Abstract
Two forms of phospholipase D (PLD) have been found to be present in nuclei isolated from rat hepatocytes by measuring phosphatidylbutanol produced from exogenous radiolabeled phosphatidylcholine in the presence of butanol. In nuclear lysates from either rat liver or ascites hepatoma AH 7974 cells, the PLD activity was markedly stimulated by a recombinant ADP-ribosylation factor (rARF) in the presence of the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and phosphatidylinositol 4, 5-bisphosphate. ATP and phorbol-12-myristate 13-acetate had no synergistic effect on this PLD activity. On the other hand, the nuclear PLD was stimulated by unsaturated fatty acids, especially by oleic acid. The ARF-dependent nuclear PLD activity was increased in the S-phase of the regenerating rat liver after partial hepatectomy and also was much higher in AH 7974 cells than in the resting rat liver. In contrast, the levels of the oleate-dependent PLD activity remained constant throughout the cell cycle in liver regeneration. The intranuclear levels of the stimulating proteins of the nuclear PLD activity, e.g. ARF, RhoA, and protein kinase Cdelta increased in the S-phase of the regenerating liver. These results suggested that the nuclear ARF-dependent PLD activity may be associated with cell proliferation.
Highlights
Two forms of phospholipase D (PLD) have been found to be present in nuclei isolated from rat hepatocytes by measuring phosphatidylbutanol produced from exogenous radiolabeled phosphatidylcholine in the presence of butanol
PLD activity was examined in nuclei isolated from rat resting liver and AH7974 cells using the exogenous radiolabeled PC substrates in the presence or absence of recombinant ADP-ribosylation factor (rARF) (5 M) and guanosine 5O-(3-thiotriphosphate) (GTP␥S) (30 M) or oleate (0.5 mM)
We have demonstrated here that two forms of PLD activity, GTP␥S-dependent and oleate-dependent, were present in the isolated nuclei from rat resting, regenerating liver cells, and rat ascites hepatoma AH7974 cells
Summary
Materials—[2-palmitoyl-9,10-3H]Dipalmitoylphosphatidylcholine (DPPC) (37.5 Ci/m mol) was obtained from DuPont NEN. For the assay of oleate-dependent PLD activity, egg PC vesicles (20 l) containing 15 nmol of egg PC and 400,000 dpm of [3H]DPPC were prepared by sonication and mixed with 5 l of 12 mM sodium oleate. For the assay of ARF-dependent PLD activity, mixed lipid vesicles (PE/PIP2/egg PC, 160/14/10 M) containing [3H]DPPC to yield 400,000 dpm/assay were prepared. The substrates (25 l) were added to 10 ml of nuclear lysates in a total of 120 l containing 50 mM HEPES/NaOH buffer, pH 7.5, 3 mM EGTA, 80 mM KCl, 2.5 mM MgCl2, 1 mM dithiothreitol, 1.2 mM CaCl2, 5 M recombinant ARF (rARF), 30 M GTP␥S, and 0.3% butanol and incubated at 37 °C for 1 h. Protein preparation showed only one band on Coomassie-stained SDS-polyacrylamide gels
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