Separation and characterization of the membrane-bound and aqueously dispersed phosphatidate phosphatidic acid phosphohydrolase activities in rat lung

Abstract

1. 1. The properties of the phosphatidic acid phosphohydrolase activities responsible for the degradation of membrane-bound phosphatidic acid (PA mb) and aqueously dispersed phosphatidic acid (PA aq) have been examined in rat lung microsomal and cytosol preparations. 2. 2. The bulk of the PA mb phosphohydrolase activities in the cytosolic and microsomal fractions could be differentiated from the bulk of the PA aq phosphohydrolase activities by their susceptibility to proteolysis and thermal inactivation. 3. 3. EDTA inhibited the PA mb phosphohydrolase activity in the cytosol by 65% but the PA aq phosphohydrolase activity by only 10%. MgCl 2 stimulated the cytosolic PA mb phosphohydrolase activity by 7% and the PA aq phosphohydrolase activity by 80%. Thermal inactivation studies indicated that the PA mb phosphohydrolase activity remaining in the presence of EDTA may be related to the bulk of the PA aq phosphohydrolase activity. Furthermore a portion of the PA mb phosphohydrolase activity may be related to the Mg 2+-dependent PA aq phosphohydrolase activity. 4. 4. Fractionation of rat lung cytosol on Bio-Gel A-5m revealed the presence of a moderate PA mb phosphohydrolase peak (35% total) in the void volume ( V 0), a major PA mb phosphohydrolase activity peak (55% total) with an apparent M r 390 000, and a minor PA mb phosphohydrolase activity peak (12% total) with an apparent M r 110 000. The PA aq phosphohydrolase activities eluted as a moderate peak (25% total) in the void volume, a major peak (50% total) with an apparent M r 130 000 followed by a broad shoulder of PA aq-dependent activity which coeluted with the 110 kdalton PA mb phosphohydrolase peak and accounted for approx. 25% of the total activity. 5. 5. The 390 kdalton PA mb phosphohydrolase activity peak accounted both for most of the EDTA-sensitive PA mb phosphohydrolase activity and for most of the Mg 2+-dependent PA aq phosphohydrolase activity in the eluates. 6. 6. Treatment of rat lung microsomes with Triton X-100 produced a 5.5-fold increase in the PA mb phosphohydrolase activity but a slight depression of the PA aq phosphohydrolase activity. Filtration of Triton X-100-solubilized microsomes and Triton X-100-treated cytosol on Bio-Gel A-1.5m revealed in both cases PA mb and PA aq phosphohydrolase activity peaks in the V 0 and at 200 ml. With both fractions, PA aq phosphohydrolase activity was also eluted at 295 ml. These results suggest a potential relation between the PA mb phosphohydrolase activities in the microsomes and cytosol. Part of the PA aq phosphohydrolase activities in these fractions may also be related. However, the major PA aq 130 kdalton phosphohydrolase activity peak isolated from untreated cytosol was completely inhibited by Triton X-100 and presumably could not be detected in eluates containing this detergent.