A fluorimetric method for determination of calcineurin activity.
Calcineurin is a calcium-dependent, serine/threonine phosphatase that is involved in a variety of signaling pathways. Calcineurin is distinct among phosphatases because its activity requires calcium and is not sensitive to inhibition by compounds that block the related phosphatases PP1A and PP2A. Therefore, the most common methods to measure calcineurin activity rely on calcium-dependent dephosphorylation of a substrate derived from the RII subunit of protein kinase A in the presence of PP1A/PP2A inhibitors. However, current techniques quantify activity by measurement of released radioactive phosphate or detection of free phosphate with malachite green. Both methods involve technical challenges and have undesirable features. We report a new calcineurin fluorimetric assay that utilizes a fluorescently labeled phosphopeptide substrate and separation of dephosphorylated peptide product by titanium-oxide. The method is rapid, quantitative, involves no radioactivity and is suitable for high throughput assays. Furthermore, with the use of a standard curve, precise measurements of calcineurin activity can be obtained.
Roberts
Brian
B
Department of Medicine, Emory University School of Medicine, United States.
Pohl
Jan
J
Gooch
Jennifer L
JL
D000818:Animals; D019703:Calcineurin / Q000032:analysis* / Q000378:metabolism; D002478:Cells, Cultured; D005470:Fluorometry / Q000379:methods*; D013058:Mass Spectrometry; D051379:Mice; D010748:Phosphopeptides / Q000378:metabolism
D010748:Phosphopeptides; D019703:Calcineurin