Calcium release from intact calmodulin and calmodulin fragment 78–148 measured by stopped‐flow fluorescence with 2‐p‐toluidinylnaphthalene sulfonate

Abstract

Calcium release from high and low-affinity calcium-binding sites of intact bovine brain calmodulin (CaM) and from the tryptic fragment 78-148, purified by high-pressure liquid chromatography, containing only the high-affinity calcium-binding sites, was determined by fluorescence stopped-flow with 2-p-toluidinylnaphthalene sulfonate (TNS). The tryptic fragments 1-77 and 78-148 each contain a calcium-dependent TNS-binding site, as shown by the calcium-dependent increase in TNS fluorescence. The rate of monophasic fluorescence decrease in endogenous tyrosin on calcium dissociation from intact calcium-saturated calmodulin (kobs 10.8 s-1 and 3.2 s-1 at 25.degree. C and 10.degree. C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence (kobsslow 10.6 s-1 and 3.0 s-1 at 25.degree. C and 10.degree. C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78-148 (kobs 10.7 s-1 and 3.5 s-1 at 25.degree. C and 10.degree. C respectively), were identical, indicating that the rate of the conformational change associated with calcium release from the high-affinity calcium-binding sites on the C-terminal half of calmodulin is not influenced by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed with intact calmodulin only (kobsfast 280 s-1 at 10.degree. C) but not with fragment 78-148, ismost probably due to the conformational change assocaited with calcium release from low-affinity sites on the N-terminal half. The calmodulin fragments 1-77 and 78-148 neither activated calcium/calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum nor inhibited calmodulin-dependent activation at a concentration approximately 1000-fold greater (5 .mu.M) than that of the calmodulin required for half-maximum activation (5.9 mM at 0.8 mM Ca2+ and 5 mM Mg2+) of calmodulin-dependent phosphoester formation.