The supernatant was used to determine the activities of PFK and PK, two key regulatory enzymes of glycolysis, and the pellet was resuspended in 25 ^l of ho-mogenization buffer to determine the activity of malate dehydrogenase (MDH), an important enzyme of the Krebs cycle. All chemicals for enzyme assay were from Sigma Chemical Company (St. Louis, MO) except those that were from Boehringer-Mannheim GmbH (Mannheim, Germany). antibiotic levaquin
Determination of PFK Activity
PFK is one of the major regulatory enzymes of glycolysis and regulates the second important control point in glycolysis. Therefore, measurement of its activity in cell-free follicular preparation will indicate the status of this control point in glycolysis at that time. Enzyme activity was assessed essentially as described by Ling et al. with minor modification to fit 1-ml reaction volume. The reaction mixture contained (final concentrations) 33 mM Tris-HCl, pH 8.0, 2 mM ATP, 5 mM MgSO4, 2 mM fructose-6 phosphate (potassium salt), 0.16 mM nAdH, 1 mM dithi-threitol, 0.05 mM KCl, and 66.6 ^l of an auxiliary enzyme solution (aldolase, triose phosphate isomerase, and glycerophosphate dehydrogenase).