ABCMdb

ABCC7 p.Lys464His

None has been submitted yet.

Publications

PMID: 12523935 [PubMed] Annereau JP et al: "Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate."

No. Sentence Comment

44 To obtain ht-NBF1jR mutants in the Walker A region (K464H and K464A), cDNA cassettes were exchanged between BamHI and Bst1107I sites with homologous cDNA cassettes excised from the mutant pMALCR1 MBP-NBF1 plasmids, constructed previously in our laboratory [31]. Login to comment
48 Overexpression of the recombinant ht-NBF1TR (Gly-404-Lys-830) 'wild-type` and mutant proteins (K464H or K464A) Escherichia coli BL21 (DE3) strain, freshly transformed with the 'wild-type` (i.e. recombinant) and mutant ht-NBF1jR plasmids, were grown in 10 ml of LB medium containing 50 µg\ml kanamycin. Login to comment
103 The Walker A motif is underlined, and Lys-464 within this motif is shown in bold to denote the site of mutations (K464H, K464A) made in this study. Login to comment
105 (C) Expression in E. coli of the 'wild-type` ht-NBF1jR protein and mutant forms K464H and K464A. Login to comment
109 Lanes 2, 4 and 6, E. coli cells transformed with plasmids containing, respectively, cDNA encoding 'wild-type` ht-NBF1jR, mutant-form K464H and mutant-form K464A prior to induction with IPTG; lanes 3, 5 and 7, as for lanes 2, 4 and 6 respectively but after induction with 0.3 mM IPTG. Login to comment
110 (D) SDS/PAGE pattern obtained at different stages of purification of the 'wild-type` ht-NBF1jR protein and mutant forms K464H and K464A. Login to comment
113 SDS/PAGE (10%) was used to analyse 10 µl aliquots at each step as follows: lane 1, molecular-mass markers as defined in (C); lanes 2-4, supernatant from guanidine hydrochloride-extracted E. coli overexpressing the 'wild-type` ht-NBF1jR protein and its mutant forms K464A and K464H respectively; lanes 5-7, flow-through fractions of the same extracts after their application to a Ni-NTA column (note that the $ 50 kDa proteins, i.e. the 'wild-type` ht-NBF1jR protein and its mutant forms, were retained); lanes 8-10, eluates derived from Ni-NTA columns containing, respectively, the 'wild-type` ht-NBF1jR protein and its K464A and K464H mutant forms after including 10 mM imidazole in the elution buffer; lanes 11-13, eluates derived from the same Ni-NTA columns after adding 200 mM imidazole to the elution buffer. Login to comment
120 E. coli transformed with the pET-28a(T) expression plasmid encoding NBF1TR residues 404-830, and a short His-tagged N-terminal region, markedly overproduce both 'wild-type` and mutant forms (K464H and K464A) of the protein Results presented in Figure 1(C) summarize SDS\PAGE patterns of E. coli cells harbouring the 'wild-type` and mutant ht-NBF1jR-containing plasmids before and after induction with IPTG. Login to comment
122 The same applies to lane 5 (mutant K464H with IPTG) versus lane 4 (mutant K464H without IPTG), and to lane 7 (mutant K464A with IPTG) versus lane 6 (mutant K464A without IPTG). Login to comment
127 The ht-NBF1TR 'wild-type` protein and its mutant forms can be purified to apparent homogeneity on a Ni-NTA column and show cross-reactivity with a monoclonal antibody to CFTR exhibiting specificity for NBF1 As the overexpressed 'wild-type` and mutant (K464H and K464A) proteins were tagged with six histidines at their N-terminus (see the Materials and methods section and Figure 1B), they were amenable to purification on a column containing bound Ni#+. Login to comment
128 For this reason, the wild-type and mutant E. coli cell pellets derived from the overexpression experiments described above were treated with a buffered solution containing the denaturant guanidine hydrochloride (6 M), and after centri- on the SDS/PAGE gels for the 'wild-type` ht-NBF1jR protein (lane 11) and its mutant forms K464A (lane 12) and K464H (lane 13). Login to comment
152 (B) ATPase activity versus [ATP] plot for the purified renatured 'wild-type` ht-NBF1jR protein and mutant forms K464H and K464A. Login to comment
154 The assay contained, in 75 µl of renaturation buffer, ATP concentrations of 0-100 µM, 5 mM MgCl2 and 0.47 µg of either the 'wild-type` ht-NBF1jR protein (WT) or its mutant forms K464H and K464A, pH 7.5. Login to comment
159 (D) Relative ATPase activities obtained for the 'wild-type` NBF1jR Protein (WT) and mutant forms K464H and K464A using the [32 P]Pi release assay. Login to comment
160 The assay was carried out in 200 µl of renaturation buffer, pH 7.5, containing 3.7 µg of the 'wild-type` ht-NBF1jR protein or its mutant forms (K464H, or K464A), and supplemented with 250 µM [γ-32 P]ATP (1.4i106 c.p.m.) and 1 mM divalent cation. Login to comment
200 Subsequent studies carried out with ht-NBF1jR proteins with mutations in the Walker A consensus region (K464H and K464A) showed that the Vmax was reduced about 50% in each case (Figure 2B). Login to comment

PMID: 9511928 [PubMed] Seibert FS et al: "Cystic fibrosis: channel, catalytic, and folding properties of the CFTR protein."

No. Sentence Comment

101 The observed Vmax of ~30 nmol/mg/min is very low compared to other ATPases such as the Ca++ -ATPase (600 nmol/mg/min; Racker, 1985) or P-glycoprotein (300-1650 nmol/mg/min; Sharom et al., 1995), although an indication of the significance of the data is given by a negative effect due to Walker A mutations K464H and K464L. Login to comment

PMID: 7545672 [PubMed] Ko YH et al: "The first nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator can function as an active ATPase."

No. Sentence Comment

4 Significantly, the mutations K464H and K464L in the Walker A consensus motif of NBF1 markedly impair its catalytic capacity. Login to comment
43 K464H: 5Ј-ACT GGA GCA GGC CAC ACT TCA CTT CTA-3Ј K464L: 5Ј-ACT GGA GCA GGC CTG ACT TCA CTT CTA-3Ј The identity of the two base changes was confirmed by DNA sequencing (20). Login to comment
113 B, comparison of the purity of wild type and mutant MBP-NBF1 proteins (K464H and K464L). Login to comment
115 C and D, the effect of the mutations K464H and K464L within NBF1 on the ATP hydrolytic activity of MBP-NBF1. Login to comment
125 Even more convincing are results presented in Fig. 4, C and D, where it is seen that the mutations, K464H and K464L, in the Walker A nucleotide binding motif (GX4GKT) of NBF1 reduce the ATP hydrolytic capacity of purified mutant MBP-NBF1 proteins by over 80%. Login to comment
133 The additional experimental results demonstrating that MBP alone has no catalytic capacity (Fig. 2C) and that mutations (K464H and K464L) within the Walker nucleotide binding motif GX4GKT markedly inhibit ATPase activity (Fig. 4, C and D) localize the catalytic site to NBF1. Login to comment
42 K464H: 59-ACT GGA GCA GGC CAC ACT TCA CTT CTA-39 K464L: 59-ACT GGA GCA GGC CTG ACT TCA CTT CTA-39 The identity of the two base changes was confirmed by DNA sequencing (20). Login to comment
112 B, comparison of the purity of wild type and mutant MBP-NBF1 proteins (K464H and K464L). Login to comment
114 C and D, the effect of the mutations K464H and K464L within NBF1 on the ATP hydrolytic activity of MBP-NBF1. Login to comment
124 Even more convincing are results presented in Fig. 4, C and D, where it is seen that the mutations, K464H and K464L, in the Walker A nucleotide binding motif (GX4GKT) of NBF1 reduce the ATP hydrolytic capacity of purified mutant MBP-NBF1 proteins by over 80%. Login to comment
132 The additional experimental results demonstrating that MBP alone has no catalytic capacity (Fig. 2C) and that mutations (K464H and K464L) within the Walker nucleotide binding motif GX4GKT markedly inhibit ATPase activity (Fig. 4, C and D) localize the catalytic site to NBF1. Login to comment