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K Aki

Publications and source records attributed to K Aki.

At least 37 records · Page 2Linked to original sources
PubMed1985

Effect of dinitrophenyl modification on oxidation-reduction of glutathione reductase from yeast.

Covalent modification of glutathione reductase (GR) from yeast with 1-fluoro-2,4-dinitrobenzene (FDNB) inhibited the NADPH-GSSG reductase activity completely. This modification also decreased the NADPH-thio-NADP+ transhydrogenase activity, stimulated the NADPH-oxidase activity, and induced the NADPH-cytochrome c reductase activity. Spectrophotometric titration showed that one tyrosine residue per FAD was modified with a dinitrophenyl group. The modified enzyme showed conversion of the two-electron reduced form (EH2) to the four-electron reduced form (EH4) in anaerobic conditions and conversion of EH2 to the oxidized form (E) in aerobic conditions. These results indicate that the modification of one tyrosine residue of the active site induces the instability of EH2.

Binding Sites↗
PubMed1984

Interaction of the fluorescence-labeled secretory component with human polymeric immunoglobulins.

The secretory component (SC) isolated from human milk was labeled with 2 mol of the fluorescent thiol reagent N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide (DACM) per mol of SC through the reactive disulfide bond of SC. The binding of the labeled SC to polymeric immunoglobulins was examined by gel filtration by measuring the fluorescence of DACM at 478 nm. The labeled SC was bound to immunoglobulin M (IgM) and its (Fc)5 mu fragment and to dimeric immunoglobulin A (IgA). When the labeled SC was bound to IgM or the (Fc)5 mu fragment, the fluorescence of DACM increased about 30%. By use of this fluorescence change, quantitative studies were made on the equilibrium and kinetics of the reversible interactions of the labeled SC with two IgM proteins and their (Fc)5 mu fragments at pH 7.0 and 25 degrees C. All the IgM proteins and their (Fc)5 mu fragments had one binding site per mole of polymers. The affinity constant (6 X 10(8) M-1), the association rate constant (7 X 10(7) M-1 min-1), and the dissociation rate constant (0.1 min-1) of one IgM were different from those of the other IgM (1.7 X 10(9) M-1, 1.0 X 10(8) M-1 min-1, and 0.06 min-1, respectively). However, the values for the (Fc)5 mu fragments of the two proteins were the same (1.9 X 10(9) M-1, 1.1 X 10(8) M-1 min-1, and 0.06 min-1, respectively) and were very similar to those of the IgM with the higher affinity constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites, Antibody↗
PubMed1984

Effect of nicotinamide adenine dinucleotide on the oxidation-reduction potentials of lipoamide dehydrogenase from pig heart.

The effect of NAD+ on lipoamide dehydrogenase from pig heart was investigated physicochemically. The observed and theoretical oxidation-reduction mid-point potentials for the oxidized lipoamide dehydrogenase (E)/two-electron-reduced lipoamide dehydrogenase (EH2) couple in the presence on NAD+ were -218 mV and -251 mV, respectively, at pH 6.0. Therefore, unexpectedly the mid-point potential of the enzyme became more positive on NAD+ binding. Decreases in the fluorescence lifetime and intensity and increase in the degree of polarization of enzyme-bound FAD were observed in the presence of NAD+. Fluorescence quenching of bound FAD by NAD+ was released by phenobarbital. The results suggest that NAD+ strengthens the intramolecular dynamic interaction between the isoalloxazine moiety and adenine moiety of bound FAD, and so alters the mid-point potential of the enzyme. These findings indicate that NAD+ acts not only as an acceptor of electrons from EH2, but also as an effector in the flavin-disulfide interaction of EH2.

Animals↗
PubMed1983

Fluorescence studies on lipoamide dehydrogenases of pig heart. II. Microenvironments of tryptophan residues.

The tryptophan residues of two forms of pig heart lipoamide dehydrogenase (LD(I) and LD(II] were investigated fluorometrically. The tryptophan contents of LD(I) and LD(II) determined by the fluorescence method were 3 mol and 2 mol per mol of FAD, respectively. These values were in good agreement with those found by the MCD method. The microenvironments of the tryptophan residues were investigated by fluorescence quenching titration with acrylamide. The tryptophan residues of both enzymes were in heterogeneous microenvironments, and CD spectra showed some differences between these microenvironments in the two enzymes. Energy transfer from tryptophan residues to bound FAD was equally efficient in the two enzymes. It seems probable that the three tryptophan residues in LD(I) are all in different microenvironments, but that two of them are in microenvironments almost identical to those of the corresponding residues in LD(II).

Animals↗
PubMed1981

Electron transfer between horse heart and Candida krusei cytochromes c in the free and bound states.

Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540-560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20 degrees C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2-3).10(4) M-1.s-1 under the same conditions. The transfer rate for the bound form increased with increase in the binding ratio at ratios below half the maximum, and was almost constant at higher ratios up to the maximum. The maximum electron exchange rate was about 2.10(6) M-1.s-1, which is 60-70 times that for the free form at a given concentration of cytochrome c. The activation energy of the reaction for the bound cytochrome c was equal to that for the free form, being about 10 kcal/mol. The dependence of the exchange rate on temperature, cytochrome c concentration and solvent viscosity suggests that enhancement of the electron transfer rate between cytochromes c on binding to phosvitin is due to increase in the collision frequency between cytochromes c concentrated on the phosvitin molecule.

Animals↗
PubMed1980

Relation of the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin.

The relationship between the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin was investigated. The rates of reduction of phosvitin-bound ferricytochrome c by cytochrome b2, ascorbate and the superoxide radical generated by xanthine oxidase wer repressed where the binding ratio was less than half the maximum, but at higher ratios they were restored gradually with increase in the ratio. The affinity of cytochrome b2 for cytochrome c was not affected by binding of cytochrome c to phosvitin. The redox potential of the bond form was lower than that of the free form and only decreased with decrease in the ratio. The conformatin around the heme moiety and the electronic structure of the heme group of bound ferricytochrome c were similar to those of free ferricytochrome c, but the conformational stability in the vicinity of the prosthetic group was related to the binding ratio as ratios above half the maximum and was well correlated with the reduction rate. Since the binding of cytochrome c to phosvitin is much stronger at binding ratios below half the maximum, these results suggest that this binding strength exclusively affects the conformational flexibility of the heme crevice in the cytochrome molecule, thus altering the reduction rate.

1-Propanol↗
PubMed1980

Biochemical studies on energy metabolism in the conduction system of bovine heart.

The conduction system of bovine heart was studied to clarify the characteristics of its energy metabolism. Results showed that its mitochondrial oxygen uptake was much lower than that of ordinary heart muscle with succinate as substrate but similar to that of ordinary heart muscle with glutamate + malate as substrate. The activity of succinate dehydrogenase was lower than that in ordinary heart muscle. The total activity of isozymes of lactate dehydrogenase was lower in the specialized heart muscle than in ordinary heart muscle. The main isozyme of lactate dehydrogenase in the specialized heart muscle was LDH 1 (H4). The cytoplasmic NAD+/NADH ratio as much higher in the specialized heart muscle than in ordinary heart muscle. These results suggest that in the specialized heart muscle energy is mainly derived from aerobic metabolism.

Animals↗
PubMed1979

Interaction of cytochrome c with the phosphorprotein phosvitin.

Candida krusei cytochrome c forms a molecular complex with phosphorprotein phosvitin in weakly alkaline solution of low ionic strength. At most, about 22 molecules of cytochrome c bind to a phosvitin molecule. The complex at the binding ratio below about 11 (half of the maximum ratio) as a much higher binding strength. Several lines of evidence indicate that the marked difference in the binding strength is due to the difference in negative charges on phosvitin molecule concerned in the binding of a cytochrome c molecule. The phosvitin-bound cytochrome c seems to have a preferred orientation with the front surface of the molecule containing the exposed heme edge in contact with the phosvitin molecule.

Animals↗
PubMed1979

Fluorescence studies on lipoamide dehydrogenases of pig heart. I. Conformational dynamics of enzyme.

The dynamic structures of two major forms (LD(I) and LD(II) of pig heart lipoamide dehydrogenase, resolved by TEAE-cellulose column chromatography, were studied by fluorescence depolarization. FAD and ANM were used as an intrinsic and an extrinsic fluorescent probe, respectively. In the experiments with bound FAD of lipoamide dehydrogenase, no thermal dependence of the fluorescence depolarization of either enzyme was observed and the values of polarization were close to the theoretical maximum value of 0.5. Both enzymes contained two reactive thiol groups which differed in their reactivities toward ANM. When the enzymes were labeled with one mol of ANM per mol of enzyme, the rotational relaxation times of LD(I) and LD(II) were found to be 18 ns and 196 ns, respectively. These findings indicate that the sement of LD(I) labeled with ANM fluctuates in the order of nanoseconds, whereas this segment of LD(II) is fixed rigidly. On the other hand, when the enzymes were labeled with two mol of ANM per mol of enzyme, both enzymes showed the composite result of fluorescence depolarization due to the motilities of the segment of enzyme and the whole enzyme molecule. These findings indicate that both LD(I) and LD(II) have the non-equivalent motilities of segments containing one reactive thiol group between the two monomers. In other words, the segment containing the ANM binding site of the one monomer is flexible and this segment of the other monomer is fixed rigidly in both enzymes.

Animals↗
PubMed1977

Formation of a complex between yeast L-lactate dehydrogenase (cytochrome b2) and cytochrome c. Ultracentrifugal and gel chromatographic analyses.

Yeast L-lactate dehydrogenase formed a stable complex with cytochrome c in weakly alkaline solution of low ionic strength. The binding ratio of cytochrome c to the enzyme depended on whether free cytochrome c was present: In the presence of a micromolar concentration of cytochrome c the enzyme formed a complex with about two molecules of cytochrome c, whereas the enzyme was in a 1:1 molecular complex after removal of free cytochrome c. This suggests that the binding of one molecule of cytochrome c changes the affinity of the other binding site on the enzyme for cytochrome c. The enzyme consists of four presumably identical subunits, each containing a binding site for cytochrome c. Thus, present data confirm the concept of negative cooperativity between the subunits of the enzyme molecule in their interaction with cytochrome c.

Candida↗
PubMed1976

Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochonria. I. Physicochemical properties of holo- and apo-NADPH-adrenodoxin reductase and interaction between non-heme iron proteins and the reductase.

A crystalline NADPH-adrenodoxin reductase was obtained from bovine adrenocortical mitochondria and its properties were investigated. Its molecular weights and isoelectric point were estimated to be 51 000 and 5.4, respectively. Amino acid and sugar contents and the interaction between the apo-reductase and flavin of NADPH-adrenodoxin reductase were investigated. Formation of a complex of bovine NADPH-adrenodoxin reductase with adrenodoxin, its apoadrenodoxin, or other non-heme iron proteins caused quenching of fluorescence of the tryptophanyl residue and bound FAD of the NADPH-adrenodoxin reductase. The results obatined suggest that adrenodoxin and apoadrenodoxin bind functionally to a site close to the tryptophanyl residue and the bound FAD of the reductase. The circular dichroism spectrum of oxidized NADPH-adrenodoxin reductase was measured in the ultraviolet and visible regions. This spectrum showed negative absorption in the visible region and was not appreciably influenced in either the ultraviolet or visible region by formation of a complex with adrenodoxin or apoadrenodoxin.

Adrenal Cortex↗