Phosphorylase kinase can be labeled specifically on the c1 subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421 -4251. Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein-labeled tryptic peptide is Lys-Met-Gln-Asp-Gly-Tyr-Phe-Gly-Gly-Ala-Arg. The environment of this fluorescein-labeled lysine has been determined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purified ['4C]carboxymethylated u subunit. The partial sequences obtained have then been localized in the primary structure of the CI subunit Proc. Nut1 Acad. Sci. USA 85,2929-29331. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728 100 pM has been determined.Phosphorylase kinase (ATP : phosphorylase phosphotransferase, EC 2.7.1.38) is a key regulatory enzyme in glycogen metabolism which catalyzes the phosphorylation of phosphorylase b to make phosphorylase a (for review see [l] ADP has been demonstrated to be an activator of phosphorylase kinase [18]. This nucleotide binds probably to the fl subunit; however, the structure of its binding domain is still unknown.Corresporzdenct. to L.