Modification of adenylate cyclase by photoaffinity analogs of forskolin

L. T. Ho, Z. M. Nie, T. J. Mende, S. Richardson, A. Chavan, E. Kolaczkowska, D. S. Watt, B. E. Haley, R. J. Ho

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

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 μM forskolin, the less active forskolin photoaffinity probes at 100 μM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may compete with F for the same binding site(s). After cross-linking [125I]PF-M 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 μM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (M(r) = 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 weakly 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.

Original languageEnglish
Pages (from-to)209-223
Number of pages15
JournalSecond Messengers and Phosphoproteins
Volume12
Issue number5-6
StatePublished - 1988

ASJC Scopus subject areas

  • Biochemistry

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