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When protein is assayed in human placental microsomes by the fluorescamine procedure, 25--30% of the observed fluorescence results from labeling of phospholipids.

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.76 microM for NADH and 24.0 microM for NAD. Vmax values with 9.28 microM 5-pregnene-3,20-dione were 22.0 nmol/min/mg protein with NADH and 65.8 nmol/min/mg protein with NAD. When isomerase was assayed as a function of 5-pregnene-3,20-dione concentration, NAD increased Vmax but had no effect on the Km value for steroid. NADP, NADPH, acetylpyridine NAD and deamino NAD did not activate nor did they compete with NAD. Exposure of microsomes to trypsin, phospholipase A2 or phospholipase C resulted in the loss of isomerase activity. Approximately 30% of the initial activity was recovered after detergent solubilization of microsomes. Hydrogen peroxide did not affect activation by NAD. The data are consistent with nucleotide enhancement of a step in the isomerization reaction other than substrate binding.

When human placental microsomes were heated in boiling water or exposed to trypsin, 30 to 40% of the 5-ene,3-ketosteroid isomerase activity was stable. Aqueous suspensions of chloroform:methanol extracts of microsomes also catalyzed isomerization of 5-pregnene-3,20-dione, activity being associated with the polar lipid fraction. The trypsin- and heat-stable activities, as well as that of resuspended microsomal lipids, showed a dependence on buffer composition and concentration. Little activity was detected in water at pH 7.0. Relative activities in various buffers were Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) greater than Pipes (1,4-piperazinediethanesulfonic acid) greater than potassium phosphate greater than Mes(4-morpholineethanesulfonic acid). The data suggest that the occurrence of membrane lipid-dependent nonenzymatic catalysis could contribute to the isotope exchange with solvent observed in previous studies of the mechanism of isomerization catalyzed by placental microsomes. The ability of the membrane lipid phase to catalyze steroid isomerization under certain conditions and the fact that this activity is subject to modifications by exogenous agents may have more general implications for an understanding of possible effects of xenobiotics on steroid hormone formation and action in vivo.