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TM Suarez-Cortes, EE Biswas, SB Biswas; Biochemical Analysis Of Genetic Mutations Related To Visual Disorders In The NBD1 Domain Of ABC Transporter (ABCR) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1402.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mutations on the ABCR gene have been clearly related to inherited human visual diseases such as Stargardt disease, fundus flavimaculatus, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. We have analyzed genetic mutations located in α and ß segments of the cytoplasmic nucleotide binding domain 1 (NBD1), and delineated the nucleotide binding and hydrolysis properties, as well as the structure and function of ABCR protein. Here, we describe the results obtained for NBD1 proteins carrying mutations that are implicated in visual diseases as reported in earlier genetic studies. Methods: Based on the sequence of human ABCR gene mutations, and their related phenotypes, we selected several mutations, which vary in their pathogenicity level in humans. We designed mutagenic primers representing each disorder and carried out in vitro mutagenesis. The nucleotidase activity of each protein was evaluated as described by our group earlier (Biochemistry (2000) 39, 15879-86 and (2001) 40, 8181-7. The ATP binding property was sutied using fluorescence anisotropy. Results: The mutant proteins (G863A, P940R, R943Q, K116A+T117A, N965S, E1087K) were expressed in E. coli (BL21). All proteins were extracted from inclusion bodies and refolded, obtaining full activity. The G863A together with P940R mutant proteins showed the lowest ATPase and CTPase activity in comparison with the wild type NBD1 protein. However, the R943Q mutant protein exhibited only a modest decrease of the ATPase and CTPase activity. The fluorescence anisotropy data indicate that ATP binding ability was also affected similarly. Conclusion: We have successfully utilized quantitative structure function analysis such as enzyme kinetics and fluorescence spectroscopy to characterize ABCR mutations and correlate with disease pathogenesis. Our results appear to relate well with previous genetic studies involving ABCR genetic mutations and severity of disease pathogenesis (Briggs C., et al., (2001) IOVS 42, 2229-2236). CR: None
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