V Shirhatti, E Sokoloski, S Eng, S Hench, F Riccardi, G Krishna
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A simple method for the assay of Bordetella pertussis adenylate cyclase employing 31P nuclear magnetic resonance spectroscopy.
A simple method for the simultaneous assay of both substrate utilization and product formation by Bordetella pertussis adenylate cyclase has been developed. This method involves measurement of ATP remaining in the reaction mixture and cyclic 3',5'-AMP (cAMP) formation by 31p-NMR spectroscopy. No separation of the nucleotides is required. The measurement of the rate of cAMP formation compared very well with other methods that require separation of product from the substrate. With this method it has been possible to show calmodulin activation of B. pertussis adenylate cyclase and to demonstrate an inhibition of calmodulin activation by melittin. The inhibition of calmodulin-activated adenylate cyclase by melittin is not permanent and can be overcome by long-term incubation.
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