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, 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.
D015720:5'-Nucleotidase; D000818:Animals; D002645:Chickens; D005895:Gizzard, Avian / Q000201:enzymology; D008051:Lipid Bilayers; D009130:Muscle, Smooth / Q000201:enzymology*; D009708:Nucleotidases / Q000302:isolation & purification / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology*
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D009708:Nucleotidases; C018775:adenosine monophosphatase; D015720:5'-Nucleotidase