Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

N-(5,5-Diacetoxypent-1-yl)doxorubicin (DAPDOX) (3), a new, water-soluble analogue of doxorubicin, has been synthesized by coupling doxorubicin with 5-oxopentane-1,1-diacetate in the presence of NaBH3CN. This analogue was designed to be converted to the corresponding aldehyde, N-(5-oxopent-1-yl)doxorubicin, in the presence of carboxylate hydrolases, enzymes that are ubiquitous in tissue. DAPDOX had a half-life of several days in 0.05 M phosphate or 0.05 M acetate buffer solution at pH 4.0. However, in 0.05 M phosphate buffer at pH 7.4 in the presence of 20 unit equiv of porcine liver carboxylate esterase, the half-life of DAPDOX was less than 1 min. N-(5-acetoxypent-1-yl)doxorubicin (4), which should give rise to N-(5-hydroxypent-1-yl)doxorubicin on esterase-mediated hydrolysis, and N-(pent-1-yl)doxorubicin (5), were also prepared for comparative biological studies. DAPDOX was 150 times more potent than doxorubicin at inhibiting the growth of Chinese hamster ovary (CHO) cells in culture. The compound retained the same degree of potency against a CHO subline 100-fold resistant to doxorubicin (CHO/DOX) that expressed elevated levels of P-glycoprotein. Compounds 4 and 5, on the other hand, were no more effective than doxorubicin at inhibiting the growth of CHO cells and were 4-7-fold less potent against the CHO/DOX subline. DAPDOX is representative of a new structural class of doxorubicin analogues with unique chemical and biological properties.
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PMID:N-(5,5-diacetoxypent-1-yl)doxorubicin: a new intensely potent doxorubicin analogue. 135 50

In the present study we have analyzed the involvement of phosphorylation in the function of P-glycoprotein and have also examined sites of phosphorylation along the P-glycoprotein polypeptide chain. The results show that in HL60 cells isolated for resistance to vincristine the protein kinase inhibitor staurosporine induces a major inhibition in the phosphorylation of P-glycoprotein. Further studies show that under the same conditions in which staurosporine inhibits P-glycoprotein phosphorylation there is a concomitant increase in cellular drug accumulation and a major inhibition in drug efflux. Additional studies using pulse-chase experiments show that the P-glycoprotein phosphate groups are metabolically active and that the protein undergoes rapid cycles of phosphorylation and dephosphorylation in the cell. Structural analyses demonstrate that cleavage of 32P-labeled P-glycoprotein at Asp-Pro linkages with formic acid results in the formation of a major phosphorylated peptide of 35 kDa and a minor peptide of 42 kDa. Western blot analysis using site-specific anti-sera against P-glycoprotein suggests that P35 represents a phosphorylated fragment containing P-glycoprotein amino acids 446-744. Analysis of tryptic peptides using site-specific antisera identifies a second major phosphorylated region of P-glycoprotein which contains amino acids 745-1088. These studies thus suggest that phosphorylation plays an important role in the biological activity of P-glycoprotein. The results also indicate that two adjacent internal regions are highly phosphorylated in the P-glycoprotein molecule.
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PMID:Analysis of P-glycoprotein phosphorylation in HL60 cells isolated for resistance to vincristine. 167 14

Studies were undertaken to identify the protein kinase(s) responsible for P-glycoprotein phosphorylation in multidrug-resistant (KB-V1) human carcinoma cells and to elucidate the functional role of phosphorylation. P-glycoprotein migrated on sodium dodecyl sulfate gels with apparent Mr 150,000 and is termed P150. When KB-V1 membrane vesicles were incubated with [gamma-32P] ATP, P150 was phosphorylated by an endogenous kinase that exhibited properties of membrane-inserted protein kinase C (PKC). Both membrane-bound P150 and purified P150 served as effective substrates for highly purified rat brain PKC which incorporated approximately 0.6 mol of phosphate/mol of P150. Enzyme assays showed that KB-V1 cells exhibit 4-fold higher PKC activity compared with the drug-sensitive KB-3 cell line. The basal phosphorylation of P150 observed in 32P-labeled cells was increased 2-fold by phorbol ester (PMA) treatment and reduced 30% by treatment with the isoquinolinsulfonamide H-7. Phosphopeptide maps of partially digested P150, phosphorylated either in vitro with PKC or in intact 32P-labeled control or PMA-stimulated cells, were indistinguishable from one another. Drug accumulation assays revealed that PMA treatment of KB-V1 cells significantly reduced [3H]vinblastine accumulation induced by verapamil or by tetrandrine. The results suggest that PKC is primarily responsible for P150 phosphorylation in KB-V1 cells and that phosphorylation may play a modulatory role in the drug transport process.
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PMID:Protein kinase C phosphorylates P-glycoprotein in multidrug resistant human KB carcinoma cells. 197 May 71

An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1. Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1. Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein. Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K. The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H. These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid. P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.
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PMID:Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells. 290 69

The pharmacokinetics of mitomycin C are reviewed from reports using specific and sensitive high-performance liquid chromatography (HPLC) assays. These studies demonstrate a rapid, biphasic elimination pattern for the drug: alpha half-life of 8 minutes and beta, or terminal, half-life of 48 minutes. Urinary elimination of intact drug is minimal (8% to 10% of a dose), whereas biliary drug levels may exceed those in the plasma. There was no evidence of dose dependent pharmacokinetics over a wide range of doses and patient populations, including pediatric solid tumor patients. Metabolic studies with mitomycin C have demonstrated an absolute requirement for reductive enzymatic activation of the drug to mono- and bifunctional alkylating species. The preferred DNA target for covalent attachment by mitomycin C was found to be the N2 position of guanine. Chemical metabolites of activated mitomycin C were also demonstrated to include 2, 7 diaminomitosene and its cis- and trans-1-hydroxy or 1-phosphate analogues. Also, there was no evidence for hypoxic cell selectivity for mitomycin C in several human tumor cell lines, although some animal tumors unequivocally display this phenomenon. Finally, there are new observations of the development of the multidrug-resistance phenotype in mitomycin C-treated L-1210 cells in vitro. These cells became collaterally resistant to anthracyclines and vinca alkaloids and expressed the P-glycoprotein in cell membranes. The implications of these findings for mitomycin C use in solid tumors in humans is discussed.
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PMID:New findings in the pharmacokinetic, metabolic, and drug-resistance aspects of mitomycin C. 313 96

P-glycoprotein (Pgp or multidrug-resistance protein) shows drug-stimulated ATPase activity. The catalytic sites are known to be of low affinity and specificity for nucleotides. From the sequence, two nucleotide sites are predicted per Pgp molecule. Using plasma membranes from a multidrug-resistant Chinese hamster ovary cell line, which are highly enriched in Pgp, we show that vanadate-induced trapping of nucleotide at a single catalytic site produces stably inhibited Pgp, with t 1/2 for reactivation of ATPase activity of 84 min at 37 degrees C and >30 h at 4 degrees C. Reactivation of ATPase correlated with release of trapped nucleotide. Concentrations of MgATP and MgADP required to produce 50% inhibition were 9 and 15 microM, respectively, thus the apparent affinity for nucleotide is greatly increased by vanadate-trapping. The trapped nucleotide species was ADP. Divalent Cation was required, with magnesium, manganese, and cobalt all effective: cobalt yielded a very stable inhibited species, t1/2 at 37 degrees C = 18 h. No photocleavage of Pgp was observed after vanadate trapping with MgATP, nor was UV-induced photolabeling of Pgp by trapped adenine nucleotide observed. Vanadate-trapping with 8-azido-ATP followed by UV irradiation caused permanent inactivation and specific labeling of Pgp. Vanadate-induced inhibition was also shown with pure, reconstituted Pgp, with similar characteristics to those in plasma membranes. Vanadate trapping overcomes technical difficulties posed by lack of high affinity nucleotide-binding site(s) or a covalent enzyme-phosphate catalytic intermediate in Pgp. The finding that vanadate trapping of nucleotide at just one site/Pgp is sufficient to give full inhibition at ATPase activity shows that the two predicted nucleotide sites can not function independently as catalytic sites.
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PMID:P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site. 764 18

Specific sites in the linker region of human P-glycoprotein phosphorylated by protein kinase C (PKC) were identified by means of a synthetic peptide substrate, PG-2, corresponding to residues 656-689 from this region of the molecule. As PG-2 has several sequences of the type recognized by the cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a substrate for PKA. PG-2 was phosphorylated by purified PKC in a Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide. Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC identified Ser-661, Ser-667 and Ser-671 as the three sites of phosphorylation. PG-2 was also found to be phosphorylated by purified PKA in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667, Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2 were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was PKA-specific. Further studies showed that PG-2 acted as a competitive substrate for the P-glycoprotein kinase present in membranes from multidrug-resistant human KB cells. The membrane kinase phosphorylated PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human P-glycoprotein can be phosphorylated by at least two protein kinases, stimulated by different second-messenger systems, which exhibit both overlapping and unique specificities for phosphorylation of multiple sites in the linker region of the molecule.
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PMID:Phosphorylation by protein kinase C and cyclic AMP-dependent protein kinase of synthetic peptides derived from the linker region of human P-glycoprotein. 790 31

Phosphorylation of P-glycoprotein (Pgp) by protein kinase C occurs on apparently the same sites in vitro and in intact cells (in situ) and is implicated in modulation of Pgp function. The region of the molecule which contains the in vitro phosphorylation sites and two specific sites within this region are now determined by peptide sequencing. Membrane vesicles from multidrug-resistant human KB-V1 cells were incubated with purified protein kinase C and [gamma-32P]ATP, and Pgp (containing 1 mol of phosphate/mol of protein) was purified to apparent homogeneity. Phosphorylation occurred exclusively on serine residues. Phosphopeptides were generated by digestion with Lys-C endoproteinase or trypsin, partially purified by high performance liquid chromatography, and further purified with strategies developed for individual phosphopeptides. Sequence analysis by Edman degradation and comparison with the deduced amino acid sequence of human (mdr 1) Pgp identified serines 661 and 671, and one or more of serines 667, 675, and 683, as sites of phosphorylation. These sites are clustered in the linker region located between the two homologous halves of Pgp. Our results identify a previously undefined, phosphorylatable domain of Pgp, smaller in size but analogous in location to the R-domain of the cystic fibrosis transmembrane conductance regulator. These data provide a basis for a better understanding of the role of phosphorylation in the mechanism of action and regulation of this important multidrug pump protein.
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PMID:Identification of specific sites in human P-glycoprotein phosphorylated by protein kinase C. 809 61

Binding protein-dependent transport systems mediate the accumulation of several ions, sugars, amino acids, and peptides in Gram-negative bacteria by using the energy of ATP hydrolysis and belong to a superfamily of membrane proteins which extends to eukaryotic cells and includes the multidrug resistance P-glycoprotein and the cystic fibrosis transmembrane conductance regulator. The binding protein-dependent galactose transport system of Salmonella typhimurium comprises four proteins which have been characterized previously by molecular cloning experiments (51,000-dalton MglA protein, with a stable proteolytic product of 38,000 daltons, 33,000-dalton MglB protein, 29,000-dalton MglC protein, 21,000-dalton MglE protein). By using a MglA hyperproducing strain, we have purified a galactose-stimulated ATPase which shows a single band in polyacrylamide gels under nondenaturing conditions and shows three bands at 51,000, 38,000, and 15,000 daltons on sodium dodecyl sulfate-polyacrylamide gels (our results suggest that the bands at 38,000 and 15,000 daltons represent proteolytic products of the 51,000-dalton protein). The ATPase activity coincides with the purified protein during the two last chromatographic steps of the purification procedure, and it cannot be isolated from a strain which does not contain the mglA gene. The MglA ATPase is stimulated 3-fold by galactose and hydrolyzes ATP to ADP and Pi (Km ATP = 60 microM, Ka galactose = 0.3 mM, Vmax = 140 nmol/min/mg of protein). The gamma-phosphate of ATP is transferred neither to galactose nor to the protein itself. Vanadate, N-ethylmaleimide and 5-methoxyindole-2-carboxylic acid, a specific inhibitor of binding protein-dependent transport systems, inhibit the MglA ATPase.
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PMID:The MglA component of the binding protein-dependent galactose transport system of Salmonella typhimurium is a galactose-stimulated ATPase. 838 96

Recent advances in the field of carrier-mediated intestinal absorption of of amino acids, oligopeptides, monosaccharides, monocarboxylic acids, phosphate, bile acids and several water-soluble vitamins across brush-border and basolateral membranes are summarized. An understanding of the molecular and functional characteristics of the intestinal membrane transporters will be helpful in the utilization of these transporters for the enhanced oral delivery of poorly absorbed drugs. Some successful examples of the synthesis of prodrugs recognized by the targeted transporters are described. Functional expression of the multidrug resistance gene product, P-glycoprotein, as a primary active transporter in the intestinal brush-border membrane leads to net secretion of some drugs such as anticancer agents in the blood-to-luminal direction, serving as a secretory detoxifying mechanism and as a part of the absorption barrier in the intestine.
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PMID:Carrier-mediated intestinal transport of drugs. 884 32


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