Characterization of phospholipase D activity in bovine photoreceptor membranes.
Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2(3)H]glycerol-3-phosphocholine (PC), or [14C]ethanol. Using [3H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [3H]phosphatidic acid and phosphatidylethanol [3H]PtdEt; whereas with [14C]ethanol or [3H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [14C]PtdEt or [3H]phosphatidylglycerol, respectively). The use of varying concentrations of [3H]PC and 400 mM of ethanol gave an apparent Km value for PC of 0.51 mM and a Vmax value of 111 nmol x h(-1) x (mg protein)(-1). The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent Km of 202 mM and a Vmax value for ethanol of 125 nmol x h(-1) x (mg protein)(-1). A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.
Salvador
G A
GA
Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur, CONICET, Buenos Aires, Argentina.
Giusto
N M
NM
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology*; D003902:Detergents / Q000737:chemistry / Q000494:pharmacology; D000431:Ethanol / Q000737:chemistry; D020404:Glycerophospholipids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D019301:Oleic Acid / Q000737:chemistry / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010739:Phospholipase D / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000201:enzymology*; D012160:Retina / Q000737:chemistry; D012374:Rod Cell Outer Segment / Q000201:enzymology; D013997:Time Factors
D003902:Detergents; D020404:Glycerophospholipids; D010713:Phosphatidylcholines; C051521:phosphatidylethanol; D019301:Oleic Acid; D000431:Ethanol; D010739:Phospholipase D; D008274:Magnesium; D002118:Calcium