ACTIVATION OF THE GLYCOSYL-PHOSPHATIDYLINOSITOL-ANCHORED MEMBRANE DIPEPTIDASE UPON RELEASE FROM PIG-KIDNEY MEMBRANES BY PHOSPHOLIPASE-C

Incubation of pig kidney microvillar membranes with Bacillus thuringie nsis or Staphylococcus aureus phosphatidylinositol-speciiic phospholip ase C (PI-PLC) resulted in the release of a number of glycosyl-phospha tidylinositol (GPI)-anchored hydrolases, including alkaline phosphatas e (EC 3.1.3.1), aminopeptidase P (EC 3.4.11.9), membrane dipeptidase ( EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28 ). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentrati on 10-fold less than that required for maximal release. In contrast so lubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competiti ve e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indi cated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleave d the GPI anchor of the affinity-purified amphipathic form of pig memb rane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar mem branes hydrolysed Gly-D-Phe with a K-m of 0.77 mM and a V-max. of 602 nmol/min per mg of protein. However, in the presence of a concentratio n of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the K-m was decreased to 0.07 mM w hile the V-max. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of t he GPI anchor on membrane dipeptidase may relax conformational constra ints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.