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METABOLISM OF ASPARAGINE AND GLYCINE IN MITOCHONDRIA AND LYMPHOMA CELLS: METABOLIC BASES FOR RESISTANCE TO L-ASPARAGINASE
Abstract
A method for simultaneously assaying asparagine synthetase and its glutaminase activity was developed. The method was based on the derivatization of the amino acids, aspartate, asparagine, glutamate, and glutamine with o-phthaldialdehyde. These fluorescent derivatives were analyzed by reversed-phase high-performance liquid chromatography. The sensitivity of the assay was in the sub-picomol range. Rat liver mitochondria were found to catabolize asparagine to oxaloacetate mostly via an intermediate of (alpha)-ketosuccinamate, or to a lesser extent via aspartate. In addition, a portion of the oxidation of asparagine to carbon dioxide was accomplished in the hepatocytic mitochondria through a previously unrecognized route which was not sensitive to inhibition by a transaminase inhibitor. Factors that influenced the Krebs cycle and oxidative phosphorylation also had a great effect on the extent of mitochondrial asparagine catabolism. Amino acid content was studied in vitro under various conditions in mouse lymphoma cells either sensitive (L5178Y) or resistant (L5178Y/L-ASE) to L-asparaginase treatment. The concentration of asparagine and essential amino acids was approximately 1.5-fold higher in the resistant cells. Although asparagine synthetase activity was found only in resistant cell extracts treatment with L-asparaginase depleted asparagine from both cell types. However, only the resistant cells had the ability to "rebound". L-asparaginase dose and time response patterns were indicative of the existence of more than a single intracellular asparagine pool. Radiolabel conversion studies indicated that asparagine-dependent glycine synthesis was diminished in sensitive, but not in resistant cells when exposed to L-asparaginase. An L-asparaginase resistant subclone (L5178J-D10) derived from its sensitive parent cell line (L5178Y) showed increased asparagine synthetase activity, elevated ability to extract respiratory energy from the medium and a mechanism for conservation of the cellular serine content presumably to ensure protein synthesis. During L-asparaginase treatment, both of these features were maintained by resistant cells while sensitive cells were further impaired by the loss of any detectable malic enzyme-mediated products.
Subject Area
Biochemistry
Recommended Citation
MORAGA AMADOR, DAVID ALFREDO, "METABOLISM OF ASPARAGINE AND GLYCINE IN MITOCHONDRIA AND LYMPHOMA CELLS: METABOLIC BASES FOR RESISTANCE TO L-ASPARAGINASE" (1986). ETD collection for University of Nebraska-Lincoln. AAI8704537.
https://digitalcommons.unl.edu/dissertations/AAI8704537