Membrane lipid dynamics and enzymic activity in bovine adrenal cortex microsomes

Abstract

The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C16 as compared to the C2 region, while the rotational motion appeared to be the most hindered at the C7–C9 level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C16 region. The extent to which the acyl chain reacted to this perturbation at the C12 level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the 3-oxosteroid Δ5−Δ4-isomerase, the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the 3-oxo-Δ4-steroids in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant (2–5 kcal · mol−1) in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy (20 kcal · mol−1) was observed at temperature below 16°C at pH 7.5. A correlated change of the pKESapp as function of temperature was detected; at 36°C pKESapp = 8.3 while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the 3β-hydroxy-Δ5-steroid dehydrogenase, the 3-oxosteroid Δ5−Δ4-isomerase was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids.