Characterization of a second myosin from Acanthamoeba castellanii

T.D Pollard,W.F Stafford,M.E Porter

doi:10.1016/s0021-9258(17)30460-x

Abstract

We purified a 400,000 molecular weight myosin, myosin-II, from Acanthamoeba castellanii. The sequence of ion exchange chromatography, actomyosin precipitation, actin extraction, and gel permeation chromatography yields per 100 g of cells about 11 mg of myosin-II which is 90 to 96% pure. ATPase activity is highest in the presence of Ca2+, but the enzyme is also active in EDTA provided high concentrations of K+ are present. The molecule consists of two 175,000 molecular weight heavy chains, one or two 17,500 molecular weight light chains, and two 16,500 molecular weight light chains. Myosin-II is rich in acidic residues and contains about 32 residues of cysteine/mol. The sedimentation coefficient is 5.9 S. Intrinsic viscosity is 126 cc/g. By equilibrium ultracentrifugation, the molecular weight averages depended upon the initial loading concentration in a way that suggested a 400,000 molecular weight species is in equilibrium with a 200,000 molecular weight species. By electron microscopy the molecule was seen to have two globular heads at one end of a tail 90 nm long. In KCl solutions of less than 0.25 M, the myosin-II tails self-associate to form the backbone of very small (6.6 x 205 nm) bipolar filaments with central bare zones 97 nm long. Myosin-II binds to actin filaments, forming periodic arrowhead-shaped complexes, but its Mg2+ ATPase activity is activated only 50% or less by actin. When radioactive myosin-II is incubated up to 90 min in unlabeled Acanthamoeba homogenates, it is not degraded into smaller fragments, such as the 190,000 molecular weight myosin-I. Our observations and the detailed enzymatic data presented by Maruta and Korn ((1977) J. Biol. Chem. 252, 6501-6509) argue that the smaller Acanthamoeba myosin-I (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem, 248, 4682-2690) does not arise by fragmentation of myosin-II in the homogenate or extract.

Highlights

We purified a 400,000 molecular weight myosin, myosin-II, from Acanthamoeba castellanii
The first purification step is chromatography on DEAEcellulose, which partially separates the peak of Ca2+ATPase containing the myosin-II heavy chain from a peak of K’EDTA-ATPase activity (Fig. 2). (The K+-EDTA-ATPase is thought to be myosin-I, because on this column and on further purification by ammonium sulfate precipitation and gel filtration, it behaves exactly like the myosin-I purified by Pollard and Korn (1973, a and b).) The myosin-II fractions overlap the large peak of actin which follows
Myosin-II is separated from the remaining actin and most of the other contaminants by chromatography on 4% agarose using a discontinuous KI buffer system (Pollard et al, 1974) designed to keep the actin depolymerized and to minimize the exposure of the myosin to KI (Fig. 3)

Summary

Introduction

We purified a 400,000 molecular weight myosin, myosin-II, from Acanthamoeba castellanii. In KC1 solutions of less than 0.25 M, the myosin-II tails self-associate to form light chains, because by gel permeation chromatography it was considerably smaller than both muscle myosin and heavy meromyosin and slightly larger than a dimer of serum albumin. During these early studies, it was realized that there were two or three additional high molecular weight Ca’+ATPases in Acanthamoeba extracts but, at least one of these cosedimented with actin filaments in the absence of ATP, they were not considered to be active forms of myosin because their Mg*‘ATPase activities were not stimulated by actin

Methods

Results

Conclusion