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E T Rakitzis

Publications and source records attributed to E T Rakitzis.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1988

On reductionism.

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Biochemical Phenomena↗
PubMed1988

Modification and inactivation of rhodanese by 2,4,6-trinitrobenzenesulphonic acid.

Bovine liver rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is modified by 2,4,6-trinitrobenzenesulphonic acid, by the use of modifying agent concentrations in large excess over enzyme protein concentration. The end-point of the reaction, viz., the number, n, per enzyme protein molecule, of modifiable amino groups was determined graphically by the Kézdy-Swinbourne procedure. It was found that the value for n depends on the pH of the reaction medium, and ranges from 2, at pH 7.00, to 10.66, at pH 9.00. Again, the value for n increases with an increase in the concentration of 2,4,6-trinitrobenzenesulphonic acid used, with values ranging from 3.52, at 0.10 mM modifying agent, to 8.96, at 2 mM modifying agent. Rhodanese primary amino groups modification by 2,4,6-trinitrobenzenesulphonic acid is described by a summation of exponential functions of reaction time at pH values of 8.00 or higher, while at lower pH values it is described by a single exponential function of reaction time. However, the log of the first derivative, at initial reaction conditions, of the equation describing protein modification, is found to be linearly dependent on the pH of the reaction. An identical linear dependence is also found when the log of the first derivative, at the start of the reaction, of the equation describing modification-induced enzyme inactivation is plotted against the pH values of the medium used. In consequence, the fractional concentration of rhodanese modifiable amino groups essential for enzyme catalytic function is equal to unity at all reaction pH values tested. It is accordingly concluded that, when concentrations of 2,4,6-trinitrobenzenesulphonic acid in excess of protein concentration are used, all rhodanese modifiable amino groups are essential for enzyme activity. A number of approaches were used in order to establish a mechanism for the modification-induced enzyme inactivation observed. These approaches, all of which proved to be negative, include the possible modification of enzyme sulfhydryl groups, disulphide bond formation, enzyme inactivation due to sulphite released during modification, modification-induced enzyme protein polymerization, syncatalytic enzyme modification and hydrogen peroxide-mediated enzyme inactivation.

Alcohol Dehydrogenase↗
PubMed1987

Kinetics of protein modification reactions: analysis of modification-induced protein unfolding.

A mathematical treatment of a two-sited, modification-induced protein unfolding model is presented, and it is shown that the dependence of the concentration of modified protein groups on reaction time is described by a linear, second-order, differential equation with nonzero right hand side. The analytic solution of this equation consists of a summation of exponential functions of reaction time. By assigning arbitrary values to the modification and isomerization rate constants of these equations, simulated cases of protein modification are presented, and the apparent end-point of the reaction is determined graphically. It is found that the apparent end-point of the reaction is, in most cases studied, different from the true value of two groups modified per protein molecule, and is a function of both the modification, and isomerization rate constants of the model. The first derivative of the protein modification reaction, at the start of the reaction, [E]'mod (0), is determined, for the same simulated cases of protein modification, by two different analytical methods. It is found that the [E]'mod(0) value, obtained from graphical and numerical analysis data, is in most cases in good agreement with the value expected from first principles. Finally, the different irreversible enzyme inhibition forms, contingent upon the different kinds of the enzyme inactivation-protein modification relationships of the protein modification model under consideration, are presented and discussed.

Kinetics↗
PubMed1986

Kinetics of protein-modification reactions. Determination of the fractional concentration of enzyme protein groups, or group reactivities, essential for catalytic function.

A mathematical treatment of modification-induced enzyme protein inactivation is presented, and it is shown that, at initial reaction conditions, the ratio of the first derivative of the equation describing enzyme activity loss to the first derivative of the equation describing protein groups modification is equal to the fractional concentration of enzyme protein reactive groups, or group reactivities, essential for catalytic function.

Catalysis↗
PubMed1985

Kinetics of protein-modification reactions. Stoichiometry of modification-produced enzyme inactivation: modification of rhodanese by 2,4,6-trinitrobenzenesulphonic acid.

A mathematical treatment is presented for the dependence of enzyme activity loss on the numbers and reactivities of the groups essential for catalytic function, when enzyme protein modification is carried out by the use of concentrations of protein reactive groups well in excess of that of modifying agent. Experimentally obtained data on the modification of rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) by 2,4,6-trinitrobenzenesulphonic acid are presented, and it is shown that, at pH9.00, the fractional concentration of rhodanese groups, or of rhodanese group reactivities, essential for enzyme catalytic function is 0.88; this value is found to decrease with decreasing pH of the reaction medium. The possibility that rhodanese inactivation by 2,4,6-trinitrobenzenesulphonic acid is brought about by modification of groups other than amino groups is ruled out by a comparison of the enzyme-inactivation and protein-modification stoichiometries, for putative reaction models for enzyme and modifying agent.

Enzymes↗
PubMed1984

Kinetics of protein modification reactions. Plot of fractional enzyme activity versus extent of protein modification in cases where all modifiable groups are essential for enzyme activity.

The plot of fractional enzyme activity versus extent of protein modification, for cases where all enzyme modifiable groups of a certain kind are essential for activity, is found to be nearly independent of the number, per enzyme active site, of modifiable groups involved. Such plots usually, by a fallacious extension of the initial portion of the plot on the extent-of-modification axis, are interpreted to mean the modification of one single group per enzyme active site (or per enzyme molecule). The possible relevance of these findings to cases in the literature is discussed.

Binding Sites↗
PubMed1983

Kinetic analysis of biphasic protein modification reactions. Cooperative effects.

A mathematical treatment of protein modification reactions is presented, and it is shown that in these cases protein modification is described by a summation of exponential functions of reaction time, the number of exponentials being equal to the number of modified protein species. It is shown that, in cases of protein modification cooperativity, there is a strict dependence of the coefficients of the multiexponential modification equation on the constants of the same equation. The conditions necessary for a reduction of a multiexponential protein modification equation to one of a summation of two exponentials only are examined. The possible formulae for the coefficients of a two-exponential-summation equation, used to describe the modification of protein models with two, three or four modifiable residues (as well as some aspects of models with five and six modifiable residues) per protein molecule are derived. It is seen that the number of such coefficients is severely limited. The most frequently obtained formula for the lower stoichiometric coefficient of a two-exponential-summation equation Is Aka/(ka-kb), where ka and kb are the constants of the two exponentials of the equation, and A is a constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymes↗
PubMed1982

Effect of diethylpyrocarbonate on human gastric mucosa acid proteinases: zymogen activation.

Proteolytic activity (with hemoglobin as the substrate, and at a pH of 3.8) of human gastric mucosa homogenates can be separated into two peaks by DEAE-cellulose column chromatography. One of the peaks contains a proteolytic enzyme that is activated some 11-fold by exposure to pH 3.5, and is also activated by preincubation with diethylpyrocarbonate. The other peak contains a proteolytic enzyme that is not activated at low pH values, and is inactivated by preincubation with diethylpyrocarbonate. The enzyme activity arising out of acid activation is partially lost by exposure to pH 8.5 (pepsin-like enzyme). The nonactivated enzyme is resistant to alkaline pH (cathepsin-like enzyme).

Aspartic Acid Endopeptidases↗
PubMed1980

Kinetic analysis of biphasic protein modification reactions.

A mathematical analysis of biphasic protein modification reactions is presented, and it is shown that, in addition to the protein species modification reactions, one more time-dependent step must be postulated to exist in the reaction process. This step involves the interconversion of the different protein species, such as binding of ligand with protein, or the change in the isomerization state of the protein. The kinetic description of the reaction process is effected through a second order homogeneous linear differential equation, with time as the independent variable, and unmodified protein concentration as the dependent variable. A simple procedure of graphical analysis of the experimental data is described, and it is shown that, by a process of elimination, the nature of the protein species interconversion time-dependent step may be recognized, and also the dependence of the protein species inactivation rate constants on various parameters in the preparation may be evaluated. The method is illustrated by the detailed analysis of one example from the literature, the inactivation of phosphorylase b by 5,5-dithiobis (2-nitrobenzoic acid).

Chemical Phenomena↗
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↗