A high yield preparation for rat kidney brush border membranes. Different behaviour of lysosomal markers

Biochim Biophys Acta. 1981 Oct 2;647(2):169-76. doi: 10.1016/0005-2736(81)90243-1.

Abstract

Rat kidney cortex slices were homogenized with a polytron in a isoosmotic medium containing 5 mmol/l EGTA. By two precipitations with MgCl2 (12 mmol/l) and differential centrifugation, brush border membranes were purified. The brush border marker enzymes alkaline phosphatase and aminopeptidase M were found to be enriched 17.0 +/- 5.3-fold and 16.7 +/- 3.7-fold, respectively. By this method, a high yield of brush border membranes was obtained (48.3 +/- 7.9% for alkaline phosphatase; 47.0 +/- 9.5% for aminopeptidase M). The acid phosphatase was enriched 5-fold, whereas other lysosomal enzymes (glucosaminidase, glucuronidase, cathepsin D) were enriched only 0.2-fold. Acid phosphatase activity could not be washed out, but could be separated from alkaline phosphatase and leucine aminopeptidase by means of free flow electrophoresis and sucrose density gradient centrifugation. Vesicles prepared by the presently described Mg/EGTA-method show better transport properties, compared to vesicles prepared by the calcium method of Evers et al. (Evers, C., Haase, W., Murer, H. and Kinne, R. (1978) Membrane Biochem. 1, 203-219), whereas by SDS-polyacrylamide gel electrophoresis, no differences in the protein patterns were observed.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid Phosphatase / metabolism
  • Alkaline Phosphatase / metabolism
  • Aminopeptidases / metabolism
  • Animals
  • Biological Transport, Active
  • CD13 Antigens
  • Cell Fractionation / methods
  • Cell Membrane / enzymology*
  • Freeze Fracturing
  • Kidney Cortex / ultrastructure*
  • Lysosomes / enzymology*
  • Male
  • Microscopy, Electron
  • Microvilli / enzymology*
  • Rats
  • Rats, Inbred Strains
  • Sodium-Potassium-Exchanging ATPase / metabolism

Substances

  • Alkaline Phosphatase
  • Acid Phosphatase
  • Aminopeptidases
  • CD13 Antigens
  • Sodium-Potassium-Exchanging ATPase