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

P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis. However, little is known about the structural and functional variations of P-gp in these tissues. With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa. To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2). We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa. In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed. Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form. N-Glycanase almost completely removed [3H]glucosamine labeling from P-gp. These data indicate that the major modification of P-gp is N-linked glycosylation. P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity. There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.
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PMID:Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues. 167 8

P-glycoprotein, a hydrophobic 170-kDa integral protein overexpressed in the plasma membrane of multidrug-resistant cells, is proposed to function as an ATP-dependent drug efflux pump. Plasma membrane preparations highly enriched in P-glycoprotein were isolated from multidrug-resistant cells by discontinuous sucrose gradient and Ca2+ precipitation methods. Several strategies were used for P-glycoprotein purification, with the goal being to achieve both good yields and purity, while keeping experimental manipulation to a minimum. P-glycoprotein was solubilized from the plasma membrane using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Immunoaffinity chromatography using C219 monoclonal antibody produced low yields of moderately pure protein. Sequential lectin affinity chromatography on RCA-120 followed by lentil lectin resulted in a P-glycoprotein preparation that showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A fraction of P-glycoprotein did not bind to RCA-120, most likely as a result of heterogeneous glycosylation. A combination of chromatography on RCA-120 followed by immunoaffinity chromatography on C219 resulted in low yields of very pure P-glycoprotein.
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PMID:Strategies for the purification of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. 168 82

The predicted cytoplasmic orientation and two-domain structure of the multidrug efflux pump P-glycoprotein were demonstrated with sequence-specific antibodies. We synthesized peptides corresponding to amino acid residues, Glu393-Lys408 (anti-P) and Leu1206-Thr1226 (anti-C) in P-glycoprotein from human mdr1 cDNA and used these peptides to produce polyclonal antibodies. From the primary structure of P-glycoprotein, and anti-C antibody is expected to recognize another position, Leu561-Thr581, in the duplicate structure of P-glycoprotein, but anti-P recognizes only one site. These antibodies bind to multidrug-resistant cells (KB-C2) with permeabilized plasma membrane but do not bind to nonpermeabilized KB-C2 cells or parental KB cells, supporting the predicted cytoplasmic orientation of these sequences. With immunoblotting of the membrane fractions from KB-C2 cells, a major 140-kDa polypeptide of the P-glycoprotein was detected with both anti-P and anti-C. Two minor polypeptides with molecular mass of 95 and 55 kDa were also detected. When membrane vesicles were digested mildly with trypsin, the amount of these two polypeptides increased. Anti-P detected only the 95-kDa polypeptide, and anti-C detected both 95- and 55-kDa polypeptides. Achromobacter lyticus protease I (lysyl endopeptidase) and Staphylococcus aureus V8 protease also produced two polypeptides with similar molecular weights. Absorption into lectin-agarose beads and labeling with [3H]glucosamine indicated that the 95-kDa polypeptide was glycosylated but that the 55-kDa polypeptide was not. These two polypeptides as well as P-glycoprotein were photoaffinity-labeled with a calcium channel blocker, [3H]azidopine, but most of the label was found in the 55-kDa polypeptide. The yield of labeled fragments from membrane vesicles photolabeled after digestion with trypsin was similar to that from membrane vesicles digested with trypsin after photolabeling. These data indicate 1) that the 95-kDa polypeptide is the fragment corresponding to the amino-terminal half of P-glycoprotein containing sugar chains; 2) that the 55-kDa polypeptide is the carboxyl-terminal half which was mainly labeled with [3H]azidopine; and 3) that P-glycoprotein has a relatively rigid structure with a small number of protease-sensitive sites and its global structure is not destroyed by tryptic cleavage.
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PMID:Cytoplasmic orientation and two-domain structure of the multidrug transporter, P-glycoprotein, demonstrated with sequence-specific antibodies. 247 41

A monoclonal antibody (C219) that recognizes the P-glycoprotein (Mr = 170,000) in plasma membranes of multidrug-resistant Chinese hamster ovary (CHO) cell lines was used to assay renal brush border membrane (BBM) and basolateral membrane (BLM) fractions for the presence of a cross-reactive polypeptide. The C219 antibody bound to a 155,000 dalton protein in immunoblots of rat BBM but not BLM proteins resolved by sodium dodecyl sulfate gel electrophoresis. The corresponding human kidney BBM and dog kidney BBM proteins had molecular weights of 170,000 and 160,000 respectively. The glycoprotein nature of the renal protein was shown by its sensitivity to N-glycanase treatment which reduced the apparent molecular weight of the dog protein to 120,000. In addition, dog P-glycoprotein could be bound to and eluted from immobilized wheat germ agglutinin. The molecular weight, antibody crossreactivity, glycosidase sensitivity and lectin binding show that this protein is a normal kidney analogue of the P-glycoprotein induced in multidrug resistant cell lines.
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PMID:Identification of P-glycoprotein in renal brush border membranes. 256 33

HL60 cells exhibiting a 140-fold increase in resistance to vincristine contain three surface membrane proteins with molecular weights of 210,000 (P210), 180,000 (P180), and 150,000 (P150) which are highly phosphorylated in vivo and in an in vitro system in the presence of Mn2+ and [gamma-32P]ATP. These phosphorylated proteins are either absent or present in very low levels in membranes of drug-sensitive cells. Growth of the vincristine-resistant isolate in the absence of drug results in a decrease in the level of resistance and a major reduction in the phosphorylation of P210 and P180. The phosphorylation of P150 is not altered in the revertant which still exhibits substantial levels of resistance. Further studies show that P210 and P180 are highly reactive with a monoclonal antibody against P-glycoprotein. These two proteins are present in only very low levels in revertant cells. The monoclonal antibody exhibits no reactivity with P150. In HL60 cells isolated for a 25-fold increase in vincristine resistance proteins reactive with P-glycoprotein monoclonal antibody are essentially absent. P150 is however highly phosphorylated in these cells. Additional experiments using lectin binding of 32P-labeled proteins demonstrates that P150 has properties distinct from P210 and P180. Analysis of drug uptake patterns in the vincristine-resistant isolates and the revertant shows that resistance is related to a reduced intracellular accumulation of drug. Reduced accumulation of vincristine is also found in HL60 cells isolated for resistance to Adriamycin. These cells are devoid of P-glycoprotein but contain phosphorylated P150. These results suggest that proteins P150, P180, and P210 may contribute to multidrug resistance in HL60 cells through a mechanism which involves reduced cellular accumulation of drug. P180 and P210 are structurally related whereas P150 is distinct from these two proteins.
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PMID:Mechanisms of multidrug resistance in HL60 cells: evidence that a surface membrane protein distinct from P-glycoprotein contributes to reduced cellular accumulation of drug. 289 87

HL60 cells resistant to Adriamycin contain a 32P-labeled, Mr 150,000 surface membrane protein (p150) which is not detected in cells sensitive to drug. The levels of phosphorylation of this protein increase with increasing levels of resistance. Analysis of plasma membranes prepared from cells labeled with [14C]glucosamine shows, however, that both sensitive cells and those exhibiting an 80-fold increase in drug resistance contain essentially identical levels of a highly glycosylated Mr 150,000 protein. Identical results are obtained when cells are labeled with [14C]galactose or [14C]mannose. Limited proteolytic digestion of [14C]glucosamine-labeled p150 from sensitive and resistant cells shows that the glycopeptides formed are identical. Additional studies involving binding of proteins to insolubilized lectin indicate that 32P-labeled p150 is glycosylated. Polyacrylamide gel electrophoresis of p150 followed by silver staining shows no difference in the levels of this protein in sensitive and 80-fold drug-resistant cells. Further studies show that two-dimensional tryptic peptide maps of 125I-labeled p150 of sensitive and resistant cells are essentially the same. It has also been found that treatment of cells with 12-O-tetradecanoylphorbol-13-acetate followed by [14C]glucosamine labeling results in a selective decrease in the glycosylation of p150 of sensitive and resistant cells. TPA has an identical effect on the phosphorylation of p150 in cells resistant to drug. HL60 cells have also been examined for the presence of the Mr 170,000 to 180,000 P-glycoprotein. Using immunoblot analysis with a monoclonal antibody directed against the P-glycoprotein we did not detect the presence of this protein in membranes of drug-sensitive or -resistant HL60 cells. The results of this study suggest that Adriamycin resistance in HL60 cells may be related to a modified form of a protein contained in cells sensitive to drug. Proteins active in drug resistance in this system may be distinct from those described for other cell lines.
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PMID:Adriamycin resistance in HL60 cells and accompanying modification of a surface membrane protein contained in drug-sensitive cells. 362 Nov 92

The overexpression of the P-glycoprotein, the MDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [3H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.
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PMID:Purification and reconstitution of functional human P-glycoprotein. 762 47

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.
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PMID:SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics. 898 3

Efflux-pumps mediated by P-glycoprotein increase the level of resistance to antibiotics in bacteria and to cytostatics in tumor cells due to decreased drug accumulation, and are also involved in the operation of blood brain barrier. Different compounds are able to enhance drug retention in the cells by inhibiting the efflux-pump mechanism of multidrug resistant (mdr) cancer cells and bacteria. The effects of substituted chlorpromazines were studied on a hemolysin producing and antibiotic resistant plasmid carrying E coli, and rhodamine uptake of multidrug resistant (mdr 1 gene expressing) mouse lymphoma cells. Hemolysin transporter protein encoding plasmids were eliminated from E. coli by a representative phenothiazine namely promethazine. Minimal inhibitory concentrations of tetracyclin and promethazine were lower for plasmidless bacteria as compared to the parent, plasmid carrying strains. The antibiotic resistance plasmid was cured of the R-plasmid of E. coli JE 2571, however, the ring substituted derivatives were less effective then parent compounds. The effect of some substituted phenothiazines on P-glycoprotein efflux-pump of mouse lymphoma cells were studied. The majority of ring substituted derivatives reversed the mdr of tumor cells. The 3,7,8-trihydroxy- and 7,8-dihydroxy derivatives of chlorpromazine were effective as P-glycoprotein blockers, however, 7,8-diacetoxy-, 7,8dimetoxy-, 7-semicarbazone-, and 5-oxo-chlorpromazine derivatives had only moderate effect. A tomato lectin, specific for blood brain capillary endothelium was able to modify the activity of P-glycoprotein in tumor cells. Phenothiazine and tomato lectin had some antagonism in tumor cells. Our results suggest that the inhibition of P-glycoprotein function in murine tumor cells and inhibition of transporter protein in E. coli bacteria may depend on pi-electron superdelocalizibility and electrophile binding of the compounds to the transporter proteins. The intracellular accumulation of antibiotics or chemotherapeutics increased as a consequence of decreased drug efflux in both bacterial and tumor cell systems. The inhibition of the drug effux-pump is the same for all individual cells of the population. These results can be realized by combination chemotherapy, however, antiplasmid effect itself cannot be exploited in this respect because the resistance was reversed in a part of the population only. The similarity with mdr P-glycoprotein in tumor cells and brain capillary endothels provides a good model for molecules opening the blood brain barrier.
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PMID:Inhibition of the transport function of membrane proteins by some substituted phenothiazines in E. coli and multidrug resistant tumor cells. 906 99

P-glycoprotein (P-gp) is a member of the ATP binding cassette superfamily of active transporters and can confer multidrug resistance on cells and tumors by pumping chemotherapeutic drugs from the cytoplasm. P-gp was purified from CHrB30 cells and retained the ability to bind substrates and hydrolyze ATP. Labeling of P-gp with lectin-gold particles suggested it is monomeric. An initial structure of purified P-gp was determined to 2.5 nm resolution by electron microscopy and single particle image analysis of both detergent-solubilized and lipid-reconstituted protein. The structure was further refined by three dimensional reconstructions from single particle images and by Fourier projection maps of small two-dimensional crystalline arrays (unit cell parameters: a, 14.2 nm; b, 18.5 nm; and gamma, 91.6 degrees ). When viewed from above the membrane plane the protein is toroidal, with 6-fold symmetry and a diameter of about 10 nm. There is a large central pore of about 5 nm in diameter, which is closed at the inner (cytoplasmic) face of the membrane, forming an aqueous chamber within the membrane. An opening from this chamber to the lipid phase is present. The projection of the protein perpendicular to the membrane is roughly rectangular with a maximum depth of 8 nm and two 3-nm lobes exposed at the cytoplasmic face of the membrane, likely to correspond to the nucleotide binding domains. This study provides the first experimental insight into the three-dimensional architecture of any ATP binding cassette transporter.
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PMID:Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. 909 18


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