Light-dependent DAGK activity in a purified photoreceptor nuclear fraction from bovine retinas.

NATALINI, P.M.; ZULIAN, S.E.; GIUSTO, N.M.; ILINCHETA DE BOSCHERO, M.G.

Potrero de los Funes, San Luis

Congreso; XLVII Reunión Anual de la SAI B; 2011

Sociedad Argentina de Investigación de Bioquímica y Biología Molecular

Diacylglycerol kinase (DAGK) activity, which synthetizes phosphatidic acid (PA) from 3H-diacylglycerol and ATP, was observed in isolated nuclei from bovine retina (AIVO, 2010). The purpose of this study was to analyze the effect of light on DAGK activity in isolated nuclei from photoreceptor cells. To this end, bovine eye cups were exposed to light (dark/light model). Nuclei isolation procedure was slightly modified to purify nuclei from photoreceptor cells. Isolated retinas were homogenized in 0.25 M sucrose, filtrated and combinated with a sucrose solution to obtain 1.6 M sucrose as a final concentration (H1), layered on the top of a discontinous gradient of 2.3 and 2.5 M sucrose and centrifuged during 70 min at 25000 rpm. N1 and N2 (interphases) and N3 (pellet) were thus obtained. Small nuclei from photoreceptor cells were found in N3. Results derived from immunofluorescence (IF) with CRX, a transcription factor from photoreceptor cell nuclei, revealed purification. A detergent-lipid mixed micelar assay was used to evaluate DAGK activity. An increased activity (two times) was observed in the N3 nuclei fraction from light-exposed retinas with respect to that found in dark-adapted retinas. IF studies revealed an increased light-dependent signal for anti-DAGK dzeta and épsilon. These data indicate that light induces an increased presence and activity of DAGK in nuclei from photoreceptor cells.