Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.

We wished to determine if the two nucleotide-binding domains (NBD) of P-glycoprotein are functionally equivalent and interchangeable, and if not, which segments and amino acids are important for proper function of each NBD within the context of the C- or N-terminal P-glycoprotien halves. For this, we constructed and tested the biological activity in yeast and mammalian cells of a series of chimeric mdr3 cDNAs in which discrete domains of the N-terminal NBD (NBD1) were replaced by the homologous segments of the C-terminal NBD (NBD2). Although most NBD1 segments could be replaced without loss of P-glycoprotein function, exchange of small segments near the Walker B motif caused a dramatic reduction in Adriamycin, actinomycin D, and colchicine resistance in LR73 cells, as well as in FK506 resistance and STE6 complementation in yeast. Site-directed mutagenesis identified amino acid positions 522-525 (ERGA-->DKGT) and 578 (Thr-->Cys) as essential for proper function of NBD1 in the context of the N-terminal half P-glycoprotein. In addition, the observed phenotype of the mutants (altered drug resistance profile) suggests that these residues may participate directly or indirectly in substrate interactions and are possibly implicated in signal transduction from NBDs to transmembrane domains, the primary sites of drug binding in P-glycoprotein.

advertising feature an8 | December 2008 | nature methods application notes cell Biology biotinylated antibody bound to streptavidin-coated donor beads and a second antibody conjugated to AlphaLISA acceptor beads. The binding of the two antibodies to the analyte brings donor and acceptor beads into proximity. Laser irradiation of donor beads at 680 nm generates a flow of singlet oxygen, triggering a cascade of chemical events in nearby acceptor beads, which results in a chemiluminescent emission at 615 nm. In competitive AlphaLISA immunoassays, a biotinylated analyte bound to streptavidin donor beads is used with an antibody conjugated to AlphaLISA acceptor beads. Rapid and simple quantification of analytesAlphaLISA assays are performed following simple 'mix-and-measure' protocols with reduced hands-on and total assay times compared to ELISAs (Fig. 2). Homogeneous AlphaLISA assays eliminate the need for multiple washes to separate bound from unbound assay components. Miniaturization and automationMiniaturization is a key consideration for reducing screening cost and increasing throughput during the drug-discovery process.AlphaLISA assays are truly miniaturizable and automatable, with ELISA is the most widely used detection platform for the quantification of analytes in biological samples. Because they require multiple washes, ELISAs are difficult to adapt to high throughput and automation. Their relatively narrow dynamic range often requires testing more than one sample dilution. There is clearly a need for simple and more robust alternatives for the quantification of biomarkers in a high-throughput screening format. The new AlphaLISA platform has been specifically designed for that purpose for both the research and drug-discovery fields.The AlphaLISA bead-based technology relies on PerkinElmer's exclusive amplified luminescent proximity homogeneous assay (AlphaScreen ® ) and uses a luminescent oxygen-channeling chemistry 1 . AlphaLISA protocols can be set up as sandwich or competition immunoassays. In a sandwich assay (Fig. 1), an analyte is captured by a AlphaLISA immunoassays: the no-wash alternative to ELISAs for research and drug discovery PerkinElmer's bead-based AlphaLISA® immunoassays are designed for the detection of analytes in biological samples. These chemiluminescent, no-wash assays are ideally suited for miniaturization and automation. They exhibit remarkable sensitivity, wide dynamic range and robust performance that compares advantageously with conventional enzyme-linked immunosorbent assay (ELISA).

We have determined the complete nucleotide sequence of the mouse gene encoding the neurofilament NF-H protein. The C-terminal domain of NF-H is very rich in charged amino acids (aa) and contains a 3-aa sequence, Lys-Ser-Pro, that is repeated 51 times within a stretch of 368 aa. The location of this serine-rich repeat in the phosphorylated domain of NF-H indicates that it represents the major protein kinase recognition site. The nfh gene shares two common intron positions with the nfl and nfm genes, but has an additional intron that occurs at a location equivalent to one of the introns in non-neuronal intermediate filament-coding genes. This additional nfh intron may have been acquired via duplication of a primordial intermediate filament gene.

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