SOLUBILIZATION AND PARTIAL-PURIFICATION FROM MOUSE SPERM MEMBRANES OFTHE SPECIFIC BINDING-ACTIVITY FOR 3-QUINUCLIDINYL BENZILATE, A POTENTINHIBITOR OF THE ZONA-PELLUCIDA-INDUCED ACROSOME REACTION

3-Quinuclidinyl benzilate (QNB), a potent antagonist of muscarinic ace tylcholine receptors, has been demonstrated to inhibit specifically th e zona pellucida (ZP)-induced acrosome reaction (AR) in mouse sperm (F lorman and Storey, 1982; Dev Biol 91:121-130). In this study we descri be the solubilization and partial purification of the mouse sperm QNB binding activity which may represent a component of the putative recep tor complex for ZP on the sperm plasma membrane. Sperm membranes were isolated from cell homogenates of washed, capacitated, epididymal mous e sperm. Scatchard plots of QNB binding to these membranes indicated a single class of binding sites with K-D = 7.2 nM and B-max = 8700 site s/cell. These binding characteristics are similar to those seen with Q NB binding to whole cells (Florman and Storey, 1982, J Androl 3:157-16 4). Sperm membranes were solubilized using 1% digitonin/0.2% cholate, and the resultant detergent-soluble fraction possessed QNB binding act ivity similar to that of intact membranes. The detergent-soluble fract ion maintained intact ZP receptor(s)-G protein coupling in that treatm ent of this fraction with either ZP or mastoparan resulted in a 35% or 65% increase in specific GTP gamma S binding, respectively. The solub ilized membrane preparation was fractionated by gel permeation HPLC. A majority of specific QNB binding activity was confined to one HPLC fr action. Analysis of this fraction by SDS-PAGE revealed a complex of ap proximately 5 proteins unique to this fraction. The most prominent pro tein had a M(r) of 72 kDa, which is within the M, range for muscarinic receptors. A protein with M(r) = 41 kDa was also present within this fraction. Subsequent pertussis toxin (PTX)-catalyzed ADP-ribosylation of this fraction revealed this protein to be the cr, subunit of the Gi class of G proteins. Although the QNB binding activity could not be p ositively identified, we propose that it is contained in one or more o f the proteins unique to this fraction and that these proteins, includ ing G(i), may act as part of a sperm receptor complex for the ZP. (C) 1994 Wiley-Liss, Inc.