Journal of Experimental Medicine, Vol 159, 89-102, Copyright © 1984 by Rockefeller University Press
Subcellular localization of the PGE2 synthesis activity in mouse resident peritoneal macrophages
C Darte and H Beaufay
The aim of this work was to establish, on a quantitative basis, the
subcellular distribution of the enzyme system that converts arachidonic
acid into prostaglandin (PG) E2 in mouse resident peritoneal (MRP)
macrophages. Kinetic studies were conducted on cell-free extracts derived
from cells cultivated for 1 d, using [1-14C]arachidonic acid as substrate
and measuring the label in PGE2 after extraction and thin layer
chromatography. The activity was synergistically enhanced by L- adrenaline
and reduced glutathione, inhibited by indomethacin, and linearly related to
the concentration of the cell-free extract. It was labile at 0 degrees C in
the medium used for homogenization and fractionation of the cells
(half-life less than 2 h). Addition of catalase (0.15 mg/ml) to the
suspension medium increased the initial activity (by congruent to 70%) and
the stability (half-life congruent to 6 h) of the enzyme in cytoplasmic
extracts. It enabled us to establish the density distribution after
isopycnic centrifugation in a linear gradient of sucrose. The sample
centrifuged consisted of untreated cytoplasmic extracts, or cytoplasmic
extracts treated with digitonin and Na pyrophosphate. Comparison of the
centrifugation behavior of PGE2 synthesis activity with that of various
enzymes used as reference for the major subcellular entities has revealed
that PGE2 synthesis fairly fits the density profile of sulfatase C in each
case. The conclusion is that at least the rate-limiting reaction in the
conversion of arachidonic acid into PGE2 is catalyzed by an enzyme
associated with the endoplasmic reticulum.
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