Purification and characterization of novel plasma membrane phosphatidate phosphohydrolase from rat liver.
An N-ethylmaleimide-insensitive phosphatidate phosphohydrolase, which also hydrolyzes lysophosphatidate, was isolated from the plasma membranes of rat liver. The specific activity of an anionic form of the enzyme (53 kDa, pI < 4) was increased 2700-fold. A cationic form of enzyme (51 kDa, pI = 9) was purified to homogeneity, but the -fold purification was low because the activity of the highly purified enzyme was unstable. Immunoprecipitating antibodies raised against the homogeneous protein confirmed the identity of the cationic protein as the phosphohydrolase and were used to identify the anionic enzyme. Both forms are integral membrane glycoproteins that were converted to 28-kDa proteins upon treatment with N-glycanase F. Treatment of the anionic form with neuraminidase allowed it to be purified in the same manner as the cationic 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 protein. An immunoreactive 51-53-kDa protein was detected in rat liver, heart, kidney, skeletal muscle, testis, and brain. Little immunoreactive 51-53-kDa protein 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 activation.
Waggoner
D W
DW
Signal Transduction Laboratory, University of Alberta, Edmonton, Canada.
Martin
A
A
Dewald
J
J
Gómez-Muñoz
A
A
Brindley
D N
DN
D000818:Animals; D000838:Anions; D000906:Antibodies / Q000378:metabolism; D002412:Cations; D002462:Cell Membrane / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008099:Liver / Q000201:enzymology*; D008297:Male; D010711:Phosphatidate Phosphatase / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011233:Precipitin Tests; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley
D000838:Anions; D000906:Antibodies; D002412:Cations; D010711:Phosphatidate Phosphatase