Kawakami N, Shimohama S, Hayakawa T, Sumida Y, Fujimoto S
Department of Environmental Biochemistry, Kyoto Pharmaceutical University, Japan.
Biochim Biophys Acta. 1996 Nov 8;1314(1-2):167-74. doi: 10.1016/s0167-4889(96)00077-8.
We examined in detail the tyrosine phosphorylation of proteins, especially inositol phospholipid-specific phospholipase C (PLC) gamma 2, during activation of respiratory burst of guinea pig polymorphonuclear leukocytes (PMNs) by pervanadate. The pervanadate, generated from a combination of H2O2 and orthovanadate, induced concomitantly tyrosine phosphorylation of 145, 120, 104, 76, 68, 60, 53, 42, 37, 28, and 25 kDa proteins and superoxide anion (O2-) production of PMNs. The pretreatment of PMNs with genistein caused an inhibition of tyrosine phosphorylation of these proteins, and also markedly depressed O2- production. Among the above proteins, a 145 kDa protein was found to be identical with the protein recognized by the anti-PLC gamma 2 antibody on Western blots. PLC gamma 2 was detected in the cytosol fraction but not in the membrane fraction of resting PMNs, whereas it was detected in both cytosol and membrane fractions of pervanadate treated PMNs. PLC activity of pervanadate treated PMNs was higher than that of resting cells. In addition, the enzyme activity of the cytosol fraction from the former cells was significantly lower than that from the latter cells, whereas the enzyme activity of membrane fraction from the former cells was significantly higher than that from the latter cells. These findings suggest that the tyrosine residue(s) of PLC gamma 2 is phosphorylated and the enzyme is translocated from the cytosol to membrane fractions in PMNs by pervanadate treatment.
我们详细研究了在过氧钒酸盐激活豚鼠多形核白细胞（PMN）呼吸爆发过程中蛋白质的酪氨酸磷酸化情况，特别是肌醇磷脂特异性磷脂酶C（PLC）γ2。由过氧化氢和原钒酸盐组合生成的过氧钒酸盐，可诱导PMN中145、120、104、76、68、60、53、42、37、28和25 kDa蛋白质的酪氨酸磷酸化以及超氧阴离子（O2-）的产生。用染料木黄酮预处理PMN会抑制这些蛋白质的酪氨酸磷酸化，并且还会显著降低O2-的产生。在上述蛋白质中，发现一种145 kDa的蛋白质与Western印迹上抗PLCγ2抗体识别的蛋白质相同。在静息PMN的胞质溶胶部分中检测到PLCγ2，但在膜部分中未检测到，而在过氧钒酸盐处理的PMN的胞质溶胶和膜部分中均检测到。过氧钒酸盐处理的PMN的PLC活性高于静息细胞。此外，前者细胞胞质溶胶部分的酶活性明显低于后者细胞，而前者细胞膜部分的酶活性明显高于后者细胞。这些发现表明，PLCγ2的酪氨酸残基被磷酸化，并且通过过氧钒酸盐处理，该酶在PMN中从胞质溶胶转运至膜部分。
