Relationship between the Activity of the 3P-Hydroxysteroid Dehydrogenase from Bovine Adrenal Cortex Microsomes and Membrane Structure INFLUENCE OF PROTEINS AND STEROID SUBSTRATES ON LIPID “MICROVISCOSITY”*

In order to describe the relationship between lipid dynamics and enzyme function in endoplasmic reticulum membranes of the adrenal cortex, a study of temperature dependence of the 3P-hydroxysteroid dehydrogenase activity has been carried out at three different pH values, in relation to the change of the rotational motion of the membrane lipids. At pH 8.5, corresponding to the highest value of the enzyme maximum velocity, the Arrhenius plot for the enzymatic activity was a straight line with the steroid substrate 17a-OH-pregnenolone, but exhibited a break at 30°C with dehydroepiandrosterone and pregnenolone as substrates. Activation energy of the enzyme reaction was lowered above the transition temperature (Tt = 30°C) from 20 kcal mol” to 8 kcalmol” for dehydroepiandrosterone and from 21 kcal-mol” to 12 kcal-mol” for pregnenolone. At pH 7.5, as well as at pH 6.3, where the enzyme activity was very low, the Arrhenius plot was linear whatever the steroid substrate. Conversely, the pH dependence of the enzyme velocity was a function of the thermal transition. With dehydroepiandrosterone as substrate, which exhibited a thermal transition in the activation energy of its enzymatic transformation, an apparent p K ~ s of 7.6 was detected at 26”C, while at 36°C the shape of the Dixon plot was different, and the apparent ~ K E S value was 7.8. With 17a-OH-pregnenolone which presented a constant activation energy for its enzymatic transformation throughout the whole temperature range studied, a single apparent pKEs of 7.3 was found at both temperatures, the two curves being identical. The dynamics of lipid organization was evaluated by measurements of fluorescence anisotropy and nanosecond decay of all-tmns-1,6-diphenyl-1,3,5-hexatriene embedded in the microsomal membranes and in the lipid-derived liposomes. The estimation of the “microviscosity” as a function of temperature indicated a change in the lipid dynamic organization at a temperature around 30°C at pH 8.5 in presence of dehydroepiandrosterone and pregnenolone, but not with l7a-OHpregnenolone. This change is correlated with the break in the Arrhenius plot of the enzymatic reaction which arises in the same conditions. Comparison of the lipid dynamic properties in the total membranes and in the