PHOSPHORYLATION OF A 25 KDA PROTEIN IS INDUCED BY THERMOSTABLE DIRECTHEMOLYSIN OF VIBRIO-PARAHAEMOLYTICUS

Thermostable direct hemolysin (TDH) is a possible virulence factor pro duced by Vibrio parahaemolyticus, Although TDH has a variety of biolog ical activities, including hemolytic. activity, the biochemical mechan ism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found t hat TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using [gamma-P-32] ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, t o determine which types of kinases were involved in the phosphorylatio n events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecul ar weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22 .5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte me mbranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyt e membranes. Copyright (C) 1996 Elsevier Science Ltd