ABSTRACT
Bovine brain calmodulin activated adenylate cyclase in calmodulin-deficient rat anterior pituitary membranes. This activation appeared to be specific by the following criteria: 1) calmodulin activation was Ca2+ dependent and responded biphasically to calcium, displaying activation at low and inhibition at higher concentrations; 2) calmidazolium, a potent calmodulin antagonist, inhibited calmodulin activation of adenylate cyclase; 3) activation of the enzyme occurred in a dose-dependent manner, at calmodulin concentrations normally found in most cells (1- to 20-microM range). However, this response was not saturated using calmodulin concentrations as high as 50 microM. The data suggest that endogenous calmodulin can be dissociated from normal anterior pituitary adenylate cyclase, that the enzyme can be subsequently stimulated by addition of micromolar concentrations of calmodulin, and that this enzyme appears to be at least 50-fold less sensitive to calmodulin than is the brain adenylate cyclase.
Subject(s)
Adenylyl Cyclases/analysis , Calmodulin/pharmacology , Pituitary Gland, Anterior/enzymology , Animals , Calcium/pharmacology , Colforsin/pharmacology , Enzyme Activation/drug effects , Imidazoles/pharmacology , Male , Rats , Rats, Inbred StrainsABSTRACT
Bordetella pertussis produces an extracellular adenylate cyclase activity that is present in the culture medium of exponentially growing cells. We have determined that calmodulin (CaM) stimulates this enzyme both in the presence and in the absence of free Ca2+. In the presence of 90 micron Ca2+ half-maximal stimulation of the enzyme occurred at 95 pM calmodulin. Comparable levels of calmodulin stimulation were observed when free Ca2+ levels were minimized by using EGTA-containing buffers. However, the concentration of CaM required for half-maximal stimulation of B. pertussis adenylate cyclase in the presence of 1 nM free Ca2+ was 24 nM. The apparent affinity of the enzyme for calmodulin was also significantly enhanced by Mn2+. In addition, troponin I inhibited calmodulin stimulation of the bacterial adenylate cyclase. Photoaffinity cross-linking experiments using azido[125I]calmodulin and B. pertussis adenylate cyclase revealed only one major cross-linked product having a molecular weight of 97000. It is proposed that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is 77000.