Solubilization and partial purification from mouse sperm membranes of the specific binding activity for 3-quinuclidinyl benzilate, a potent inhibitor of the zona pellucida-induced acrosome reaction.
3-Quinuclidinyl benzilate (QNB), a potent antagonist of muscarinic acetylcholine receptors, has been demonstrated to inhibit specifically the zona pellucida (ZP)-induced acrosome reaction (AR) in mouse sperm (Florman and Storey, 1982; Dev Biol 91:121-130). In this study we describe the solubilization and partial purification of the mouse sperm QNB binding activity which may represent a component of the putative receptor complex for ZP on the sperm plasma membrane. Sperm membranes were isolated from cell homogenates of washed, capacitated, epididymal mouse sperm. Scatchard plots of QNB binding to these membranes indicated a single class of binding sites with KD = 7.2 nM and Bmax = 8700 sites/cell. These binding characteristics are similar to those seen with QNB binding to whole cells (Florman and Storey, 1982, J Androl 3:157-164). Sperm membranes were solubilized using 1% digitonin/0.2% cholate, and the resultant detergent-soluble fraction possessed QNB binding activity similar to that of intact membranes. The detergent-soluble fraction maintained intact ZP receptor(s)-G protein coupling in that treatment of this fraction with either ZP or mastoparan resulted in a 35% or 65% increase in specific GTP gamma S binding, respectively. The solubilized membrane preparation was fractionated by gel permeation HPLC. A majority of specific QNB binding activity was confined to one HPLC fraction. Analysis of this fraction by SDS-PAGE revealed a complex of approximately 5 proteins unique to this fraction. The most prominent protein had a M(r) of 72 kDa, which is within the M(r) 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 alpha subunit of the G(i) class of G proteins. Although the QNB binding activity could not be positively identified, we propose that it is contained in one or more of the proteins unique to this fraction and that these proteins, including G(i), may act as part of a sperm receptor complex for the ZP.
Ward
C R
CR
Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia.
Kopf
G S
GS
Storey
B T
BT
D000177:Acrosome / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D037342:Pertussis Toxin; D011813:Quinuclidinyl Benzilate / Q000378:metabolism* / Q000494:pharmacology*; D011976:Receptors, Muscarinic / Q000302:isolation & purification / Q000378:metabolism*; D015398:Signal Transduction; D012995:Solubility; D013084:Sperm-Ovum Interactions / Q000187:drug effects*; D013094:Spermatozoa / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D015044:Zona Pellucida / Q000187:drug effects / Q000502:physiology*
D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D009243:NAD; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011813:Quinuclidinyl Benzilate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins