Human erythrocyte myosin: Identification and purification

Abstract

Human erythrocytes contain an M(r) 200,000 polypeptide that cross-reacts specifically with affinity-purified antibodies to the M(r) 200,000 heavy chain of human platelet myosin. Immunofluorescence staining of formaldehyde-fixed erythrocytes demonstrated that the immunoreactive myosin polypeptide is present in all cells and is localized in a punctate pattern throughout the cell. Between 20-40% of the immunoreactive myosin polypeptide remained associated with the membranes after hemolysis and preparation of ghosts, suggesting that it may be bound to the membrane cytoskeleton as well as being present in the cytosol. The immunoreactive myosin polypeptide was purified from the hemolysate to ~85% purity by DEAE-cellulose chromatography followed by gel filtration on Sephacryl S-400. The purified protein is an authentic vertebrate myosin with two globular heads at the end of a rod-like tail ~150-nm long, as visualized by rotary shadowing of individual molecules, and with two light chains (M(r) 25,000 and 19,500) in association with the M(r) 200,000 heavy chain. Peptide maps of the M(r) 200,000 heavy chains of erythrocyte and platelet myosin were seen to be nearly identical, but the proteins are distinct since the platelet myosin light chains migrate differently on SDS gels (M(r) 20,000 and 17,000). The erythrocyte myosin formed bipolar filaments 0.3-0.4-μm long at physiological salt concentrations and exhibited a characteristic pattern of myosin ATPase activities with EDTA, Ca++, and Mg++-ATPase activities in 0.5 M KCl of 0.38, 0.48, and <0.01 μmol/min per mg. The Mg++-ATPase activity of erythrocyte myosin in 0.06 M KCl (<0.01 μmol/min per mg) was not stimulated by the addition of rabbit muscle F-actin. The erythrocyte myosin was present in about 6,000 copies per cell, in a ratio of 80 actin monomers for every myosin molecule, which is an amount comparable to actin/myosin ratios in other nonmuscle cells. The erythrocyte myosin could function together with tropomyosin on the erythrocyte membrane (Fowler, V.M., and V. Bennett, 1984, J. Biol. Chem., 259:5978-5989) in an actomyosin contractile apparatus responsible for ATP-dependent changes in erythrocyte shape.