Purification of the surface cAMP receptor in Dictyostelium.
We have previously identified and demonstrated reversible ligand-induced modification of the major cell surface cAMP receptor in Dictyostelium discoideum. The receptor, or a subunit of it, has been purified to homogeneity by hydroxylapatite chromatography followed by two-dimensional preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification was monitored by following 32Pi incorporated by photoaffinity labeling with 8-azido-[32P]cAMP or by in vivo labeling with 32Pi. Two interconvertible forms of the receptor, designated R (Mr 40,000) and D (Mr 43,000), co-purified. Two-dimensional peptide maps of independently purified and 125I-iodinated R and D forms of the receptor were nearly identical but did have several distinct peptides. The estimated 6000-fold purification required is consistent with the number of cell surface binding sites assuming there are not multiple binding sites/polypeptide. In the accompanying article we report the generation of a monospecific polyclonal antiserum which has helped to further elucidate the physical properties and developmental regulation of the cAMP receptor.
D000345:Affinity Labels; D001386:Azides; D002462:Cell Membrane / Q000032:analysis; D002845:Chromatography; D002918:Chymotrypsin; D000242:Cyclic AMP / Q000031:analogs & derivatives; D004023:Dictyostelium / Q000032:analysis*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D010446:Peptide Fragments / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010777:Photochemistry; D011953:Receptors, Cyclic AMP / Q000302:isolation & purification*
D000345:Affinity Labels; D001386:Azides; D006882:Hydroxyapatites; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D011953:Receptors, Cyclic AMP; C017042:8-azidoadenosine-3',5'-monophosphate; D017886:Durapatite; D000242:Cyclic AMP; D002918:Chymotrypsin