A high-performance liquid chromatography assay of brain adenylate cyclase using [3H]ATP as substrate.
A sensitive, reproducible assay for adenylate cyclase is described which separates labeled cyclic AMP from ATP and other nucleotides by high-performance liquid chromatography (HPLC) on reverse-phase columns. The technique utilizes [3H]ATP as substrate, and the principal compound contaminating the [3H]cyclic AMP peak, adenosine, is removed by incubation of assay tubes with small amounts of adenosine deaminase. The HPLC elution utilizes high resolution (3 microns) short (10 cm) C-18 columns for increased resolution and decreased flow rates. Since cyclic AMP elutes at 4 min following injection, this procedure can easily process large numbers of samples per day when combined with automated techniques of sample injection and collection.
D000241:Adenosine / Q000032:analysis; D000243:Adenosine Deaminase; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D002462:Cell Membrane / Q000166:cytology / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003342:Corpus Striatum / Q000166:cytology / Q000201:enzymology; D000242:Cyclic AMP / Q000032:analysis; D008297:Male; D009994:Osmolar Concentration; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D014316:Tritium
D014316:Tritium; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000243:Adenosine Deaminase; D000262:Adenylyl Cyclases; D000241:Adenosine