. Differential role of soluble and peripheral proteins in phosphatidic acid metabolization in rod outer segments. Light Effect.

PASQUARE, SUSANA; SALVADOR, GABRIELA; GIUSTO NORMA,

Buenos Aires

Congreso; International Congress of Eye Research; 2006

Society of Eye Research

We have reported the presence of phosphatidate phosphohydrolase 2 isoform (PAP2) and its inhibition in ROS by light. PAP2 hydrolyzed not only phosphatidic acid (PA) but also other phosphorylated substrates such as lysophosphatidic acid (LPA), ceramide-1-phosphate (C-1-P) and sphingosine-1-phosphate (S-1-P) producing different intracellular mediators. The purpose of this work was to evaluate the preference of PAP2 isoforms in ROS by alternative substrates and to analyze the enzymatic activity when soluble and peripheral proteins were absent. Bovine retinas were maintained in dark (DROS) or they were bleached for 20 min (BROS) and ROS were depleted of soluble and peripheral proteins by low (5 mM) ionic strength. The membranes obtained following this procedure were used as source of the enzymes. [3H]PA (100 µM) was used as substrate either presence or absence of LPA, S-1-P and C-1-P, 100 µM, and the reaction products either [3H]DAG and [3H]MAG, were determined. The presence of PAP2b was observed by Western Blot and no differences were found between DROS and BROS. PAP2b was not detected in the supernatants obtained from membranes extracted by low ionic strength. PAP2 activity was inhibited by 58% in BROS with respect to the activity determined in DROS. DAG production in depleted membranes from DROS was diminished by the presence of LPA and S-1-P in the same percentage, 43% and 17% when C-1-P was present. In BROS 5 mM membranes, the most important competitive effect was observed in the presence of C-1-P (44%). This effect was lower (33%) in the presence of LPA and S-1-P. [3H]MAG production in 5 mM DROS was diminished by 45% in the presence of LPA, C1P and S1P whereas in membranes from BROS this product formation was diminished by 20% when LPA and S-1-P were present and by 48% in the presence of C-1-P. Our results are indicative of a differential preference of PAP2 by its alternative substrates in DROS and BROS.