Table 2. Kinetic Parameters of C2 Domain Activation and Deactivation Time Courses^a.

			membrane dissociationc			Ca2+ dissociationd
	membrane associationb		kdiss (s−1)			kdiss (s−1)
C2 domain	kform (s−1)	(n)	kdiss1(s−1)	kdiss2 (s−1)	(n)	kdiss1(s−1)	kdiss2 (s−1)	(n)	stoichiometry (mol/mol)e	(n)
	cPLA2-α	26 ± 1	(6)	14 ± 1	2.5 ± 0.1	(10)	24 ± 8	2.9 ± 0.4	(9)	2.0 ± 0.2	(5)
PKC-β	180 ± 10	(6)	132 ± 1		(12)	67 ± 3		(10)	3.0 ± 0.1	(10)
Syt-IA	230 ± 20	(7)	520 ± 20		(12)	590 ± 30		(11)	3.1 ± 0.3	(9)

^aResults represent the average (±SEM) of the indicated n independent stopped-flow fluorescence experiments. Experimental conditions: 25 °C; 100 mM KCl, 20 mM HEPES, pH 7.4, 5 mM DTT.

^bThe observed rate constant for equilibration of the membrane association reaction, initiated by rapidly mixing a solution of C2 domain and PS–PC–dPE vesicles (47.5%:47.5%:5%, 250 μM phospholipid final) with a solution of Ca²⁺ (200 μM final), as in Figure 6. The resulting time course was best fit by a monoexponential equation (eq 3) to yield k_form as defined in the text.

^cThe rate constant for C2 domain dissociation from membranes, measured by mixing a solution of C2 domain, Ca²⁺, and phospholipid vesicles (PS– PC–dPE, 47.5%:47.5%:5%) with a solution of EDTA, as in Figure 7A. The dissociation rate constants k_diss1 and k_diss2 were determined by best fitting the data with a biexponential equation (eq 4) representing a two-step mechanism (cPLA₂-α) or a monoexponential equation (eq 3) representing a one-step mechanism (PKC-β, Syt-IA).

^dThe rate constant for Ca²⁺ dissociation from the membrane-bound domain, measured by rapidly mixing a solution of C2 domain, Ca²⁺, and phospholipid vesicles (PS–PC, 50%:50%) with a solution of the fluorescent Ca²⁺ chelator Quin-2, as in Figure 7B. The dissociation rate constants k_diss1 and k_diss2 were extracted as in the previous footnote.

^eCa²⁺ stoichiometry (mol of Ca²⁺ bound/mol of C2 domain) was determined at pH 7.0.