Purification and Na+ uptake by human placental microvillus membrane vesicles prepared by three different methods

Biochim Biophys Acta. 1988 Nov 22;945(2):127-34. doi: 10.1016/0005-2736(88)90475-0.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / metabolism
  • Amiloride / pharmacology
  • Carrier Proteins / metabolism*
  • Cell Fractionation / methods*
  • Cell-Free System
  • Humans
  • Hydrogen-Ion Concentration
  • Microscopy, Electron
  • Microvilli / metabolism
  • Placenta / metabolism*
  • Placenta / ultrastructure
  • Sodium / metabolism*
  • Sodium-Hydrogen Exchangers


  • Carrier Proteins
  • Sodium-Hydrogen Exchangers
  • Amiloride
  • Sodium
  • Alkaline Phosphatase