Abstract
The cDNA encoding the C-terminal nucleotide-binding domain (NBD2) from mouse P-glycoprotein involved in multidrug resistance was obtained from adrenal cell mRNA and amplified by reverse transcriptase polymerase chain reaction. NBD2 was highly overexpressed in Escherichia coli in fusion with glutathione S-transferase and could be purified after efficient thrombin cleavage. Both fused and purified NBD2 bound TNP (2',3'-O-(2,4,6-trinitrophenyl))- derivatives of nucleotides with high affinity. TNP-ATP or TNP-ADP binding at micromolar concentrations produced a characteristic blue-shifted enhancement of extrinsic fluorescence and was specifically prevented or chased by ATP or ADP at millimolar concentrations. A similar affinity binding was monitored by quenching of intrinsic fluorescence. The spectrum of fusion protein, containing 5 tryptophan residues, was maximally quenched at 328 nm upon interaction with TNP-nucleotides. TNP-GTP exhibited a lower affinity than TNP-ATP but produced a higher maximal quenching (44% instead of 28%). The intrinsic fluorescence of purified NBD2, containing a single tryptophan residue, exhibited a narrow spectrum with a maximum at 328 nm characteristic of a hydrophobic tryptophan environment. A high quenching was observed upon nucleotide interaction with similar affinity. The results put forward a functional role for the tryptophan-containing sequence of P-glycoprotein NBD2 that was not detected up to now.
Highlights
ThecDNA encoding the C-terminal nucleotide-bind- with overexpression of P-glycoprotein, a membrane transporter ing domain (NBD2) from mouse P-glycoprotein involvtehdat actively extrudes thecytotoxic drugs usingATP hydrolysis in multidrug resistance was obtained from adrenal celals an energysource.This membrane m R N A and amplifiedby reverse transcriptase polymer- protein is encoded by the mdr gene family composed of two ase chain reaction.NBD2 was highly overexpressed in members in man, mdrl and mdr2o,r three in mouse, m d r l
23) was linearized with BamHI and EcoRI and ligated with cDNA to generatea plasmid coding for a n in-frame fusion proteincom- Overexpression and Purification of NBD2-From current posed of glutathione S-transferase and nucleotide-binding domain 2 (GST-NBD2).The designof pGEX-KTincluded a thrombin cleavage and an upstream glycine kinkerengineeredbetweentheGSTand site models of P-glycoprotein derived from sequence C-terminal NBDB is predicted to beextrinsic, analysis
Ex- plified by reversetranscriptase PCR frommouse adrenal pression of the fusion protein was induced wit0h.4 mM IPTG for 2 h at 30 "C; cells were harvestedby centrifugation at 5,500 x g for 10 min at 4 "C, and resuspended in PBS buff(e1r50 mM NaCl, 16 mM Na,HPO, 4 mM NaH,PO, pH 7.3) containing 1%(v/v) Triton X-100, 1mM MgCl, and 1 mM phenylmethylsulfonyl fluoride
Summary
Weight markers were obtainedfrom Pharmacia Biotech Inc. The TNP- The yeast mitochondrial F,-ATPase taken as a reference gave the exnucleotides (sodiumsalts)came from Molecular Probes, InIPc.TG,ATP, pected specific activity under the experimental conditions usead,20%. RNA was extracted in guanidinium thiocyanate using the Total RNA Fluorescence Emission Measurements-Experiments were performed isolation kitfrom Promega. Murine leukemiavirus reverse transcriptase) and subsequent amplifi- Fluorescence measurements were performedby diluting protein socation (using the GeneAmp polymerase chain reaction (PCR) process lutions inl ml (final volumeo)f 50 mM Tris-HC1, 10%glycerol, a t pH 8.0 and AmpliTaq DNA polymerase) were performed with the GeneAmp for GST-NBDP fusion protein or pH 7.5for purified NBD2. The spectra were corrected for buffer contribution to for 30 s, annealing a t 55 "C for 30s and elongationat 72 "C for 60 s plus fluorescence and nucleotide inner fieltfeferct, by control experiments in.
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