Fluorescently labeled myosin subfragment 1: identification of the kinetic step associated with the ATP-induced fluorescence decrease

Abstract

Marsh, and Lowey [Marsh, D. J., & Lowey, S. (1980) Biochemistry 19, 774-784] introduced a fluorescence label, 5-(iodoacetamido)fluorescein (IAF), into the alkali 1 light chain of myosin subfragment 1 (S-1) without perturbing the K+-Ca2+, or actin-activated ATPase activities of the labeled S-1. They also found that the addition of MgATP to the labeled S-1 led to a 6-10% decrease in the fluorescence of the bound IAF label. In the present study, we investigated the pre-steady-state kinetics of the fluorescence decrease of the IAF label caused by the binding of MgATP to the labeled S-1. Our results show that most of the decrease in IAF fluorescence is caused by the ATP-hydrolysis step. This implies that a conformational change in the S-1 molecular associated with ATP hydrolysis has led to a change in the microenvironment of the IAF fluorophore bound to the alkali 1 light chain.