Study of the interaction between rabbit cardiac contractile and regulatory proteins. An in vitro motility assay

A series of experiments was performed in an in vitro motility assay with reconstructed thin filaments to obtain pCa-force relationships for cardiac isomyosins V1 and V3. Two concentrations of each isomyosin (200 and 300 microg/ml) on the surface of a flow cell were tested. Isometric force was estimated as the amount of actin-binding protein, alpha-actinin, stopping thin filament movement. It was found that the amount of alpha-actinin stopping the movement at saturating calcium concentration for V3 was twice higher than for V1 at both concentrations of isoforms. Hill coefficients of cooperativity (h) were determined for pCa-force relationships. The value of h did not differ significantly for isoforms at 300 microg/ml of protein (h was 1.56 for V1 and 1.54 for V3). However, the Hill coefficient was higher for V3 isoform at 200 microg/ml (h = 2.00 and 1.76 for V3 and V1, respectively). Importantly, the Hill coefficient increased for both isoenzymes when their concentrations were decreased. The connection between Hill coefficient and cooperative interactions between cardiac contractile and regulatory proteins is analyzed in detail.