THE N-TERMINUS OF A1-TYPE MYOSIN ESSENTIAL LIGHT-CHAINS BINDS ACTIN AND MODULATES MYOSIN MOTOR FUNCTION

There are two isoforms (A1 and A2) of the myosin essential light chain (ELC) and consequently two isoenzymes of myosin subfragment 1(S1), S1 (A1) and S1(A2). The two isoenzymes differ in their kinetic properties with S1(A1) having a lower apparent K-m for actin and a slower turnov er of MgATP (k(cat)) than S1(A2). The two forms of the ELC differ only at their N-termini where A1 has an additional 40-odd amino acids that are not present in A2. The human atrial ELC (an A1-type ELC) was over expressed in Escherichia coli and purified by ammonium sulphate fracti onation and ion-exchange chromatography, The recombinant ELC had actin activated MgATPase kinetics similar to those for rabbit skeletal S1(A 1) under the same conditions. Deletion of the first 45 amino acid resi dues resulted in an ELC similar to the rabbit skeletal A2 isoform anti , when hybridised into S1, in S1(A2)-like kinetic properties. Results obtained with an ELC mutant that lacks the first 11 residues were inte rmediate between these two extremes but tending towards the S1(A2)-lik e phenotype. The wild-type ELC (both hybridised into S1 or free in sol ution) could be cross-linked to F-actin, whereas the deletion mutant l acking the first 45 amino acids could not. The deletion mutant lacking the first 11 amino acids cross-linked only poorly under the same cond itions, consistent with the MgATPase data. We therefore conclude that these N-terminal eleven amino acids predominantly encode an actin-bind ing site which modulates the kinetics of the myosin motor. Furthermore , while free A1-type ELC cross-linked to both polymeric F-actin and th e monomeric G-actin:DNase-I complex, the same ELC in S1(A1) could only cross-link to F-actin. This suggests that the light chain binds to a different actin monomer than the heavy chain.