Purification of smooth-muscle myosin free of calmodulin and myosin light-chain kinase. Susceptibility to oxidation.
Smooth-muscle myosin purified as described by Persechini & Hartshorne [(1983) Biochemistry 22, 470-476] contains trace amounts of calmodulin and myosin light-chain kinase, which can be removed by Ca2+-dependent hydrophobic-interaction chromatography followed by calmodulin-Sepharose affinity chromatography. The resultant column-purified myosin exhibits properties similar to those of the non-purified myosin, e.g. actin activation of the Mg2+-ATPase requires Ca2+/calmodulin-dependent phosphorylation of the two 20 kDa light chains. However, unlike the non-purified myosin, the column-purified myosin undergoes a time-dependent transition to a form which no longer requires phosphorylation for actin activation of the myosin Mg2+-ATPase. This transition is identified as a time-dependent change in conformation of the column-purified myosin from a 10 S to 6 S form and is caused by slow oxidation of the column-purified myosin, since it could be prevented by storage under N2 and reversed by 5 mM-dithiothreitol.
D000199:Actins / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000302:isolation & purification; D002645:Chickens; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D009130:Muscle, Smooth / Q000032:analysis*; D009219:Myosin-Light-Chain Kinase / Q000302:isolation & purification; D009218:Myosins / Q000302:isolation & purification*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011487:Protein Conformation
D000199:Actins; D002147:Calmodulin; D009219:Myosin-Light-Chain Kinase; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium