Chromatography of Myosin on Diethylaminoethyl-Sephadex A-50^*

Abstract

A chromatographic system using DEAE-Sephadex A-50 with 0.04 m pyrophosphate buffer, pH 7.5, and a linear KC1 gradient to 0.50 m KC1 separates monomeric myosin from aggregated myosin, other unidentified proteins, and ribonucleic acid (RNA).The procedure has been applied to myosin preparations from skeletal muscle of rabbit, chicken, and K-zeveral investigators have employed column chromatography for the purification of myosin. Brahms (1959) and Perry (1960) used DEAE-cellulose with 0.2 m KC1 buffered with Tris to pH 7.4, and obtained some degree of fractionation, but the bulk of the material passed directly through the column without retention. Perry (1960) also employed a buffer system (0.16 m KC1-0.02 m Tris, pH 8.2) with a KC1 gradient that resulted in a separation of myosin from ribonucleoprotein and other proteins that passed through unretarded. However, the adenosine triphosphatase (ATPase)* 1 **activity varied considerably across the myosin peak, and dimers not present in the starting material were seen in the myosin purified by this procedure. Based on the work of Brahms and Brezner (1961), who showed that myosin was soluble in polyphosphates at low ionic strength, Asai (1963) used DEAE-cellulose columns, ATP in the solvent, and a KC1 gradient to obtain chromatograms that were similar to those obtained with the pH 8.2 system of

Isolation and some physicochemical properties of requiem shark myosin.

“…Otherwise, the muscle was coarsely minced, mixed with an equal volume of glycerol, myosin, the yield being 1.4 times as much as that with 0.04M pyrophosphate and matching those of other myosins so far reported.19-24) However, the leading edge of this large myosin peak might be contaminated with aggregated myosin as sug gested by RICHARDS et al 19) On the other hand, the replacement by 0.02M pyrophosphate (pH 7.5) resulted in less satis factory and less reproducible separation of shark myosin, as shown in Fig. 3.…”

Light Chains of Myosins from White, Red, and Cardiac Muscles

“…Myosin was further purified by column chromatography on DEAE-Sephadex A-50, essentially by the methods of Richards et al (30). 150-200 mg of myosin, dissolved in a solution containing 0.04 M Na4P207 (pH 7.5), 0.1 mM EDTA, and 0.1 mM dithiothreitol (buffer I), were applied to a column (2.5 X 35 cm) of DEAE-Sephadex A-50 equilibrated with the same buffer.…”

A Novel Variant of Cardiac Myosin-binding Protein-C That Is Unable to Assemble into Sarcomeres Is Expressed in the Aged Mouse Atrium

“…Crude myosin was prepared from rabbit ventricles as described previously (Perry, 1955), and the myosin was purified using DEAESephadex A-50 (Richards et al, 1967). Reconstitution of myosin filaments was performed during a dialysis against the binding buffer.…”