Evidence for the direct interaction of chicken gizzard 5'-nucleotidase with laminin and fibronectin.
The ectoenzyme 5'-nucleotidase purified from chicken gizzard is shown to specifically interact with laminin and fibronectin, components of the extracellular matrix, by a number of different techniques: (i) cosedimentation with laminin by sucrose gradient centrifugation; (ii) affinity adsorption to both laminin- and fibronectin-Sepharose 4-B; (iii) specific binding to both laminin and fibronectin dotted onto cellulose filters; and (iv) monoclonal antibodies against 5'-nucleotidase are shown to interfere with the interaction of 5'-nucleotidase with laminin and fibronectin. For all the techniques employed, the interactions were found to be specific, since 5'-nucleotidase did not bind to unrelated proteins such as bovine serum albumin or to monomeric actin. The interaction of purified chicken gizzard 5'-nucleotidase could be demonstrated for the hydrophobic enzyme solubilized in detergent and after its reconstitution into artificial phospholipid vesicles. The affinity adsorption experiments indicate that reconstituted enzyme binds more strongly to both laminin and fibronectin. The 5'-nucleotidase employed in this study is anchored to the plasma membrane by a glycan-phosphatidylinositol linker. After treatment with phosphatidylinositol-specific phospholipase C, the enzyme is transformed into a hydrophilic form, for which interactions with laminin and fibronectin could also be demonstrated by the dot-blot technique. Thus controlled cleavage of the phosphatidylinositol linker of 5'-nucleotidase could enable cells to rapidly alter their adhesiveness to certain components of the extracellular matrix.
Stochaj
U
U
Institut für Anatomie und Zellbiologie, Abteilung Zellbiologie, Philipps-Universität, Marburg, F.R.G.
Dieckhoff
J
J
Mollenhauer
J
J
Cramer
M
M
Mannherz
H G
HG
D015720:5'-Nucleotidase; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002499:Centrifugation, Density Gradient; D002645:Chickens; D002846:Chromatography, Affinity; D005353:Fibronectins / Q000302:isolation & purification / Q000378:metabolism*; D005895:Gizzard, Avian / Q000201:enzymology; D007797:Laminin / Q000302:isolation & purification / Q000378:metabolism*; D009130:Muscle, Smooth / Q000201:enzymology*; D009708:Nucleotidases / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D005353:Fibronectins; D007797:Laminin; D009708:Nucleotidases; D015720:5'-Nucleotidase