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'-nucleotidase 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. & 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.
Dieckhoff
J
J
Knebel
H
H
Heidemann
M
M
Mannherz
H G
HG
D015720:5'-Nucleotidase; D000199:Actins / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000918:Antibody Specificity; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005895:Gizzard, Avian / Q000201:enzymology; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology; D009708:Nucleotidases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D012910:Snake Venoms / Q000032:analysis; D013045:Species Specificity
D000199:Actins; D000596:Amino Acids; D012910:Snake Venoms; D009708:Nucleotidases; C018775:adenosine monophosphatase; D015720:5'-Nucleotidase