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

EMBO J. 1985 Dec 30;4(13B):3675-9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclases / isolation & purification*
  • Adenylyl Cyclases / metabolism
  • Animals
  • Azides
  • Binding Sites
  • Cattle
  • Cerebral Cortex / enzymology*
  • Cross-Linking Reagents
  • Kinetics
  • Macromolecular Substances
  • Molecular Weight

Substances

  • Azides
  • Cross-Linking Reagents
  • Macromolecular Substances
  • hydroxysuccinimidyl-4-azidobenzoate
  • Adenylyl Cyclases