26] Preparation of smooth muscle myosin

Abstract

Publisher Summary This chapter presents procedure for preparation of smooth muscle myosin from porcine aortic smooth muscle. Myosins can be isolated from a variety of smooth muscles. Almost all of these tissues contain considerable quantities of extracellular collagen and elastin. As a consequence, they are difficult to disrupt. Rather harsh methods are usually necessary for tissue homogenization. Measures to minimize myosin proteolysis and denaturation are, therefore, especially important after the violent disruption of the smooth muscle. All operations are performed in the cold (4-5°); protease inhibitors are included in each buffer; frothing and vigorous stirring are avoided at each step in the procedure. The result is purification of a homogeneous, enzymically active myosin in reasonable yield. Additionally, purification of phosphorylated myosin and unphosphorylated myosin is presented. Polyacrylamide gel electrophoresis is used for phosphorylated aortic myosin. Crude myosin was phosphorylated with [γ 32 P]ATP and used to prepare aortic myosin. The myosin issubjected to electrophoresis in sodium dodecyl sulfate and to subsequent assay for 32 P in 2-mm slices of the stained gel.