Separation of radiolabeled orthophosphate and adenosine 5'-triphosphate by 20% polyacrylamide gel electrophoresis: an assay for brain microsomal Mg2+/Ca2+ ATPase activity.

Measuring orthophosphate is an important tool in biochemical analyses used to study membrane transport ATPases essential for calcium homeostasis. Current techniques involve extraction of radioactive phosphate with organic solvents, a technique that results in large quantities of hazardous radioactive waste. Other colorimetric assays are less sensitive and are complicated by interference of background absorbance from membrane tissue and unutilized ATP. This report describes a unique assay for the detection of inorganic phosphate and its application to the study of rat brain microsomal Mg2+/Ca2+ ATPase from a membrane fraction. The technique involves the separation of radioactive phosphate from unused gamma-radiolabeled ATP by resolution on 20% polyacrylamide gels. Both are visualized with X-ray film and quantitated by liquid scintillation counting after extraction from the gels. The assay can detect as little as 4.1 pmol of radiolabeled ATP and ATPase activity in 3.5 ng/microliter of membrane protein. This method offers the advantage of simultaneous quantitation of radiolabeled ATP and radioactive orthophosphate without the generation of large quantities of radioactive waste. The results demonstrate the development of a novel assay procedure for quantitating orthophosphate that is extremely sensitive, reproducible, and applicable to the study of any phosphate liberating enzyme.