[Binding of the antileukemia drug Escherichia coli L-asparaginase to the plasma membrane of normal human mononuclear cells]. / Asociación de la droga antileucémica L-asparraginasa de Escherichia coli a la membrana plasmática de células mononucleares humanas normales.

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 microns. 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 aminotransferase, nor with extracts of blood cells, which would make it a specific tool for the detection of EcA in whole cells and in homogenates electrotransfered 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 patient's own isolated immune cells.

L-aspartate aminotransferase and L-asparaginase in rat lymph node: a histoenzymological and immunohistochemical study.

L-asparaginase and L-aspartate aminotransferase are both involved in the synthesis of L-aspartic acid. It has been observed that L-asparaginase is involved in the immunosuppressor morphine-dependent syndrome in lymphoid cells whereas L-aspartic acid blocks the development of this syndrome. The aim of the present study was to clarify the localization of L-AATase activity and L-asparaginase in rat lymph nodes using histoenzymological and immunohistochemical methods, respectively. No positive reaction was demonstrated for L-AATase while L-asparaginase shown to be present in lymphocytes and lymphoblastic cells. These observations lead us to suggest that L-asparaginase is the enzyme mainly responsible for the synthesis of the L-aspartic acid necessary for satisfying the living requirements of lymphoid cells. Therapeutically administered L-asparaginase could exert its action intracellularly after crossing the cell membrane.

Bacterial aspartate kinase-like activity in human platelet.

One form of a group of enzymes known as aspartate kinases, primarily reported in prokaryotes and plants, might also exist in animal cells. Here we report the immunodetection of an aspartate kinase-like activity in human platelets using antibodies against the pure form of the enzyme purified from Escherichia coli. Moreover, the enrichment of platelet extracts with the bacterial kinase results in the phosphorylation of discrete forms mainly of membrane-bound endogenous polypeptides.

Light and electron microscopic immunolocalization of L-asparaginase in the rat central nervous system.

The investigation on the localization of L-asparaginase, the enzyme involved in the synthesis of L-aspartic acid, has been carried out using the immunohistochemical method. Antibodies against this enzyme were obtained immunizing BALB/c mice with purified Escherichia coli L-asparaginase. Light microscopic observation revealed positive immunoreactivity in the great majority of neurons and glial cells, and electron microscopic analysis demonstrated immunological localization of the enzyme in the cytosol. The ubiquitous distribution of L-asparaginase suggests its involvement in many important functions of the central nervous system.

Exogenously added free phosphotyrosine induces aggregation of circulating platelets in rabbits.

When free phosphotyrosine is injected into rabbits, circulating aggregated platelets are readily observed in concomitance with altered electrocardiographic profiles. Since phosphotyrosine is also able to induce platelet aggregation in vitro, altogether these results suggest that free phosphotyrosine in blood could be meaningful for in vivo platelet activation.

Immunolocalization of cytosolic aspartate aminotransferase (cAAT) in axon terminals that form synapses in the rat cerebellar cortex. A study at the electron microscopic level.

This study was conducted to determine the ultrastructural localization of cytosolic aspartate aminotransferase (cAAT)-like immunoreactivity in the cerebellar cortex in the rat. The isoenzyme was found both in excitatory and inhibitory axon terminals, but not in the climbing fibers of the molecular layer. These findings suggest that cAAT may have a different role in the excitatory and inhibitory synapses, and that climbing fibers of the molecular layer do not appear to use aspartate as neurotransmitter.

Aspartate kinase as an enzyme able to phosphorylate aspartate in the nervous system. Immunocytochemical study.

An immunohistochemical method using antibodies against bacterial aspartate kinase has been carried out in order to localize this enzyme in the cerebellar cortex of rat. The results obtained with light microscope have demonstrated a positive immunoreaction in both the granular and molecular layers. Electron microscopic observation has also revealed the reaction in several dendrites, axons and myelinated fibers. These facts lead us to think that aspartate kinase could be involved in the phosphorylation of aspartate in the nervous tissue.

Partial characterization of a highly conserved aspartyl kinase (AK) in normal human liver.

Subcellular fractions from human liver were assayed for aspartyl kinase (AK) activity measured by standard spectrophotometric methods. Along the purification procedure three different fractions displayed the expected enzyme activity. Interestingly, two of these fractions were specifically recognized by antibodies raised against E. coli aspartate kinases, suggesting a high degree of evolutionary conservation for these ubiquitous enzymes for prokaryotes. Since their known function in bacteria is not strictly required in eukaryotes, these observation imply that the presence and activity of aspartyl kinase(s) in mammals might represent putative regulatory roles for these enzymes in eukaryotic cell metabolism.

Thiamine-like molecules in the cerebellar cortex of the rat: light and electron microscopic immunocytochemical investigation.

Immunocytochemical methods were used for studying the location of thiamine-like molecules in the cerebellar cortex of the rat. A positive reaction was observed in several synapses and in several dendrites. This reaction was associated with tubular formations and synaptic vesicles. A positive reaction was also found in several mitochondria and in glial cells. The possible role of thiamine-like molecules in cerebellar cortex is discussed.

Light and electron microscope observations of aspartate aminotransferase (AATase) in the hippocampus revealed by histoenzymological and immunocytochemical methods.

Immunocytochemical and histoenzymological were used to determine the localization and activity of AATase respectively at light and electron microscopic levels. Ths histoenzymological method revealed AATase activity in synaptic vesicles and in mitochondria. Both methods detected AATase presence and activity in pre and post-synaptic sites. No simultaneous positive reaction was observed at pre and post-synaptic zones of a given synapses. The significance of these results are discussed in the present paper.