DEVELOPMENT AND APPLICATION OF A SENSITIVE RADlOlMMUNOASSAY*

Calmodulin has been radioiodinated by the BoltonHunter procedure. This procedure results in incorporation of 1.5 mol of ‘251/mol of protein and yields a specific radioactivity of 2400 Ci/mmol. The radioiodinated calmodulin retains complete biological activity as determined by its ability to activate calmodulindeficient cyclic nucleotide phosphodiesterase from rat brain. A sensitive radioimmunoassay for calmodulin has been developed using this ‘2Sl-calmodulin as tracer. The assay exhibits a limit of detection of 15 pg and an assay sensitivity of 115 pg with intraand interassay variabilities of <3% and t5%, respectively. Purified calmodulins from bovine brain, rat testis, Renilla reniformis (sea pansy) and Arachis hypogaea (peanut) demonstrate identical immunological cross-reactivities. These findings support the structural data revealing the highly conserved amino acid sequence of this protein. A homologous calcium-binding protein, troponin C, required a 665-fold greater protein concentration to cause 50% reduction in binding of ‘“51-calmodulin. The slope of the troponin C competition curve was different than that for calmodulin suggesting divergence between these two proteins. No immunological crossreactivity was observed by the muscle calcium-binding protein, parvalbumin, at 50,000-fold excess protein. Adaptation of the radioimmunoassay to cell and tissue extracts reveals that calmodulin levels in various tissues and cells were always greater when assayed by the radioimmunoassay as compared to the phosphodiesterase assay. In addition, significant levels of calmodulin were detected in Dictyostelium discoideum and Chlamydomonas reinhardii by the radioimmunoassay whereas no calmodulin was demonstrable using the phosphodiesterase assay.