Reaction of fluorescein isothiocyanate with an ATP-binding site on the phosphorylase kinase alpha subunit.
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 [14C]carboxymethylated alpha subunit. The partial sequences obtained have then been localized in the primary structure of the 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 reveals a competition between ATP and FITC; a Ki for ATP of 728 +/- 100 microM has been determined.
Zaman
N
N
Abteilung für Biochemie Supramolekularer Systeme, Ruhr-Universität Bochum, Federal Republic of Germany.
Varsányi
M
M
Heilmeyer
L M
LM
Sotiroudis
T G
TG
Johnson
C M
CM
Crabb
J W
JW
D000255:Adenosine Triphosphate / Q000032:analysis* / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D001667:Binding, Competitive; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010764:Phosphorylase Kinase / Q000032:analysis* / Q000037:antagonists & inhibitors; D013861:Thiocyanates / Q000494:pharmacology
D000345:Affinity Labels; D000596:Amino Acids; D005452:Fluoresceins; D010446:Peptide Fragments; D013861:Thiocyanates; D000255:Adenosine Triphosphate; D010764:Phosphorylase Kinase; D016650:Fluorescein-5-isothiocyanate