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

Abstract

5′‐Nucleotidase, purified to homogeneity from chicken gizzard using published procedures [Dieckhoff, J., Knebel, H., Heidemann, M. and Mannherz, H. G. (1985) Eur. J. Biochem. 151, 377–383] was incorporated into artificial phospholipid vesicles after prolonged dialysis against detergent‐free buffer or by a gel filtration procedure. After dialysis the obtained liposomes exhibit a mean diameter of 80 nm and contain 5′‐nucleotidase at random orientation, demonstrated by finding up to 50% of the total liposome‐incorporated AMPase activity to be cryptic, i.e. could only be measured after their permeabilization by addition of detergent. By affinity chromatography a phospholipid vesicle fraction could be obtained containing almost exclusively cryptic AMPase activity, thus representing the inside‐out orientation of 5′‐nucleotidase. Comparative analysis of physiochemical and enzymatic properties of 5′‐nucleotidase reveals differences between the detergent‐solubilized and the liposome‐incorporated 5′‐nucleotidase including a changed accessibility of the enzyme to polyclonal and monoclonal antibodies. Binding and AMPase inhibition studies with different polyclonal antibodies strongly indicate to the existence of a cytoplasmic domain of chicken gizzard 5′‐nucleotidase. F‐actin appears preferentially to interact with the cytoplasmic domain of liposome‐incorporated 5′‐nucleotidase.

Purification and Characterization of Membrane-Bound 5'-Nucleotidase of Vibrio parahaemolyticus1

“…Thus, a single protein catalyzes the hydrolyses of nucleoside 5'-monophosphates, -diphosphates, and -triphosphates. 5'-Nucleotidases that hydrolyze only nucleoside 5'-monophosphates have been purified from mammalian cell membranes, and reported to have molecular mass of 79 kDa (chicken gizzard) (17), 73 kDa (human placenta) (18), and 62 kDa (Torpedo electric organ) (19).…”

Current State of the Structural Analysis of the Actin: DNase I Complex

“…5 ' -Nucleotidase purified from chicken gizzard exhibits a molecular mass of 79 kDa after SDS-PAGE and behaves like an integral membrane protein, i.e. it is only soluble in the presence of detergents and can be reconstituted into artificial phospholipid vesicles (Dieckhoff et al , 1987 Recently evidence has been presented indicating a specific interactin of purified chicken gizzard 5 ' -nucleotidase with laminin and fibronectin, both components of the extracellular matrix (ECM).…”

“…Reconstitution of 5 ' -nucleotidase into artificial unilamellar phospholipid vesicles produces a mixed liposome population containing the enzyme in either inside-out or outside-out orientation (Dieckhoff et al 1987). These liposomes can be fractionated into inside-out liposomes by affinity adsorption of the outside-out liposomes.…”

Inhibition of lymphocyte 5′-nucleotidase by lectins: effects of lectin specificityand cross-linking ability

“…This system thus provides a unique opportunity to study the modulation of the catalytic function of a membrane glycoprotein by binding of an external agent. However, despite successful purification and reconstitution of 5'-nucleotidase (Campbell et al 1983;Sharom et al 1985;Dieckhoff et al 1987), the mechanism of lectininduced enzyme inhibition is still unknown. Earlier studies have shown that dimeric derivatives of concanavalin A fail to inhibit the enzyme, either in the intact cell membrane or in reconstituted lipid bilayer systems (Slavik et al 1977;Dornand et al 1978aDornand et al , 1978bCampbell et al 1983).…”