山梨医科大学雑誌　第10巻1号　011-019(1995)

Enzymatic Fluorometric Assay for Adenylyl Cyclase Activity
Comparison with Radioimmuno

Atsushi SUGIYAMA and Keith G. LURIE

Abstract: An enzymatic fluorometric assay was developed to assess the adenylyl cyclase activity in membrane preparations. The assay consists of two parts: 1) pharmacological stimulation or inhibition of adenylyl cyclase, and 2) measurement of newly synthesized cyclic AMP. The critical step of cyclic AMP measurement is initial enzymatic destruction of non-cyclic adenine nucleotides and phosphorylated metabolites, which can interfere with later assay steps. This is accomplished using a combination of apyrase, 5' nucleotidase, adenosine deaminase and alkaline phosphatase. The diester linkage of cyclic AMP is then cleaved and newly generated AMP is measured fluorometrically. The adenylyl cyclase activity was measured in rabbit cardiac membrane preparations (n=6) and compared with radioimmunoassay and original [α-32P] ATP Salomon assay. With the enzymatic fluorometric assay, the basal activity and those after exposure to isoproterenol (10^-7M and 10^-6M), NaF (10^-2M), guanylyl-5'-imidodiphosphate (10^-4M), carbachol (10^-6M) and adenosine (10^-3M) were 67±4, 88±5, 147±9, 297±12, 117±6, 56±3 and 34±2 (cyclic AMP production pmof/mg of protein/min, mean±S.E.M.), respectively. The total assay duration including sample reading procedure was 6.5 hrs. The results were virtually identical to those obtained using radioimmunoassay or Salomon method. We conclude that this new assay is highly sensitive, safe, versatile, inexpensive, and has multiple potential applications.

Key words: adenylyl cyclase, non-radioactive, enzymatic cycling, cAMP, radioimmunoassay




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