Adenylate cyclase is the mediator of the action of numerous hormones and neurotransmitters and it is central to the control of metabolism and function in probably all tissues. Its activity is modulated by numerous agents of physiological and biochemical interest, including calcium and some bacterial toxins, and consequently a better understanding of the control of cellular cAMP metabolism is dependent on an understanding of the regulation of adenylate cyclase. As with numerous other enzymes important to cell regulation, calcium activation of a brain adenylate cyclase is mediated by calmodulin, though the basis of the sensitivity of the brain cyclase to calmodulin is not known. There is only an emerging picture of the number of components of the hormone receptor-adenylate cyclase system, without any clear understanding of the mechanisms of their interaction which regulate cyclase activity. The proposed research focuses on the mechanisms of calmodulin-induced activation of adenylate cyclase and will ascertain whether it: a) involves the participation of the stimulatory and inhibitory guanine nucleotide regulatory components; b) involves other unidentified components, unique to the calmodulin-sensitive enzyme; c) resides in unique properties of the catalytic unit of the calmodulin-sensitive enzyme; or d) involves a combination of these possibilities. The two principal approaches will be to purify the Ca2+/calmodulin-sensitive adenylate cyclase and to identify with labelled calmodulin and crosslinking agents calmodulin binding proteins associated with this enzyme. The potential role of Ns and/or Ni on calmodulin activation of the cyclase will be determined through indirect and direct assessments and through reconstitution studies. Given that divalent cation has an established role in the regulation of adenylate cyclase activity, character of their involvement in calmodulin-activation of the brain enzyme will be determined. Polyclonal and/or monoclonal antibodies will be prepared against the cyclase catalytic unit and/or the associated calmodulin-binding protein. These will be used: a) to develop immunopurification adsorbants; and b) to clarify questions of localization of this form of cyclase or of the associated calmodulin-binding protein. In the narrow sense, the studies will aid our understanding of how individual agents change adenylate cyclase activity, but in the broader sense will give insights into the integration of cell regulatory processes involving both cAMP and calcium.
Showing the most recent 10 out of 28 publications
