the transfer rate suggest a close correlation between the rate of molecular axial rotation and the transfer rate.View full abstractDownload PDF (370K) -
Koei HAMANA, Koichi IWAI1979 Volume 86 Issue 3 Pages 789-794
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHigh mobility group (HMG) nonhistone chromosomal proteins have been shown to exist also in the ciliated protozoan Tetrahymena pyriformis. One or two histone-like components were extracted with 0.25M HCl from the chromatin, in addition to five histone species. These proteins were also extracted selectively with 0.5M HClO4, 0.35M NaCl, or 4mM spermidine, together with H1 histone, and were characterized as HMG proteins on the basis of the following criteria: high mobilities on polyacrylamide gel electrophoresis, relatively low molecular weights, amino acid compositions rich in lysine and glutamic acid, and relative contents in chromatin. This extends the distribution of the HMG proteins to all four eukaryotic kingdoms, and suggests the possibility that they have some universal role in chromatin structure and function.View full abstractDownload PDF (1076K) -
Masanobu JANADO, Reiko NAKAYAMA, Yuki YANO, Hiroko NAKAMORI1979 Volume 86 Issue 3 Pages 795-802
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn frontal gel chromatography on Bio-Gel P-2, sodium dodecyl sulfate (SDS) at the concentrations below its critical micelle concentration (cmc) showed anomalously high partition coeffics (Kobsav) indicative of strong interactions with the swollem gel phase; further, Kobsav was found to increase with concentration and temperature. This preferential partition of SDS in the Bio-Gel phase was analyzed in analyzed in terms of the transfer free energy of SDS from the mobile phase (0.1M NaCl) to the swollen Bio-Gel phase. The results showed that the overall transfer process is promarily governed by hydrophobic free energy arising from the anomalous nature of hydrated water in the gel matrix; that is, in highly hydrated water “iceberg” formation is evidently limited and the hydrophobic free energy is accordingly lowered, resulting in the preferential patition of SDS in the swollen Bio-Gel phase. The increase in the negativity of transfer free energy with concentration, though relatively small, indicated a definite tendency for the formation of SDS clusters in the gel phase. Finally, a model illustrating the states of SDS molecules in the gel matrix is preseented, which may also be pertinent to SDS-protein and SDS-amylose complexes.View full abstractDownload PDF (577K) -
Takeshi YAMAZAKI, Akemi SUZUKI, Shizuo HANDA, Tamio YAMAKAWA1979 Volume 86 Issue 3 Pages 803-809
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA quantitative consecutive method was developed for analysis of sphingoglycolipids in bio-logical materials by high performance liquid chromatography (HPLC). Crude lipid extracts were separated into neutral and acidic fractions on a DEAE-Sephadex column. Glycolipid fractions were obtained by acetylation and Florisil column chromatography, and the acetylated glycolipids were N-p-nitrobenzoylated by treatment with p-nitrobenzoyl chloride in pyridine at 60°C for 6 h. Excess reagent and by-products were removed by solvent partition and gel filtration. The glycolipid derivatives were analyzed by their absorption at 254 nm on Zorbax SIL, a silica gel column, with a gradient of 0.5-7 % isopropanol in hexane-chloroform (2 : 1, v/v) at a flow rate of 0.5 ml/min. The detector response was linear with up to 60 nmol of injected glycolipids. The practical lower limit of detection was about 50 pmol. The deriva-tives were separated on the basis of their sugar chains. Effluents corresponding to each peak were collected and analyzed further on the basis of their lipid portion on μ-Bondapak C18, a reversed phase column. This combined procedure was applied to the analysis of erythrocyte glycolipids. Samples containing as little as 20μg of glycolipids could be analyzed by this method.View full abstractDownload PDF (448K) -
Kazuhiro KOHAMA1979 Volume 86 Issue 3 Pages 811-820
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. New methods of preparing troponins from slow skeletal and cardiac muscle of the chicken have been developed. The electrophoretic mobilities of slow skeletal muscle troponin sub-units were different from those of the corresponding fast skeletal muscle subunits.
2. A new method for determining the amount of divalent cations bound to troponin was developed. The principle of the method is to immobilize troponin by conjugating it with Sepharose 4B resin, thus making it readily sedimentable.
3. The numbers of Sr and Ca ions bound to slow muscle troponin at concentrations sufficient to produce maximum contraction were 1.73 and 1.36 mol per mol, respectively, being nearly equal to those of cardiac troponin but half of those of fast muscle troponin.
4. The concentrations of Sr and Ca ions giving half-maximal ion binding to slow muscle troponin (K50%) were 5.5 × 10-6 M and 4.6 × 10-7 NI, respectively.
5. K50% for Sr of cardiac troponin was significantly higher than that of slow muscle troponin. Although K50% for Sr of cardiac troponin was the same as that of fast muscle troponin, cardiac troponin bound more Sr ions than fast muscle troponin at lower Sr ion concentrations. The mechanism underlying the high sensitivity of cardiac muscle contraction to Sr ions is discussed in comparison with that of slow muscle.View full abstractDownload PDF (1558K) -
Tateo YAMANAKA, Keiko FUJII, Yuhsuke KAMITA1979 Volume 86 Issue 3 Pages 821-824
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
The T. novellus enzyme is composed of two kinds of subunits of 32, 000 and 23, 000 daltons and its minimum molecular weight is 55, 000 on the basis of heme content and amino acid conposition. The N. agilis enzyme also has two kinds of subunits of 40, 000 and 27, 000 daltons and its minimum molecular weight is 66, 000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.View full abstractDownload PDF (1547K) -
Yukuo YOSHIDA, Eisuke FURUYA, Kunio TAGAWA1979 Volume 86 Issue 3 Pages 825-828
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA sensitive colorimetric method for quantitative determination of phosphatidylcholine is described. The procedure is very simple, not involving lipid extraction or acid digestion, and can be used for direct estimation of phosphatidylcholine in blood plasma. The method depends on the formation of a stable hydrophobic complex of phosphatidylcholine with tetrathiocyanatocobaltate. Similar complexes are formed by other species of diacylglycero-phosphoryl esters, but the absorbance coeffcients of these complexes are only 10 to 20% of that of the phosphatidylcholine complex. Phospholipids without unsaturated fatty acids, such as dipalmitoyl phosphatidylcholine, do not form hydrophobic complexes. Lysophosphatidyl-choline and sphingolipids also do not form similar complexes.View full abstractDownload PDF (276K) -
Joan WIKMAN-COFFELT, Sudhir SRIVASTAVA1979 Volume 86 Issue 3 Pages 829-832
Published: September 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrog skeletal muscle myosin light chains readily dissociate from the myosin oligomer in the absence of divalent cations, and unlike rabbit skeletal muscle myosin light chains, the released light chains of frog skeletal muscle myosin have a high Ca2+ binding affinity. Whereas each Ca2+ binding light chain of frog skeletal muscle myosin, when in association with the heavy chains bound 1 mol of Ca2+, when in the dissociated state bound 0.5 mol of Ca2+; the latter were readily displaced with low Mg2+ concentrations. Whereas 10-5M Mg2+ displaced all of the Ca2+ biding sites on the released light chains at Ca2+ concentration ranges of 10-7 to 10-4M, there was negligible displacement of the Ca2+ binding sites with native frog skeletal muscle myosin under these same conditions.View full abstractDownload PDF (299K)
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