Catalytic unit of adenlyate cyclase: purification and identification by affinity crosslinking.
The guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG)-activated adenylate cyclase from rabbit myocardial membranes was purified approximately equal to 60,000-fold to a specific activity of 15 mumol X mg-1 X min-1 by Lubrol PX extraction, affinity chromatography, and gel permeation HPLC. The major purification (greater than 2000-fold) was achieved by affinity chromatography on forskolin-Sepharose, a method previously developed in this laboratory. The final product appeared as two major peptides of Mr 150,000 and 42,000 and one minor peptide of Mr 45,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis. It is suggested that the Mr 42,000 and 150,000 components represent the alpha-subunit of the stimulatory guanine nucleotide binding regulatory protein (GS) and the catalytic unit, respectively, because upon crosslinking of a reconstituted adenylate cyclase containing the [32P]ADP-ribosylated alpha-subunit of GS (Mr, 42,000), a single radiolabeled product of Mr 190,000 appeared on NaDodSO4/polyacrylamide gels. Further identification is based on the correlation of the Mr 150,000/42,000 bands with enzymatic activity when the purified enzyme was analyzed by various chromatographic procedures.
D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D002846:Chromatography, Affinity; D005576:Colforsin; D003432:Cross-Linking Reagents; D004224:Diterpenes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004224:Diterpenes; D046911:Macromolecular Substances; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases