Isolation of 5'-nucleotidase from chicken gizzard, its properties, and subcellular localization in chicken tissues using monospecific antibodies.
After solubilization with zwitterionic detergents 5'-nucleotidase was purified to homogeneity from chicken gizzard. Purified 5'-nucleotidase appeared to be composed of a single polypeptide chain of 79 kDa as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); gel filtration studies in the presence of detergents, however, indicated that the native enzyme is a homodimer. Antisera against purified chicken gizzard 5'-nucleotidase were raised in rabbits, they were shown to inhibit the enzymic activity of different 5'-nucleotidase preparations in a species and tissue specific manner. On frozen sections the antisera stained the cell periphery of chicken gizzard smooth and skeletal muscle and faintly stained cardiac muscle cells when using the indirect immunofluorescent technique. In chicken cardiac tissue, a prominent staining of the vascular system also became apparent. In avian and rat non-muscle tissues (hepatic and pancreatic tissue) the vascular system was always found to be brightly stained, i.e. the vascular smooth muscle and endothelial cells. On frozen sections of chicken liver the sinusoidal region of the hepatocytes was brightly stained, the bile canalicular region, however, only faintly. Using the immunocytochemical technique, a more prominent tissue specificity rather than species specificity of the available antisera became apparent. This may therefore reflect the existence of tissue-specific isoforms of the enzyme.
Heidemann
M
M
Dieckhoff
J
J
Hlavsa
C
C
Mannherz
H G
HG
D015720:5'-Nucleotidase; D000818:Animals; D000906:Antibodies; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005895:Gizzard, Avian / Q000166:cytology / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology*; D009708:Nucleotidases / Q000302:isolation & purification* / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
D000906:Antibodies; D009708:Nucleotidases; D015720:5'-Nucleotidase