Affinity purification of seminalplasmin and characterization of its interaction with calmodulin

Abstract

Bull seminalplasmin antagonizes with high potency and selectivity the activations effect of calmodulin ion target enzymes [Gietzen and Galla (1984 Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinith purification of seminalplasmin was developed. The high specificity of seminal plasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% .alpha.-helix is induced on interaction with calmodulin. These fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changes on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin and to ascertain the structural homology between semialplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem J. (1986) 233, 5.