Selective labelling of phosphorylase kinase with fluorescein isothiocyanate

Fluorescein isothiocyanate (FITC) is a highly specific inhibitor of rabbit muscle phosphorylase kinase. The rapid inhibition process is accompanied by an almost exclusive incorporation of fluorescein into the α sub-unit. A molar ratio of 0.8 mol FITC per mol α subunit for a 60% inhibited kinase was calculated. Mg²⁺ and Mg²⁺-ATP completely block the inhibitory effect of FITC, but ATP, ADP and Ca²⁺ have no significant effect on FITC inhibition. Trypsin-activated phosphorylase kinase is not inactivated by FITC, while the fluorescein-modified enzyme can be activated by digestion with trypsin to the same level of activity of trypsin-activated unmodified enzyme.