Limited proteolysis of chicken gizzard 5'-nucleotidase.
Chicken gizzard 5'-nucleotidase represents an ectoenzyme which is linked to the plasma membrane via a phosphatidylinositol glycan. We have characterized the possible domain-like organization of 5'-nucleotidase by limited proteolysis. A hydrophobic proteolytic fragment carrying the intact C-terminus, as well as two major hydrophilic products, were identified. We developed procedures for specific radiolabelling of the active center of 5'-nucleotidase. This allowed us to locate the catalytic site within hydrophilic fragments obtained after limited proteolysis. We demonstrate that removal of N-linked carbohydrate chains increases the sensitivity of 5'-nucleotidase to proteolytic attack, indicating that sugar moieties protect against proteolysis. 5'-Nucleotidase represents a binding protein for components of the extracellular matrix. The interaction between 5'-nucleotidase and the laminin/nidogen complex unmasked proteolytic cleavage sites in the C-terminal portion of the enzyme. This resulted in the specific production of a hydrophilic form of 5'-nucleotidase. In summary, we have further characterized chicken gizzard 5'-nucleotidase: (1) the protein is organized into two domain-like structures, (2) the N-terminal domain harbors the active center; (3) N-linked carbohydrates protect the protein against proteolytic degradation; (4) interaction with components of the extracellular matrix alters the conformation of 5'-nucleotidase.
Stochaj
U
U
Institut für Anatomie und Zellbiologie, Universität zu Marburg, Germany.
Cramer
M
M
Mannherz
H G
HG
D015720:5'-Nucleotidase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D005109:Extracellular Matrix / Q000378:metabolism; D005895:Gizzard, Avian / Q000201:enzymology*; D006031:Glycosylation; D007797:Laminin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008958:Models, Molecular; D010206:Papain; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D014357:Trypsin
D007797:Laminin; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C029967:nidogen; D015720:5'-Nucleotidase; D002918:Chymotrypsin; D014357:Trypsin; D003546:Cysteine Endopeptidases; D010206:Papain; C019913:clostripain