Porcine Aorta Smooth‐muscle Myosin Contains Three Species Made of Different Combinations of two 17‐kDa Essential Light‐chain Isoforms

Porcine aorta myosin was reacted with a bifunctional cross-linking reagent, N,N'-o-phenylenedimaleimide. The 17-kDa essential light chain (LC17) in each myosin head was intramolecularly cross-linked within a single myosin molecule. The 34-kDa cross-linked LC17 dimer was isolated and its peptide map, after lysylendopeptidase digestion, was obtained by reverse-phase HPLC. Based on the amino acid compositions of peptide fragments, the N-terminal Cys residues of LC17 subunits were assigned to be cross-linked to each other. To study the distribution of two LC17 isoforms, LC17nm and LC17gi [Hasegawa, Y., Ueda, Y., Watanabe, M. & Morita, F. (1992) J. Biochem. 111, 798-803], aorta myosin was reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2). The LC17 dimer cross-linked with Nbs2 was resolved into three distinct bands on urea/PAGE using a 4% acrylamide gel. Densitometric analysis of the three band intensities showed that three pairs of LC17 isoforms in aorta myosin are present in the ratio of LC17nm-LC17nm/LC17nm-LC17gi/C17gi-LC17g i = 22:46:32. This ratio is consistent with the random combination of two LC17 isoforms with myosin heavy chains.