Phosphatidate phosphatase activity in isolated rod outer segment from bovine retina.
Phosphatidate phosphohydrolase (EC 3.1.3.4) was detected in isolated bovine rod outer segments and its properties investigated. The enzyme activity was assayed using aqueously dispersed 1,2-diacyl-sn-[2-3H]glycerol 3-phosphate as substrate. The phosphatidic acid concentration was optimal at 1 mM and the estimated Km value was 6.7 X 10(-4) M. The activity was linear for 60 min with a protein concentration of 0.3 mg. A clear pH optimum was observed at 7.5. When the enzyme activity was measured using a substrate phosphatidic acid containing 15% lyso compound, the production of diacylglycerols was inhibited by about 70-75% at all concentrations studied. In rod outer segment preparations containing 0.2 mM Mg2+, further additions of the ion (0.2-2.5 mM) only produced a slight inhibition of the activity at 2.5 mM. F- (50 mM), Ca2+ (1 mM) and EDTA (50 mM) inhibited the dephosphorylation of the substrate by 80, 10 and 70%, respectively. The aqueously dispersed phosphatidic acid-dependent activity present in rod outer segments was stimulated by Triton X-100 and taurocholate. Similar values were observed in the enzyme activity of entire rod outer segments and in that of disks obtained from them, showing the enzyme to be associated with disk membrane. The [3H]diacylglycerol production from [3H]phosphatidic acid was analyzed in synaptosomal-mitochondrial and microsomal fractions and in a crude pigment epithelium preparation. The degree of dephosphorylation of phosphatidic acid in these subcellular fractions was as follows: microsomes greater than synaptosomal-mitochondrial fraction greater than rod outer segment greater than crude pigment epithelium. The present report is the first evidence of phosphatidic acid phosphohydrolase activity associated with rod outer segment membranes.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D004075:Diglycerides / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D017830:Octoxynol; D010711:Phosphatidate Phosphatase / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010786:Photoreceptor Cells / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013997:Time Factors
D004075:Diglycerides; D010712:Phosphatidic Acids; D011092:Polyethylene Glycols; D017830:Octoxynol; D004492:Edetic Acid; D010744:Phosphoric Monoester Hydrolases; D010711:Phosphatidate Phosphatase; D008274:Magnesium; D005459:Fluorides; D002118:Calcium