SELECTIVE RELEASE OF APICAL MEMBRANE ENZYMES FROM CULTURED RENAL EPITHELIA BY PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C

Membrane proteins can be attached to the plasma membrane in several wa ys. Recently, a mechanism has been described, by which a number of cel l surface proteins are anchored to the exoplasmic side of the plasma m embrane by covalent linkage to glycosyl-phosphatidylinositol (GPI). Th e growth properties of renal epithelial cells in tissue culture enable free access to apical cell surface and brush border membrane proteins . To study the nature of membrane anchoring of apical plasma membrane enzymes in cultured renal epithelial cells, confluent LLC-PK1, OK, NRK , and MDCK epithelia were treated in tissue culture dishes with bacter ial phosphatidyl-inositol-specific phospholipase C (PI-PLC), and the P I-PLC-specific release into the tissue culture medium of the apical me mbrane enzymes akaline phosphatase (AP), gamma-glutamyl transpeptidase , leucine aminopeptidase, trehalase, and maltase was determined. Of th e five enzymes tested, AP and trehalase, already described as GPI-anch ored membrane proteins, were specifically released by PI-PLC from inta ct cell monolayers. Of the four cell lines investigated, LLC-PK1 cells express AP and trehalase which were released by PI-PLC-releaseable en zyme activity. MDCK cells, on the other hand, express AP activity, rel easeable by PI-PLC, but no trehalase activity. In studies on the time course of synthesis and reinsertion of AP into the apical membrane of LLC-PK1 cells after removal by PI-PLC, a 60% recovery of AP activity w as obtained only after 7 days. Analysis of protein release by sodium d odecyl sulfate-polyacrylamide gel electrophoresis of culture supernata nts after surface labeling with biotin and subsequent Western blotting with streptavidin revealed four protein bands at approximately 130, 9 0, 30 and 20 kD in LLC-PK1 cells and five GPI-anchored proteins at 110 , 85, 65, 40, and 26 kD in OK cultures. The finding of a PI-PLC-specif ic release of apical membrane enzymes from renal tubular cell lines of different species (pig, opossum, rat, and dog) and of different nephr on origin indicates a high conservation of the GPI anchor of renal bru sh border membrane proteins and further proves the high degree of diff erentiation retained by the cell lines in tissue culture. In addition, this method may provide a possible tool for isolating GPI-anchored ap ical membrane proteins from intact epithelial monolayer cultures.