ABCMdb

PMID: 15737336

Yang R, McBride A, Hou YX, Goldberg A, Chang XB
Nucleotide dissociation from NBD1 promotes solute transport by MRP1.
Biochim Biophys Acta. 2005 Mar 1;1668(2):248-61., 2005-03-01 [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn The replacement of D793 with a non-acidic residue, such as D793L or D793N, increases the rate of ATP-dependent LTC4 transport. Login to comment 42 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln The definition of D793E means that the D793E mutated N-half is co-expressed with wild-type C-half and E1455Q, the wild-type N-half co-expressed with E1455Q mutated C-half. Login to comment 44 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln Membrane vesicles were prepared from Sf21 cells infected with viral particles expressing pDual without MRP1 cDNA insertion (lane 1), wild-type N-half+wild-type C-half (Wild-type, lanes 2-4), D793E mutated N-half+wild-type C-half (D793E, lanes 5-7), D793L mutated N-half+wild-type C-half (D793L, lanes 8-10), D793N mutated N-half+wild-type C-half (D793N, lanes 11-13) and wild-type N-half+E1455Q mutated C-half (E1455Q, lanes 14-16). Login to comment 49 ABCC1 p.Asp793Glu The ratios of the band intensities in the same amount of total membrane proteins, for example, 300 ng of wild-type N-half (co-expressed with wild-type C-half) versus 300 ng of the D793E mutated N-half (co-expressed with wild-type C-half), were determined, considering the amount of wild-type N-half (or C-half) as 1.000. Login to comment 50 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln Since the ratio of N-half, for example, D793E mutated N-half, is similar to that of the C-half co-expressed with D793E mutated N-half, the mean ratios of the protein expressions including N-half and C-half are: 0.993F0.168 (D793E), 0.991F0.073 (D793L), 1.151F0.186 (D793N) and 0.921F0.108 (E1455Q). Login to comment 64 ABCC1 p.Glu1455Gln This mutation in NBD2 (E1455Q) almost completely eliminates ATP-dependent LTC4 transport. Login to comment 78 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln Generation of constructs The oligo-nucleotides to introduce the mutations in MRP1 are: MRP/D793E/forward, 5V-CT GAC ATT TAC CTC TTC GAT GAA CCC CTC TCA GCA GTG GAT GCC-3V; MRP/D793E/reverse, 5V-GGC ATC CAC TGC TGA GAG GGG TTC ATC GAA GAG GTA AAT GTC AG-3V; MRP/D793N/forward, 5V-CT GAC ATT TAC CTC TTC GAT AAT CCC CTC TCA GCA GTG GAT GCC -3V; MRP/D793N/reverse, 5V-GGC ATC CAC TGC TGA GAG GGG ATT ATC GAA GAG GTA AAT GTC AG-3V; MRP/ E1455Q/forward, 5V-G AAG ATC CTT GTG TTG GAT CAG GCC ACG GCA GCC GTG GAC CTG G-3V; MRP/ E1455Q/reverse, 5V-C CAG GTC CAC GGC TGC CGT GGC CTG ATC CAA CAC AAG GAT CTT C-3V. Login to comment 81 ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn The aspartic acid residue at position 793 was mutated to either glutamic acid or asparagine (Fig. 1B, D793E or D793N) by using the forward/reverse primers and the QuikChange site directed mutagenesis kit from Stratagene [28]. Login to comment 82 ABCC1 p.Asp793Leu D793L was introduced into the cDNA in the pNUT expression vector already [29]. Login to comment 83 ABCC1 p.Glu1455Gln E1455Q was also introduced into the cDNA by the same strategy. Login to comment 92 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Gln This strategy was also used to generate constructs expressing D793E, L, and N mutated N-half and E1455Q mutated C-half. Login to comment 93 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Asp793Asn ABCC1 p.Asp793Asn To make constructs expressing D793E, D793L and D793N mutated N-half and wild-type C-half simultaneously, the KpnI- DraIII fragment from pDual/N-half/C-half and the DraIII- XhoI fragments from pNUT/D793E, pNUT/D793L or pNUT/D793N were cloned into the KipI-XhoI fragment from pDual/N-half/C-half, named as pDual/D793E-N-half/ C-half, pDual/D793L-N-half/C-half or pDual/D793N- N-half/C-half. Login to comment 94 ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln To make a construct expressing wild-type N-half and E1455Q mutated C-half simultaneously, the ClaI-BspEI fragment derived from pNUT/E1455Q and the RsrII-ClaI fragment from pDual/N-half/C-half were cloned into the RsrII-BspEI fragment derived from pDual/N-half/ C-half, named as pDual/N-half/E1455Q-C-half. Login to comment 156 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln Substitution of the Asp residue with a non-acidic amino acid in NBD1 increased the Km and Vmax values for LTC4 in MRP1 mediated transport In order to test whether these Walker B mutations, D793E, D793L and D793N in NBD1 and E1455Q in R. Yang et al. Login to comment 158 ABCC1 p.Glu1455Gln Fig. 3 shows that E1455Q is almost completely inactivated, consistent with the previously reported result [43]. Login to comment 159 ABCC1 p.Asp1454Leu ABCC1 p.Asp1454Leu ABCC1 p.Glu1455Leu ABCC1 p.Glu1455Leu Interestingly, this result is also similar to that of the double mutant D1454L/E1455L [28] including the mutations of the D1454 residue in the Walker B motif and the putative catalytic base E1455 residue directly adjacent to the D1454, implying that the mutation of the putative catalytic base E1455 to a non-acidic amino acid affecting ATP hydrolysis [43] has the same effects as the D1454L/E1455L double mutant affecting ATP binding [28] and hydrolysis. Login to comment 160 ABCC1 p.Asp793Glu The mutation of the corresponding residue D793 in NBD1 to the longer arm acidic glutamic acid (D793E) changed the kinetics of ATP-dependent LTC4 transport (Fig. 3). Login to comment 161 ABCC1 p.Asp793Glu The transport activity of D793E is lower than that of wild-type at lower LTC4 concentrations, consistent with the previously reported results [43]. Login to comment 163 ABCC1 p.Asp793Glu These might be the consequence of the altered properties of this D793E mutated NBD1 [43]. Login to comment 164 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn Interestingly, the substitution of the putative catalytic base D793 in NBD1 with a non-acidic amino acid, such as D793L or D793N, increases ATP-dependent LTC4 transport activity (Fig. 3 and Table 1), implying that ATP hydrolysis at NBD1 might not be essential for the transport. Login to comment 169 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln Since the amounts of MRP1 proteins in membrane vesicles containing wild-type, D793E, D793L, D793N and E1455Q are similar (Fig. 1C), the much lower Vmax value of E1455Q than that of the wild-type (Table 1), although the amount of E1455Q (ratio of 0.921) is slightly less than wild-type, indicates a greatly decreased k2 value, which is perhaps directly associated with the greatly diminished ATPase activity at Fig. 3. Login to comment 174 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln The samples are: wild-type, wild-type N-half+wild-type C-half; D793E, D793E mutated N-half+wild-type C-half; D793L, D793L mutated N-half+wild-type C-half; D793N, D793N mutated N-half+wild-type C-half and E1455Q, wild-type N-half+E1455Q mutated C-half. Login to comment 175 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln Table 1 Km and Vmax values (LTC4) of wild-type and mutant MRP1s Sample Km (nM LTC4)a Vmax (pmol LTC4 mgÀ1 minÀ1 )N-half C-half Wild-type Wild-type 59F1 287.5F7.5 D793E Wild-type 110F10 365.0F25.0 D793L Wild-type 100F0 560.0F0.0 D793N Wild-type 105F5 575.0F75.0 Wild-type E1455Q 50F0 37.5F0.5 a The Km values (n=2) and Vmax values (n=2) were derived from Fig. 3. Login to comment 183 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln / Biochimica et Biophysica Acta 1668 (2005) 248-261 253 the E1455Q mutated NBD2 as shown in Fig. 7M and O; whereas the higher Vmax value (Table 1) of D793L (a ratio of 0.993 indicates that the amount of D793L is slightly less than wild-type) or D793N (ratio of 1.151) indicates a slightly increased k2 value, leading to a higher Km (LTC4) value and a higher rate of ATP-dependent LTC4 transport. Login to comment 185 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln Combination of D793E, D793L or D793N mutated NBD1 with E1455Q mutated NBD2 does not enhance ATP-dependent LTC4 transport activity The k2 values should be directly associated with the rates of ATP hydrolysis by variant MRP1 mutants. Login to comment 186 ABCC1 p.Glu1455Gln The greatly decreased k2 value for E1455Q is interpreted as that mutation of the putative catalytic E1455 residue to a non-acidic amino acid greatly diminishes the rate of ATP hydrolysis at the mutated NBD2. Login to comment 187 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn Whereas the moderately increased k2 values for D793L and D793N could be interpreted in the following two ways: (1) The mutation of the putative catalytic base D793 to a non-acidic amino acid, such as L or N, somehow increases the rate of ATP hydrolysis at the mutated NBD1 and enhances ATP-dependent LTC4 transport; (2) The mutation of the putative catalytic base D793 to a non-acidic amino acid, such as L or N, decreases the affinity for ATP and increases the release rate of the bound nucleotide from the mutated NBD1. Login to comment 189 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln In order to test these two possibilities, the D793E, D793L or D793N mutated N-half was co-expressed with the E1455Q mutated C-half. Login to comment 191 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn The results in Fig. 4B show that all the mutants, including wild-type N-half+E1455Q mutated C-half, D793E mutated N-half+E1455Q mutated C-half, D793L mutated N-half+E1455Q mutated C-half and D793N mutated N-half+E1455Q mutated C-half, have similar ATP-dependent LTC4 transport activities. Login to comment 194 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln D793E, D793L or D793N mutated NBD1 does not enhance the ATP-dependent LTC4 transport activity of E1455Q mutated NBD2. Login to comment 197 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln The mean ratios of the protein expressions including N-half and C-half are: 1.33F0.11 (E1455Q), 1.49F0.13 (D793E/E1455Q), 0.98F0.05 (D793L/ E1455Q) and 1.88F0.29 (D793N/E1455Q). Login to comment 204 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln The samples are: wild-type, wild-type N-half+wild-type C-half; D793E, D793E mutated N-half+wild-type C-half; D793L, D793L mutated N-half+wild-type C-half; D793N, D793N mutated N-half+wild-type C-half and E1455Q, wild-type N-half+E1455Q mutated C-half. Login to comment 205 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln Table 2 Km values (ATP) of wild-type and mutant MRP1s Sample Km (AM ATP)a N-half C-half Wild-type Wild-type 72.2F1.6 D793E Wild-type 106.0F9.7 D793L Wild-type 107.0F7.5 D793N Wild-type 92.0F12.5 Wild-type E1455Q 55.0F0.0 a Km values (for wild-type, D793E, D793L and D793N, n=5; for E1455Q, n=) were derived from corresponding Michaelis-Menten curves shown in Fig. 5. R. Yang et al. Login to comment 210 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln The Km (ATP) value of E1455Q, the putative catalytic base mutant in NBD2, is slightly less than that of wild-type (Table 2), whereas the Km (ATP) values of D793E, D793L and D793N are slightly higher than that of wild-type (Table 2). Login to comment 220 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln Fig. 6A, D, G, J and M show the autoradiograms reflecting [a-32 P]-8-N3ATP labeling of wild-type, D793E, D793L, D793N and E1455Q. Labeling was quantified by Packard Instant Imager and plotted against the concentration of [a-32 P]-8-N3ATP (Fig. 6B, C, E, F, H, I, K, L, N and O). Login to comment 222 ABCC1 p.Asp793Glu The Kd (ATP) value for D793E mutated NBD1, co-expressed with wild-type NBD2, is slightly less than that of wild-type NBD1 (Table 3), implying moderately increased affinity for ATP. Login to comment 223 ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu However, the Kd (ATP) value for wild-type NBD2 co-expressed with D793E mutated NBD1 is slightly higher than that of the wild-type NBD2 co-expressed with wild-type NBD1, implying that the D793E mutated NBD1 has an effect on the wild-type NBD2. Login to comment 224 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn The Kd (ATP) values for D793L and D793N mutated NBD1 co-expressed with wild-type NBD2 are almost three fold higher than that of wild-type NBD1 (Table 3), implying that the mutation of this acidic D793 residue to a non-acidic amino acid decreased k1 (lower rate of binding) and/or increased kÀ1 (higher rate of releasing), i.e. lower affinity. Login to comment 225 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn The mutation of D793L or D793N does not have a significant effect on the wild-type NBD2 (Table 3). Login to comment 226 ABCC1 p.Glu1455Gln The Kd for E1455Q mutated NBD2 co-expressed with wild-type NBD1 is almost five fold higher than that of wild-type NBD2 (Table 3), implying that the substitution of the putative catalytic E1455 residue with a non-acidic amino acid decreased k1 (lower rate of binding) and/or increased kÀ1 (higher rate of releasing), i.e. lower affinity. Login to comment 227 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn In contrast to the counterpart NBD1 mutants D793N and D793L, the substitution of the E1455 with a non-acidic amino acid has an effect on the wild-type NBD1 (Table 3). Login to comment 229 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn The substitution of the putative catalytic D793 residue with a non-acidic amino acid increases release rate of the bound ATP The higher Kd (ATP) values of D793L and D793N mutated NBD1 were interpreted as that the binding rate was decreased whereas the release rate of the bound ATP was increased. Login to comment 237 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln Fig. 7A, D, G, J and M show the autoradiograms reflecting [g-32 P]-8-N3ATP labeling of wild-type, D793E, D793L, D793N and E1455Q. Labeling was quantified by Packard Instant Imager and plotted against the incubation time (Fig. 7B, C, E, F, H, I, K, L, N and O). Login to comment 240 ABCC1 p.Glu1455Gln / Biochimica et Biophysica Acta 1668 (2005) 248-261 255 expected, most of the labeling disappeared within a short time, except for the E1455Q mutated NBD2 (Fig. 7M and O). Login to comment 241 ABCC1 p.Glu1455Gln The labeling with [g-32 P]-8-N3ATP at the C-half containing E1455Q mutated NBD2 is increased almost three fold within 2 min incubation at 37 8C and the T1/2 of this mutated NBD2 is much longer than that of wild-type (Table 4). Login to comment 243 ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln / Biochimica et Biophysica Acta 1668 (2005) 248-261256 decreased Kd) at the E1455Q mutated NBD2 at 37 8C, consistent with our previous finding [51]; (2) the bound ATP at the E1455Q mutated NBD2 cannot be efficiently hydrolyzed due to the substitution of the putative catalytic acidic residue with a non-acidic amino acid (greatly decreased k2); (3) the release rate of the bound ATP from this mutated NBD2 is much lower (decreased kÀ1) than that of wild-type, leading to a longer T1/2 value (Table 4). Login to comment 245 ABCC1 p.Glu1455Gln In addition, the T1/2 value for the wild-type NBD1 co-expressed with E1455Q mutated NBD2 is slightly longer than that of wild-type N-half co-expressed with the wild-type C-half (Table 4), perhaps reflecting the stabilization effect of the bound nucleotide at NBD1 by the occluded nucleotide at NBD2 [28]. Login to comment 246 ABCC1 p.Glu1455Gln These results are consistent with the lower Km (ATP) value of E1455Q than that of the wild-type (Table 2) and the low ability to transport LTC4 (Figs. 3 and 5). Login to comment 247 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln In the cases of D793E, D793L and D793N, the T1/2 values (for NBD1 and NBD2) are shorter than that of the wild-type and E1455Q (Table 4). Login to comment 259 ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln The substitution of the counterpart putative catalytic residue E1455 with a non-acidic amino acid (E1455Q) almost completely abolished the ATP-dependent LTC4 transport (Figs. 3 and 5), presumably due to the greatly decreased ATPase activity of this E1455Q mutated NBD2 (Fig. 7M and O). Login to comment 263 ABCC1 p.Asp793Glu ABCC1 p.Glu1455Gln In addition, the substitution of this putative catalytic residue D793 with a longer spacer-arm negatively charged Glu enhances its hydrolytic capacity [43], but does not increase the ATP-dependent solute transport activity of D793E/E1455Q mutated MRP1 (Fig. 4) or markedly Fig. 6. Login to comment 264 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn D793L and D793N mutated NBD1s have higher Kd values than that of wild-type. Login to comment 267 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn A, D, G, J, and M: Autoradiograms of [a-32 P]-8-N3ATP labeled wild-type N-half co-expressed with wild-type C-half; D793E mutated N-half+wild-type C-half; D793L mutated N-half+wild-type C-half; D793N mutated N-half+wild-type C-half; and wild-type N-half+E1455Q mutated C-half. Login to comment 270 ABCC1 p.Asp793Glu E and F: D793E mutated N-half (E) co-expressed with wild-type C-half (F). Login to comment 271 ABCC1 p.Asp793Leu H and I: D793L mutated N-half (H) co-expressed with wild-type C-half (I). Login to comment 272 ABCC1 p.Asp793Asn K and L: D793N mutated N-half (K) co-expressed with wild-type C-half (L). Login to comment 273 ABCC1 p.Glu1455Gln N and O: Wild-type N-half (N) co-expressed with E1455Q mutated C-half (O). Login to comment 274 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln Table 3 Substitution of D793 with a non-acidic amino acid decreases affinity for ATP Sample Kd of NBD1 (AM ATP)a Kd of NBD2 (AM ATP)N-half C-half Wild-type Wild-type 11.7F2.8 32.7F2.3 D793E Wild-type 7.8F4.1 41.0F8.1 D793L Wild-type 30.5F2.5 32.9F1.9 D793N Wild-type 28.4F4.5 33.7F0.7 Wild-type E1455Q 19.4F3.3 155.8F9.0 a The Kd (AM ATP) values (for wild-type, n=12; D793E, n=9; D793L and E1455Q, n=; D793N, n=8) were derived from Fig. 6. R. Yang et al. Login to comment 283 ABCC1 p.Glu1455Gln The substitution of the putative catalytic E1455 with a non-acidic amino acid Q greatly increased the Kd (ATP) of the E1455Q mutated NBD2 (Fig. 6 and Table 3), meaning increased kÀ1 and decreased k1 on ice. Login to comment 284 ABCC1 p.Glu1455Gln This E1455Q mutation at NBD2 also has a negative effect on the co-expressed wild-type NBD1, leading to the increased Kd (Table 3), indicating the increased kÀ1 and decreased k1 on ice. Login to comment 285 ABCC1 p.Glu1455Gln However, the properties of the E1455Q mutated protein at 37 8C are different from that on ice. Login to comment 286 ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln The [g-32 P]-8-N3ATP bound to the E1455Q mutated NBD2 cannot be efficiently hydrolyzed (greatly decreased k2) but occluded there (decreased kÀ1) (Fig. 7M and Table 4); in the meantime, the occluded [g-32 P]-8-N3ATP at the E1455Q mutated NBD2 stabilized the bound [g-32 P]-8-N3ATP [28] at the wild-type NBD1 co-expressed with the E1455Q mutated NBD2 (Fig. 7N), meaning decreased kÀ1; all these changes lead to a decreased Km (ATP) value and an inability to transport LTC4 (Table 1 and 2). Login to comment 287 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn In contrast, interestingly, the substitution of the putative catalytic residue D793 in NBD1 with a non-acidic amino acid, such as D793L or D793N, increased the rate of ATP-dependent LTC4 transport ([29] and Fig. 3). Login to comment 288 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn This radical substitution increased the Kd (ATP) values of the D793L or D793N mutated NBD1 (Fig. 6 and Table 3), where Kd=kÀ1/k1, since ATP hydrolysis on ice is limited. Login to comment 289 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn The increased Kd of D793N or D793L means increased kÀ1, in other words, increased releasing rate of the bound ATP, and/or decreased k1, in other words, decreased the rate of ATP binding. Login to comment 290 ABCC1 p.Asp793Leu ABCC1 p.Asp793Asn The time required to lose 50% of the bound ATP from the D793L or D793N mutated NBD1 and the co-expressed wild-type NBD2 is much shorter than that of the wild-type (Fig. 7 and Table 4), implying the increased kÀ1 and/or decreased k1 values. Login to comment 295 ABCC1 p.Glu1455Gln E1455Q mutated NBD2 greatly diminishes the rate of ATP hydrolysis at the mutated NBD2. Login to comment 301 ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn A, D, G, J, and M: Autoradiograms of [g-32 P]-8-N3ATP labeled wild-type N-half co-expressed with wild-type C-half; D793E mutated N-half+wild-type C-half; D793L mutated N-half+wild-type C-half; D793N mutated N-half+wild-type C-half; and wild-type N-half+E1455Q mutated C-half. Login to comment 306 ABCC1 p.Asp793Glu E and F: D793E mutated N-half (E) co-expressed with wild-type C-half (F). Login to comment 307 ABCC1 p.Asp793Leu H and I: D793L mutated N-half (H) co-expressed with wild-type C-half (I). Login to comment 308 ABCC1 p.Asp793Asn K and L: D793N mutated N-half (K) co-expressed with wild-type C-half (L). Login to comment 309 ABCC1 p.Glu1455Gln N and O: Wild-type N-half (N) co-expressed with E1455Q mutated C-half (O). Login to comment 310 ABCC1 p.Asp793Leu ABCC1 p.Asp793Leu ABCC1 p.Asp793Glu ABCC1 p.Asp793Glu ABCC1 p.Asp793Asn ABCC1 p.Asp793Asn ABCC1 p.Glu1455Gln ABCC1 p.Glu1455Gln Table 4 Release rate of the bound nucleotide at the wild-type and mutated NBDs Sample T1/2 of NBD1 (min)a T1/2 of NBD2 (min)N-half C-half Wild-type Wild-type 5.3F0.3 3.7F2.0 D793E Wild-type 3.0F0.7 3.4F1.0 D793L Wild-type 2.3 2.3 D793N Wild-type 2.5F0.5 2.2F0.7 Wild-type E1455Q 6.1F0.3 25.6F2.4 a The T1/2 value (for wild-type, D793E, D793N and E1455Q, n=3; for D793L, n=1) is the time required to release 50% of the bound nucleotide and was derived from Fig. 7. R. Yang et al. Login to comment