INVESTIGADORES
GIUSTO Norma Maria
artículos
Título:
Phospholipase D activity in rod outer segment of retinal photoreceptor cells
Autor/es:
SALVADOR, G.A.; GIUSTO, N.M.
Revista:
LIPIDS
Editorial:
SPRINGER HEIDELBERG
Referencias:
Año: 1998 vol. 33 p. 853 - 860
ISSN:
0024-4201
Resumen:
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.