Adenylate cyclase from bovine brain cortex: purification and characterization of the catalytic unit.
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.
D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology*; D003432:Cross-Linking Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight
D001386:Azides; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; C029534:hydroxysuccinimidyl-4-azidobenzoate; D000262:Adenylyl Cyclases