ABCMdb

PMID: 16500904

Takahashi K, Kimura Y, Kioka N, Matsuo M, Ueda K
Purification and ATPase activity of human ABCA1.
J Biol Chem. 2006 Apr 21;281(16):10760-8. Epub 2006 Feb 24., [PubMed]

Sentences

No. Mutations Sentence Comment

20 ABCA1 p.Lys939Met Like these xenobiotic transporters, ABCA1 K939M mutant, in which lysine, indispensable for the hydrolysis of ATP by various ABC proteins (8-11), was substituted by methionine, was impaired in apoA-I-dependent PL and cholesterol efflux (3, 5). Login to comment 22 ABCA1 p.Lys939Met However, because the ABCA1 K939M mutant is defective in its interaction with apoA-I (3, 5), it is also possible that ATP binding and/or ATP hydrolysis cause conformational changes of ABCA1, which are required for the interaction with apoA-I, and ABCA1 functions as a regulator in HDL formation (12) as SUR does in the ATP-sensitive potassium channel complex (13). Login to comment 32 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met 2 The abbreviations used are: ABCA1, ATP-binding cassette protein A1; ABCA1 MM, ABCA1 K939M-K1952M; MDR, multidrug resistance; MRP, multidrug resistance-related protein; NBF, nucleotide-binding fold; apo, apolipoprotein; HDL, high density lipoprotein; FC, free cholesterol; PL, phospholipid; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; SM, sphingomyelin; PG, phosphatidylglycerol; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; DPPC, 1,2-dipalmitoylphosphatidylcholine; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; DPPE, 1,2- dipalmitoylphosphatidylethanolamine; DPPG, 1,2-dipalmitoylphosphatidylglycerol; Sf9, Spodoptera frugiperda 9; HEK, human embryo kidney; NGF, N-glycosidase F; DDM, n-dodecyl-beta-D-maltoside; NTA, nitrilotriacetic acid. Login to comment 33 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met 2 The abbreviations used are: ABCA1, ATP-binding cassette protein A1; ABCA1 MM, ABCA1 K939M-K1952M; MDR, multidrug resistance; MRP, multidrug resistance-related protein; NBF, nucleotide-binding fold; apo, apolipoprotein; HDL, high density lipoprotein; FC, free cholesterol; PL, phospholipid; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; SM, sphingomyelin; PG, phosphatidylglycerol; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; DPPC, 1,2-dipalmitoylphosphatidylcholine; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; DPPE, 1,2- dipalmitoylphosphatidylethanolamine; DPPG, 1,2-dipalmitoylphosphatidylglycerol; Sf9, Spodoptera frugiperda 9; HEK, human embryo kidney; NGF, N-glycosidase F; DDM, n-dodecyl-beta-D-maltoside; NTA, nitrilotriacetic acid. Login to comment 46 ABCA1 p.Lys939Met ABCA1 p.Lys939Met To construct the NBF1 Walker A lysine mutant, ABCA1 K939M, DNA fragments containing a K939M missense mutation were generated by a two-step PCR method with two pairs of primers, 5b18;-GGGCCACAATGGAGCGGGGATGACGACCAC-3b18; and 5b18;-CTGTCCCCCAGGACGTCCGCTTCATCCATG-3b18; and 5b18;-GTG- GTCGTCATCCCCGCTCCATTGTGGCCC-3b18; and 5b18;-CTCAGTG- GCTGTGATCATCAAGGGCATCG-3b18;. Login to comment 49 ABCA1 p.Lys939Met ABCA1 p.Lys939Met To construct the NBF1 Walker A lysine mutant, ABCA1 K939M, DNA fragments containing a K939M missense mutation were generated by a two-step PCR method with two pairs of primers, 5Ј-GGGCCACAATGGAGCGGGGATGACGACCAC-3Ј and 5Ј-CTGTCCCCCAGGACGTCCGCTTCATCCATG-3Ј and 5Ј-GTG- GTCGTCATCCCCGCTCCATTGTGGCCC-3Ј and 5Ј-CTCAGTG- GCTGTGATCATCAAGGGCATCG-3Ј. Login to comment 114 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 K939M-K1952M protein, expressed and purified in the same procedure, was as pure as the wild type (Figs. 1B and 2E). Login to comment 117 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met ABCA1 K939M-K1952M protein, expressed and purified in the same procedure, was as pure as the wild type (Figs. Login to comment 145 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met To confirm that ATPase activity is derived from purified ABCA1, a baculovirus for ABCA1 K939M-K1952M mutant, in which lysine 939 and lysine 1952 in the Walker A motif of nucleotide binding folds were replaced by methionines, was constructed. Login to comment 147 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met Purified ABCA1 K939M-K1952M protein showed little ATPase activity, less than 10 nmol/min/mg of protein (Fig. 5). Login to comment 149 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met To confirm that ATPase activity is derived from purified ABCA1, a baculovirus for ABCA1 K939M-K1952M mutant, in which lysine 939 and lysine 1952 in the Walker A motif of nucleotide binding folds were replaced by methionines, was constructed. Login to comment 151 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met Purified ABCA1 K939M-K1952M protein showed little ATPase activity, less than 10 nmol/min/mg of protein (Fig. 5). Login to comment 162 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met E, purified ABCA1 K939M-K1952M mutant, 0.5 òe;g (Coomassie Brilliant Blue R-250 staining). Login to comment 166 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met E, purified ABCA1 K939M-K1952M mutant, 0.5 ␮g (Coomassie Brilliant Blue R-250 staining). Login to comment 208 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met A, ATPase activity was measured in the presence of various concen- trationsofMgATP.F,wildtype;E,K939M-K1952M mutant. Login to comment 214 ABCA1 p.Lys939Met ABCA1 p.Lys1952Met A, ATPase activity was measured in the presence of various concen- trationsofMgATP.F,wildtype;E,K939M-K1952M mutant. Login to comment 262 ABCA1 p.Lys939Met Another critical issue in ABCA1-mediated pre-betaHDL formation is the role of apoA-I. Cellular cholesterol and PL efflux mediated by ABCA1 is fully dependent on lipid-free apoA-I. As the ABCA1 K939M mutant, whose Walker A lysine residue is substituted by methionine, is reported to be defect in its interaction with apoA-I, we expected that apoA-I would affect the ATP hydrolysis of ABCA1; however, we found no clear effects of apoA-I on the ATP hydrolysis of purified ABCA1 either before or after reconstitution in liposomes under our examination conditions (Fig. 8). Login to comment 270 ABCA1 p.Lys939Met As the ABCA1 K939M mutant, whose Walker A lysine residue is substituted by methionine, is reported to be defect in its interaction with apoA-I, we expected that apoA-I would affect the ATP hydrolysis of ABCA1; however, we found no clear effects of apoA-I on the ATP hydrolysis of purified ABCA1 either before or after reconstitution in liposomes under our examination conditions (Fig. 8). Login to comment