Chemical and immunological analysis of prey-derived vomeronasal stimulants

We have isolated seven proteins from earthworm preparations that are chemoattractive to garter snakes. Three of these proteins have been purified to homogeneity: two from aqueous earthworm wash (EWW) and one from electric shock-induced earthworm secretion (ESS). One of the two highly purified proteins from EWW has a relative molecular mass of 20 kDa and contains free sulfhydryl groups that appear to play a functional role in its chemoattractivity. The other purified protein from EWW has a molecular mass of 3 kDa (low molecular weight protein, LMW). The highly purified chemoattractive protein (ES20) from ESS is a glycoprotein having a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents. It consists of a single polypeptide chain. The sequence of terminal 15 amino acid residues from its amino (NH2-) terminal has been determined. It binds specifically to the membranes of vomeronasal sensory epithelium in a saturable and reversible fashion with a Kd value of about 0.3 microM and Bmax value of 0.4 nmol/mg of protein. This protein causes an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal neurons. All the isolated chemoattractive proteins from both earthworm preparations can be divided immunologically into three groups: (i) those closely related to the ES20 snake-attractive protein, (ii) those closely related to the LMW snake-attractive protein, and (iii) those unrelated to either ES20 or the LMW protein.