Preparation and characterization of an enzymatically active immobilized derivative of myosin.
Purified skeletal muscle myosin (EC 3.6.1.3) has been covalently bound to Sepharose 4B by the cyanogen bromide procedure. The resulting complex, Sepharose-Myosin, possesses adenosine triphosphatase activity and is relatively stable for long periods of time. Under optimal binding conditions, approximately 33% of the specific ATPase activity of the bound myosin is retained. Polyacrylamide gel electrophoresis of polypeptides released from denatured Sepharose-Myosin indicates that 85% of the myosin is attached to the agarose beads through the heavy chains and the remainder through the light chains, in agreement with predictions of binding and release based upon either the lysine contents or molecular weights of themyosin subunits. The adenosine triphosphatase of the immobilized myosin has been investigated under conditions of varying pH, ionic strength, and cation concentration. The ATPase profiles of immobilized myosin are quite similar to those for free myosin, however subtle differences are found. The Sepharose-Myosin ATPase is not as sensitive as myosin to alterations in salt concentration and the apparent KM is approximately two-fold higher than that of myosin. These differences are probably due to chemical modification in the region of the attachment site(s) to the agarose beads and hydration and diffusion limitations imposed by the polymeric agarose matrix.
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002645:Chickens; D002846:Chromatography, Affinity; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D012685:Sepharose
D000199:Actins; D011189:Potassium Chloride; D012685:Sepharose; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium