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Measurement of F-actin activated ATPase activity

Identification/characterization of proteins or small molecules that affect heavy meromyosin ATPase activity

Identification/characterization of proteins or small molecules that affect heavy meromyosin F-actin interaction

Material：
       Heavy meromyosin has been produced by α-chymotrypsin proteolytic cleavage of full-length myosin II protein isolated from rabbit skeletal muscle. Heavy meromyosin consists of an active motor fragment consisting of two head domains connected by their subfragment-2 (S2) regions and two pairs of light chains, essential light chain (ELC) and regulatory light chain (RLC), see Figure 1. Heavy meromyosin has been determined to be biologically active in an F-actin activated ATPase assay. Heavy meromyosin is supplied as a white lyophilized powder. When reconstituted in nanopure water, the protein will be in the following buffer: 10 mM Imidizole pH 7.0, 50 mM KCl, 3 mM MgCl2, 1 mM DTT, 5% sucrose and 1% dextran. The lyophilized protein is stable at 4°C desiccated (<10% humidity) for 1 year. 

Figure 1. Diagrammatic representation of the myosin II protein and its subfragments. Myosin II or conventional myosin is a hexameric protein consisting of two heavy chains and two light chains. Myosin II can be proteolytically cleaved into heavy meromyosin (HMM) and light meromyosin(LMM) by α-chymotrypsin. Heavy meromyosin consists of the myosin head subfragment-1 domain (S1), its associated light chains (essential light chains and regulatory light chains), and the coiled-coil subfragment -2 domain. Light meromyosin consists of coiled-coil protein structure. The myosin S1-subfragment is produced by papain digestion of HMM.

Purity：
       Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% gradient polyacrylamide gel. Heavy meromyosin protein was determined to be 70% pure (see Figure 2). 

Figure 2. Heavy meromyosin purity determination. A 10 μg sample of MH01 was separated by electrophoresis in a 4-20% SDS-PAGE system, and stained with Coomassie Blue. Arrow indicates the truncated myosin head domain (approx. 150 kDa), arrowheads indicate the RLC (approx. 20 kDa) and ELC (approx. 17 kDa). Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat. # ADV02).

Biological Activity：
       The biological activity of rabbit heavy meromyosin is determined from its rate of F-actin activated ATP hydrolysis. A standard biological assay for monitoring ATP hydrolysis by heavy meromyosin consists of an in vitro F-actin ATPase assay (Cat. # BK054). Stringent quality control ensures that in the presence of F-actin, rabbit heavy meromyosin will have a minimum hydrolysis rate 500 fold greater than in the absence of F-actin.