Table 2 ATPase activity.

From: Replacing the eleven native tryptophans by directed evolution produces an active P-glycoprotein with site-specific, non-conservative substitutions

 

WT Pgp

W(3Cyto)

p

WL-Pgp

p

Specific verapamil-stimulated ATPase activity (μmol/min/mg)a

5.6 ± 1.0 (7)b

6.3 ± 1.1 (7)

0.28

5.5 ± 0.71 (7)

0.83

Km(MgATP)a

0.75 ± 0.20 (5)

0.40 ± 0.07 (5)

0.006

0.65 ± 0.07 (5)

0.32

Basal ATPase activity (μmol/min/mg)

0.21 ± 0.15 (6)

1.03 ± 0.48 (6)

0.002

0.70 ± 0.26 (6)

0.011

Verapamil EC50 (μM)c

2.0 ± 0.36 (5)

2.0 ± 0.67 (5)

0.10

2.5 ± 0.17 (5)

0.043

Valinomycin EC50 (μM)

0.33 ± 0.08 (8)

0.23 ± 0.03 (6)

0.02

0.34 ± 0.09 (6)

0.84

FK506 EC50 (μM)

0.084 ± 0.03 (6)

0.14 ± 0.03 (5)

0.009

0.16 ± 0.04 (5)

0.004

Cyclosporin A IC50 (μM)d

0.81 ± 0.37 (5)

0.99 ± 0.20 (5)

0.35

0.67 ± 0.04 (5)

0.42

  1. aVmax and Km were determined in the presence of 30 μM verapamil to maximally stimulate ATP hydrolysis using simple Michaelis-Menten kinetics with the SigmaPlot Software package; no cooperativity for MgATP hydrolysis was observed.
  2. bMeans ± standard deviations are given with the number of independent experiments (n) indicated in brackets; p-values were calculated using the Paired Student’s T-Test.
  3. cThe concentration required for 50% stimulation of the ATPase activity (EC50) was calculated from individual fits of which the average ± SD are shown in Fig. 7A,C, and D.
  4. dInhibition of the verapamil-stimulated ATPase activity (IC50) by Cyclosporin A was determined from individual fits of which the average ± SD are shown in Fig. 7B.
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