B. Mammalian P-glycoproteins[31] Baculovirus-mediated expression of human multidrug resistance cDNA in insect cells and functional analysis of recombinant P-glycoprotein
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Does perfluorooctane sulfonate (PFOS) act as chemosensitizer in zebrafish embryos?
2016, Science of the Total EnvironmentCitation Excerpt :Cells were incubated for 72 h, then harvested, disrupted by nitrogen cavitation, and cell membranes were pelleted by centrifugation and ultracentrifugation steps. The pellet containing the membrane fractions was dissolved in ice cold TMEP buffer (Germann, 1998; Sarkadi et al., 1992), homogenized using syringes, and total protein was determined with the BCA™ Protein Assay Kit (Thermo Scientific). ATPase assays were performed in 96-well plates with 20 μg of total protein per well.
Current advances on ABC drug transporters in fish
2014, Comparative Biochemistry and Physiology Part - C: Toxicology and PharmacologyCitation Excerpt :In contrast, in dye accumulation assays with drug-resistant cell lines, both substrates and inhibitors increase the cellular accumulation of fluorescent dyes that are substrates of the studied transporter (Homolya et al., 1993; Holló et al., 1994). Another approach to investigate chemical interaction with ABC pumps at high sample throughput is based on the measurement of transporter ATPase activity in cell membrane fractions obtained from cell lines overexpressing specific transporters (Ambudkar et al., 1992; Doige et al., 1992) or generated in recombinant baculovirus/insect cell expression systems (Sarkadi et al., 1992; Germann, 1998). In these experimental systems, ABCB1 (P-glycoprotein) shows basal ATPase activity in the absence of added substrates of the transporter, which is dependent on the presence of cholesterol in the cell membrane (Garrigues et al., 2002).
Improved expression and purification of human multidrug resistance protein MDR1 from baculovirus-infected insect cells
2009, Protein Expression and PurificationExistence of catalase-less peroxisomes in Sf21 insect cells
2003, Biochemical and Biophysical Research CommunicationsProteolytic Cleavage of the Linker Region of the Human P-glycoprotein Modulates Its ATPase Function
2002, Journal of Biological ChemistryCorrelation between steady-state ATP hydrolysis and Vanadate-induced ADP trapping in human P-glycoprotein: Evidence for ADP release as the rate-limiting step in the catalytic cycle and its modulation by substrates
2001, Journal of Biological ChemistryCitation Excerpt :Briefly, His6-tagged Pgp was expressed using Trichoplusia ni (High FiveTM; Invitrogen, San Diego, CA) insect cells grown in monolayer cultures and infected with a recombinant baculovirus BV-MDR1(H6) that contains a humanMDR1 that encodes Pgp. Crude Pgp-containing membranes were prepared by Dounce homogenization under hypotonic conditions (23), and membrane proteins were solubilized with octyl β-d-glucopyranoside (1.25%) in the presence of 20% glycerol and lipid mixture (0.1%) (see “Routine Procedures”). Solubilized proteins were subjected to metal affinity chromatography (Talon resin from CLONTECH, Palo Alto, CA) in the presence of 0.95% octyl β-d-glucopyranoside and 0.04% lipid; 80% purified Pgp was eluted with 100 mm imidazole (11).