Dw. Waggoner et al., PURIFICATION AND CHARACTERIZATION OF A NOVEL PLASMA-MEMBRANE PHOSPHATIDATE PHOSPHOHYDROLASE FROM RAT-LIVER, The Journal of biological chemistry, 270(33), 1995, pp. 19422-19429
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.