L-asparaginasa de Escherichia coli induce fosforilación de polipéptidos endógenos en células inmunitarias humanas

Purpose: To detect patterns of endogenous polypeptide phosphorylation in monocyte, lymphocyte, and polymorphonuclear leukocyte populations, induced by the products of the catalytic action of L-asparaginase (EcA). Materials and methods: Monocytes, polymorphonuclear cells and lymphocytes were isolated from heparinized blood from healthy, volontary donors. The samples were incubated in 0.4 mCi/ml of [^γ-32P]H₃PO₄,with: 1 μg/μl of EcA, EcA and the substrate or with the products of EcA's catalytic activity: NH₄⁺ and aspartate. The cells were lysated and electrophoresed using denaturing polyacrylamide gels that were then exposed on radiographic plates. The levels of polypeptide phosphorylation were quantified by computer densitometric analysis. Results: The autoradiographs and the densitometric quantification of the electrophoretic profiles of monocytes, polymor-phonuclear leukocytes, and lymphocytes revealed an increase in polypeptide phosphorylation when the cells were incubated with the enzyme and its substrate, ammonium and aspartate, or ammonium, which demonstrates that the NH₄⁺ triggers intracellular phosphotransferase activity. A 58 kDa phosphoprotein outstood, it being common to the three cell populations studied. There were also specific phosphorylable polypeptides in monocytes, polymorphonuclear leukocytes, and lymphocytes. Conclusions: Escherichia coli L-asparaginase, binds the plasma membrane in normal human immune cells, catalyzing the L-asparagine substrate. The products of its activity: aspartate and NH₄⁺ modify the extracellular environment, particularly the latter since it could diffuse into the cytosol and modify the pH, which would activate signal transduction pathways associated with the phosphorylation of substrates.