Oleic acid promotes the activation and translocation of phosphatidate phosphohydrolase from the cytosol to particulate fractions of isolated rat hepatocytes

Abstract

The incubation of hepatocytes with 1-4mM-oleate increased the total activity of phosphatidate phosphohydrolase that was measured in the presence of Mg2+ to about 2-fold. This was accompanied by an increase in the proportion of the enzyme that was isolated with the particulate fractions. Conversely, the addition of up to 4mM-oleate decreased the recovery of phosphatidate phosphohydrolase in the cytosolic fraction from about 70% to 3% when hepatocytes were lysed with digitonin. Most of the increase in the membrane-associated phosphohydrolase activity was isolated after cell fractionation in the microsomal fraction that was enriched with the endoplasmic-reticulum marker arylesterase. It is proposed that the translocation of phosphatidate phosphohydrolase facilitates the increased synthesis of triacylglycerols in the liver when it is presented with an increased supply of fatty acids.

Conserved Residues in the N Terminus of Lipin-1 Are Required for Binding to Protein Phosphatase-1c, Nuclear Translocation, and Phosphatidate Phosphatase Activity

“…Our group demonstrated the ability of Mg 2ϩ -dependent PAP activity (now attributed to the lipins) to translocate from the cytosol onto the membranes of the endoplasmic reticulum when stimulated with unsaturated fatty acids due to the increase in negative charge on the membrane surface (15)(16)(17)37). This was essentially corroborated after the lipins were discovered to be responsible for PAP activity (4,5,9).…”

A rapid assay for measuring the activity and the Mg2+ and Ca2+ requirements of phosphatidate phosphohydrolase in cytosolic and microsomal fractions of rat liver

“…la). This stimulation was also seen in five independent experiments with subcellular fractions from liver, and with five membrane and soluble fractions prepared from cultured hepatocytes after digitonin treatment [16,33]. Higher concentrations of EDTA inhibited the reaction.…”

“…This latter method gave almost complete removal of glycerol and glycerol phosphate, but this was not essential, since the recoveries of these compounds in Table 1 It ought to be possible to determine the activity of the Mg2+-dependent PAP activity by measuring the activity in the presence of either EDTA or an optimum concentration of Mg2+. The difference between these values should therefore reflect the Mg2+-dependent activity [12,23,33]. This approach was therefore tested with two preparations of phosphatidate that contained different concentrations of Ca2+, to see whether this cation affected the interpretation of the results.…”

Purification of Mg2+-dependent phosphatidate phosphohydrolase from rat liver: new steps and aspects

“…-PAP on the basis of known properties of HSP90b, such as its ability to bind to calmodulin Minami et al, 1993), to form large protein aggregates not entering a polyacrylamide gel under native conditions (Nemoto and Sato, 1998) and to bind to actin under conditions of Mg 2q concentration and temperature Nishida et al, 1986) very similar to those used in this study for the precipitation of Mg 2q -PAP from the particle-free cytosolic supernatant. The results presented here not only provide new steps for purification of the enzyme, but also open new aspects for the understanding of previous findings, such as the translocation of Mg 2q -PAP from the cytosolic to the microsomal compartment (Cascales et al, 1984;Martín-Sanz et al, 1984;Hopewell et al, 1985) and the effect of calcium on Mg 2q -PAP activity (Pollard and Brindley, 1984;Martin et al, 1986). According to the evidence presented, it may well be that the (direct or indirect) relationship of Mg2 q -PAP activity to HSP90b represents a common basis to characterise these phenomena as actin/calmodulin-dependent interactions.…”