Crosslinking of the skeletal myosin subfragment-1 heavy chain to the N-terminal actin segment of residues 40-113

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Abstract
Glutaraldehyde (GA) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), a hydrophobic, carboxyl group directed, zero-protein cross-linker, were employed for the chemical cross-linking of the rigor complex between F-actin and the skeletal myosin S-1. The enzymatic properties and structure of the new covalent complexes obtained with both reagents were determined and compared to those known for the EDC-acto-S-1 complex. The GA- or EEDQ-catalyzed covalent attachment of F-actin to the S-1 heavy chain induced an elevated Mg2+-ATPase activity. The turnover rates of the isolated cross-linked complexes were similar to those for EDC-acto-S-1 (30 s-1). The solution stability of the new complexes is also comparable to that exhibited by EDC-acto-S-1. The proteolytic digestion of the isolated AEDANS-labeled covalent complexes and direct cross-linking experiments between actin and various performed proteolytic S-1 derivatives indicated that, as observed with EDC, the COOH-terminal 20K and the central 50K heavy chain fragments are involved in the cross-linking reactions of GA and EEDQ. KI-depolymerized acto-S-1 complexes cross-linked by EDC, GA, or EEDQ were digested by thrombin which cuts only actin, releasing S-1 heavy chain-actin peptide cross-linked complexes migrating on acrylamide gels with Mr 100K (EDC), 110K and 105K (GA), and 102 (EEDQ); these were fluorescent only when fluorescent S-1 was used. They are identified by immunostaining with specific antibodies directed against selected parts of the NH2-terminal actin segment of residues 1-113. Only the peptide of residues 1-28 was cross-linked by EDC. In contrast, the actin peptides including residues 1-28 and 40-113 were cross-linked after GA or EEDQ treatment of acto-S-1. These studies extend further our knowledge on the interaction sites between actin and the myosin head heavy chain.