The essential light chains constitute part of the active site of smooth muscle myosin

Abstract

Myosin, a major contractile protein, characteristically possesses a long coiled-coil α-helical tail and two heads. Each head contains both an actin binding site and an ATPase site and is formed from the NH₂-terminal half of one of the two heavy chains (relative molecular mass, M_r, 200,000) and a pair of light chains; the so-called regulatory and essential light chains of approximately M_r 20,000 each. Recently we have identified¹ Trp 130 of the myosin heavy chain from rabbit skeletal muscle as an active-site amino-acid residue after labelling with a new photoaffinity analogue of ADP, N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate (NANDP)². Nonspecific labelling was eliminated by first trapping NANDP at the active site with thiol crosslinking agents³. Exclusive labelling of the heavy chains with no labelling of the light chains agreed with previous findings^4,5 that the heavy chains alone contain the actin-activated Mg-ATPase activity of rabbit skeletal myosin. Here we report similar photolabelling experiments with smooth muscle myosin (chicken gizzard) in which ³H-NANDP is trapped at the active site with vanadate⁶ and which show that both the heavy chains and the essential light chains are labelled. The results indicate that both chains contribute to the ATP binding site and represent the first direct evidence for participation of the essential light chains in the active site of any type of myosin.