Catalytic unit of adenlyate cyclase: purification and identification by affinity crosslinking.

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Abstract
The guanosine 5''-[.beta.,.gamma.-imido]triphosphate (p[NH]ppG)-activated adenylate cyclase from rabbit myocardial membranes was purified .apprxeq. 60,000-fold to a specific activity of 15 .mu.mol .cntdot. min-1 .cntdot. min-1 by Lubrol PX extraction, affinity chromatography, and gel permeation HPLC [high performance liquid chromatography]. The major purification (> 2000-fold) was achieved by affinity chromatography on forskolin-Sepharose, a method previously developed in this laboratory. The final product appeared as 2 major peptides of MW 150,000 and 42,000 and 1 minor peptide of MW 45,000 when analyzed by NaDodSO4/polyacrylamide gel electrophoresis. The MW 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 (MW, 42,000), a single radiolabeled product of MW 190,000 appeared on NaDodSO4/polyacrylamide gels. Further identification is based on the correlation of the MW 150,000/42,000 bands with enzymatic activity when the purified enzyme was analyzed by various chromatographic procedures.

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