SYNTHESIS AND USE OF A BIOTINYLATED 3-AZIDOPHENOTHIAZINE TO PHOTOLABEL BOTH AMINO-TERMINAL AND CARBOXYL-TERMINAL SITES IN CALMODULIN

The biotinylated probe, -(4-(4-biotinyl-1-piperazinyl)butyl)phenothiaz ine, was used to examine the phenothiazine binding domains in calmodul in (CaM) by photolabeling. This phenothiazine, synthesized from 3-azid o-10-(4-(1-piperazinyl)butyl)phenothiazine and d-biotinyl tosylate, in hibited the CaM-mediated activation of phosphodiesterase (PDE) with an I-50 of 12.5 (+/-2.8) mu M. Photolabeling of CaM produced covalent ad duds in excellent yield (32%) in a light- and Ca2+-dependent manner. S tudies performed over a range of drug concentrations suggested a 2:1 s toichiometry for the binding of the phenothiazine probe to CaM. Limite d trypsin digestion and purification of the resulting fragments by eit her SDS-PAGE or HPLC provided two principal phenothiazine-containing p eptides. Amino acid composition and sequence analyses performed on the se two peptides established that both the N- and C-terminal domains in CaM, particularly the regions amino terminal to Ca2+-binding loops 1 and 3, were modified by the photoactivated phenothiazine derivative. T hese data, particularly for the C-terminal domain, are in excellent ag reement with the X-ray structure analysis of a 1:1 CaM-trifluoperazine complex.