Affinity chromatography of myosin, heavy meromyosin, and heavy meromyosin subfragment one on F-actin columns stabilized by phalloidin

Abstract

A method of affinity chromatography based on the trapping of actin filaments within agarose gel beads is described. This method can be used for the purification of myosin and its active proteolytic subfragments, as well as for studies on the interaction between actin and these proteins. Actin columns stabilized by phalloidin bind myosin, heavy meromyosin (HMM), and heavy meromyosin subfragment 1 (HMM-S1) specifically and reversibly. The effect of pyrophosphate and KCl on the dissociation of actomyosin, acto-HMM, or acto-HMM-S1 complex is reported. We also describe the single-step purification of myosin from a crude rabbit psoas muscle extract.

Cellular Adaptations in the Diaphragm in Chronic Obstructive Pulmonary Disease

“…To purify myosin, part of the myofibrillar suspension was dialyzed for five to six hours against 200 volumes of 20 mM TRIS-hydrochloric acid (pH 7.5), 0.5 M potassium chloride, 1 mM magnesium chloride, and 0.5 mM b -mercaptoethanol, and then subjected to a "bath-flow" procedure with a resin of phalloidin-stabilized, actin-coated Sepharose B prepared according to the method of Grandmont-Leblanc and Gruda. 15 This resin was first equilibrated against the dialysis buffer and then incubated for 30 minutes at 25°C with the myofibrillar solution in a ratio of 1 mg of myofibril per gram of resin. The resin was washed twice with 10 volumes of dialysis buffer, and the myosin was then diluted by 2 M potassium chloride.…”

Human plasma actin‐depolymerizing factor

“…They were purified using an F-actin affinity column stabilized by phalloidin or virosin (kindly donated by Dr T. Staron, INRA, Luck, France). The preparation of the column was as described by Grandmont-Leblanc and Gruda for the isolation of myosin by affinity chromatography [26]. The column was washed with potassium phosphate buffer followed by this buffer containing 1 M KCl, checking spectrophotometrically at 280 nm that no actin was released from the column.…”

“…The column was washed with potassium phosphate buffer until the Azso of the filtrate dropped practically to zero and anti-actin antibodies were then eluted with 1 M KCI in potassium phosphate buffer. Between uses, the column was kept at 4°C in the binding buffer [26] (0.5 M KCl, 0.02 M Tris/HCl pH 7.6, 1 mM MgC12, 0.5 mM 2-mercaptoethanol) containing 0.1 % NaN3.…”