| Abstract: | Radiolabeled forskolin, 7-(3H-acetyl)-forskolin, was synthesized to explore interactions between forskolin and bovine brain membrane preparations. The radiolabeled derivative was chemically characterized, and found to be indistinquishable from unlabeled forskolin in its ability to stimulate bovine brain adenylate cyclase. Preliminary binding data demonstrated that binding of 7-(3H-acetyl)-forskolin to membranes was concentration dependent. However, competition binding studies using a constant concentration of 7-(3H-acetyl)-forskolin with increasing levels of unlabeled forskolin showed enhanced binding of the labeled derivative. This suggested that 7-(3H-acetyl)-forskolin was degraded by membranes and protected by native forskolin. Incubation of forskolin with membranes and analysis of the products by thin layer chromatography and mass spectroscopy showed the formation of 7-desacetylforskolin. The deacetylation of forskolin was monitored by quantitating the release of [3H]acetate from 7-(3H-acetyl)-forskolin. The reaction was linear with time and protein concentration. These data illustrate that forskolin can be degraded by membranes and indicate that ligand binding studies using labeled forskolin and membrane preparations should be cautiously interpreted. |
