Detection of adenylyl cyclase activity using a fluorescent ATP substrate and capillary electrophoresis.
A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CE-LIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or Galpha(s)-GTPgammaS as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected EC50 values for forskolin and Galpha(s)-GTPgammaS of 27 +/- 6 microM and 317 +/- 56 nM, respectively. These EC50 values compared well to those previously reported using [alpha-32P]ATP as substrate. When AC was concurrently activated with 2.5 microM forskolin and 25 nM Galpha(s)-GTPgammaS, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2'-(or-3')-O-(N-methylanthraniloyl) guanosine 5'-triphosphate with an IC50 of 9 +/- 6 nM. The use of a fluorescent substrate combined with CE separation has enabled development of a rapid and robust method for detection of AC activity that is an attractive alternative to the AC assay using radioactive nucleotide and column chromatography. In addition, the assay has potential for high-throughput screening of drugs that act at AC.
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Jennifer M
JM
Cunliffe
Roger K
RK
Sunahara
Robert T
RT
Kennedy
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry*; D019075:Electrophoresis, Capillary; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013997:Time Factors
C518935:3'-O-(N-methylanthraniloyl) guanosine 5'-triphosphate; D000067956:Adenylyl Cyclase Inhibitors; D004791:Enzyme Inhibitors; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases