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Abstract
The localization of phospholipase C delta3 (PLC delta3) in the cell and its regulatory properties has been investigated. Western blotting showed that human platelet PLC delta3 is located in the membrane and cytosolic fraction. The enzyme amount in the cytosolic fraction was significantly lower than that in the membrane fraction. In rat liver, PLC delta3 was present in both the membrane and cytosolic fraction and was absent in nuclei. Examination of the effects of phospholipids on PLC delta3 revealed that this enzyme is inhibited by phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Similar inhibition was observed in the presence of sphingomyelin and phosphatidylserine (PtdSer). This is in contrast to PLC delta1, which is activated by PtdCho and PtdEtn. In a detergent assay, PLC delta1 is activated by spermine and sphingosine, whereas PLC delta3 was inhibited by both these compounds at concentrations that maximally stimulated PLC delta1. A deletion mutant of PLC delta3, lacking the entire pleckstrin homology (PH) domain (residues 1-137), was fully active in the detergent assay, and it was inhibited by spermine, sphingosine and phospholipids to the same extent as the native enzyme. PLC delta3 activation required calcium ions. The relationship between the Ca2+ concentration and enzymatic activity was almost identical for the deletion mutant and the native enzyme. However, in the liposome assay, PLC delta3 was less sensitive to Ca2+ stimulation. This is in contrast to PLC delta1, which is equally sensitive to Ca2+ stimulation in both the detergent and liposome assays. We conclude that Ca2+ is necessary to induce specific conformational changes of PLC delta3, which leads to a productive orientation of the catalytic domain relative to the membrane. The regulatory properties of PLC delta3 described in this report suggest that PLC delta3 has a relatively low activity in cellular conditions that fully activate PLC delta1.
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