Rosenblat_2010_Atherosclerosis_213_408

OBJECTIVE: To analyze the ability of macrophage sub-cellular fractions to stabilize paraoxonase 2 (PON2).
METHODS: Nuclei, mitochondria, lysosomes, endoplasmic reticulum (ER) and cytosol were isolated from J774A.1 macrophage cell line and incubated with recombinant PON2.
RESULTS: Among the fractions analyzed the ER contains the highest PON2 lactonase activity, and was the most potent one in stabilizing recombinant PON2 (rePON2). Whereas control rePON2 activity was decreased by 40% after 20 h of incubation at 37 degrees C, in the presence of ER it decreased by only 15%. This effect could be attributed to the ER aqueous phase, and not to the ER lipids. The ER proteins fraction was responsible for PON2 stabilization, since heated ER or proteinase K-treated ER was not able to protect rePON2 from inactivation, while the protein fraction (after ammonium sulfate precipitation) completely prevented rePON2 inactivation. Since in the macrophage ER, there are increased levels of NADPH, secondary to glutathione reductase deficiency, we next studied the effect of the redox environment on PON2 inactivation. Incubation of rePON2 with DTT protected PON2 from inactivation. Similarly, NADPH, but not NADP, significantly increased rePON2 lactonase activity by up to 19%, after 20h of incubation as compared to control rePON2. Unlike ER from non-treated macrophages, ER harvested from oxidized-, or from cholesterol loaded-macrophages showed a significant lower basal PON2 lactonase activity, and did not protect PON2 from inactivation but rather increased it. CONCLUSION: Under normal conditions macrophage ER stabilizes PON2 activity, and this effect could be attributed to ER proteins and redox status.