Reaction of fluorescein isothiocyanate with an ATP‐binding site on the phosphorylase kinase α subunit
- 1 July 1989
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 182 (3) , 577-584
- https://doi.org/10.1111/j.1432-1033.1989.tb14866.x
Abstract
Phosphorylase kinase can be labeled specifically on the .alpha. subunit with fluorescein 5''-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T.G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421-425]. 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 structure of the .alpha. methylated .alpha. subunit. The partial sequences obtained have then been localized in the primary structure of the [14C]carboxymethylated .alpha. subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929-2933]. 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 revealed a competition between ATP and FITC; a Ki for ATP of 728 .+-. 100 .mu.M has been determined.Keywords
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