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ENZYMOLOGY OF ASPARAGINE METABOLISM (ASPARAGINE SYNTHETASE, ALKALINE PHOSPHATASE, LEUKEMIA, CHEMICAL MODIFICATION)
Abstract
This study deals with different aspects of asparagine metabolism. First, a highly sensitive method for assaying asparagine synthetase and its glutaminase activity simultaneously was developed using reversed-phase high performance liquid chromatography. The amino acids L-aspartate, L-asparagine, L-glutamate, and L-glutamine were separated after derivatization with O-phthaldialdehyde. This assay can detect picomoles of asparagine and glutamate when a fluorescence detector is used. Second, chemical modification of asparagine synthetase was carried out. Beef pancreatic asparagine synthetase was inactivated by treatment with the sulfhydryl reagent 5,5'-dithiobis(2-nitro benzoic acid), DTNB. Asparagine protected the glutamine-dependent and ammonia-dependent asparagine synthetase activities as well as the glutaminase activity. Kinetic studies with partially inactivated asparagine synthetase showed that the K(,m) values for aspartic acid and glutamine did not change when compared with the K(,m) values of the control enzyme. The stoichiometry of DTNB inhibition was measured using ('14)C DTNB. It was calculated to be one sulfhydryl group per molecule of asparagine synthetase. Next, differences in the enzymology of L-asparaginase sensitive and resistant L5178Y leukemia cells were examined. The L-asparaginase resistant cells were found to have a 3-7 fold higher alkaline phosphatase activity than L-asparaginase sensitive cells. Asparagine synthetase was much higher in the resistant cells, whereas the sensitive cells had none. Glyoxylate asparagine aminotransferase was five-fold higher in resistant cells. It was also found that L-asparaginase resistant cells showed a slight increase in glycine content upon L-asparaginase treatment, whereas the sensitive cells lost a large portion of their cellular glycine. Finally, the transport characteristics of L-asparaginase sensitive and resistant cells were observed. The L-asparaginase sensitive cells showed a higher rate of transport of the amino acids glycine, serine, asparagine and aspartate into the cytoplasmic pool.
Subject Area
Biochemistry
Recommended Citation
UNNITHAN, SHASHIKALA, "ENZYMOLOGY OF ASPARAGINE METABOLISM (ASPARAGINE SYNTHETASE, ALKALINE PHOSPHATASE, LEUKEMIA, CHEMICAL MODIFICATION)" (1985). ETD collection for University of Nebraska-Lincoln. AAI8526634.
https://digitalcommons.unl.edu/dissertations/AAI8526634