CHARACTERIZATION OF PHOSPHOLIPASE-D ACTIVITY IN BOVINE PHOTORECEPTOR-MEMBRANES

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod ou ter 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 [C-14]ethanol. Using [H-3]PC and ethanol as a substrate, we were able to detect the hydrolytic pro perties as well as the transphosphatidylation reaction catalyzed by ph ospholipase D (PLD): formation of [H-3]phosphatidic acid and phosphati dylethanol [H-3]PtdEt; whereas with [C-14]ethanol or [H-3]glycerol in the absence of exogenous PC, only transphosphatidylation reactions wer e detected (formation of [C-14]PtdEt or [H-3]phosphatidylglycerol, res pectively). The use of varying concentrations of [H-3]PC and 400 mM of ethanol gave an apparent K-m value for PC of 0.51 mM and a V-max valu e of 111 nmol x h(-1) x (mg protein)(-1). The activity was linear up t o 60 min of incubation and up to 0.2 mg of protein. The optimal ethano l concentration was determined to be 400 mM, with an apparent K-m of 2 02 mM and a V-max value for ethanol of 125 nmol x h(-1) x (mg protein) (-1). A clear pH optimum was observed around 7. PLD activity was incre ased in the presence of olamidopropyl)dimethylammonio]-1-propane-suifo nate or sodium deoxycholate and inhibited with Triton X-100. The enzym e activity was also activated in the presence of Ca2+ or Mg2+ (1 mM) a lthough these ions were not required for measuring PLD activity. The h igh specific activity of PLD found in purified ROS compared to the act ivity found in other subcellular fractions of the bovine retina sugges ts that this enzymatic activity is native to ROS. The present report i s the first evidence of PLD activity associated with photoreceptor ROS .