AN IMPROVED PROCEDURE FOR PURIFYING 5'-NUCLEOTIDASE FROM VARIOUS SOURCES - EVIDENCE FOR TISSUE AND SPECIES-DIFFERENCES IN THEIR MOLECULAR MASS AND AFFINITY FOR F-ACTIN

5''-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5''-nucletoidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E. M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. and Luzio, J. P. (1984) Biochem. J. 221, 369-377]. Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5''-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5''-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5''-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.