Affinity chromatography of immobilized actin and myosin.
Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder.
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D009124:Muscle Proteins / Q000302:isolation & purification*; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010206:Papain; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D012685:Sepharose
D000199:Actins; D009124:Muscle Proteins; D011189:Potassium Chloride; D012685:Sepharose; D010206:Papain; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium