Small pieces of pig testis were homogenized at 4°C in the same buffer used to prepare SF. The homogenized samples were further lysed by two cycles of freeze-thawing in liquid N2. The suspension was then processed in the same manner as the SF preparation but without sonication or ammonium sulfate precipitation. Samples were aliquoted and frozen at — 80°C.
Inhibitor Preparations and Loading Conditions
A stock solution of 1-[6-[17p-3-methoxyestra-1,3,5-trien-17-yl]aimno]hexyl]-1H-pyrrole-2,5-dione (U73122; Calbiochem, La Jolla, CA) was prepared by dissolving the compound in chloroform to a final concentration of 5 mM. The solution was then aliquoted, dried under a stream of N2, and stored at —20°C. On the day of use, U73122 aliquots were reconstituted in dimethylsulfoxide and diluted to working concentrations of 10-30 ^M in TL-Hepes supplemented with 5% FCS. Eggs were preincubated in 50-^l drops containing 10-20 ^M U73122 under paraffin oil for 15 min at 37°C prior to the injection of SF or IP3, and injection drops were also supplemented with the appropriate concentration of U73122. For some experiments, SF was incubated in 10-30 ^M U73122 for 15 min at 4°C prior to injection into eggs. In these experiments, eggs were not exposed to the inhibitor. Dithiothreitol (DTT) was used in reversal experiments either by mixing U73122 with DTT prior to SF addition or by adding DTT following the 15-min incubation of SF in U73122. In all experiments, DTT was used at a final concentration of 10 mM. A 5-mM stock solution of U73343, the inactive analogue, was prepared and used in a manner analogous to U73122.