ABCMdb

PMID: 18241200

Ramjeesingh M, Ugwu F, Stratford FL, Huan LJ, Li C, Bear CE
The intact CFTR protein mediates ATPase rather than adenylate kinase activity.
Biochem J. 2008 Jun 1;412(2):315-21., 2008-06-01 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Gly551Asp As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Login to comment 5 ABCC7 p.Glu1371Gln Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Login to comment 19 ABCC7 p.Glu1371Gln The channel function of the CFTR-E1371Q mutant has been studied, and it exhibits a prolonged channel open time, consistent with the idea that ATPase activity promotes channel closure [6,7]. Login to comment 26 ABCC7 p.Gly551Asp ABCC7 p.Glu1371Gln Furthermore, the consequences of the disease-causing mutation G551D and the catalytic base mutation E1371Q on the enzymatic activity of the full-length protein were also evaluated. Login to comment 36 ABCC7 p.Gly551Asp ABCC7 p.Glu1371Gln Purification and reconstitution of wt (wild-type), CFTR-G551D and CFTR-E1371Q Detailed protocols regarding the generation of wt and mutant CFTR-His10 proteins are described elsewhere [29,30]. Login to comment 118 ABCC7 p.Gly551Asp ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Interestingly, the addition of AMP (400 μM) to PKA-treated CFTR did not exert any significant effect on its ATPase activity (Figures 3A and 5B), a finding which is consistent with previous Figure 4 The disease-causing mutant CFTR-G551D and the putative catalytic base mutant CFTR-E1371Q exhibit reduced ATPase activity (A) Silver-stained gel showing purification of CFTR-E1371Q. Login to comment 119 ABCC7 p.Glu1371Gln (B) Phosphoimage shows Pi production by proteoliposomes containing CFTR-E1371Q (right panel) compared with empty liposomes in the absence (-) or presence (+) of 400 μM AMP. Login to comment 120 ABCC7 p.Gly551Asp ABCC7 p.Glu1371Gln (C) ATPase activity (Results are means + - S.E.M.) of CFTR-G551D (n = 6) or CFTR-E1371Q (n = 6). Login to comment 125 ABCC7 p.Gly551Asp ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln The enzymatic activity of full-length mutant CFTR proteins: CFTRG551D and CFTR-E1371Q Gly551 is located in the signature motif of NBD1 [1] and is thought to exist at the interface through which NBD1 and NBD2 interact to form the catalytic site for ATP hydrolysis [4,7]. Login to comment 126 ABCC7 p.Gly551Asp The mutation, CFTR-G551D, is associated with a severe form of CF. Login to comment 127 ABCC7 p.Gly551Asp We reported previously that the enzymatic activity of purified PKA phosphorylated CFTR-G551D, measured as the generation of [α-32 P]ADP from [α-32 P]ATP, was severely abrogated [4]. Login to comment 132 ABCC7 p.Glu1371Gln Therefore we expressed the full-length mutant protein CFTR-E1371Q and purified it from Sf9 cells (Figure 4A) for the purpose of studying its ATPase activity. Login to comment 136 ABCC7 p.Gly551Asp ABCC7 p.Glu1371Gln As for the wt and CFTR-G551D proteins, there was no measurable adenylate kinase activity stimulated in CFTR-E1371Q by the addition of 400 μM AMP (Figure 4B). Login to comment 198 ABCC7 p.Glu1371Gln Substitution of Glu1371 with a glutamine residue has been shown to delay the rate of channel closing, providing support for the idea that ATPase activity promotes closure of the channel gate [6,7]. Login to comment 200 ABCC7 p.Glu1371Gln Interestingly, the ATPase activity of CFTR was not completely inhibited in the CFTR-E1371Q mutant, which exhibited approx. Login to comment 207 ABCC7 p.Gly551Asp The CF disease-causing mutant, CFTR-G551D, exhibits defective ATPase activity, a result which is consistent with the putative location of the signature motif (L548 SGGQ552 ) relative to ATP in the NBD dimer interface [7,21]. Login to comment