Analysis of the PKC-γ-Related Immunocrossreactive Region of a Novel Leukocyte Protein γ-rp

Abstract

The newly discovered gamma-PKC-related-protein of human leukocytes (γ-rp) crossreacts with a polyclonal antibody preparation originally designed to be specific for PKC-γ (γMb-Ab). As this antibody is currently the only suitable probe for γ-rp, we sought to characterize the binding of the two proteins. We determined that the γMg-Ab does not recognize the native form of γ-rp. However, with denaturing immunoblots of γ-rp, we found that 1) the crossreactive γ-rp epitope differs somewhat from that of classic rat brain PKC-γ, but probably only to the degree of the rat/human PKC species difference; 2) the previously reported doublet bands of γ-rp represent a single protein with cell-stimulus inducible modifications; 3) antibodies present in the γMg-Ab pool bind to two separate sites within the γ-rp epitope; 4) access to one binding site is conformationally restricted, even after protein denaturation; 5) agonist-induced modification of γ-rp does not significantly affect the total amount of γMg-Ab that it can bind, but 6) does significantly affect the rate of antibody binding to one site. This investigation defines the appropriate experimental use of our antibody, and the significance of these findings for the future study and cloning of γ-rp is discussed.