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[摘要]：In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits G alpha(q) and G beta(1) respectively. When FLAG-tagged G alpha(q) or G beta(1) was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg(2+) dependency and substrate specificity of G alpha(q) were similar to those of lysoPLD purified from the rat brain. Mutation of G alpha(q), at amino acids Lys(52), Thr(186) or Asp(205), residues that are predicted to interact with nucleotide phosphates or catalytic Mg(2+), dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged G alpha(q) overexpressed in COS-7 cells was similar to 4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated K(m) and V(max) values for lysoPAF (1-O-hexadecyl-snglycero-3-phosphocholine) obtained from Neuro2A cells were 21 mu M and 0.16 mu mol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for G alpha(q) and G beta(1) as an enzyme with lysoPLD activity. Tag-purified G alpha(11) also exhibited a high lysoPLD activity, but G alpha(i) and G alpha(s) did not. The lysoPLD activity of the G alpha subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with G alpha(q) and G alpha(11) siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies. |
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