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 the monophasic fluorescence decrease in endogenous tyrosine on calcium dissociation from intact calcium-saturated calmodulin (kobs 10.8 sand 3.2 s-' at 25°C and 10°C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence (kt:: 10.6 s -l and 3.0 s-l at 25°C and 10°C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78-148 (kobs 10.7 s-' and 3.5 s-' 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 z ; 280 s-l 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 pM) than that of the calmodulin required for half-maximum activation (5.9 nM at 0.8 mM Ca2 , but a biphasic pattern was obtained in a recent study with Quin 2 or ANS [lo], which was suggested to reflect the dissociation of calcium from the high and low-affinity calcium-binding sites, located in the C-terminal half and N-terminal half, respectively, as determined from NMR studies [11][12][13][14].Calcium release and fluorescence changes associated with calcium release have been determined only with intact calmodulin but not with calmodulin fragments. The use of calmodulin fragments [I 51 gives information on the distribution of binding sites of fluorescence probes and may answer the question as to whether the rate of the conformational change associated with calcium release from the high-affinity calcium-binding sites, measured by intrinsic tyrosine fluorescence or fluorescence probes, is determined solely by the calcium release from the C-terminal half of the protein, i.c. is independent of the N-terminal half. For this reason fluorescence changes of calmodulin fragments I -77 and 78 -148 [15] as well as stopped-flow fluorescence on calcium release from intact calmodulin and the calmodulin fragment 78 -148 were measured using the naphthalenesulfonic acid derivative 2-p-toluidinylnapht...

Effects of Myosin Light Chain Kinase and Peptides on Ca2+ Exchange with the N- and C-terminal Ca2+ Binding Sites of Calmodulin

“…5 (trace A) shows the time course of the decrease in TNS fluorescence when the Ca 2ϩ ⅐CaM⅐TNS complex is mixed with EGTA. Similar to Suko et al (1985), we observed a biphasic process in which 54% of the TNS fluorescence decrease occurs at 405 Ϯ 20/s (Fig. 5, trace A (N-EGTA)), and 46% occurs at 2.1 Ϯ 0.1/s (Fig.…”

“…2ϩ Binding Sites-The hydrophobic polarity probe TNS undergoes a large fluorescence increase when it binds to the Ca 2ϩ -dependent hydrophobic pockets in both the N-and Cterminal halves of CaM (Suko et al, 1985;Johnson et al, 1986).…”

Interactive properties of calmodulin

“…However, from studies using 43Ca2+-n.m.r. (Andersson et al, 1982;Teleman et al, 1986) and stopped-flow fast kinetics (Martin et al, 1985;Suko et al, 1985) it appears that CaM has two pairs of sites: one pair in the Nterminal half of CaM has a koff of 300-500 s-' and the other (in the C-terminal half) a k0,r of 10-40 s-'.…”

Specificity and Symmetry in the Interaction of Calmodulin Domains with the Skeletal Muscle Myosin Light Chain Kinase Target Sequence

“…the C-terminal and the N-terminal Ca 2ϩ binding domain (20,21). The equilibrium (22)(23)(24) and kinetic properties (25,26) of intact calmodulin in the Ca 2ϩ binding and dissociation reactions, as well as the secondary structure (24,27), are well represented by a summation of the properties of these fragments, suggesting that the two domains are effectively independent structures. The isolated domains are capable of activating target proteins, but the degree of activation varies with the target protein (26, 28 -35).…”