Calcium release from intact calmodulin and calmodulin fragment 78-148 measured by stopped-flow fluorescence with 2-p-toluidinylnaphthalene sulfonate. Effect of calmodulin fragments on cardiac sarcoplasmic reticulum.
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 the monophasic fluorescence decrease in endogenous tyrosine on calcium dissociation from intact calcium-saturated calmodulin (kobs 10.8 s-1 and 3.2 s-1 at 25 degrees C and 10 degrees 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 degrees C and 10 degrees 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 degrees C and 10 degrees 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 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 degrees C) but not with fragment 78-148, is most probably due to the conformational change associated 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 microM) than that of the calmodulin required for half-maximum activation (5.9 nM at 0.8 mM Ca2+ and 5 mM Mg2+) of calmodulin-dependent phosphoester formation.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D009206:Myocardium / Q000201:enzymology*; D009282:Naphthalenesulfonates; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D002147:Calmodulin; D009282:Naphthalenesulfonates; D010446:Peptide Fragments; C047030:calmodulin (78-148); C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D011494:Protein Kinases; D002118:Calcium