The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3 beta-hydroxy-delta 5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary.

A technique which allows for the continuous microdensitometric monitoring (at 3.3 s intervals) of an enzyme reaction has been applied to a study of 3 beta-hydroxy-delta 5-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5 micron thick) of the pro-oestrous rat ovary. Initial reaction rates for NAD+ reduction by the activity of the enzyme were maintained for less than 3 min at all the concentrations (0-500 mumol/l) of co-factor employed. The relationship between NAD+ concentration and enzyme activity under initial velocity rate conditions was hyperbolic and maximum enzyme activity was seen when the NAD+ concentration was greater than 250 mumol/l. The apparent Michaelis-Menten constant (Km) was 29 mumol/l and Vmax 0.63 mumol NAD+ reduced/min/cm3 corpora lutea.