Emmanuel T. Rakitzis

Reactivity of 6-phosphogluconolactone with hydroxylamine: the possible involvement of glucose-6-phosphate dehydrogenase in endogenous glycation reactions.

The reactivity of 6-phosphogluconolactone and of delta-gluconolactone with hydroxylamine (a model compound in electrophilicity determination studies) was examined and compared with the reactivity of several other electrophiles, such as acid anhydrides and esters, some of which exhibit adverse biological effects (e.g. carcinogenicity). At pH 7.6 and 30 degrees C, and with an excess of hydroxylamine concentration, most of the compounds tested disappear from the medium in a monoexponential reaction. On the other hand, the reaction of 6-phosphogluconolactone with hydroxylamine is biexponential. This finding indicates the existence of 6-phosphogluconolactone in two interconvertible, isomeric forms. The reactivity, towards hydroxylamine, of 6-phosphogluconolactone and, to a lesser extent of delta-gluconolactone, is on the upper scale of reactivity of the electrophiles tested. It is concluded that 6-phosphogluconolactone (and in particular, one of its isomeric forms) is a highly electrophilic compound, and may possibly react with sundry intracellular nucleophiles, thereby exerting untoward metabolic effects. In this connection, it is of interest that a positive correlation has been found to exist between glucose-6-phosphate dehydrogenase activity and cell proliferation.

Kinetics of irreversible enzyme inhibition: co-operative effects.

Abstract A mathematical treatment of multiple irreversible enzyme-inhibitor binding is presented. The model considered consists of an enzyme molecule with one active site, and with two inhibitor binding sites. Equations are derived which describe the dependence of the concentration of active enzyme species, as well as the concentration of irreversibly enzyme bound inhibitor, on the rate constants characteristic of the various enzyme-inhibitor complex species.

Kinetic analysis of biphasic protein modification reactions

SummaryA 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).

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.

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)

The concentration of modified protein species as an index of protein modification cooperativity

Abstract A mathematical treatment of stoichiometric protein modification reactions is presented, and a formula is derived for the determination of the concentration of the i -fold modified protein species as a function of the concentration of the unreacted protein moiety. The method is applied to a case from the literature: the carbamylation of amino-terminal valine residues of human hemoglobin.

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.

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).