title: Purification and Na+ uptake by human placental microvillus membrane vesicles prepared by three different methods.
abstract: Three methods were used to prepare microvillus membrane vesicles from each of six human placentas. Two of these incorporated an agitation stage to preferentially remove microvilli and either Ca2+ (Method 1) or Mg2+ (Method 2) aggregation of non-microvillus membrane. The third method involved homogenisation of the tissue followed by Mg2+ aggregation of non-microvillus membrane (Method 3). Enrichment of alkaline phosphatase activity (27.6 +/- 1.9, 25.3 +/- 2.7) and ouabain binding (5.9 +/- 2.6, 5.3 +/- 2.2, respectively) was similar in vesicles prepared by Methods 1 and 2, respectively. Method 3 vesicles showed a significantly (P less than 0.01) lower alkaline phosphatase enrichment (18.1 +/- 1.2), but ouabain binding enrichment (6.3 +/- 1.3) was not different and vesicle protein recovery (mg/g placenta) was 5-fold greater. Na+ uptake in the presence of an outwardly directed proton gradient was significantly inhibited in all microvillus membrane vesicles by amiloride (0.5 mM). However, the amiloride sensitive component of Na+ uptake was 3-6-fold greater in Method 3 vesicles than in Method 1 and 2 vesicles, and showed overshoot above equilibrium in the former but not the latter. Further experiments using the pH sensitive dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein suggested that the proton gradient dissipated faster from Method 1 than from Method 3 vesicles. Thus methodological differences can have a marked effect on transport processes in microvillus membrane vesicles prepared from the human placenta.
authors.affiliation: Department of Child Health, University of Manchester, St Mary's Hospital, U.K.
authors.forename: J D
authors.initials: JD
authors.lastname: Glazier
authors.forename: C J
authors.initials: CJ
authors.lastname: Jones
authors.forename: C P
authors.initials: CP
authors.lastname: Sibley
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods*; D002474:Cell-Free System; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008871:Microvilli / Q000378:metabolism; D010920:Placenta / Q000378:metabolism* / Q000648:ultrastructure; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
chemical_list: D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium; D000469:Alkaline Phosphatase