Quantitative immunochemical studies of myosin in Dictyostelium discoideum

We have isolated monoclonal antibodies against myosin from the eukaryotic microorganism Dictyostelium discoideum. Immunoblot analysis using chymotryptic fragments of myosin has shown that the 17 antibodies are directed against at least five different sites on the myosin heavy chain. Using an antibody (M342) that reacts with an epitope on the tail portion of the myosin molecule, we have developed an assay to quantitate myosin in whole cells and subcellular fractions. Samples are deposited on nitrocellulose paper using a filtration manifold and are probed with metabolically labeled M342 antibodies. The assay is rapid and sensitive; it permits the measurement of myosin even in crude cell lysates that contain detergent. By use of the filtration immunoassay, we have found that myosin constitutes 0.72% of the total protein in vegetative amoebae. We have also determined that Triton-resistant cell residues (cytoskeletons or cortical actin matrices) prepared in the absence of ATP contain nearly half of the cell's myosin. If ATP is present, 98% of that myosin is released, although actin levels do not diminish.