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Effects of phosphatidylinositol 4,5-bisphosphate and neomycin on phospholipase D: Kinetic studies

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Abstract

The kinetics of phosphatidylcholine-specific phospholipase D activated by phosphatidylinositol 4,5-bisphosphate (PIP2) and inhibition by neomycin were studied in an enzyme preparation partially purified from human hepatocarcinoma cell line. It was found that phospholipase D was marginally activated by phosphatidyl-4-phosphate (PIP) and phosphatidylethanolamine (PE). In contrast, it was considerably activated by PIP2 in different concentration of phosphatidylcholine (PC). Sphingomyelin (SM), lysophosphatidylcholine (LPC) and phosphatidylserine (PS) were neither substrates nor inhibitors of the phospholipase D. PIP2 induced an allosteric effect on phospholipase D and a negative cooperative effect with respect to phosphatidylcholine as indicated in the Lineweaver-Burk plot. In the absence of PIP2, a straight line was obtained, whereas a downward concave curve was observed in the presence of 25 μM of PIP2. The Hill coefficient and the apparent Km of phosphatidylcholine in the presence of 25 μM PIP2 were calculated to be 0.631 and 10.79 mM, respectively. PIP2 also increased the maximal velocity (Vmax) of the phospholipase D reaction, suggesting that the affinity of substrate to enzyme was decreased, and the turnover number of the enzyme (kcat) was increased by PIP2. The activation of phospholipase D by PIP2 was dose dependent up to 50 μM of PIP2. The Ka of PIP2 was 15.8 mM. Neomycin, a polycationic glycoside, was shown to be an uncompetitive inhibitor of phospholipase D, and revealed the formation of a neomycin-PIP2 complex. The Ki of neomycin was estimated to be 8.7 mM.

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Huang, Y., Qureshi, A. & Chen, H. Effects of phosphatidylinositol 4,5-bisphosphate and neomycin on phospholipase D: Kinetic studies. Mol Cell Biochem 197, 195–201 (1999). https://doi.org/10.1023/A:1006930706311

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