Open AccessCalcium release from intact calmodulin and calmodulin fragment 78–148 measured by stopped‐flow fluorescence with 2‐ p ‐toluidinylnaphthalene sulfonate
Author(s) -
SUKO Josef,
PIDLICH Johann,
BERTEL Oswald
Publication year - 1985
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1985.tb09323.x
Subject(s) - calmodulin , calcium , fluorescence , chemistry , biophysics , binding site , tyrosine , chromatography , biochemistry , biology , organic chemistry , physics , quantum mechanics
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 ( K obs 10.8 s −1 and 3.2 s −1 at 25°C and 10°C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence ( K siow obs 10.6 s −1 and 3.0 s −1 at 25°C and 10°C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78–148 ( K obs 10.7 −1 and 3.5 s −1 at 25°C and 10°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 ( K fast obs 280 s −1 at 10°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 μM) than that of the calmodulin required for half‐maximum activation (5.9 nM at 0.8 mM Ca +2 and 5 mM Mg 2+ ) of calmodulin‐dependent phosphoester formation.