Calcium dissociation kinetics

Orlowski, S. Champeil, P. (1991a). Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase. Biochemistry 30, 352-361. 

Kinetic of calcium dissociation Fast Phase 663 s"Slow Phase 9s- ... 

The enzyme maintains the energy of the activated phosphate and calcium of E-P Ca2 in a simple manner by utilizing an ordered kinetic mechanism for the reactions of this key intermediate, as shown in (11). The ordered mechanism requires that in order for calcium to dissociate to the outside the phosphate must first be transferred to ADP to give ATP. If calcium dissociated first and the interaction energy was lost, the phosphate would no longer be activated and could not be transferred to ADP. On the inside calcium dissociates first, against the high chemi-... 

These rules avoid the designations Ei and E2, for which there is not a universally accepted definition. The different chemical and vectorial specificities appear to be mediated by the different chemical forms of the enzyme - by E and E Ca2 for the chemical specificity and by E and E-P for the vectorial specificity. The rules and the ordered kinetic mechanism of the enzyme are consistent with well known chemical properties of the enzyme, including the occluded calcium of the key intermediate E-P Ca2 that is exposed to neither side of the membrane. This intermediate behaves as if the calcium ion is simply covered up by covalently bound phosphate, or as if phosphorylation induces a conformation change that covers it up and prevents calcium dissociation to the outside it also makes possible a further conformation change that allows its dissociation inside. This dissociation occurs in a slow step that is affected by the properties of the membrane and is rate determining under most conditions. 

Lobster hemocyanin in glycine-sodium hydroxide buffer at pH 9.6 undergoes a reversible whole molecule-half molecule dissociation which is very sensitive to the level of free calcium ion present (12). This dissociation process is also very sensitive to pH (12). Cann and colleagues have developed, by simulation techniques (13,14), a general picture of the kinds of partial or complete boundary resolution that may be expected for such coupled systems in various types of transport experiments. Kinetic investigations of the lobster hemocyanin system under the conditions of the present study were developed by using stopped-flow... 

Orlowski, S. Champeil, P. (1991b). The two calcium ions initially bound to nonphosphorylated sarcoplasmic reticulum Ca2+-ATPase can no longer be kinetically distinguished when they dissociate from phosphorylated ATPase toward the lumen. Biochemistry 30,11331-11342. 

Figure 3. Simulation of the kinetic scheme from Figure 2 using the constants from Table 3. The top figure represents the kinetics of Ca dissociation which has a biphasic response (fast phase 663 s and slow phase 9 s ). The middle figure represents the titration of calmodulin by Ca ". The signal rising between 0 and 2-3 Ca " ions is associated with the occupancy of the sites from the COOH terminus and the other signal is associated with the occupancy of the N-terminal sites. The bottom figure is a Scatchard representation of the direct calcium binding isotherm.

Fig. 9. Kinetics of Ca2+ dissociation from the cPLA, C2 domain in the absence and presence of phosphatidylcholine vesicles, (a) Stopped-flow measurement of the Ca2t off rate using the fluorescent calcium indicator Quin-2, (b) Conformational changes triggered by Ca- removal from the C2 domain monitored using the intrinsic fluorescence of Trp71. (c) Membrane release of the C2 domain followed by protein-to-membrane FRET. (From Nalefski et al. 1997 with permission.)...

Calcium entry into a cell frequently has a number of secondary effects such as initiation of contraction, release of neurotransmitters, and modulation of membrane ion channels. This is usually accomplished by the binding of calcium ions to calcium receptors inside the cell. MichaeHs-Menten kinetic schemes with steady-state assumptions are used to model this binding and therefore expressions of the form / = [Cfl++]"/([Cfl++]" + K) are frequently employed. Here / is the fraction of calcium that is bound to the receptor and K is the dissociation constant for the reaction, and n is the number of calcium ions that bind to each receptor molecule. 

The ultrasonic technique has been used to study the binding of calcium to sorbitol the interaction is weak and the formation rate constant (40 C) could only be approximately determined as (1-2) x 10 dm mol" s . The kinetics have also been studied of the dissociation of calcium cryptates in various solvents " (see Table 9.4) and of cryp tand exchange... 

The kinetics of the binding of oxygen to the haemocyanin from the meirine gastropod Buccinum undatum under various conditions have been reported. In the presence of calcium ions the association rate constant is 7.8 x 10 1 mol s (25 "Q, and it is almost independent of pH. On the other hand, the dissociation rate constant increases significantly over the pH range 7.0 (A d=13 s ) to 8.0 (56 s ). The rate constant for the reaction of O2 with (undissociated) haemocyanin from Panulirus... 

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