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 GTPbinding protein) that was [32P]ADP-ribosylated by choleratoxin/[32P]NAD: cross-linking with disuccinimidyl suberate gave products with mol. wts. of 160 000, -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, photoaffinity 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.

The guanosine 5'-[3,y-imido]triphosphate (p[NH]ppG)-activated adenylate cyclase from rabbit myocar-

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An ~ZsI]iodoazidosalicylic acid derivative of forskolin was synthesized for identification of the ditetpene's binding sites on the catalytic subunit of adenylate cyclase and on glucose transport proteins. The affinity label was selectively incorporated into proteins of M, 4000&60000 in membranes from human erythrocytes and from various other tissues. The iodoazidosalicylic acid derivative also specifically labeled the catalytic moiety of adenylate cyclase from rabbit myocardial membranes. However, the structural requirements of the two forskolin-binding sites must be different, since the affinity of the photolabel for the glucose carriers is much higher than that for the cyclase catalyst. Furthermore, the label is readily competed with by D-glucose and cytochalasin B for its binding site on the glucose carrier but not on adenylate cyclase.

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