CHARACTERIZATION OF A NOVEL ZINC-BINDING SITE OF PROTEIN-KINASE-C INHIBITOR-1

The zinc-binding properties of an endogenous protein inhibitor of protein kinase C was studied. Equilibrium gel penetration revealed that 1 mol of this protein binds 0.97 mol of zinc with a dissociation constant of 4.3-mu-M. The site of zinc-binding, MVVNEGSDGGQSVYHVHLHVLGGR, was identified by a multi-step process consisting of tryptic digestion, fragment isolation, transfer to nitrocellulose, and hybridization with (ZnCl2)-Zn-65. Binding of (ZnCl2)-Zn-65 to selected synthetic fragments further localized the site of interaction to the sequence QSVYHVHLHVL. This region contains 3 closely positioned histidine residues and represents a novel zinc-binding site.