A novel approach to the ultrastructural localization of cell surface receptors: affinity-gold labeling of Na, K-ATPase.
We describe here a novel method of affinity-gold labeling for the ultrastructural localization and biochemical characterization of functional cell surface receptors. This approach combines the widely used colloidal gold technique, a previously published method for coating the gold with a matrix of derivatized dextran, small receptor-specific ligands, and a photoactivatable cross-linker. The resulting gold-affinity probe directed to a selected receptor by the ligand, is subsequently attached to the receptor by light activation of the cross-linker. As a specific example, a gold affinity probe prepared with ouabain, a selective inhibitor of Na,K-ATPase, as the directing ligand was used to investigate the ultrastructural localization of this enzyme complex in cell membranes. The biological activity of ouabain covalently linked to derivatized dextran containing the photoactivatable cross-linker was examined by its action on ion transport across dog trachea epithelium and on the enzyme activity of Na,K-ATPase preparations obtained from the rectal gland of the elasmobranch, Mustelus californicus. By these tests the probe mimics the effects of free ouabain. Electron micrographs of labeled human erythrocytes and cultured human foreskin fibroblasts showed an apparent random distribution of Na,K-ATPase on the plasma membranes of these cells. Binding of the probe was blocked in the presence of excess ouabain, a result demonstrating that the affinity probe binds at the same sites as free ouabain. Covalent attachment of probe by light activation of the nitroarylazido groups greatly enhanced retention during washing and standard procedures of fixation and dehydration. The high density of the gold probe was utilized to isolate the covalently attached membrane components from labeled human foreskin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)
Mrsny
R J
RJ
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Birrell
G B
GB
Volwerk
J J
JJ
Widdicombe
J H
JH
Griffith
O H
OH
D000345:Affinity Labels; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D002478:Cells, Cultured; D004285:Dogs; D004910:Erythrocyte Membrane / Q000201:enzymology; D005399:Fishes; D006046:Gold; D006801:Humans; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D010042:Ouabain / Q000494:pharmacology; D012491:Salt Gland / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis*
D000345:Affinity Labels; D010042:Ouabain; D006046:Gold; D000254:Sodium-Potassium-Exchanging ATPase