Synthesis and application of a photoaffinity analog of nicotinamide adenosine dinucleotide: Identification of the active sites of glutamate and lactate dehydrogenases
A photoaffinity analog of NAD{sup +} has been synthesized by chemically coupling ({sup 32}P)2-azido-AMP and NMN{sup +} to produce ({sup 32}P)nicotinamide 2-azidoadenosine dinucleotide (2-azido-NAD{sup +}). The utility of 2-azido-NAD{sup +} as an effective active-site-directed photoprobe was demonstrated using bovine liver glutamate dehydrogenase and porcine muscle lactate dehydrogenase as model enzymes. In the absence of ultraviolet light 2-azido-NAD{sup +} is a substrate for these enzymes. The specificity of active site labeling was demonstrated by photoinhibition, saturation and competition experiments. The active sites of these enzymes were identified utilizing 2-azido-NAD{sup +}. The immobilized boronate column chromatography was used to isolate the photolabeled peptides. The results demonstrate that the photoaffinity analog of NAD{sup +} has potential application as a probe to characterize NAD{sup +}binding proteins and to identify the active sites of these proteins.
- Research Organization:
- Kentucky Univ., Lexington, KY (United States)
- OSTI ID:
- 5475883
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
NUCLEOTIDES
CHEMICAL PREPARATION
OXIDOREDUCTASES
RECEPTORS
CHEMICAL COMPOSITION
CATTLE
GLUTAMIC ACID
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MUSCLES
NAD
PHOSPHORUS 32
SWINE
TRACER TECHNIQUES
AMINO ACIDS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARBOXYLIC ACIDS
CHROMATOGRAPHY
COENZYMES
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ENZYMES
GLANDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MEMBRANE PROTEINS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
RUMINANTS
SEPARATION PROCESSES
SYNTHESIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques