USE OF SYNTHETIC PEPTIDES AND COPOLYMERS TO STUDY THE SUBSTRATE-SPECIFICITY AND INHIBITION OF THE PROTEIN-TYROSINE KINASE PP60(C-SRC)

The ability of synthetic peptides and polypeptides to act as substrates and/or inhibitors of pp60(c-scr) was examined. The random copolymer, poly(K4Y) had a threefold lower specificity than poly(E(4)Y). Peptides containing lysine vs. glutamate were also found to have a lower substrate specificity (V-max:K-m ratio). In order to assess the substrate specificity of acidic peptides, an assay protocol using DEAE-membranes was developed. Peptides containing a (YXE)(5)YXD motif (X = G, A, V, P, or norvaline) were tested as inhibitors and substrates of pp60(c-scr). The glycine-containing peptide was the best substrate having a specificity 16 000-fold higher than (5)Val-angiotensin II, the most commonly used peptide substrate. Most of the peptides, except for the proline containing peptide, had K-i values of 20-100 mu M. In a series of (XGE)(5)XGD peptides, where X = Y or F, tyrosine at position 10 was found to be the preferred site for accepting a phosphate. Analogs in which the glycine was replaced with alanine indicated that loss of flexibility around position 10 was detrimental to substrate specificity. Results suggest that conformational requirements of the peptides tested was important and substrate specificity was a more sensitive parameter than binding as measured by K-i values.