Abstract
Publisher Summary Adenylate cyclase and guanylate cyclase catalyze the formation of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), respectively, from adenosine triphosphate (ATP) and guanosine triphosphate (GTP) in the presence of a metal. With the exception of testes, mammalian adenylate cyclase is exclusively membrane bound and guanylate cyclase is distributed in particulate and cytosolic compartments. This chapter focuses on two highly sensitive assay methods applicable to both enzyme systems. The radiometric assay using [ α - 32 P]ATP or [ α - 32 P]GTP is extremely rapid, whereas the second method employing nonlabeled substrate and radioimmunoassay (RIA) of the product is relatively slow. However, it offers the most sensitive, convenient, and least expensive method to measure cyclic nucleotides in adenylate and guanylate cyclase incubations. After incubation with the cyclase, the excess substrate [ α - 32 P]ATP or [ α - 32 P]GTP is separated from the product cyclic[ 32 P]AMP or cyclic[ 32 P]GMP. This procedure combines chromatography of nucleotides on Dowex-50 columns and neutral alumina (aluminum oxide). The amount of labeled cyclic nucleotide formed is then determined.
Published Version
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