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)

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

Lysyl endopeptidase (LE) from Achromobacter lyticus M497-1 (EC 3.4.21.50) was utilized to prepare F(ab')2 fragments from mouse anti-P-glycoprotein IgG2a obtained from the UIC2 hybridoma. This report describes a novel single step purification procedure for F(ab')2 fragments that eliminates residual LE activity responsible for secondary cleavage of F(ab')2 to Fab fragments. The purification of F(ab')2 and Fc fragments was accomplished utilizing protein G affinity chromatography and either gradient or step changes in the pH/ionic strength for elution of the Fc and F(ab')2 fragments. Residual LE was eluted from the protein G column with buffer containing 200 mM L-lysine prior to elution of F(ab')2 and Fc fragments. The activity of LE was monitored using the fluorogenic substrate Boc-Val-Leu-Lys-7-amido 4-methyl coumarin. A similar purification procedure for F(ab')2 fragments produced following pepsin digestion of IgG2a is also outlined. The ability of Fab' fragments, from reduced F(ab')2 fragments following LE digestion of IgG2a, to conjugate to thiol reactive groups was demonstrated using N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-meso chlorin e6 mono (N-2-aminoethylamide) (Mce6) conjugates containing reactive maleimide groups. The biological activity of the Fab' targeted HPMA copolymer-Mce6 conjugates was tested against the P-glycoprotein expressing human ovarian carcinoma A2780/AD cell line utilizing a cell survival assay. Fab' targeted HPMA copolymer-Mce6 conjugate demonstrated significantly higher cytotoxicity than either a monoclonal antibody (mAb) targeted HPMA copolymer-Mce6 conjugate or a non-targeted HPMA copolymer-Mce6 conjugate, p < 0.05.
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PMID:Preparation of Fab' from murine IgG2a for thiol reactive conjugation. 1169 31