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Volume 93 (1983)
- Issue 6 Pages 1463-
- Issue 5 Pages 1221-
- Issue 4 Pages 943-
- Issue 3 Pages 669-
- Issue 2 Pages 323-
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Volume 93, Issue 4
Displaying 1-32 of 32 articles from this issue
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Atsushi MATSUMOTO, Fumi MORITA1983 Volume 93 Issue 4 Pages 943-953
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTyr 180 of chicken breast muscle alkali light chain A1 was nitrated with tetranitromethane. The nitroA1 was incorporated into chicken breast muscle subfragment-1 (S-1) by exchange with the intrinsic alkali light chain. In the presence of adenylyl-imidodiphosphate (AMPPNP) or ADP, the S-1 containing nitroA1 showed a difference visible absorption spectrum by Mg2+ or Ca2+. The difference spectrum has a trough around 435nm, indicating a blue shift of the absorption spectrum due to the nitrophenol chromophore of the modified A1. The plot of ΔA at 435nm versus concentration of free Mg2+ fitted a single binding curve, independent of the total concentration of AMPPNP. These results reveal that free Mg2+ binds to the active site of S-1 ATPase, but not as Mg-AMPPNP complex. The dissociation constants of magnesium from S-1 complex were different with the two nucleotides and were 1.25×10-8M and 1.24×10-7M with AMPPNP and ADP, respectively.
The difference spectrum was also obtained in the presence of ATP. The Δε value after adding ATP changed with the ATPase reaction. The steady state rate of S-1 ATPase was measured at various concentrations of free Mg2+. The dissociation constant of magnesium from the steady state complex, EPADPP (α), was estimated as 6×10-8M. These results suggest that the affinity of magnesium at the active site of ATPase changes with the intermediate states of ATPase reaction. The affinity of calcium was lower than that of magnesium.View full abstractDownload PDF (1708K) -
Nanaya TAMAKI, Keiko KUBO, Hidenori AOYAMA, Akimi FUNATSUKA1983 Volume 93 Issue 4 Pages 955-959
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmong uracil derivatives investigated, 6-azauracil, 6-azathymine, and 5-iodouracil were found to be potent inhibitors of purified rabbit liver 4-aminobutyrate aminotransferase while 6-azauridine and 6-azauridine 5'-phosphate were not. The enzyme inhibited by 6-azauracil was reactivated by dialysis but not by addition of pyridoxal 5'-phosphate. 6-Azauracil acted as a non-competitive inhibitor with respect to β-alanine as well as 2-oxoglutaric acid, and had a K1 of approximately 0.7mM at pH 7.3.
The kinetic data suggested that 2-oxoglutaric acid acted as an inhibitor as well as an amino acceptor for the enzyme; a catalytic site was associated with an apparent Km of 0.15mM for 2-oxoglutaric acid and a low affinity site was associated with an I50 of approximately 5mM for the 2-oxo acid. With inhibitory concentrations of 2-oxoglutaric acid as substrate the inhibitory effect of 6-azauracil was considerably diminished.
From these findings, the inhibitory effect of 6-azauracil was revealed to be different from that of structural analogs of 4-aminobutyric acid showing that 6-azauracil is a new type of 4-aminobutyrate aminotransferase inhibitor.View full abstractDownload PDF (399K) -
Isao MATSUI, Shuzo OTANI, Ayako KURAMOTO, Seiji MORISAWA, Anthony E. P ...1983 Volume 93 Issue 4 Pages 961-966
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn increased activity of spermidine N1-acetyltransferase was induced in bovine lymphocytes by stimulation with either sodium n-butyrate or phytohemagglutinin (PHA). The acetylase activity was elevated by 2- to 3-fold 24h after the addition of butyrate, whereas a similar increase of the enzyme activity was found 48h after stimulation with PHA. When butyrate and PHA were added to the lymphocyte suspensions simultaneously, the peak of enzyme induction was observed at 24h after the addition and the increase (9-fold) was found to be more than additive. Since the increases in acetylase activity by both butyrate and PHA were markedly inhibited by actinomycin D or cycloheximide, they appear to have resulted from the synthesis of new protein rather than the release of the enzyme from a cryptic inactive form. The enzyme induced by the two agents was most active with spermidine as a substrate, but spermine was also acetylated to a smaller extent (25% of spermidine). Virtually no acetylation was observed with putrescine. Also, the product formed from spermidine and acetyl CoA was more than 90% N1-acetyl sperm idine. These results indicate that the acetylases induced by the two agents are similar. Although the addition of indomethacin or 8-brornocyclic AMP to PHA-stimulated lymphocytes caused an inhibition of the enzyme induction, no such inhibitory effect was found in the case of enzyme induction by butyrate. These results indicate that both sodium n-butyrate and PHA induce spermidine N1-acetyltransferase in bovine lymphocytes, but the induction mechanisms are different from each other.View full abstractDownload PDF (524K) -
Atsushi TAKEDA, Kouichi HIRANO, Yoko SHIROYA, Tatsuya SAMEJIMA1983 Volume 93 Issue 4 Pages 967-975
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe dissociation of porcine erythrocyte catalase [EC 1. 11. 1. 6] into subunits on denaturation with alkali, GuHCI and urea was investigated by following the changes in hydrodynamic properties, absorption and CD spectra in the Soret region and inactivation of the enzyme. It was found that dissociation proceeded in an “all or none” manner from the native tetramer (molecular weight, ca. 250, 000) into identical 1/4-sized monomers (molecular weight, ca. 54, 000 with alkali, 65, 000 with urea and 71, 000 with GuHCl) as estimated by ultracentrifugal analyses. On this dissociation, the sedimentation coefficient decreased from about 11S to 5.1-3.7 S, and absorption spectra in the Soret region decreased to about 40 of the native level and showed a broad band around 365-375 nm and a shoulder around 415-420 nm; these changes were accompanied by complete loss of enzyme activity. The change in enzyme activity correlated well with that of absorption and CD spectra in the Soret region, depending on denaturation time, alkaline pH used and concentration of both denaturants. The reassociated catalase obtained by removing urea by dialysis was characterized by recovery of distinct CD bands in the Soret and near ultraviolet regions, although the partial refolding of a-helical conformation occurred without recovery of enzyme activity. These results indicate that the conformational changes and dissociation process of catalase into subunits can be monitored spectrophotometrically in relation to enzyme activity, and that subtle conformations near the heme groups and polypeptide backbone play an important role in maintaining full enzyme activity of the catalase molecule.View full abstractDownload PDF (810K) -
Tadashi SHIMO-OKA, Kazuo OHNISHI, Yoshio WATANABE1983 Volume 93 Issue 4 Pages 977-987
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCrude actomyosin fraction from porcine brain contained a large amount of high molecular weight actin-binding protein (BABP). The molar ratio of BABP to actin (BABP/actin) in the fraction was estimated to be 0.22. From this fraction, BABP and actin were solubilized with a molar ratio of 0.25, suggesting the existence of an interaction between BABP and brain actin. BABP was finally purified to 90%, purity. The purified BABP was negatively stained and observed by electron microscopy; it appeared to be a slender, flexible, two-stranded molecule whose contour length was about 200 nm. The structure was very similar to those of fodrin and other high molecular weight actin-binding proteins such as filamin, spectrin, and ABP. Lattice cage-like structures composed of BABP molecules were occasionally observed at high BABP concentrations. The addition of BABP to actin filaments resulted in the appearance of many branching, filamentous bundles. The electron microscopic observations suggested that a single BABP molecule could crosslink actin filaments, that is, one BABP molecule has two actin binding sites.View full abstractDownload PDF (5403K) -
III. Isolation of RNA Polymerase-RNA Complexes from Influenza Virus PR8Kiyoshi KAWAKAMI, Akira ISHIHAMA1983 Volume 93 Issue 4 Pages 989-996
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRibonucleoprotein (RNP) cores with RNA-synthesizing activity were prepared in two fractions, M protein-free and M protein-associated, from detergent-treated influenza virus PR 8 by centrifugation through a discontinuous triple gradient of cesium sulfate, glycerol, and NP-40. The M-free RNP was fractionated by phosphocellulose column chromatography into two major RNP forms, A and B, which differed in the content of P proteins, while the M-associated RNP gave only the low P-content Form-B RNP. Starting from the high P-content Form-A RNP, an RNA-P proteins complex virtually free from NP protein was isolated by cesium sulfate equilibrium centrifugation. The complex, containing only three P proteins (P 1, P 2, and P 3), was still active in catalyzing RNA synthesis in vitro without addition of exogenous template, indicating that NP protein is not required for the catalysis of RNA synthesis. RNA synthesis by the isolated RNA-P proteins complex was dependent on either ApG or capped RNA primers, and required four ribonucleoside triphosphates as substrates. The RNA product in this reaction was hybridizable to viral RNA. A complex of one each of the three P proteins was separated from RNA by glycerol gradient centrifugation after ribonuclease treatment or cesium chloride equilibrium centrifugation.View full abstractDownload PDF (1845K) -
Shigeo HORIE, Tomino WATANABE, Kumiko AVE1983 Volume 93 Issue 4 Pages 997-1010
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conditions for the preparation of the ferricytochrome a-ferrocytochrome a3-carbon monoxide complex (a3+, a32+CO) of cytochrome oxidase [EC 1. 9. 3. 1] by the ferricyanide-reoxidation method and some properties of the prepared complex were studied.
The addition of a small volume of concentrated ferricyanide solution to the dithionite-reduced and carbon monoxide-treated cytochrome oxidase preparation was required to obtain the (a3+, a32+CO) spectrum showing absorption maxima at 590, 545, and 429 nm. The addition of larger volumes of ferricyanide solution, thus introducing larger amounts of oxygen into the preparation, caused decomposition of the carbon monoxide complex. A part of the added ferricyanide was immediately reduced by dithionite whereas the remainder was gradually reduced by partial oxidation product(s) of dithionite. The (a3+, a32+CO) complex was stable only when excess ferricyanide remained in the reaction mixture. The formation of the (a3+, a32+CO) spectrum was observed when sodium citrate, phosphate or borate buffer containing either cholate or a non-ionic detergent was employed as the solvent buffer, but not with the buffers containing sodium dodecyl sulfate (SDS) or cetyltrimethyl-ammonium bromide (CETAB). The formation was considerably inhibited by trishydroxymethyl-aminomethane(Tris)-HCI buffer.
The (a3+, a32+CO) spectrum appeared with maximal intensity at around pH 7. The pH-dependency of the intensity of the spectrum was not in parallel with the pH-dependent change of the polymerization state of the cytochrome oxidase preparation. On freezing to liquid nitrogen temperature, the (a3+, a32+CO) complex prepared in usual solvent buffers was mostly converted to the oxidized form of cytochrome oxidase (a3+, a33+). However, when prepared in the phosphate buffer, pH 8. 0, containing 1.2% (w/v) sodium cholate and with 20% saturation with ammonium sulfate, the complex mostly remained unchanged after the freezing.
Based on the results obtained, the stability of the juxta-heme structure of cytochrome a3 was also discussed.View full abstractDownload PDF (1146K) -
Eisuke NISHIDA, Hikoichi SAKAI1983 Volume 93 Issue 4 Pages 1011-1020
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetics of actin polymerization were analyzed by measuring the changes in absorbance which accompany the G-F transformation of actin. In these studies, gel-filtered actin was polymerized in the absence of shearing stress under physiological ionic conditions. Self-polymerization was found to be characterized as a process having a lag phase followed by a pseudo-first-order decay process, suggesting that actin polymerization consists of distinct nucleation and elongation phases. The size of the nucleus was estimated to be two to four monomer units by analyzing the actin concentration-dependences of the rate constant for the pseudo-first-order process, the maximal rate of polymerization and the half-polymerization time. The elongation reaction was induced by mixing actin filament seeds with actin monomers under conditions where spontaneous nucleation is slow. This elongation was also found to be a pseudo-first-order process, indicating that spontaneous nucleation was negligible. Our data suggested that the initial rate of elongation was proportional to both the number concentration of actin filaments and the actin monomer concentration above the critical concentration, and that the depolymerization rate was proportional to the number concentration of actin filaments but independent of monomer concentration. The results of direct analysis of the depolymerization reaction were consistent with this suggestion. These studies strongly support the condensation polymerization mechanism as a model for actin polymerization.View full abstractDownload PDF (867K) -
Eisuke NISHIDA, Shohei MAEKAWA, Hikoichi SAKAI1983 Volume 93 Issue 4 Pages 1021-1026
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetics and thermodynamics of microtubule assembly were studied using outer doublet tubulin isolated from starfish sperm. Purified tubulin was gel-filtered just prior to the experiments. The self-polymerization process was found to be characterized by a lag followed by a growth phase that did not show a single first-order decay reaction. This suggested that the nucleation and elongation steps are not completely separate. The maximal rate of polymerization and the reciprocal of the duration of lag time increased approximately in proportion to the third and second powers of tubulin concentration above the critical concentration, respectively. Measurement of temperature dependence of the critical concentration gave a linear van't Hoff plot with ΔH°=13.5 kcal/mol and ΔS°=69 e. u.View full abstractDownload PDF (537K) -
Masato NOGUCHI, Tadashi YOSHIDA, Goro KIKUCHI1983 Volume 93 Issue 4 Pages 1027-1036
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn heme degradation catalyzed by the reconstituted heme oxygenase system, 8 to 9 mol of dioxygen and 11 to 12 mol of NADPH were consumed per mol of heroin lost, and about half the amount of dioxygen consumed could be accounted for by the production of hydrogen peroxide, which accumulated in the reaction mixture. Production of hydrogen peroxide in the heme oxygenase reaction did not appear to be due to the bimolecular dismutation of superoxide anions but rather seemed to be due to dissociation of a “peroxo” species formed on heme or intermediates of heme degradation. The hydrogen peroxide produced appeared to cause a considerable degree of non-specific degradation of heme (not leading to the formation of biliverdin) and also caused an inactivation of heme oxygenase. By taking into account the amount of dioxygen incorporated into hydrogen peroxide and some other factors, it could be deduced that 3 mol of dioxygen is consumed for the formation of 1 mol of biliverdin in the heme oxygenase reaction.View full abstractDownload PDF (953K) -
Akiyoshi TANAKA, Takashi YAMASHITA, Masatake OHNISHI, Keitaro HIROMI1983 Volume 93 Issue 4 Pages 1037-1043
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding mechanism of Rhizopus glucoamylase with maltooligosaccharides was investigated at pH 4.5 and 0.5°C by the steady-state and the transient kinetic methods.
By the steady-state kinetic study, the Michaelis constant Km and the molecular activity Ko were evaluated for maltooligosaccharides with degree of polymerization (DP) of 2 to 7. The subsite affinities of seven subsites in the active site were determined on the basis of the subsite theory [Hiromi, K., et al. (1973) Biochim. Biophys. Acta 302, 362-375] by using the Km and Ko values of the linear substrates. It was found that Subsite 2 has the highest affinity (4.7 kcal/mol), which is even larger than the sum of the six other subsite affinities, and Subsite I has a small negative affinity (-0.12 kcal/mol).
The dissociation constant Kd of the enzyme (E)-substrate (S) complex for each substrate, determined by monitoring the decrease in the enzyme fluorescence caused by the substrate binding, was found to decrease with increasing DP.
Fluorescence stopped-flow kinetic studies on the binding of these substrates suggested a two-step mechanism which consists of a fast bimolecular process and a subsequent slow unimolecular process:
E+Sk+1_??_k-1ESk+2_??_k-2ES*
where ES and ES* are the loosely and tightly bound enzyme-substrate complexes, respectively. For all the substrates examined, it was confirmed that the fluorescence intensity decrease occurs solely in the unimolecular process. The dissociation constant of ES, K-1 (=k-1/k+1), and k+2 were almost independent of DP, whereas k-2 decreased with increasing DP. The molecular binding affinity (the unitary part of the standard affinity) for the fast bimolecular process was estimated to be about 4.9 kcal/mol for each substrate, which is close to the subsite affinity of Subsite 2.
These results were interpreted as follows: In the initial bimolecular process, the enzyme and a substrate form a loosely bound complex (ES) with lower specificity, irrespective of DP. In the ES complex, mainly Subsite 2 may be involved in the binding. In the subsequent unimolecular process, the ES complex transforms into a more tightly bound complex (ES*) with higher specificity. The ES* complex can probably be the productive complex, in which Subsite 1 is occupied by the nonreducing end glucose residue of the substrate. The decrease in k-2 with DP is most likely accounted for by the fact that the number of interactions to be broken in the ES* complex increases with DP. The decrease in Kd (=K-1k-2/k+2) with DP arises mainly from the decrease in k-2.View full abstractDownload PDF (566K) -
Keiko KITAGISHI, Keitaro HIROMI, Masanobu TOKUSHIGE1983 Volume 93 Issue 4 Pages 1045-1054
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe tryptophan residue of talopeptin, which is a specific inhibitor for thermolysin, was converted into kynurenine by ozonization followed by acid-catalyzed hydrolysis, and (Trp→Kyn) talopeptin (Kyn-talopeptin) thus obtained was purified with gel-chromatography.
The inhibitor constant of Kyn-talopeptin, K1, and the dissociation constant of thermolysin-Kyn-talopeptin complex, Kd, directly obtained by fluorometric titration were in good agreement with each other. These values were found to be about 10 times larger than those of intact talopeptin, but both inhibitors showed a similar pH dependence. Upon the binding of Kyn-talopeptin with thermolysin, the protein fluorescence of thermolysin decreases by about 60%, while the kynurenine fluorescence (measured at 450 nm when excited at 360 nm) of the inhibitor increases about 14 times. The measurements of the excitation and fluorescence spectra of EI complex strongly indicated the energy transfer from tryptophan residue(s) (the donor) of the enzyme to kynurenine residue (the acceptor) of the inhibitor. The distance between the donor and the acceptor was roughly estimated to be 18 Å. This value is in good agreement with the one expected from the crystallography of phosphoramidon-thermolysin complex.
The binding process was studied kinetically with the stopped-flow method over the pH range between 4.5 and 8.5, by monitoring the decrease in the fluorescence intensity of the enzyme tryptophan caused by the complex formation. Comparison of the data with those previously obtained for talopeptin-thermolysin system revealed that the replacement of the tryptophan residue by kynurenine of the inhibitor does not affect the apparent second-order association rate constant, kon, seriously.View full abstractDownload PDF (787K) -
Kaoru OMICHI, Tokuji IKENAKA1983 Volume 93 Issue 4 Pages 1055-1060
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new substrate of α-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-α-n-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-gIucopy-ranosyl-(1→4)-D-glucopyranose, was prepared using dextrin as a starting material. Compared with other substrates so far reported, the fluorogenic substrate is unique in that it is resistant to exo-α-glucosidases due to the blocking group introduced into the non-reducing end glucose residue. The product of α-amylase digestion was rapidly separated from the substrate and was detected very sensitively by HPLC and a fluorescence detector. This method for α-amylase assay was also applied for determination of α-amylase in human serum.View full abstractDownload PDF (1754K) -
Kazuhiko YAMAMOTO1983 Volume 93 Issue 4 Pages 1061-1069
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C.
2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity.
3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations.
4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase.
5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.View full abstractDownload PDF (1486K) -
Masao TSUDA, Tadashi KITAZAKI, Yoshio IMAI1983 Volume 93 Issue 4 Pages 1071-1077
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProfiling of plasma lipoprotein distribution and apolipoprotein analysis were carried out semi-quantitatively by strictly controlled gel-filtration and SDS slab-gel electrophoresis for rats (Sprague-Dawley rats and ExHC rats), mice and hamsters. The effects of cholesterol feeding on lipoprotein and apolipoprotein patterns of these animals were also studied.
HDL were major lipoproteins in all the animals. Hamsters had the most abundant lower density lipoproteins among the rodents with the ratio of cholesterol content between high density lipoproteins and lower density lipoproteins being about 1:1. After cholesterol feeding, the cholesterol content of lower density lipoproteins increased in all the animals. The increase was the greatest in ExHC rats and the least in mice.
The profiling methods employed should be of value in determining the changes in the lipoprotein and apolipoprotein patterns of both experimental animals and man.View full abstractDownload PDF (2275K) -
Kunio YONEMASU, Takako SASAKI, Akira SHIMIZU1983 Volume 93 Issue 4 Pages 1079-1084
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman, bovine, and mouse C1q, a subcomponent of the first complement component, were purified, and both globular (GF) and collagen-like fragments (CLF) were isolated from human and bovine C1q. Antisera were produced in rabbits with these C1q or fragments, and F(ab')2 of immunoglobulin G (IgG) was purified from the antisera in order to avoid the possible non-specific binding of C1q of these animals to the Fe portion of rabbit IgG. Immunodiffusion analyses and radioimmune inhibition tests with these F(ab')2 showed that the definitive antigenic crossreactivity was among C1q molecules of these animals, and that the regions participating in interspecies cross-reactions were located in both GF and CLF of C1q. These results suggest that both the C-terminal non-collagenous globular and the N-terminal collagen-like domains of C1q molecules may have remained highly conserved during evolution.View full abstractDownload PDF (3286K) -
I. Presence of Two Distinct ATPases and Their Partial PurificationShin-ichi TOMINAGA, Yoshito KAZIRO1983 Volume 93 Issue 4 Pages 1085-1092
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBrain microtubules purified by cycles of assembly and disassembly contained an ATPase activity in the fraction of microtubule-associated proteins (MAPs). This ATPase activity was found to be stimulated by 6 S tubulin in the presence of Ca2+ ions, suggesting its functional association with brain microtubules (Ihara et al. (1979) J. Biochem. 86, 587-590). On further purification by DEAF-cellulose column chromatography, two peaks of ATPase activity were separated; one, eluted at 0.2M KCl (ATPase I), was dependent on added 6 S tubulin but the other, eluted at 0.5M KCl (ATPase II), was not. ATPase I was highly unstable but could be stabilized by the addition of 0.1mM ADP, 50% (v/v) glycerol or 0.3mg/ml tubulin. ATPase I was further purified by CM-cellulose column chromatography, and by gel filtration on Sephacryl S-300. Its molecular weight, estimated by gel filtration, was 33, 000. ATPase II had a high molecular weight and appeared to be associated with membrane vesicles. It sedimented on glycerol density gradient centrifugation with an s value of 27 S. It was purified by high speed sedimentation and hydrophobic chromatography, and was observed under an electron microscope to consist of membrane vesicles of about 70 nm in diameter containing knob-like structures similar to those of H+-pump ATPase.View full abstractDownload PDF (640K) -
II. Properties of ATPases I and IIShin-ichi TOMINAGA, Yoshito KAZIRO1983 Volume 93 Issue 4 Pages 1093-1100
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe catalytic properties of two ATPases which had been purified from bovine brain microtubules (Tominaga, S. & Kaziro, Y. (1983) J. Biochem. 93, 1085-1092) were studied. ATPase I, which had a molecular weight of 33, 000, required the presence of 1.0 μm tubulin, 0.2mM Mg2+, and 10mM Ca2+ for maximal activity. The activation of ATPase I by tubulin was specific to the native form of tubulin, which could not be replaced by F-actin or tubulin denatured either by heat or more mildly by dialysis in the absence of glycerol. ATPase I was not specific to ATP, and GTP, and to a lesser extent, UTP and CTP were also hydrolyzed. Km for ATP of ATPase I was about 0.04mM. ATPase I was inhibited by 5mM Mg2+, 0.04M K+, 10-3M vanadate, 10mM N-ethylmaleimide, or 20%. (v/v) glycerol.
ATPase II, which was associated with membrane vesicles, required the presence of 0.2-2.0mM Mg2+ and 20mM KCl for activity. Tubulin stimulated the reaction of ATPase II only partially, and the addition of Ca2+ was rather inhibitory. ATPase II was specific to ATP with a Km value of 0.14mM. It was inhibited by 1.6mM N-ethylmaleimide and 20% (v/v) glycerol, but was not very sensitive to vanadate. Instead, ATPase II was inhibited by trifluoperazine, chlorpromazine, and nicardipin at 10-3M.View full abstractDownload PDF (611K) -
A Deletion Mutant Lacking Three Out of Four Transfer RNA Genes Upstream of the Coding Region of tufBAtsushi MIYAJIMA, Takashi YOKOTA, Yutaka TAKEBE, Morihiko NAKAMURA, Yo ...1983 Volume 93 Issue 4 Pages 1101-1108
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOf the two plasmids pTUBI and pTUB 2 constructed by cloning of the 8.9 kb EcoRI fragment carrying tufB (Miyajima, A., Shibuya, M., & Kaziro, Y. (1979) FEBS Lett. 102, 207-210), pTUB 2 possesses a deletion of about 0.3 kb. Restriction and sequence analyses have located the deletion in the region of the four tRNA genes thrU-tyrU-glvT-thrT upstream of the tufB structural gene. As a result of homologous recombination between thrU and thrT, the four tRNA genes have been replaced by a single thrU-thrT hybrid gene.
The deletion of the three tRNA genes does not significantly alter the in vivo expression of tufB as assessed by the kirromycin-sensitive phenotype of the transformant cells and by the synthesis of EF-Tu in mini-cells. Nor does the deletion affect the synthesis of β-galactosidase in lysogens carrying a λ transducing phage with a tufB-lacZ fusion.
Transcription of tufB and synthesis of EF-Tu in a cell-free transcription-translation coupled system were essentially the same, regardless of whether pTUB 1 or pTUB 2 DNA was used as a template. Likewise, 0.2mM ppGpp inhibits the synthesis of tufB mRNA on both pTUB 1 and pTUB 2 templates to the same extent.
We concluded that the replacement by thrU-thrT hybrid gene of the four tRNA genes upstream of the tufB coding region does not significantly affect either in vivo or in vitro expression of tufB.View full abstractDownload PDF (1843K) -
Shuichi HASHIZUME, Tsutomu HONJOH, Masahiro SHOJI1983 Volume 93 Issue 4 Pages 1109-1118
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA heat-labile, non-dialyzable factor (s) in soluble fractions from Escherichia coli strains and Bacillus subtilis was found to incorporate the radioactivity of [14C]-glutamic acid into 95°C CCl3COOH-insoluble fraction. Incorporation catalyzed by a partially purified factor from E. coli B required ATP, Mg2+, tRNA, casein, carbonate, and 2-mercaptoethanol. A mixture of nineteen amino acids other than glutamic acid had no effect on the incorporation. Heparin, spermine and monovalent cations were inhibitory. Incorporation proceeded via glutamyl-tRNA. The incorporation from [14C]glutamyl-tRNA required Mg2+, casein, carbonate, and 2-mercaptoethanol, and there was no incorporation from [14C]aspartyl-tRNA. The reaction product was identified as protein. The incorporated moiety was the glutamyl moiety of glutamic acid and it retained a free α-amino group in the product protein. The incorporating factor of E. coli B was demonstrated to be glutamyl-tRNA synthetase.View full abstractDownload PDF (802K) -
Takae TOWATARI, Nobuhiko KATUNUMA1983 Volume 93 Issue 4 Pages 1119-1128
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe selective cleavage of peptide bonds by cathepsin L from rat liver was examined with a hexapeptide, luteinizing hormone releasing hormone, neurotensin and oxidized insulin A chain as model substrates. The specificity of cathepsin L was compared with that of cathepsin B. Cathepsin L cleaved peptide bonds that have a hydrophobic amino acid, such as Phe, Leu, Val, and Trp or Tyr, in position P2. A polar amino acid, such as Tyr, Ser, Gly, Glu, Asp, Gin, or Asn, in position P1 enhanced the susceptibility of the peptide bond to cathespin L, though the importance of the amino acid residue in position P1' was not as great as that of the amino acid in position P2 for the action of cathepsin L. These results suggest that, in contrast to cathepsin B, cathepsin L shows very clear specificity.View full abstractDownload PDF (1739K) -
Nobuhiko KATUNUMA, Takae TOWATARI, Masaharu TAMAI, Kazunori HANADA1983 Volume 93 Issue 4 Pages 1129-1135
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEfficient methods were developed for synthesizing synthetic substrates for assays of cathepsin B and cathepsin L. Several 2-naphthylamide compounds with a blocked NH2;-terminus, Suc-Tyr-Met-NA, β-Ala-Tyr-Met-NA, and D-Leu-Tyr-Met-NA, were specific and sensitive substrates for cathepsin L and cathepsin B; they were not specific for cathepsin L only, because all of them were also hydrolyzed by cathepsin B. Some kinetic constants for the hydrolyses of these three synthetic substrates by cathepsin B and cathepsin L are given.View full abstractDownload PDF (580K) -
Kazunobu MATSUSHITA, Mamoru YAMADA, Emiko SHINAGAWA, Osao ADACHI, Mino ...1983 Volume 93 Issue 4 Pages 1137-1144
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThere was found to be a KCN-insensitive, alternate oxidase chain branching from the ordinary oxidase chain in the respiratory chain of Pseudomonas aeruginosa grown aerobically. The alternate oxidase activity was highly resistant to KCN, and had a lower affinity for oxygen than ordinary cytochrome oxidase did. The branching point of the alternate oxidase chain from the ordinary oxidase chain was shown to be localized behind cytochrome b. The KCN-insensitive alternate oxidase chain was inhibited slightly with antimycin A and intensively with 2-thenoyltrifluoroacetone. The former inhibited the respiration behind cytochrome b and the latter before cytochrome b.
N, N, N', N'-Tetramethyl-p-phenylenediamine oxidase-negative mutant (T 105) was prepared from P. aeruginosa. The mutant clearly lacked a functional ordinary cytochrome oxidase, but had the KCN-insensitive alternate oxidase chain and could grow aerobically. The KCN-insensitive alternate oxidase chain had a H+/O ratio of 4, suggesting the existence of two energy-coupling sites in the chain. Under the conditions where both ordinary oxidase and alternate oxidase chains were functioning, the H-/O ratio of the parent strain was 5.6. From these data, we also discuss the energetics of the ordinary oxidase chain in the respiratory chain of aerobic P. aeruginosa.View full abstractDownload PDF (657K) -
Isamu TAKAGAHARA, Jun-iti YAMAUTI, Katsumi FUJII, Jinpei YAMASHITA, Ta ...1983 Volume 93 Issue 4 Pages 1145-1157
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The kinetics of a coupled enzyme reaction in the following generalized from
_??_
was analyzed theoretically by computer calculation without any approximation. Two assumptions were made; 1) the rate (v1) of the primary enzyme (E1) is constant, and 2) the rates (v j) of the other enzymes (Ej) having Km; j (Michaelis constant) and Vmax; j (maximum rate) (j=2, 3, 4, …, k) obey the Michaelis-Menten equation. When the reaction is started by adding the substrate (S1) of E1, the rate (Vk) of the last enzyme (Ek) increases till rk (=vk/v1) reaches 1; if nj (=Vmax;j/v1) is sufficiently high, rk passes any value from 0 to 1. The reaction time (t) required rk to reach an arbitrarily fixed value (X) is called the “response time” (tr) (=tX), and at this time vk reaches (X×100)% of v1. Equations for the relation among rk, t (min), v1 (M/min), Km; j (M), and Vmax; j (M/min) were obtained.
2. In the two-enzyme system (k=2), Vmax: 2=F2×Km; 2/t+v1, where F2 is a function of r2 and n2. The nomogram for F2 is presented; for example, F2=4.61 at r2=0.99 and n2_??_10. The above equation with F2=4.61 was rearranged empirically, so as to be correct at n2>2; Vmax; 2=4.61×Km; 2/t0.99+1.4×v1.
3. The above equations described well the experimental kinetics of the two-enzyme system composed of yeast hexokinase and yeast or Leuconostoc inesenteroides glucose-6-phosphate dehydrogenase.
4. In the two-enzyme system, both the consumption of S1 by E1 (E1 reaction) and the formation of P2 by E2 (E2 reaction) can be plotted on the ordinate with the reaction time on the abscissa. The asymptote on the curve for the E2 reaction (E2 asymptote), which is parallel to the straight line for the E1 reaction (E1 asymptote), intersects the abscissa at T2(min) (delay time); t=T2×F2. In the multi-enzyme system, Tj gives the delay time between the Ej-1, and the Ej reaction, and is a convenient parameter for calculation with systems composed of more than three enzymes, because calculation can be easily achieved with t=(T2+T3+……+Tk)×Fk, which is analogous to the equation for the two-enzyme sytem. For the threeenzyme system (k=3), t=(T2+T3)×F3. If E2 and E3 are equally available, the equations for the three-enzyme system are Vmax; 2=F3×Km; 2/(0.5×t)+v1 and Vmax; 3=F3×Km; 3/(0.5×t)+v1. F3 is the function of r3, n2, and n3View full abstractDownload PDF (951K) -
Yasuko KAWAMURA, Satoshi NAKAMURA1983 Volume 93 Issue 4 Pages 1159-1166
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetics of assembly of oxyhemoglobin from isolated α and β chains was investigated by the use of a circular dichroism (CD) stopped-flow apparatus. The CD change in the Soret region was observed after mixing equivalent concentrations of the isolated chains. The intensity of the CD change was proportional to the protein concentration. The dilution of the isolated chains did not produce any detectable CD change. These results indicate that the CD change could be ascribed to the combination of a and β monomers into αβ dimer.
The time courses of the CD change showed a rapid phase and a slow phase. The slow phase was a first-order reaction with a rate constant of 2.8×10-3 s-1 (independent of the protein concentration), which suggested that the slow phase reflected the dissociation of self-associated β chain. The rapid phase depended on the protein concentration: (1) the ratio of the rapid phase to the total CD change decreased with increase in the protein concentration, and (2) the half-life of the rapid phase decreased with increasing protein concentration. The ratio of the rapid phase coincided with the fraction of β monomer which was calculated from the self-association constant of β chain. The constant was estimated to be 2.4×1016 M-3 by frontal gel chromatography on the assumption that the isolated β chain was in a monomer-tetramer equilibrium. This result indicated that the rapid phase could be ascribed to the combining of a and β monomers initially present. Therefore, the half-life of the rapid phase was analyzed on the basis of a scheme which included the monomer-tetramer equilibrium of the β chain and a second-order combination reaction of α and β monomers. The analysis yielded a second-order rate constant of 7.5×105M-1•S-1.
These results suggest that α and β monomers rapidly combine to form αβ dimer followed by assembly into Hb, though at high protein concentration the rate of the assembly is limited by the dissociation of self-associated β chain.View full abstractDownload PDF (592K) -
Takao OHYASHIKI, Tetsuro MOHRI1983 Volume 93 Issue 4 Pages 1167-1173
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpectrofluorometric studies on the interaction of phlorizin with rabbit intestinal brush border membranes under various conditions were carried out using 1-anilino-8-naphthalene sulfonate (ANS) and N-(1-anilinonaphthyl-4)maleimide (ANM). The fluorescence intensity of membrane-bound ANS was markedly decreased upon addition of phlorizin. The apparent dissociation constant of phlorizin and the membranes at pH 7.4 was determined to be approximately 24 μM from the slope of a plot of the fluorescence change of membrane-bound ANS against phlorizin concentration. Addition of D-glucose diminished the quenching effect of phlorizin on the ANS fluorescence by lowering the binding affinity of phlorizin for the membranes. The interaction between phlorizin and the membranes was influenced by temperature, ionic strength and divalent cations. The binding affinity of phlorizin for the membranes is markedly reduced at higher temperature, showing a transition between 25 and 30°C. Imposition of an ionic strength gradient across the membrane vesicles (out>in) increased the binding affinity. On the other hand, divalent cations decreased the affinity; Mg2+ was more effective than Ca2+. The binding affinity of ANS for the membranes was evidently decreased, and the reactivity of SH groups of the membrane proteins with ANM was apparently reduced, upon binding of phlorizin to the membranes.
Based on these results, the relationships among modification of membrane structures, the extent of phlorizin binding and phlorizin-induced conformational changes in the membrane proteins are discussed.View full abstractDownload PDF (534K) -
Koui TAKAHASHI, Satoshi OGIHARA, Mitsuo IKEBE, Yuji TONOMURA1983 Volume 93 Issue 4 Pages 1175-1183
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSElectron microscopically, the myosin molecule from the plasmodium of Physarum polycephahum has a long tail of 173 nm, having a flexible region over the range of 80 to 120 nm from the head-tail junction. In 0.6M ammonium acetate, this region of the dephosphorylated myosin molecules is more flexible than that of the thiophosphorylated ones. In 50mM ammonium acetate, the dephosphorylated myosin molecules exist in monomeric and oligomeric forms, independently of ATP and Mg2+, whereas the thiophosphorylated myosin molecules form dense aggregates of thick filaments. The tails of the monomeric dephosphorylated myosin molecules bend sharply at the flexible region at angles of more than 120°. In oligomers of the dephosphorylated myosin molecules, the molecules are all associated sideto-side with straight tails and are oriented in the same direction. Based on these results, the regulation mechanism of cell motility of the plasmodium is discussed.View full abstractDownload PDF (4723K) -
Noriyuki KUMAZAWA, Takeshi TSUJIMOTO, Masaru KANASHIRO, Retsu MIURA, Y ...1983 Volume 93 Issue 4 Pages 1185-1193
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSN-(1-Oxyl-2, 2, 5, 5-tetramethyl-3-pyrrolidinyl)maleimide (MSL) was incorporated into rat liver mitochondria and the nitroxide radical incorporated was found to decay considerably. The incorporation was blocked by a high concentration of NEM, but not by pCMB. Spin labeled fatty acid derivatives, 2-(3-carboxypropyl)-2-tridecyl-4, 4-dimethyl-3-oxazolidinyloxyl (FSL1) and 2-(14-carboxytetradecyl)-2-ethyl-4, 4-dimethyl-3-oxazolidinyloxyl (FSL2), were also incorporated and the nitroxide radical decayed. However, incorporation of FSL1 or FSL2 was not blocked by NEM or pCMB. The ESR spectrum of 3-carboxyl-2, 2, 5, 5-tetramethyl-pyrroline-loxyl (CSL) did not change on reaction with the mitochondria. The labeled MSL exhibited an ESR spectrum composed of both strongly immobilized and weakly immobilized components. A similar reaction with FSL1 gave an ESR spectrum mainly composed of a strongly immobilized component, the weakly immobilized component was negligibly small, while FSL2 exhibited an ESR spectrum in which free-like signals of the nitroxide radical were predominant. The results suggest that MSL is labeled selectively in the mitochondrial membrane through those SH groups that are not reactive to pCMB, and the labeled nitroxide radical is reduced in situ. The mode of incorporation into the mitochondria differs between MSL and the other spin labeled reagents, and labeling of MSL at the binding site may precede reduction of the nitroxide radical. The incorporation of MSL was dependent on the concentration of MSL used. ADP-acceleration of mitochondrial oxygen uptake with succinate was inhibited by labeling the mitochondria with MSL without loss of the electron transferring activity.View full abstractDownload PDF (757K) -
Yasunori NITTA, Toshiko KUNIKATA, Takehiko WATANABE1983 Volume 93 Issue 4 Pages 1195-1201
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. In order to investigate the interactions between soybean β-amylase [EC 3. 2. 1. 2] and ligands (maltotriose as substrate, and maltose and α- and β-cyclodextrins as inhibitors for the hydrolysis of maltoheptaose), the difference spectra were measured at 25°C and pH 5.4, in 0.05M acetate buffer. Each difference spectrum produced by these ligands showed a clear peak at 292-293 nm due to a tryptophan residue. In addition to this peak, the spectra of α- and β-cyclodextrins showed a specific peak at 298-299 nm, and that of maltotriose showed a shoulder at 298 nm.
2. From the concentration dependency of the difference molar extinction coefficient, Δε, at 292-293 nm or at 298-299 nm, the dissociation constant of the enzymeligand complex, Kd, was evaluated for maltotriose, and α- and β-cyclodextrins. For each ligand, the Kd values obtained at these two wavelengths were in good agreement with Michaelis constant, Km, or the inhibitor constant, K1. The Kd value for maltose obtained from the titration of Δε at 292 nm was also in good agreement with K1.
3. Maltose produced a hydrophobic change in the environment of the tryptophan residue, while the interactions of maltotriose, and α- and β-cyclodextrins with this enzyme caused an electrostatic change in the vicinity of the tryptophan residue in addition to the hydrophobic change.
Since the signal at 298-299 nm was not found in the difference spectrum of maltose, this singnal may be due to a tryptophan residue different from that which produces the signal at 292-293 nm. If both the signals are due to the same tryptophan residue, we must conclude that some conformational change is caused in the enzyme active site by the ligand binding.View full abstractDownload PDF (546K) -
Kiyotaka HIRAIWA, Toru OKA, Kunio YAGI1983 Volume 93 Issue 4 Pages 1203-1210
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBleomycin-Cu(II) complex tended to increase the lipid peroxide level in cultured lung fibroblasts, though neither free bleomycin nor free cupric ion increased the level. Simultaneous addition of DL-α-tocopherol decreased the level significantly. Bleomycin-Cu(II) complex decreased glutathione peroxidase activity remarkably, though free bleomycin reduced the activity only slightly. Collagenase activity was not decreased but rather increased by both free bleomycin and bleomycin-Cu(II) complex. Accordingly, the accumulation of collagen induced by bleomycin could be explained not by a decrease in collagenase activity, but by the occurrence of cross-linking of collagen due to the increased lipid peroxides.View full abstractDownload PDF (644K) -
Joseph Ololade FOLAYAN1983 Volume 93 Issue 4 Pages 1211-1215
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPoly-5-dimethylaminouridylic acid, (poly(Me2N5U)) has been synthesized by the conversion of 5-bromouridine-5'-monophosphate to 5-dimethylaminouridine-5'-monophosphate which was later made into the 5'-diphosphate and subsequently polymerized by PNPase. The polymer formed a 1:1 hybrid with poly(A) with the ability to induce the production of interferon in chick embryoes as certain doses of the hybrid protected chick embryoes against wesselsbron virus (H 10964).View full abstractDownload PDF (422K) -
Susumu TSUNASAWA, Tatsuhiro IMANAKA, Takashi NAKAZAWA1983 Volume 93 Issue 4 Pages 1217-1220
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acylamino acid-releasing enzyme purified from porcine liver showed peptidase activity above pH 8. Of the non-acylated peptides tested, this peptidase activity was only exerted on peptides with Gly or Ala at their N-termini. These results are consistent with the previous observations for similar enzymes from sheep red blood cells (Witheiler, J. & Wilson, D. B. (1972) J. Biol. Chem. 247, 2217-2221) and beef liver (Gade, W. & Brown, J. L. (1978) J. Biol. Chem. 253, 5012-5018). The pH dependence of the peptidase activity showed that only peptides with uncharged N-terminal amino acids such as glycyl- or alanyl-peptides act as substrates for the enzyme. These results suggest that the peptidase activity seen for the acylamino acid-releasing enzyme is an intrinsic activity of the enzyme that is triggered by misrecognition of uncharged smaller N-terminal amino acids in non-acylated peptides as acyl groups at higher pHs.View full abstractDownload PDF (269K)
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