In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein. Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted "stability" to this perturbation of bilayer organization by P-gp and moreover this led to altered protein function.
During cold storage (2°C) of Enterobacter aerogenes for 15 min, 2, 4, 6 and 8 d in glucose mineral salts medium (GMS) and skim milk (SM), substantial differences were noticed in ATP/cell levels. At the appropriate interval, ATP was determined with the firefly bioluminescent method on a portion of the culture, while another portion was activated at 30°C for 40 min and ATP determined at 10-min intervals. Initial ATP content after 15 min of storage in SM was 0.55 femtogram (fg)/cell as compared to 0.84 fg/cell in GMS. Upon cold storage for longer periods (2, 4, 6 and 8 d), the ATP/cell concentrations were relatively stable (0.82, 0.73,0.62 and 0.72 fg) in GMS, while they declined to levels below 0.2 fg in SM. However, activation of only 30 min increased cell ATP concentration in SM to 1.06, 0.92, 0.77, 0.66 and 0.72 fg, and narrowed the difference between ATP/cell levels in the two media. As a realistic ATP concentration/cell value is necessary to calculate bacterial numbers in milk by the bioluminescent technique, a 30-min activation period at 30°C is recommended to replenish the A TP lost during refrigerated storage.
The selective destruction of non-bacterial ATP and subsequent determination of bacterial ATP using the ATP bioluminescent technique was investigated. Treatments to release ATP from somatic cells and hydrolyze free ATP also significantly reduced the ATP content of Enterobacter cloacae in skim and raw milk. The reduction can mainly be ascribed to apyrase (an ATPase) affecting the ATP content of intact bacteria. Somatic cell treatments failed to completely eliminate non-bacterial ATP. Although treatment with a somatic releasing reagent, EDTA and apyrase, resulted in a 96% reduction in the ATP content of raw milk, the remaining non-bacterial ATP was still considerably more than found in the bacterial component of raw milk studied here. Until reagents are available to selectively destroy all non-bacterial ATP without affecting the bacterial ATP content, the bioluminescent technique will have limited application in determination of the bacterial quality of raw milk.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.