Differential photoaffinity labeling of catalytic subunits of NaK-ATPase with carrier-free /sup 125/I-cardiac glycosides
The authors have obtained evidence for structural differences in the cardiac glycoside binding site between the ..cap alpha.. and ..cap alpha..(+) forms of the catalytic subunit of NaK-ATPase, using three closely related photoaffinity derivatives of the cardiotonic steroid, digitoxigenin. (/sup 125/I)N-(p-azido-m-iodo-o-hydroxybenzoyl)-4-amino-4,6-dideoxy-galactosyl digitoxigenin (IA-GaD), (/sup 125/I)N-(3-(p-azido-m-iodophenyl)-propionyl)-4-amino-4,6-dideoxy-ga-lactosyl digitoxigenin (AIPP-GaD) and (/sup 125/I)N-(3-(p-azido-m-iodophenyl)-propionyl)-4-amino-4,6-dideoxy-glucosyl digitoxi-genin (AIPP-GluD) were synthesized. AIPP-GaD and AIPP-GluD are stereoisomers. Eel electroplax and dog kidney NaK-ATPase (..cap alpha.. form) and rat brain synaptosomes (rich in ..cap alpha..(+) form) were photolabelled and then analyzed by SDS-PAGE and autoradiography. Photolysis with either carrier-free IA-GaD or AIPP-GluD gave ouabain-protectable labelling of NaK-ATPase catalytic subunit from all three tissues. However, photolysis with AIPP-GaD showed protectable labelling of the enzyme from eel and kidney but not from brain. This suggests a structural difference in the ..cap alpha..(+) form which results in either an inability to bind AIPP-GaD, or, perhaps more likely, an absence of a photoinsertion site in the correct location in the ..cap alpha..(+) form, as compared with the ..cap alpha.. form. It is of interest that the labelling pattern of the enzyme in the human erythrocyte resembles that of the brain enzyme.
- Research Organization:
- Univ. of Wisconsin, Madison
- OSTI ID:
- 6943259
- Report Number(s):
- CONF-8606151-; TRN: 87-006998
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP-ASE
BIOLOGICAL VARIABILITY
MOLECULAR STRUCTURE
DIGITOXIN
BIOCHEMICAL REACTION KINETICS
BRAIN
DOGS
ELECTROPHORESIS
HEART
IODINE 125
ISOENZYMES
NERVES
RATS
RECEPTORS
STEROIDS
ACID ANHYDRASES
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CARBOHYDRATES
CARDIAC GLYCOSIDES
CARDIOTONICS
CARDIOVASCULAR AGENTS
CARDIOVASCULAR SYSTEM
CENTRAL NERVOUS SYSTEM
DAYS LIVING RADIOISOTOPES
DIGITALIS GLYCOSIDES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
GLYCOSIDES
HYDROLASES
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPES
KINETICS
MAMMALS
MEMBRANE PROTEINS
NERVOUS SYSTEM
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHOHYDROLASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RODENTS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques