Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Multidrug-resistant human tumor cells overexpress the MDR1 gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/vol) octyl beta-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCl fraction, enriched in P-glycoprotein but devoid of Na,K-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation. Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine greater than daunomycin greater than actinomycin D greater than verapamil greater than colchicine. The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 microM) but were not affected by agents that inhibit other ATPases and phosphatases. These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 mumol per min per mg of protein).
 ...
PMID:Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. 135 64

A newly synthesized isoquinolinesulfonamide named H-85; N-[2-[N-formyl-N-[[3-(4-chlorophenyl)-2-propenyl] amino] ethyl]-5-isoquinolinesulfonamide was found to reverse drug resistance in multidrug resistant P388 murine leukemic cells (P388/ADR). The energy-dependent extrusion of [3H]vinblastine from P388/ADR-cells was significantly suppressed by 10 microM H-85 but not so the efflux from the sensitive P388 cells. A 140-kDa protein overexpressed in P388/ADR cells was photoaffinity labeled with a vinblastine analog; N-(P-azid-[3-125I]salicyl-N'-(beta-aminoethyl) vindesine and H-85 selectively inhibited photolabeling of the 140-kDa protein. This 140-kDa protein was purified to apparent homogeneity by succeeding steps of phosphocellulose, DEAE-cellulose, and W-66 (a derivative of H-85)-coupled sepharose chromatography. The purified 140-kDa protein proved to be immunopositive with the P-glycoprotein-specific monoclonal antibody, C219.
 ...
PMID:Obviation of drug resistance and affinity purification of P-glycoprotein by isoquinolinesulfonamides. 168 33

Cells containing increased levels of the membrane phosphoprotein P-glycoprotein exhibit a multidrug-resistant phenotype. In the present study we have analyzed protein kinases capable of phosphorylating P-glycoprotein in membranes of HL60 cells isolated for resistance to vincristine. Analysis of this system demonstrates that in isolated membranes the protein kinase inhibitor staurosporine greatly reduces P-glycoprotein phosphorylation. In contrast, the kinase inhibitor H-7 does not affect this reaction. Fractionation of solubilized membrane proteins from sensitive and resistant cells on DEAE-cellulose reveals a major protein kinase (PK-1) which exhibits optimal activity in the presence of Mn2+ and histone H1. This enzyme fraction does not contain detectable levels of protein kinase C or cAMP-dependent protein kinase. PK-1 phosphorylation of two endogenous proteins is, however, greatly enhanced in the presence of phosphatidylserine or phosphatidyl-inositol. In reaction mixtures containing Mg2+ or Mn2+ in the absence of phospholipid, PK-1 from resistant cells phosphorylates an endogenous protein of 180 kilodaltons (P180), which exhibits an electrophoretic mobility identical to P-glycoprotein. In parallel experiments with PK-1 from sensitive cells there is no detectable phosphorylation of a P180 protein. P180 phosphorylated by PK-1 from resistant cells is immunoprecipitated by antibody against P-glycoprotein. Additional studies demonstrate that PK-1 is capable of phosphorylating specific synthetic peptides which correspond to the sequence of P-glycoprotein. Peptide phosphorylation occurs at both serine and threonine residues. These studies thus identify a novel membrane-associated protein kinase in HL60 cells which is capable of phosphorylating P-glycoprotein. This enzyme may have an important role in regulating levels of multidrug resistance.
 ...
PMID:Characterization of a membrane-associated protein kinase of multidrug-resistant HL60 cells which phosphorylates P-glycoprotein. 196 66

The anti-drug resistance effect of three derivatives (AR-1, AR-2 and AR-3) of [1,2,5-trimethyl-4-phenyl-4-beta-(N,N-disubstituted-ethylamino)] piperidines, that were evaluated as calcium and calmodulin antagonists, was studied on doxorubicin (ADM) and vincristine (VCR) resistant Sarcoma-45 inoculated rats. Treatment with ADM (5 mg/kg) or VCR (3 mg/kg) alone, as well as with AR-1, AR-2 or AR-3 (50 mg/kg) alone, had no effect on tumor growth. However, AR-2 in dose 50 mg/kg (calmodulin antagonist), but not AR-1 and AR-3 (calcium channel blocker), administered with ADM (5 mg/kg) or VCR (3 mg/kg), significantly suppressed tumor growth 80% and 70%, respectively. Two rats treated with ADM/AR-2 and one treated with VCR/AR-2 were cured. 170 kDa protein was purified from sarcoma-45 tumor cells to apparent homogeneity by successive steps of phosphocellulose, DEAE-cellulose, and AR-2-coupled sepharose chromatography. The protein proved to be immunopositive with the P-glycoprotein-specific monoclonal antibody. It is concluded that the effect of AR-2 can be explained by both hydrophobic and electrostatic interaction with a protein target (170 kDa P-glycoprotein) in resistant sarcoma-45 tumor cell's membrane.
 ...
PMID:Reversal of drug resistance in sarcoma-45 by the new calmodulin antagonist--trihydrochloride of [1,2,5-trimethyl-4-phenyl-4-beta-[N-(beta-ethylamino)-N-4'-methoxybe nzy l]-ethylamino] piperidine (AR-2). 1063 81

Membrane vesicles from the multidrug-resistant KB-V1 and KB-C1 cell lines overexpressing P-glycoprotein (Pgp), responsible for pleiotropic chemotherapeutic agents resistance, were solubilized with octyl-glucoside (OG-EX) and further fractionated on DEAE-sepharose column with increased concentrations of NaCl. The fraction containing Pgp (F3) was reconstituted into proteoliposomes (F3-PLP). Comparisons of the phosphorylation levels of Pgp achieved throughout the purification and reconstitution steps were addressed in this study. The [delta32 P] ATP-driven phosphorylation of Pgp was strongly increased in OG-EX, decreased in F3 and not detected in F3-PLP, when compared to Pgp phosphorylation in native plasma membrane vesicles. [delta32 P]ATP-phosphorylation of Pgp in F3-PLP could be restored by exogenously added PKC or by the catalytic sub-unit of PKA. The vanadate-induced hyperphosphorylation effect on Pgp by [delta32 P]ATP observed with plasma membrane vesicles was maintained in OG-EX, but was lost in F3 and did not enable labelling in F3-PLP. Enhancement of [delta32 P]-labelling of native Pgp via [delta32 P]ATP combined with GTP was maintained and also triggered phosphorylation of purified/reconstituted Pgp in F3-PLP as well. Altogether, our data suggest differential phosphorylation patterns of the transporter linked to environmental molecular composition (lipids, presence of detergent) and structure (unfolded versus embedded). In addition, restoration by GTP of Pgp phosphorylation by [delta32 P]ATP in the frame of F3-PLP suggests intra-molecular modulations and hints that other phosphorylation sites and processes, different from the classic ones involving PKC and/or PKA, may participate in the transporter's mechanism.
 ...
PMID:Differential phosphorylation patterns of P-glycoprotein reconstituted into a proteoliposome system: insight into additional unconventional phosphorylation sites. 1630 79