[2] Purification of myosin I and myosin I heavy chain kinase from Acanthamoeba castellanii

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The myosins I are structurally distinct from conventional myosins (myosins II) in that they are globular, single-headed proteins whose native molecular masses range from 140,000 to 160,000 Da. The activity of the myosin I heavy chain kinase is enhanced by its autophosphorylation which in turn is stimulated by phosphatidylserine. Myosin I and its heavy chain kinase are isolated from Acanthamoeba castellanii by conventional chromatographic methods. This chapter presents detailed procedures for isolating both. A cell extract is adsorbed to DE-52 and the kinase and myosin I are step eluted in a single pool. This material is applied to a phosphocellulose column, which is then eluted with a salt gradient in order to resolve the kinase and all three myosin I isozymes. The myosins I are further purified (individually) on ADP- or ATP-agarose and Mono Q columns, both eluted with salt gradients. Myosin I is assayed during its purification by its (K+,EDTA)-ATPase activity. A radioisotopic form of this assay utilizing [γ-32P]ATP is necessary, at least in the early stages, because of the presence of phosphate liberated from pyrophosphate. It is difficult to estimate recovery and the extent of purification, but this procedure should yield 500 μg or more of myosin I heavy chain kinase.

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