Characterization of the pulmonary N-ethylmaleimide-insensitive phosphatidate phosphohydrolase

Phosphatidate phosphohydrolase (PAPase) is a key enzyme involved in glycero lipid synthesis where it converts phosphatidic acid to diacylglycerol. Prev ious studies performed in lung have demonstrated the existence of 2 differe nt forms of PAPases, namely PAP-1 and PAP-2. the former pulmonary Mg+2-depe ndent enzyme is N-ethylmaleimide (NEM)-sensitive, heat labile, and is invol ved in phospholipid biosynthesis. However, the function of the latter lung isozyme is unknown. PAP-2 activity was selectively assayed using NEM in the absence of Mg+2. Studies employing this assay and adult rat lung microsoma l preparations demonstrated that PAP-2 activity was inhibited by amphiphili c amines, sphingoid bases, products of the PAP-2 reaction (monoacylglycerol [MAG] and diacylglycerol [DAG]), and substrate analogs such as lysophospha tidic acid (lyso-PA), ceramide-1-phosphate, and to a lesser extent, sphingo sine-1-phosphate. Purified lung plasma membranes, prepared using discontinu ous sucrose and Percoll gradients, showed that PAP-2 activity was enriched 6.9 +/- 1.6-fold over the whole homogenate and was between the enrichment f or plasma membrane markers 5'-nucleotidase (14.7 +/- 0.3) and Na+, K+-ATPas e (4.0 +/- 0.2). Both phosphatidic acid and lysophosphatidic acid were good substrates for PAP-2 activity in this purified plasma membrane fraction. I n contrast, sphingosine-1-phosphate was a relatively poor substrate. PAP-2 activity was slightly enriched in isolated type II cells and low in isolate d rat lung fibroblasts. This study shows lung contains PAP-2 activity in pl asma membranes and type II cells where it could play a role in signal trans duction.