Mg2-dependent phosphatidate phosphohydrolase of rat lung: development of an assay employing a defined chemical substrate which reflects the phosphohydrolase activity measured using membrane-bound substrate.
An assay of pulmonary phosphatidate phosphohydrolase activity has been developed that employs a chemically defined liposome substrate of equimolar phosphatidate and phosphatidylcholine. Enzyme assays employing this substrate resolved two distinct activities based upon their requirements for Mg2+. Assays were performed in the presence and absence of 2 mM MgCl2 and the Mg2+-dependent phosphatidate phosphohydrolase activity calculated by difference. The Mg2+-independent phosphatase activity resembled that found using aqueous dispersions of phosphatidate (PAaq). Approximately 90% of the Mg2+-dependent phosphatidate phosphohydrolase activity was recovered in the cytosol and the remainder was associated with the microsomal fraction. The Mg2+-dependent phosphatidate phosphohydrolase activity has kinetic parameters of Km = 55 microM, Vmax = 1.6 nmol/min/mg protein for the microsomal fraction, and Km = 215 microM, Vmax = 6.8 nmol/min/mg protein for the cytosolic fraction. These parameters resembled those found using the microsomal membrane-bound (PAmb) substrate. In addition, the pH optima and sensitivity to detergents and thermal inactivation are equal to those for the PAmb-dependent phosphatidate phosphohydrolase activity. In the course of these studies the microsomal and cytosolic activities were qualitatively equal, indicative of a single enzyme in two subcellular locations. In conclusion, the assay of Mg2+-dependent phosphatidate phosphohydrolase activity measured using equimolar phosphatidate and phosphatidylcholine liposomes is equivalent to that activity previously described using microsomal membrane-bound substrate. However, the chemically-defined system provides a more simplified starting point for further studies on this important enzyme.
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes; D008168:Lung / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D010711:Phosphatidate Phosphatase / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D008081:Liposomes; D010744:Phosphoric Monoester Hydrolases; D010711:Phosphatidate Phosphatase