ABCMdb

PMID: 11123914

Biswas EE, Biswas SB
The C-terminal nucleotide binding domain of the human retinal ABCR protein is an adenosine triphosphatase.
Biochemistry. 2000 Dec 26;39(51):15879-86., [PubMed]

Sentences

No. Mutations Sentence Comment

11 ABCA4 p.Leu2027Phe The NBD2 expression plasmid was used to generate a Leu2027Phe mutation associated with Stargardt disease. Login to comment 77 ABCA4 p.Leu2027Phe The NBD2 expression vector pET29aNBD2 was used as template, 12 cycles of PCR, and each cycle was 30 s at 95 °C, 30 s at 50 °C, and 15 min at 68 °C using the complementary oligonucleotides 5'-AGT TTG ATG CAA TCG ATG AGC TGT TCA CAG GAC GAG AAC ATC TTT AAC ATC TTT ACC TT-3' and 5'-AAG GTA AAG ATG TTC TCG TCC TGT GAA CAG CTC ATC GAT TGC ATC AAA CT-3') as mutagenic primers to generate NBD2 L2027F. Login to comment 162 ABCA4 p.Leu2027Phe Influence of a Leu2027Phe Mutation on ATP Hydrolysis by NBD2 Polypeptide. Login to comment 163 ABCA4 p.Leu2027Phe The mutation L2027F has been previously reported in some patients suffering from Stargardt macular degeneration (9, 21). Login to comment 165 ABCA4 p.Leu2027Phe Using a site-directed mutagenesis protocol, the mutation (L2027F) was introduced into the pET29aNBD2 plasmid, following which the mutant protein was expressed and purified. Login to comment 168 ABCA4 p.Leu2027Phe The L2027F point mutation in the NBD2 polypeptide increased the affinity of binding Km (46 µM) and resulted in a decrease in the rate of ATP hydrolysis, 16.6 nmol of ATP min-1 mg-1 . Login to comment 194 ABCA4 p.Leu2027Phe This protein was also able to utilize dATP as a substrate, but appeared to do so with FIGURE 5: (A) DNA sequence analysis of NBD2mut1 (L2027F). Login to comment 195 ABCA4 p.Leu2027Phe Chromatogram of DNA sequence analysis surrounding the region of the L2027F mutation of NBD2. Login to comment 197 ABCA4 p.Leu2027Phe (B) ATPase activity of the Leu2027Phe NBD2 mutant protein. Login to comment 215 ABCA4 p.Leu2027Phe As we indicated under Results, this increase in binding affinity (also observed with the Leu2027Phe mutant) was accompanied by a decrease in the rate of hydrolysis (Vmax). Login to comment 216 ABCA4 p.Leu2027Phe Functional Consequence of L2027F Mutation Associated with Stargardt Disease. Login to comment 217 ABCA4 p.Leu2027Phe The L2027F mutation in the full-length ABCR gene is associated with Stargardt disease (SGD1) (9, 21). Login to comment 219 ABCA4 p.Leu2027Phe Introduction of the point mutation L2027F in the NBD2 polypeptide was observed to increase the affinity of ATP binding 14-fold (Km ) 46 µM) while decreasing the rate of ATP hydrolysis (Vmax ) 16.6 nmol of ATP min-1 mg-1). Login to comment 222 ABCA4 p.Leu2027Phe The observed defects in retinal transport associated with the L2027F mutation may be related, at least in part, to the decreased rate of ATP hydrolysis. Login to comment 230 ABCA4 p.Leu2027Phe What we have demonstrated in this report is that the Leu2027Phe mutation does indeed lead to a significant decrease in the basal level of ATPase of the NBD2 domain of ABCR. Login to comment