Characterization of the pulmonary N-ethylmaleimide-insensitive phosphatidate phosphohydrolase.
Phosphatidate phosphohydrolase (PAPase) is a key enzyme involved in glycerolipid synthesis where it converts phosphatidic acid to diacylglycerol. Previous studies performed in lung have demonstrated the existence of 2 different forms of PAPases, namely PAP-1 and PAP-2. The former pulmonary Mg+2-dependent enzyme is N-ethylmaleimide (NEM)-sensitive, heat labile, and is involved 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 microsomal preparations demonstrated that PAP-2 activity was inhibited by amphiphilic amines, sphingoid bases, products of the PAP-2 reaction (monoacylglycerol [MAG] and diacylglycerol [DAG]), and substrate analogs such as lysophosphatidic acid (lyso-PA), ceramide-1-phosphate, and to a lesser extent, sphingosine-1-phosphate. Purified lung plasma membranes, prepared using discontinuous 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 for plasma membrane markers, 5'-nucleotidase (14.7 +/- 0.3) and Na+, K(+)-ATPase (4.0 +/- 0.2). Both phosphatidic acid and lysophosphatidic acid were good substrates for PAP-2 activity in this purified plasma membrane fraction. In contrast, sphingosine-1-phosphate was a relatively poor substrate. PAP-2 activity was slightly enriched in isolated type II cells and low in isolated rat lung fibroblasts. This study shows lung contains PAP-2 activity in plasma membranes and type II cells where it could play a role in signal transduction.
Nanjundan
M
M
Department of Biochemistry, University of Western Ontario, London, Canada.
Possmayer
F
F
D000818:Animals; D017304:Annexin A5 / Q000037:antagonists & inhibitors / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002518:Ceramides / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005989:Glycerides / Q000494:pharmacology; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008246:Lysophospholipids / Q000494:pharmacology; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D000073718:Pancreatitis-Associated Proteins; D010711:Phosphatidate Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013379:Substrate Specificity
D017304:Annexin A5; D002518:Ceramides; D004075:Diglycerides; D004791:Enzyme Inhibitors; D005989:Glycerides; D008246:Lysophospholipids; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; C065576:ceramide 1-phosphate; C060506:sphingosine 1-phosphate; C406242:phosphatidic acid phosphatase type 2; D010711:Phosphatidate Phosphatase; D013110:Sphingosine; D005033:Ethylmaleimide