PURIFICATION AND CHARACTERIZATION OF A NOVEL PLASMA-MEMBRANE PHOSPHATIDATE PHOSPHOHYDROLASE FROM RAT-LIVER

An N-ethylmaleimide-insensitive phosphatidate phosphohydrolase, which also hydrolyzes lysophosphatidate, was isolated from the plasma membra nes of rat liver. The specific activity of an anionic form of the enzy me (53 kDa, pI < 4) was increased 2700-fold, A cationic form of the en zyme (51 kDa, pI = 9) was purified to homogeneity, but the fold purifi cation was low because the activity of the highly purified enzyme was unstable. Immunoprecipitating antibodies raised against the homogeneou s protein confirmed the identity of the cationic protein as the phosph ohydrolase and were used to identify the anionic enzyme, Both forms ar e integral membrane glycoproteins that were converted to 28-kDa protei ns upon treatment with N-glycanase F. Treatment of the anionic form wi th neuraminidase allowed it to be purified in the same manner as the c ationic enzyme and yielded an immunoreactive protein with a molecular mass identical to the cationic protein, Thus, the two ionic forms most likely represent different sialated states of the protein. An immunor eactive 51-53-kDa protein was detected in rat liver, heart, kidney, sk eletal muscle, testis, and brain. Little immunoreactive 51-53-kDa prot ein was detected in rat thymus, spleen, adipose, or lung tissue. This work provides the tools for determining the regulation and function of the phosphatidate phosphohydrolase in signal transduction and cell ac tivation.