It has recently been discovered that the extracellular adenylate cyclase activity released into the culture media from Bordetella pertussis is stimulated by calmodulin (CaM). The objective of this proposal is to define the mechanism for CaM regulation of this enzyme with particular emphasis on quantitative characterization of interactions between CaM and B. pertussis adenylate cyclase. Preliminary evidence from our laboratory indicates that this bacterial enzyme is stimulated by CaM even in the absence of free CA++, although its apparent affinity for CaM is enhanced by calcium. This observation is in direct contrast with other CaM regulated enzymes where it has been established that CaM and CA++ are both required for CaM stimulation. We propose to purify the CaM sensitive adenylate cyclase from B. pertussis and directly quantitate binding of CaM to the enzyme in the presence and absence of CA++ using fluorescent CaM (AEDANS.CaM). In addition, the enzyme will be labeled with (32p)2', 3' dial ATP in the presence of sodium cyanoborohydride to identify its catalytic subunit. The adenylate cyclase CaM binding subunit will be identified using (125I)azido-CaM as a photoaffinity label. We also propose to examine the effect of various lpipds on the activity of B. pertussis adenylate cyclase since hydrophobic interactions arfe implicated for stabilization of CaM-enzyme complexes and other CaM regulated enzymes are lipid activated. This study will provide valuable data concerning the subunit composition of B. pertussis adenylate cyclase, the relationship between the enzyme's catalytic subunit and its CaM binding subunit and the quantitative influence of CA++ and other parameters on this interaction. This data should contribute to a better understanding of the molecular basis of B. pertussis pathology.
NIH ExPorter
NIH ExPorter