PubMed Health⌕ Search

Biomedical subjects

T Takabe

Publications and source records attributed to T Takabe.

At least 73 records · Page 4Linked to original sources
PubMed1983

Electron--nuclear-double-resonance studies of spinach plastocyanin.

Electron--nuclear-double-resonance (ENDOR) studies of spinach plastocyanin revealed that 14N-hyperfine couplings from its two co-ordinated imidazole groups are characteristically small in anisotropy and relatively high in ENDOR frequencies and that the two imidazole groups are magnetically equivalent. The relatively strong copper-imidazole co-ordinate bonds of plastocyanin are interpreted to be a result of the coexistence of a strong copper--thiol co-ordinate bond.

Binding Sites↗
PubMed1983

Ribulose 1,5-bisphosphate carboxylase from the halophilic cyanobacterium Aphanothece halophytica.

Various structural and functional properties of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) isolated from the halophilic cyanobacterium (blue-green alga) Aphanothece halophytica were reexamined. The ready dissociation of this algal RuBisCO during sedimentation in a linear sucrose density gradient was observed. Low NaCl concentrations promote the dissociation of small subunit (B) from the original native enzyme molecule as evidenced by the sucrose density gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is thus possible that the intracellular osmoticum of A. halophytica might influence the structural integrity and activity of RuBisCO. The low residual carboxylase activity ascribed to the catalytic core, an oligomer form of the large subunit (A) apparently deficient in small subunit (B), was found to be markedly stimulated by a protein component which appears identical to subunit B. The purification and structural characterization of the catalytic core and subunit B were attempted by step-wise column chromatography on DEAE-cellulose, Utrogel AcA 34, Sephadex G-75, and hydroxylapatite, and at the final stage each component was purified to near homogeneity, although the catalytic core is still associated with a small quantity of subunit B. The addition of subunit B to the catalytic core does not alter the Km (HCO-3, RuBP) values, but Vmax values are markedly enhanced. Sucrose density gradient centrifugation gave a value of 16 S for the catalytic core. The molecular weights of the monomeric forms of the catalytic core (subunit A) and subunit B were 5.0 X 10(4) and 1.4 X 10(4), respectively.

Centrifugation, Density Gradient↗
PubMed1983

Electron transfer between plastocyanin and P700 in highly-purified photosystem I reaction center complex. Effects of pH, cations, and subunit peptide composition.

Treatment of isolated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel chromatographies yielded a highly purified P700-chlorophyll a protein complex complex which consists of five polypeptides. The protein complex is virtually free of chlorophyll b (Ch1 alpha/Ch1 b greater than 10) with approximately 30 chlorophylls per P700, and contains iron-sulfur centers A, B, and X. At pH values higher than 6, divalent cations, but not monovalent or trivalent cations, efficiently accelerated the electron transfer from reduced spinach plastocyanin to the photooxidized P700 in the P700-chlorophyll alpha protein complex. At pH values lower than 6, the reaction rate drastically increased with decreasing pH with a maximum at about pH 4.3 without cations. Divalent salts as well as monovalent or trivalent salts decreased the P700 reduction rate at low pH, indicating the involvement of electrostatic interaction in those pH regions. The rate of electron transfer from plastocyanin to the photooxidized P700 in the reaction center protein, which consists of only the largest peptide subunit and no iron-sulfur centers, was reduced only 50% at pH 7.0 in the presence of MgCl2 as compared to the case of P700-chlorophyll alpha protein complex. Essentially similar effects of pH and metal ions on this electron transfer reaction were observed as in the case of P700-chlorophyll alpha protein complex. These results strongly suggest that plastocyanin donates electrons directly to the largest peptide of P700-chlorophyll alpha protein complex and the observed effects of pH and cations are mainly due to the interaction between the largest peptide of P700-chlorophyll alpha protein complex and plastocyanin. The four small subunits in the protein complex seemed to have only a minor role in the reaction with plastocyanin.

Cations↗
PubMed1981

pH dependence of adenosine 5'-triphosphate synthesis and hydrolysis catalyzed by reconstituted chloroplast coupling factor.

The purified ATP-synthesizing complex from chloroplasts has been reconstituted into phospholipid vesicles with bacteriorhodopsin by use of octyl glucoside. Phosphorylation rates up to 90 mmol of ATP (mg of protein)-1 min-1 have been achieved. The dependence of the steady-state kinetic parameters on external and internal pH for both synthesis and hydrolysis was determined. The Michaelis constants are independent of the magnitude of the pH gradient at external pH values of 6.6 and 8.0. The dependence of the maximum velocity for ATP synthesis on the external pH is bell shaped at a constant pH gradient with a maximum at about pH 6.7. The variation of the maximum velocity with external pH is not dependent on the magnitude of the pH gradient. At external pH values of 6.6 and 8.0, the maximum velocity for ATP synthesis varies with approximately the 2.3 power of the internal hydrogen ion concentration. The maximum velocity for ATP hydrolysis also is dependent on the external pH, with a maximum at about pH 8.4; however, most of the ATPase activity is not coupled to the proton flux. Both Mg2+ and Mn2+ are good cofactors for ATP synthesis and hydrolysis whereas Ca2+ is completely ineffective for synthesis and only about 10% as effective as Mg2+ and Mn2+ for hydrolysis. The results obtained suggest that ATP synthesis or hydrolysis may be coupled to proton pumping indirectly, as, for example, by conformational changes.

Adenosine Triphosphate↗
PubMed1981

Mechanism of glycolate transport in spinach leaf chloroplasts.

The incorporation of (14)CO(2) into glycolate by intact spinach leaf (Spinacia oleracea L. var. Kyoho) chloroplasts exposed to (14)CO(2) (NaH(14)CO(3), 1 millimolar) in the light was determined as a function of O(2) concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent K(m) (O(2)) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic (14)CO(2) fixation under an O(2) atmosphere.Glycolate transport into and out of chloroplasts was studied using the silicon oil centrifugation method. Both uptake and loss of glycolate were found to be rapid, with small temperature dependencies between 0 degrees C and 25 degrees C (Q(10) = 1.1). The reaction rate as a function of the concentration of glycolate up to 30 millimolar was linear in both directions.The effect of external pH on the reaction rate in both directions was also examined. Glycolate penetrates rapidly, even at pH 8, showing a surprisingly high permeation of the glycolate anion. This rate was about 30 micromoles per milligram Chl per hour at 0 degrees C, and the initial concentration of glycolate of 10 millimolar with a pH range of 7 to 8. The observed rate is comparable to the reported value for glycolate synthesis in chloroplasts under photorespiratory conditions. The uptake of glycolate into chloroplasts was accelerated below pH 7, while the rate of excretion was considerably lowered. It is, thus, suggested that undissociated glycolic acid penetrates the chloroplast envelopes more rapidly than does the anion.

Journal Article↗
PubMed1980

Glycolate formation catalyzed by spinach leaf transketolase utilizing the superoxide radical.

A homogeneous preparation of transketolase was obtained from spinach leaf; the specific enzyme activity was 9.5 mumolo of glyceraldehyde-3-P formed (mg of protein)-1 min-1, when xylulose-5-P and ribose-5-P were used as the donor and acceptor, respectively, of the ketol residue. Transketolase catalyzed the formation of glycolate from fructose-6-P coupled with the O2- -generating system of xanthine-xanthine oxidase. The addition of superoxide dismutase (145 units) or 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (5 mM), both O2- scavengers, to the reaction system inhibited glycolate formation 72 and 58%, respectively. The reacton was not inhibited by catalase. Mannitol, an .OH scavenger, and beta-carotene and 1,4-diazobicyclo[2.2.2]octane, 1O2 scavengers, showed little or no inhibitory effects. The rate of glycolate formation catalyzed by the transketolase system was measured in a coupled reaction with a continuous supply of KO2 dissolved in dimethyl sulfoxide, used as an O2- -generating system. The optimum pH of the reaction was above pH 8.5. The second-order rate constant for the reaction between transketolase and O2-, determined by the competition for O2- between nitroblue tetrazolium (NBT) and transketolase, was 1.0 X 10(6) M-1 s-1. Transketolase showed an inhibitory effect on the O2- -dependent reduction of NBT only if the reaction mixture was previously incubated with ketol donors such as fructose-6-P, xylulose-5-P, or glycolaldehyde. The results suggest the possibility that transketolase catalyzes O2- -dependent glycolate formation under increased steady-state levels of O2- in the chloroplast stroma.

Glycolates↗
PubMed1980

Ionic strength and pH effects on the rates of reduction of spinach plastocyanin by ascorbate.

Kinetic studies of the reduction of plastocyanin from spinach chloroplasts by ascorbate were carried out using a stopped flow apparatus. The reduction of spinach plastocyanin by ascorbate followed first-order kinetics in the reductant with a rate constant k = 415 M-1.S-1 [20 degrees C, 0.1 M NaCl, pH 8.0 (10 mM Tris-HCl)]. The second-order rate constants for the reduction of plastocyanin by ascorbate increased dramatically with increasing pH and ionic strength. The cationic micelles of cetyltrimethylammonium bromide also accelerated this electron-transfer reaction. The importance of electrostatic interactions between plastocyanin and ascorbate is discussed in relation to the photosynthetic electron-transport reactions.

Ascorbic Acid↗
PubMed1980

Autoreduction of spinach plastocyanin at alkaline pH.

The autoreduction of spinach plastocyanin has been studied by fluorescence, absorption, and paramagnetic resonance spectroscopy. Decolorization of oxidized plastocyanin, due to the autoreduction of plastocyanin, occurred rapidly at alkaline pH (half-life 20 min at pH 10.2). Its rate also increased with increasing ionic strength. In the presence of a hydrophobic fluorescent probe such as 2-p-toluidino-naphthalene-6-sulfonate (TNS), the fluorescence intensity of TNS due to binding to oxidized plastocyanin gradually increased following the autoreduction of plastocyanin, which suggests that copper is autoreduced in parallel with the exposure of a hydrophobic site (probably near Cys 84) to the solvent. The EPR spectra also supported the change of coordination geometry of copper. The reduction rate of plastocyanin by ferrocyanide at alkaline pH was almost the same as that at neutral pH, and TNS did not alter the exogenous reduction rate even at alkaline pH. These results suggest that exogenous and endogenous reduction take place independently at different reduction sites in spinach plastocyanin.

Electron Spin Resonance Spectroscopy↗
PubMed1980

Electron transfer reactions of cytochrome f from Brassica komatsuna with hexacyanoferrate.

The electron transfer reactions of membrane-bound monomeric cytochrome f from Brassica komatosuna (Brassica rapa L. var. perviridis Bailey) with hexacyanoferrate (II)-(III) have been studied as a function of pH, ionic strength and temperature. The second-order rate constant for the oxidation of cytochrome f by Fe(CN)6(3-) at pH 7.0, mu 0.1 M, and 20 degrees C is 1.7 X 10(5) M-1 X S-1, which is similar to the value of oligomeric cytochrome f from parsley. The activation parameters obtained were delta H not equal to = -0.87 kcal/mol and delta S not equal to - -38 cal/mol x deg. Respective rat constant and activation parameters obtained for the reduction of cytochrome f by Fe(CN)6(4-) were k = 1.7 X 10(4) M-1 x S-1, delta H not equal to = +6.7 kcal/mol, and delta S not equal to -16 cal/mol x deg. Both the rate constants for the oxidation and the reduction of cytochrome f markedly decreased with increasing ionic strength. The results indicate that the oxidation and the reduction take place at a positively charged site on the cytochrome f surface, and electrostatic interactions are important for these reactions. The participation of protons and specific amino acid residues in electron transfer reactions of cytochrome f is implied from the pH results. Alkaline isomerization of ferricytochrome f was not observed. The midpoint potential of cytochrome f has a constant value of 360 mV between pH 5.0-8.9, and decreases by about 55 mV per pH unit above 8.9. The results are compared with the data for horse heart cytochrome c and Euglena gracilis cytochrome c-552. These data are discussed in relation to the theories of electrostatic corrected outer-sphere electron transfer of Marcus and multiphonon nonadiabatic electron tunneling of Jortner and Hopfield.

Brassica↗
PubMed1980

Electron transfer reactions between cytochrome f and plastocyanin from Brassica komatsuna.

The protein-protein electron transfer reactions between cytochrome f and plastocyanin, both purified from Brassica komatsuna (Brassica rapa L. var. pervirdis Bailey), have been studied as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of ferrocytochrome f by plastocyanin was found to be k = 4.5 X 10(7) M-1 x S-1 at pH 7.0 mu 0.2 M, and 20 degrees C, with activation parameters delta H not equal to = 8.4 kcal/mol and delta S not equal to = 4.9 cal/mol x deg. Respective rate constant and activation parameters obtained for the reduction of ferricytochrome f by plastocyanin were k = 1.9 X 10(7) M-1 x S-1, delta H not equal to = 8.6 kcal/mol, and delta S not equal to = 3.9 cal/mol x deg. The high rate constants for these reactions and delta S not equal to = 4.9 cal/mol x deg. Respective rate constant and activation parameters obtained for the reduction of ferricytochrome f by plastocyanin were k = 1.9 X 10(7) M-1 x S-1, delta H not equal to = 8.6 kcal/mol, and delta S not equal to = 3.9 cal/mol x deg. The high rate constants for these reactions are attributable not to a low activation enthalpy but to a positive activation entropy term. The rate constants both for the oxidation and the reduction of cytochrome f by plastocyanin drastically decreased with increasing ionic strength, indicating the importance of electrostatic interactions. Divalent cations are more effective than monovalent cations in reducing the rates of these reactions. The rate constants for the oxidation of cytochrome f by plastocyanin are constant between pH 6.0 and 9.0 but decrease markedly above pH 9.0 and below pH 6.0. In the case of the reduction of cytochrome f by plastocyanin, an optimum pH around 7.0 was obtained and a biphasic feature was observed at alkaline pH. The results are discussed in relation to photosynthetic electron transport systems.

Brassica↗
PubMed1979

A fluorescence study of the effects of pH and Mg2+ on the conformation of fructose 1,6-diphosphatase from spinach chloroplasts.

2-p-Toluidino-naphthalene-6-sulfonate is a sensitive fluorescent reporter group which can be used for the detection of the conformation of fructose 1,6-diphosphatase from spinach chloroplasts. When fructose 1,6-diphosphatase was added to a dilute solution of 2-p-toluidino-naphthalene-6-sulfonate at pH 9.0, the fluorescence intensity gradually increased. At this pH, the enzyme activity decreased at the same rate. However, at neutral pH (7.5), this time-dependent fluorescence change was not observed. In the presence of Mg2+, which is an activator of the enzyme, the fluorescence intensity was increased instantly and did not change for 30 min in the pH range 8.0--9.0. From the concentration dependence of the fluorescence intensity, the dissociation constant for Mg2+ was determined, Kdis = 3 mM. The effects of pH and Mg2+ on the conformation and activity of chloroplast fructose 1,6-diphosphatase are discussed.

Chloroplasts↗
PubMed1975

Further studies on the subunit structure of Chromatium ribulose-1,5-phosphate carboxylase.

Upon alkali exposure Chromatium ribulose-1,5-bisphosphate carboxylase dissociates into constituent subunits, a catalytic oligomer of the larger subunit, A8, and monomeric form of the small subunit B. By sedimentation equilibrium molecular weights of the native enzyme and the catalytic oligomer produced by an alkali treatment were estimated to be 5.11 x 10 5 and 4.29 x 10 5, respectively. To provide information on reversibility of the dissociation by determining whether the enzymically inactive small subunit B of the whole enzyme molecule did indeed exchange with exogenously added subunit B a radioisotopic method was used. After initial alkaline dialysis at pH 9.2 of a mixture of a nonlabeled native enzyme preparation and 14C-labeled subunit B, and the subsequent dialysis at pH 7.0, incorporation of 14C into the recovered native enzyme was determined. Without the alkaline treatment there was no detectable exchange, while after alkaline dialysis for 5 and 10 hr the subunit B exchange was 89 and 82%, respectively. Rabbit antiserum prepared against the catalytic oligomer of the spinach ribulose-1,5-bisphosphate carboxylase, anti-(A) (spinach), inhibited the Chromatium carboxylase and oxygenase activities. This result together with the identical immunoprecipitation lines on an agar plate formed between the antiserum and the Chromatium carboxylase and between the antiserum and the catalytic subunit of the Chromatium enzyme strongly indicated structural near identity of the catalytic subunits of the spinach and Chromatium carboxylase molecules. Results also show that the catalytic site of the Chromatium ribulose-1,5-bisphosphate carboxylase and oxygenase exists in the large polypeptide chain.

Animals↗