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Photolabeling and radioligand binding of human erythrocyte NaK-ATPase with sup 125 I-derivatives of cymarin and digitoxigenin

Thesis/Dissertation ·
OSTI ID:7244627
NaK-ATPase is an enzyme which maintains Na{sup +} and K{sup +} gradients across the plasma membrane of eukaryotic cells, and is specifically inhibited by cardiac glycosides. The cardiac glycoside binding site is located primarily on the catalytic {alpha} subunit but the glycoprotein {beta} and proteolipid-{gamma} subunits may also contribute to the structure of the site. In order to label the cardiac glycoside binding site of human erythrocytes, four photoaffinity ligands with very high specific radioactivity were synthesized. The compounds, which are abbreviated ({sup 125}I)AISC, ({sup 125}I)AIPP-GluD, ({sup 125}I)AIPP-GalD and ({sup 125}I)IA-GalD, were all effective photolabels for NaK-ATPase as shown by ouabain-protectable, covalent labeling of the {alpha}, {beta}, and proteolipid-{gamma} subunits. In order to study the possible existence of a very high affinity binding site in erythrocyte NaK-ATPase, a carrier-free radioligand, ({sup 125}I)I-TASC, was synthesized; this compound had the same structure as ({sup 125}I)AISC except that a light-sensitive azide group was replaced with a hydroxyl group. Competitive binding assays with cymarin against 0.2 nM ({sup 125}I)I-TASC suggested two classes of erythrocyte binding sites. Scatchard analysis of direct ({sup 125}I)I-TASC binding indicated that the very high affinity, low capacity class of erythrocyte bindings sites had a K{sub D} of 54 pM and a B{sub max} of 23 fmol/mg protein.
Research Organization:
Wisconsin Univ., Madison, WI (USA)
OSTI ID:
7244627
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ANIMALS
ATP-ASE
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
ERYTHROCYTES
HYDROLASES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLING
LIGANDS
MAMMALS
MAN
MATERIALS
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PRIMATES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
TRACER TECHNIQUES
VERTEBRATES