Enzymology of asparagine metabolism
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. 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/sub m/ values for aspartic acid and glutamine did not change when compared with the K/sub m/ values of the control enzyme. The stoichiometry of DTNB inhibition was measured using (/sup 14/C)DTNB. Differences in the enzymology of L-asparaginase sensitive and resistant L5178Y leukemia cells were examined. 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.
- Research Organization:
- Nebraska Univ., Lincoln (USA)
- OSTI ID:
- 5742096
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ASPARAGINE
METABOLISM
LIGASES
ENZYME ACTIVITY
MEMBRANE TRANSPORT
AMINO ACIDS
CARBON 14 COMPOUNDS
LEUKEMIA
LIQUID COLUMN CHROMATOGRAPHY
TRACER TECHNIQUES
AMIDES
CARBOXYLIC ACIDS
CHROMATOGRAPHY
DISEASES
ENZYMES
HEMIC DISEASES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
NEOPLASMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
SEPARATION PROCESSES
550501* - Metabolism- Tracer Techniques