[Escherichia coli L-asparaginase induces phosphorylation of endogenous polypeptides in human immune cells].
OBJECTIVE
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).


METHODS
Monocytes, polymorphonuclear cells and lymphocytes were isolated from heparinized blood from healthy, voluntary donors. The samples were incubated in 0.4 mCi/ml of [gamma-32P]H3PO4, with: 1 microgram/microliter 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, polymorphonuclear 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.
Mercado
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Laboratorio de Fisiología Celular, Universidad Católica de Valparaiso, Chile.
Arenas
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D001215:Asparaginase / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007111:Immunity, Cellular; D008214:Lymphocytes / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D010750:Phosphoproteins / Q000097:blood; D010766:Phosphorylation
D001798:Blood Proteins; D010750:Phosphoproteins; D001215:Asparaginase