Sperm Plasma Membrane Isolation
SPMs obtained from fresh sperm immediately after Per-coll washing were purified at least with a factor eight (see Table 1) as the specific activity of alkaline phosphatase, acid phosphatase, and 5′-nucleotidase were respectively 8.5, 8, and 9, times higher in isolated plasma membranes when compared with complete sperm cavitate. The purified SPM isolate contained less than 7% of contamination with acrosome material as detected with the specific activity of acrosin recovered in the SPM (Table 1).
SPMs, obtained after the sperm was incubated for 4 h in one of four different Tyrode media (T-BIC, T+BIC, T+BIC+Hep, T+BIC+Hep+Glu), showed similar purification rates from plasma membrane material and identical low amounts of acrosomal contamination (P > 0.05; n = 3 for all each treatment (Table 1).
Enzyme-Linked Carbohydrate Binding Assay
Serial dilutions of isolated SPM (starting with 2 mg/ml) were immobilized on polystyrene microtiter plates and varying concentrations of biotinylated carbohydrate probes (initially from 20 mM) were allowed to bind to SPM. Both fucose-PAA-biotin and LeA-PAA-biotin were captured by immobilized SPM in a concentration-dependent manner. Fucose-PAA-biotin presented approximately 12 times higher binding capacity for the isolated SPM.
TABLE 1. Specific activity of the enzymes in sperm cells and isolated sperm plasma membranes (SPM) and purification of SPM obtained in different Tyrode media.
* Measured as nmol mg protein1 min in sperm cells or isolated SPM (n = 3, mean ± SD).
+ Ratio of the specific enzyme activities in sperm cavitate and isolated SPM (n = 3, mean ± SD).