Activity of heart and muscle lactate dehydrogenases in all‐aqueous systems and in organic solvents with low amounts of water

Abstract

The effect of urea and guanidine hydrochloride (GdmC1) on the activity of lactate dehydrogenases from heart and muscle was studied in standard water mixtures and in reverse micelles formed with n-octane, hexanol, cetyltrimethykdmmonium bromide and water in a concentration that ranged over 2.5 -6.0% (by vol.). In all water mixtures GdmCl (0.15-0.75 M) and urea (0.5-3.0 M) inhibited the activity of the enzymes at non-saturating pyruvate concentrations. At concentrations of pyruvate that proved inhibitory for enzyme activity due to the formation of a ternary enzyme-NAD-pyruvate complex, GdmCl and urea increased the activity of the enzymes. This increase correlated with a decreasc of the ternary complex, as evidenced by its absorbance at 320-325 nm. In the low-water system it was found that: (a) at all concentrations of pyruvate tested (0.74 -30 mM), GdmCl enhanced the activity of the heart enzyme to a similar extent; (b) in the muscle enzyme, GdmCl inhibited or increased the activity through a proccss that depended on the concentration of pyruvate and GdmCl; (c) under optimal conditions, the activation by GdmCl was about two times lower in the muscle than in the heart enzyme, although in all-water media the activity of the muscle enzyme was twice as high. The expression of lactate dehydrogenase activity in the low-water system was higher with the heart than with the muscle enzyme compared to their activities in all-water media (about 260 and 600 pmol min-I mg-' in the heart and muscle enzymes respectively). Apparently for catalysis, the water requirement in the heart enzyme is lower than in the muscle enzyme. It is likely that the different response of the two enzymes to solvent is due to their distinct structural features.For a long time it has been known that guanidine salts and urea disrupt the three-dimensional structure of proteins [l]. However, there are reports that indicate that the activity of an important number of enzymes is diminished [2-51 or enhanced 16-121 by denaturants at concentrations that do not produce gross changes of protein structure. The mechanisms that account for these effects are not known with certainty, but it has been suggested that they take place through modifications of the catalytic site, or to an increase in the flexibility of the enzyme. In the preceding paper [13], it was shown that, in reverse micelle systems with a low water content, the activity of four out of five enzymes tested could be increased severalfold by denaturants. Here the activities of heart and muscle lactate dehydrogenases were studied in allaqueous media and in a low-water system with and without denaturants. The studies were an attempt to ascertain if indeed, and how, thc structural differences of the isoenzymes affect their functional response to solvent modifications. The experiments werc made in all-water mixtures and in reverse micelles formed with cetyltrimethylammonium bromide, hexanol, and n-octane [14, 151. Lactate dehydrogenase has the advantage that it is one of the most thoroughly studied enzymes....

Trimethylamine-N-Oxide Counteracts Urea Effects on Rabbit Muscle Lactate Dehydrogenase Function: A Test of the Counteraction Hypothesis

“…1. Studies by us and others show that use of concentrations higher than 100 M NADH or 3 mM pyruvate results in deviations from the modified Theorell-Chance mechanism, because of high-substrate inhibition (Everse et al, 1970;Fernandez-Velasco et al, 1992;Griffin and Criddle, 1970;Yancey and Somero, 1978). To avoid complications due to deviation from the mechanism in the limit of high substrate concentrations and to ensure that Eqs.…”

“…The differences can readily be explained by the fact that, unlike Yancey and Somero, who used substrate inhibitory concentrations (3 mM) of pyruvate in all of their studies on k cat and K m , we varied the pyruvate concentration over a range that avoided the phenomenon of high-substrate inhibition. The apparent urea activation observed by Yancey and Somero was attributed by them to an abortive complex, a phenomenon described by Fernandez-Velasco et al as an enzyme-NAD-pyruvate ternary complex (Fernandez-Velasco et al, 1992;Yancey and Somero, 1978). The abortive complex is promoted whenever the pyruvate concentration in the assay is high (Ͼ2.5 mM), resulting in high-substrate inhibition observed in LDHs from a variety of species, including mammalian muscle and elasmobranchs (Everse et al, 1970;Fernandez-Velasco et al, 1992;Fromm, 1963;Stambaugh and Post, 1966;Yancey and Somero, 1978).…”

“…The apparent urea activation observed by Yancey and Somero was attributed by them to an abortive complex, a phenomenon described by Fernandez-Velasco et al as an enzyme-NAD-pyruvate ternary complex (Fernandez-Velasco et al, 1992;Yancey and Somero, 1978). The abortive complex is promoted whenever the pyruvate concentration in the assay is high (Ͼ2.5 mM), resulting in high-substrate inhibition observed in LDHs from a variety of species, including mammalian muscle and elasmobranchs (Everse et al, 1970;Fernandez-Velasco et al, 1992;Fromm, 1963;Stambaugh and Post, 1966;Yancey and Somero, 1978). It has been shown previously that urea diminishes the concentration of the abortive complex, and relief of substrate inhibition results in the apparent "activation" of LDH activity by urea at high concentrations of pyruvate (Fernandez-Velasco et al, 1992;McQueen, 1974).…”

“…The abortive complex is promoted whenever the pyruvate concentration in the assay is high (Ͼ2.5 mM), resulting in high-substrate inhibition observed in LDHs from a variety of species, including mammalian muscle and elasmobranchs (Everse et al, 1970;Fernandez-Velasco et al, 1992;Fromm, 1963;Stambaugh and Post, 1966;Yancey and Somero, 1978). It has been shown previously that urea diminishes the concentration of the abortive complex, and relief of substrate inhibition results in the apparent "activation" of LDH activity by urea at high concentrations of pyruvate (Fernandez-Velasco et al, 1992;McQueen, 1974). In the presence of both TMAO and urea, high substrate inhibition is apparently not prevented, leading to a lower LDH activity in the mixture of TMAO plus urea than in urea alone.…”

Activity and fluorescence changes of lactate dehydrogenase induced by guanidine hydrochloride in reverse micelles

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Denaturants activate several multimeric enzymes in reverse micelles [Garza-Ramos, G., Darszon, A., Tuena de G6mez-Puyou, M. & G6mez-Puyou, A. (1992) Eul: J. Biochem.

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