Osha Roopnarine and Leslie A. Leinwand
Dept of Molecular, Cellular, and Developmental Biol. Univ. of Colorado, Boulder, CO.
We are studying the functional properties of myosin mutants that cause familial hypertrophic cardiomyopathy (FHC). To determine the effect of the mutations on the actin-myosin interaction we are studying the actin- activated myosin ATPases. We have co-expressed histidine-tagged rat cardiac myosin subfragment-1 (S1) along with rat ventricular light chain 1 (VLC1) in mammalian cells (COS). The S1s studied were wildtype a and b and three FHC mutations (Arg249Gln, Arg403Gln, and Val606Met). The crystal structure of S1 indicates that these mutations are near the ATP-binding (249- S1) and actin-binding (403-S1 and 606-S1) regions. The S1s and associated VLC1 were purified from the cell lysate by an affinity resin (nickel-agarose) that binds to the His-tag at the C-terminal of the S1. We have previously established that a- and b-S1 expressed and purified from COS cells retain physiological ATPase properties (Roopnarine and Leinwand, 1996, Biophys. J, 70:A172). Our present results show that the actin-activated ATPases of the purified S1s are: a-S1 = 606-S1 b-S1 403-S1 249-S1, indicating that two of the mutations affect the activation of myosin ATPase by actin.