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Table 1 Summary of ATPase activities of V 1 complexes containing mutant A 3 B 3 heterohexamers and wild-type/mutant DF heterodimers and the binding affinities of those mutants measured by SPR assay

From: Mutant LV476-7AA of A-subunit of Enterococcus hirae V1-ATPase: High affinity of A3B3 complex to DF axis and low ATPase activity

Protein

Initial specific activity (units/mg)*

K D(nM) (using mutant A3B3as ligand and mutant/wild-type DF as analyte)

A(LV 476-7 AA) 3 B 3 DF

7.9 ± 0.3

1.1 ± 0.2

A(LV 476-7 AA) 3 B(L 389 A) 3 DF

9.1 ± 0.2

1.2 ± 0.1

A(LV 476-7 AA) 3 B 3 D(RR 165-6 AA)F

15.5 ± 1.4

50.9 ± 8.4

A(LV 476-7 AA) 3 B 3 D(L 170 N)F

13.0 ± 0.3

1.4 ± 0.3

Wild-type A 3 B 3 DF

16.0 ± 0.2

1.6 ± 0.1

  1. ATPase activities of the reconstituted mutant A3B3DF’s were measured using ATP regenerating system (Alam et al. 2013; & Murata et al. 2001). ATPase assay was started by the addition of 4 μg proteins. For SPR assays, different concentrations of analyte wild-type/mutant DF heterodimer were injected onto the sensor chip Ni-NTA surface having immobilized mutant A3B3 heterohexamers. Reconstituted mutant A3B3 heterohexamers and wild-type/mutant DF heterodimer were diluted in running buffer (20 mM MES-Tris, pH 6.5; 150 mM NaCl; 50 μM EDTA-Na; 0.005% polyoxyethylene (20) sorbitol monolaurate). Experimental details were described in “Materials and methods”.
  2. *“Initial specific activity” was calculated by measuring the specific activity during the first minute of the assay (starting from the 16th second) after adding proteins.
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