An improved procedure for purifying 5′‐nucleotidase from various sources

Abstract

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.

Purification and Properties of a 5'-Nucleotidase from Rat Renal Membranes

“…The purification factor obtained in the present study lies among the highest values reported for 5'-nucleotidase [11,[15][16][17][18]. To our knowledge our preparation shows the highest specific activity reported so far.…”

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…5′-NTs have been isolated from numerous sources, including multiple mammalian species, [254][255][256][257][258] birds, 259 arthropods, 260 reptiles, 261 plants, 262,263 and bacteria. [264][265][266] In animals, two types of 5′-NTs are present, but of these, only one form, labeled ecto-5′-NTs, has a binuclear center.…”

Reconstitution of purified chicken gizzard 5'-nucleotidase in phospholipid vesicles. Evidence for its transmembraneous character and the existence of functional domains on both sides of the phospholipid bilayer

“…5'-Nucleotidase from chicken gizzard smooth muscle was prepared from detergent extracts as described previously by affinity chromatography on AMP-Sepharose and Lens culinaris lectin-Sepharose [5]. Triton X-100, which was usually used to keep the enzyme in solution, was replaced in the second affinity-chromatographic step by Chaps or another detergent, which can be easily removed by dialysis.…”

“…Lens culinaris lectin was isolated from common lentils as described by [11] and actin from rabbit skeletal muscle as described in [5].…”

“…Isoforms of 5'-nucleotidase were also tested for their ability to associate or incorporate in phospholipid vesicles. Only a low percentage incorporation (about 1 %), however, of purified snake venom 5'-nucleotidase [5] could be obtained under otherwise identical, i.e. optimal, conditions (dialysis procedure).…”