Fractionation of renal brush border membrane proteins with Triton X-114 phase partitioning.
Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.
Vachon
V
V
Département de chimie, Université du Québec à Montréal, Canada.
Pouliot
J F
JF
Laprade
R
R
Béliveau
R
R
D015720:5'-Nucleotidase / Q000302:isolation & purification / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005959:Glucosidases / Q000302:isolation & purification / Q000378:metabolism; D007668:Kidney / Q000737:chemistry / Q000201:enzymology / Q000648:ultrastructure; D007931:Leucyl Aminopeptidase / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008871:Microvilli / Q000737:chemistry* / Q000201:enzymology; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D012995:Solubility; D014198:Trehalase / Q000302:isolation & purification / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification / Q000378:metabolism
D008565:Membrane Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D005959:Glucosidases; D014198:Trehalase; D007931:Leucyl Aminopeptidase