TABLE 4.

Kinetic parameters derived from Kintek fits of progress curves of ZPI/cZPI reactions with PZ and rPZ

Fluorescence progress curves were obtained for reactions of 20 nm NBD-ZPI or NBD-cZPI with 100–600 nm PZ or rPZ in 50 mm Tris buffer, pH 7.1, I 0.15 at 25 °C such as those in Fig. 8, corrected for buffer background, and then fitted using Kintek software by a three-step sequential binding model as described under “Experimental Procedures.” Calcium was added at 1.5 mm, and lipid was added at 10 μm when present. The last column represents the calculated overall equilibrium dissociation constant obtained from the calculated dissociation constants: K₁ = k₋₁/k₁, K₂ = k₋₂/k₂, and K₃ = k₋₃/k₃, for the three binding steps.

	k+1	k−1	K1	k+2	k−2	K2	k+3	k−3	K3	(K1K2K3)/(1 + K2K3 + K3)
NBD-ZPI-PZa	21.0 ± 0.1 (20)	0.18 ± 0.02 (0.14)	0.0086 ± 0.0010	1.3 ± 0.3 (0.52)	2.1 ± 0.2 (0.71)	1.6 ± 0.5	0.10 ± 0.02 (0.035)	0.035 ± 0.001 (0.043)	0.35 ± 0.08	0.003
+Ca2+	12.2 ± 0.6	0.5 ± 0.2	0.04 ± 0.02	0.8 ± 0.8	1.9 ± 0.4	2 ± 2	0.10 ± 0.09	0.055 ± 0.005	0.6 ± 0.5	0.02
+Ca2+ + lipid	9.4 ± 0.4	0.5 ± 0.1	0.05 ± 0.01	1.0 ± 0.3	0.9 ± 0.1	0.9 ± 0.4	0.06 ± 0.02	0.05 ± 0.01	0.8 ± 0.4	0.01
NBD-ZPI-rPZ	15.3 ± 0.1	0.26 ± 0.01	0.017 ± 0.001	0.09 ± 0.02	0.56 ± 0.08	6 ± 2	0.15 ± 0.04	0.09 ± 0.01	0.6 ± 0.2	0.01
NBD-cZPI-PZ	17.4 ± 0.3	0.40 ± 0.05	0.023 ± 0.003	0.3 ± 0.3	1.9 ± 0.2	6 ± 7	0.06 ± 0.05	0.092 ± 0.003	2 ± 2	0.02
NBD-cZPI-rPZ	20 ± 2	0.64 ± 0.03	0.032 ± 0.005	0.2 ± 0.1	0.8 ± 0.7	4 ± 6	0.3 ± 0.2	0.13 ± 0.05	0.4 ± 0.4	0.02

^a Global fit of association and dissociation kinetic data of Fig. 8. Values in parentheses are parameters obtained from fitting just association kinetic data.