Assessments of Adenylyl Cyclase Activity Methods Using Labeled ATP

These assays have been widely described by Salomon et al. [19, 20] . The first method utilizes [a32P]ATP, which is converted by adenylyl cyclase to p2P] cAMP to assess enzyme activity in cell and tissue homogenates or membranes. The second one involves incubating cells or tissues with [3H]adenine to label

LIGAN D U6AND

LIGAN D U6AND

Phosphoriiated Substrates

Figure 9.2 This cartoon represents the signaling pathways of G-- and Gj-coupled receptors. Adenylyl cyclase (AC) is at the center of Gas- and Garmediated regulation. Arrows represented by continous lines indicate activation while the arrow represented by dotted line indicates inhibition. PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; C, PKA catalytic subunit; R, PKA regulatory subunit; CREB, cAMP response element-binding protein; CRE, cAMP response element.

Phosphoriiated Substrates

Figure 9.2 This cartoon represents the signaling pathways of G-- and Gj-coupled receptors. Adenylyl cyclase (AC) is at the center of Gas- and Garmediated regulation. Arrows represented by continous lines indicate activation while the arrow represented by dotted line indicates inhibition. PDE, phosphodiesterase; PKA, cAMP-dependent protein kinase; C, PKA catalytic subunit; R, PKA regulatory subunit; CREB, cAMP response element-binding protein; CRE, cAMP response element.

intracellular pools of adenine nucleotides [21]. Accumulation of [3H]cAMP is then used as an index of adenylyl cyclase activity. Both of these methods require the separation of radioactively labeled cAMP from other components of the reaction mixture and particularly from adenine nucleotides other than cAMP. This is efficiently accomplished utilizing sequential chromatography on Dowex cation exchange and alumina columns.

When performing these assays, it is important to note that adenylyl cyclase activity can be influenced by several factors. First, degradation of ATP by enzymes (nucleotidases and hydrolases) present in cell homogenates can result in depletion of substrate. To prevent this problem, incubations are performed in an ATP regeneration system typically consisting in creatinine phosphate and creatinine phosphokinase (or phosphoenolpyruvate and pyruvate kinase). Second, adenylyl cyclase activity is dependent on the presence of divalent cation (Mg2+ or Mn2+) which must be present during incubation. Third, phosphodiesterases present in cell membranes can hydrolyze cAMP to AMP.

Therefore, incubation buffers typically contain one or more phosphodiesterase inhibitors (e.g., isobutylmethylxantine, rolipram, and RO 20-1724) to prevent cAMP breakdown. Fourth, the regulation of adenylyl cyclase by GPCRs requires GTP (or a nonhydrolyzable GTP analog) be included in the incubations. Fifth, it is important that cAMP generation is linear with respect to time and protein content. This may require modification of the amount of cellular protein, length of incubation, or incubation temperature. A factor that contributes to nonlinear (and loss) enzymatic activity is the relative instability of adenylyl cyclase owing to denaturation, especially at temperatures typically used for enzyme assays [22]. Furthermore, when studying a G; -coupled receptor signaling pathway, in order to better appreciate the reduction in cAMP levels, the enzyme must be firstly activated, and this is usually done by using the diterpene forskolin. Although these methods are very sensitive and still widely used nowadays, they rely upon dangerous and costly radioactive compounds.

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