The effects of different membrane isolation and purification techniques on D-glucose transport into rat brush-border membrane vesicles

Abstract

Current and widely used methods for the isolation and purification of brush-border membranes involve the aggregation of non-brush-border membranes with the divalent cations Ca2+ or Mg2+ with or without subsequent exposure to chaotropic agents (e.g., KSCN). Evidence suggests that these techniques yield morphologically distinct and heterogeneous populations of membranes and that functional differences exist between membrane vesicles prepared by the different procedures, presumably reflecting this heterogeneity. To investigate the effect of the various isolation techniques on the kinetic parameters of D-glucose transport, rat intestinal brush-border vesicles were prepared by each of the following four methods: (i) Ca2+ precipitation; (ii) Ca2+ precipitation with KSCN treatment; (iii) Mg2+ precipitation; and (iv) Mg2+ precipitation with KSCN treatment. Membrane purity as indicated by the enrichment of the enzyme membrane markers sucrase and alkaline phosphatase did not differ between isolation procedures. The Mg–Na–K ATPase activity showed an enrichment factor of less than 1.0 for each of the isolation techniques. D-Glucose uptake was measured with a rapid filtration method under conditions of a zero-trans, 100 mM cis-NaSCN gradient. The membrane preparations yielded similar Hofstee transformations displaying the curvilinear relationship thought to be consistent with the existence of multiple transporters for D-glucose. The average kinetic parameters calculated from the Hofstee plots for each technique were similar. It was concluded that D-glucose transport into rat jejunal membrane vesicles was unaffected by the variation in morphology arising from the technique used to purify the membranes.