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Biomedical subjects

E T Rakitzis

Publications and source records attributed to E T Rakitzis.

At least 19 recordsLinked to original sources
PubMed1992

Funding errors.

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Research Support as Topic↗
PubMed1992

Kinetic evidence for the existence of an unstable intermediate in the trinitrophenylation-induced rhodanese inactivation reaction.

1. Rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate, in the presence of n-butylamine in the reaction medium, has been studied by a kinetic analysis of the data, based on the assumption that enzyme inactivation is brought about by direct reaction of this with the modifying agent. 2. Initial reaction rates for rhodanese activity loss were determined by a mathematical analysis of the first three recorded values of rhodanese residual activity. 3. It was found that fractional rhodanese activity values, at infinite reaction time with 2,4,6-trinitrobenzenesulphonate (end-point values), were significantly lower than the values calculated on the assumption of rhodanese inactivation being entirely due to direct trinitrophenylation of enzyme protein. 4. Also, initial enzyme inactivation values were higher in the presence, rather than in the absence, of n-butylamine. 5. These results indicate that 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation, in the presence of n-butylamine in the reaction medium, is due to the generation of a highly reactive, unstable intermediate, probably a free radical species.

Animals↗
PubMed1990

Utilization of the free energy of the reversible binding of protein and modifying agent towards the rate-enhancement of protein covalent modification.

An analysis is presented of the catalytic factors responsible for the rate-enhancement that may be observed when a protein modification reaction is compared with a reaction of the same modifying agent with a model micromolecular compound exhibiting the same reactive group as the protein under study. It is seen that affinity-mediated rate-enhancement of protein modification is realized by the loss of activation entropy. On the assumption that attainment of maximal affinity-mediated rate-enhancement presents with an activation entropy of the protein modification reaction equal to zero, whereas the activation enthalpy of the reaction remains unchanged, it is shown that the value for maximal affinity-mediated rate-enhancement is equal to e-delta s++/R. Accordingly, protein modification reactions may be differentiated into (i) reactions the rate-enhancement of which (relative to the reaction of the same modifying agent with a model compound) is primarily entropy-controlled and (ii) reactions the rate-enhancement of which is primarily enthalpy-controlled. It is seen that modifying agents of low reactivity towards model compounds, but with a high, i.e. highly negative, activation entropy are better suited as prospective affinity-based protein-modifying agents, since the potential affinity-mediated rate-enhancement, and hence the selectivity, of these compounds is necessarily high. Kinetic and thermodynamic constants of the reaction of modifying agents with proteins, and with model compounds, and values of maximal affinity-mediated rate-enhancement, based on published data of the reaction of several modifying agents with model compounds, are presented and discussed.

Kinetics↗
PubMed1990

Kinetic analysis of regeneration by dilution of a covalently modified protein.

An analysis of regeneration by dilution of a covalently modified protein is presented. It is shown that, when protein regeneration is realized through the intermediacy of a protein-modifying agent adsorptive complex, the reaction is described by a summation of two exponential functions of reaction time plus a constant-term equation. The conditions whereby this equation reduces to a single-exponential equation are delineated. It is shown that, when protein regeneration is described by a single-exponential function of reaction time, the first-order protein-regeneration rate constant is a function of modifying-agent concentration and also of the microscopic reaction rate constants. Accordingly, the protein-modifying agent dissociation constant (Ki), as well as the protein-covalent-modification and -regeneration, rate constants (k+2 and K-2), may be determined by an analysis of dilution-induced protein-regeneration (or enzyme-reactivation) data obtained at different dilutions of the covalently modified protein-modifying agent preparation.

Adsorption↗
PubMed1990

Diethylbarbiturate potentiation of 2,4,6-trinitrobenzenesulphonate-induced rhodanese inactivation.

The rate of rhodanese inactivation by 2,4,6-trinitrobenzenesulphonate is increased in the presence of diethylbarbiturate in the reaction medium. A "rate saturation effect" indicates the formation of a rhodanese-diethylbarbiturate complex, prior to modification-induced enzyme inactivation. The dissociation constant of this complex is 19.0 mM. Diethylbarbiturate has no effect on the trinitrophenylation rate of the free amino groups of rhodanese. When rhodanese modification, in the presence of diethylbarbiturate in the reaction medium, is carried out by the use of a 2,4,6-trinitrobenzenesulphonate concentration much lower than the concentration of rhodanese modifiable amino groups, reaction stoichiometry indicates that 3 to 5 moles of rhodanese are rendered inactive for each mole of 2,4,6-trinitrobenzenesulphonate utilized. This finding indicates the existence of a chain-reaction type mechanism of rhodanese inactivation.

Animals↗
PubMed1989

Kinetic analysis of protein modification reactions at equilibrium.

A kinetic analysis is presented of reactions of protein modification, and/or of modification-induced enzyme inactivation, which can formally be described by a single exponential function, or by a summation of two exponential functions, of reaction time plus a constant term. The reaction schemes compatible with the kinetic formalism of these cases are given, and a simple kinetic criterion is described whereby the identification of one of these cases, strong negative protein modification co-operativity, may be carried out. The treatment outlined in this paper is applied to a case from the literature, the inactivation of glyceraldehyde-3-phosphate dehydrogenase by butane-2,3-dione [Asriyants, Benkevich & Nagradova (1983) Biokhimiya (Engl. Transl.) 48, 164-171].

Animals↗
PubMed1989

Determination of NADH2-ferricyanide oxidoreductase (cytochrome b5 reductase, diaphorase) activity of human erythrocytes by an analysis of the time-dependence of NADH2 oxidation.

The time-dependence of the reaction of human erythrocyte diaphorase activity has been studied by the use of NADH2 and ferricyanide as substrates. Reaction was found to be first-order with respect to NADH2 concentration, and zero-order with respect to ferricyanide concentration. These findings indicate that human erythrocyte diaphorase has a Km value for NADH2 by far higher than, and for ferricyanide by far lower than, the concentration of the substrates used, i.e. 0.1 and 0.2 mmol/l, respectively. The diaphorase activity determination method, described in the present communication, has been used in 19 healthy adults and children. Diaphorase activity was found to be 7.29 +/- 3.69 1 SD mumol NADH2 oxidized/ml packed cells per min, at 25 degrees C, and pH 7.00.

Adult↗
PubMed1979

Activation of cathepsin D by glycine ethyl ester.

Cathepsin D purified from bovine spleen is activated by glycine ethyl ester. A maximum of 70% activation was observed at a glycine ethyl ester concentration of 0.1 M and at pH 4.5. The activation effect appears to be reversible.

Cathepsins↗
PubMed1978

Kinetic analysis of the inhibition of sulfate transport in human red blood cells by isothiocyanates.

A kinetic analysis of anion self-exchange in human red blood cells, in the presence of an irreversible inhibitor, is presented and applied to the study of the inactivation of sulfate transport by three isothiocyanates: 3-isothiocyano-1,5-naphthalenedisulfonic acid, disodium salt (INDS), 1-isothiocyano-4-naphthalene sulfonic acid, sodium salt, monohydrate (INS), and 1-isothiocyano-4-benzenesulfonic acid, sodium salt, monohydrate (IBS). The time dependence of the inhibition of sulfate transport by the isothiocyanates used could be described by a single exponential and could be shown to contain a reversible and an irreversible component. In each case a portion of sulfate efflux was found to be resistant to inactivation. The residual portion of the sulfate efflux varied with inhibitor: 4% for INS, 16% for INDS, and 34% for IBS. INS showed the largest reversible inhibitory effect (12% of the flux remaining at 0.2 mM inhibitor concentration), while INDS showed the weakest effect (92% of the flux remaining at 0.3 mM inhibitor concentration). IBS had the highest rate of inactivation while INDS had the lowest. The kinetic analysis further suggests that all three isothiocyanates bind reversibly to an inhibitory site on the membrane before they bind covalently, and therefore irreversibly, to the same site on the membrane. The equilibrium constant for the dissociation of the reversibly-bound complex, Ki, and the rate of irreversible inactivation after all membrane sites are reversibly bound, kmax, have been computed for all three inhibitors: INDS (Ki = 420 micron, kmax = 5.04 hr-1), INS (Ki = 148 micron, kmax = 6.48 hr-1), and IBS (Ki = 208 micron, k(max) = 8.11 hr-1).

Biological Transport, Active↗