The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3β-hydroxy-Δ5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

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Abstract
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 3β-hydroxy-Δ5-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5µm 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 (0-500µmol/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 250µmol/l. The apparent Michaelis—Menten constant (K m) was 29µmol/l and Vmax 0.63µmol NAD+ reduced/min/cm3 corpora lutea.