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
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARBOXYLIC ACIDS
CATTLE
CHEMICAL COMPOSITION
CHEMICAL PREPARATION
CHROMATOGRAPHY
COENZYMES
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ENZYMES
GLANDS
GLUTAMIC ACID
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MAMMALS
MEMBRANE PROTEINS
MUSCLES
NAD
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
OXIDOREDUCTASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
RECEPTORS
RUMINANTS
SEPARATION PROCESSES
SWINE
SYNTHESIS
TRACER TECHNIQUES
VERTEBRATES