The growth-associated neuronal phosphoprotein B-50: improved purification, partial primary structure, and...

A reversed phase HPLC procedure is reported that has allowed the separation of the growth-associated, kinase C substrate protein B-50 [previously purified by isoelectric focussing (IEF)] into three components (1-, m- and rB-50). The minor form 1B-50 (probably a proteolysis product) gave a 24-residue N-terminal amino acid sequence, but the major and possibly native form (mB-50) (and also rB-50 which is probably formed during IEF) appeared to be N-terminally blocked. HPLC also separated B-60, the major proteolysis product of B-50, into three components, and the N-terminal sequence of the major B-60 was determined. HPLC peptide mapping of SAP digests of the various B-50 and B-60 protein confirmed their close relationship, and four SAP generated fragments also afforded sequence data. The amino acid sequences obtained (1B-50, B-60 and fragments) are all found in the recently predicted (based on nucleotide sequencing) B-50/GAP43 sequence (226 amino acids), further confirming the identity of B-50 and GAP43, and helping clarify the relationship of B-60 (starting at position 41 of the predicted sequence) to B-50. Correlation of amino acid analyses, SAP fragment data, and the predicted sequence provided additional information on the length of the translated products, including evidence that the N-terminus of the major (blocked) form of B-50 starts at position 1 (Met) of the predicted sequence.