Adenylate cyclase from bovine brain cortex: purification and characterization of the catalytic unit.

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Abstract
The non‐stimulated (basal) adenylate cyclase from bovine brain cortical membranes was purified 10 000‐fold to apparent homogeneity by Lubrol PX extraction and two cycles of affinity chromatography on forskolin‐agarose. The final product appears as one major band (mol. wt. 115 000) on SDS‐polyacrylamide gels. Further identification was achieved by affinity cross‐linking using Gs (stimulatory GTP‐binding protein) that was [32P]ADP‐ribosylated by cholera‐toxin/[32P]NAD: cross‐linking with disuccinimidyl suberate gave products with mol. wts. of 160 000, approximately 270 000 and higher. The distribution of these products was dependent on the concentration of cross‐linker, suggesting aggregation of two or more adenylate cyclase complexes. In contrast, photo‐affinity cross‐linking with 4‐azidobenzoyl‐[32P]Gs yielded a single product with a mol. wt. of 160 000. Purified adenylate cyclase was completely unresponsive towards stimulators (GTP‐analogs, NaF) acting via Gs suggesting that this component was removed during purification. On the other hand, stimulation by forskolin and by added activated Gs was preserved but to a smaller degree as compared with the crude enzyme. In contrast, the stimulation of Ca2+/calmodulin was only marginal. Purified adenylate cyclase reversibly bound to wheat germ agglutinin‐Sepharose. This suggests that bovine brain adenylate cyclase is a glycoprotein.