Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase.
5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.
D015720:5'-Nucleotidase; D000199:Actins / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002645:Chickens; D005455:Fluorescent Antibody Technique; D005895:Gizzard, Avian / Q000201:enzymology*; D007074:Immunoglobulin G; D007163:Immunosorbent Techniques; D009708:Nucleotidases / Q000276:immunology* / Q000378:metabolism; D013045:Species Specificity; D014018:Tissue Distribution
D000199:Actins; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D009708:Nucleotidases; D015720:5'-Nucleotidase