NOVEL CHARACTERISTICS OF A MYOSIN ISOLATED FROM MAMMALIAN RETINAL-PIGMENT EPITHELIAL AND ENDOTHELIAL-CELLS

We have isolated a novel, high M(r) protein from human retinal pigment epithelial cells and endothelial cells by affinity chromatography on Sepharose 4B. Two polypeptides are present on SDS-gels of the 8 an ure a eluent with apparent molecular mass of similar to 210 and 47 kDa. In the absence of dithiothreitol, the two polypeptides migrate as one pr otein band with an apparent molecular mass of similar to 550 kDa. ''Pi glet,'' as this molecule is tentatively named, is present in retinal p igment epithelial and endothelial cells of several species, but could not be detected in the nonepithelial cells we examined. Immunofluoresc ent localization using an antibody to the 210-kDa polypeptide revealed a filamentous network in the cytoplasm of cultured cells. This antibo dy was used to identify a cDNA for piglet in a bovine aortic endotheli al cell expression library. Sequence data indicate a high degree of id entity with non-muscle myosin II heavy chain. We subsequently found th at piglet had an actin-activated ATPase activity, colocalized with act in in cells, and reacted on Western blots with a pan-non-muscle myosin II heavy chain antiserum. The protein was also recognized by antibodi es specific for myosin heavy chain isoform A, but did not react with a nti-isoform B antibodies. Although piglet has several features in comm on with known forms of non-muscle myosin II, the distinctly unconventi onal features it displays suggest that it is a novel myosin.