Objective: To demonstrate that the enzyme L-asparaginase from Escherichia coli (EcA) binds to the plasma membranes of normal human lymphocytes and monocytes. Material and methods: Lymphocytes and monocytes were isolated from heparinized blood samples which came from healthy volunteer donors. The cells were incubated with EcA to detect a possible binding of the enzyme to the mononuclear cells by indirect immunofluorescence using confocal microscopy. Meanwhile, ultracentrifugation was used to obtain the erythrocyte ghost microsomal fraction (P100) which was then analyzed by Western blotting to determine if EcA binds the lipid bilayer unspecifically. For the immunoassays, monospecific polyclonal antibodies were obtained from ascitic tumors developed in mice immunized with commercial L-asparaginase. Results: EcA bins the lymphocyte and monocyte plasma membranes. In monocytes, there occurs a capping phenomenon, that is, the accumulation of fluorescent marker in one region. The image analyzer highlights it clearly at a depth of 3.8 μm. This binding would be unspecific, that is, there is no mediation of a specific receptor that binds EcA. This arises from the ability of the enzyme to bind to the membranes of erythrocyte ghost, as evidenced by the ability of the molecule to associate with a hydrophobic medium. The antibodies against EcA obtained from ascitic tumours developed in mice do not show cross reactivity with Na+/K+ ATPase, aspartate aminotranferase, nor with extracts of blood cells, which would make it a specific tool for the detection of EcA in whole cells and in homogenates electrotransferred to nitrocellulose membranes. Conclusion: L-asparaginase from E. coli behaves as a lipoprotein due to its ability to insert itself into hydrophobic environments, in which it resembles an isozyme present in T. pyriformis. The binding of this enzyme to lymphocytes and monocytes, demonstrated in this work, would permit the modification of the antileukemic treatment injecting doses of EcA bound to the patient's own isolated immune cells.
|Translated title of the contribution||Binding of the antileukaemic drug L-asparaginase from escherichia coli to the plasma membrane of normal human mononuclear cells|
|Number of pages||6|
|State||Published - 1 Dec 1999|