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J. Biol. Chem., Vol. 277, Issue 24, 21759-21767, June 14, 2002

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Biochemical Defects in Retina-specific Human ATP Binding Cassette Transporter Nucleotide Binding Domain 1 Mutants Associated with Macular Degeneration^*

Tatiana Suárez, Subhasis B. Biswas^§, and Esther E. Biswas^¶

From the  Department of Laboratory Sciences, Program in Biotechnology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the ^§ Department of Molecular Biology, School of Osteopathic Medicine & Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08043

The retina-specific human ABC transporter (ABCR) functions in the retinal transport system and has been implicated in several inherited visual diseases, including Stargardt disease, fundus flavimaculatus, cone-rod dystrophy, and age-related macular degeneration. We have previously described a general ribonucleotidase activity of the first nucleotide binding domain (NBD1) of human ABCR (Biswas, E. E. (2001) Biochemistry 40, 8181-8187). In this communication, we present a quantitative study analyzing the effects of certain disease-associated mutations, Gly-863  Ala, Pro-940  Arg, and Arg-943  Gln on the nucleotide binding, and general ribonucleotidase activities of this domain. NBD1 proteins, harboring these mutations, were created through in vitro site-specific mutagenesis and expressed in Escherichia coli. Results of the enzyme-kinetic studies indicated that these mutations altered the ATPase and CTPase activities of NBD1. The G863A and P940R mutations were found to have significant attenuation of the rates of nucleotide hydrolysis and binding affinities. On the other hand, the R943Q mutation had small, but detectable reduction in its nucleotidase activity and nucleotide binding affinity. We have measured the nucleotide binding affinities of NBD1 protein and its mutants quantitatively by fluorescence anisotropy changes during protein binding to ethenoadenosine ATP (ATP), a fluorescent ATP analogue. We have correlated the dissociation constant (K_D) and the rates of nucleotide hydrolysis (V_max) of NBD1 and its mutants with the available genetic data for these mutations.

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