Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking [125I]PF-M (see Figure 1 for structure) to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by [125I]PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that [a] no other AC-regulatory proteins are known to be of this size, [b] the catalytic unit of bovine brain enzyme is in the same range and [c] this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.

Adenylyl Cyclases↗

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Production and assay of antibodies to an activator of adenylate cyclase, forskolin.

Forskolin is a unique diterpene activator of adenylate cyclase which has been extensively used in the study of cAMP generating systems. This report describes the production of antibodies to forskolin and the optimization of two sensitive assay methods for such antibodies. 7-0-Hemisuccinyl 7-deacetyl forskolin, coupled to either human serum albumin or goat IgG, was injected into goats to elicit antibodies to the forskolin hapten. Two assay methods, a radioimmunoassay with [12-3H]forskolin as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) with horse radish peroxidase-labelled rabbit anti-goat IgG as an indicator, were optimized to test for the presence of forskolin antibodies in antisera and isolated IgG fractions. The titers for forskolin antisera were 4000-10000. Both assay methods can be adapted to quantify forskolin and its protein conjugates. The availability of antibodies to this diterpene will be useful in accelerating the understanding of the mechanism of adenylate cyclase activation by forskolin.

Adenylyl Cyclases↗