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Assay for inhibitors of nucleoside transport based upon the use of 5-/sup 125/I)Iodo-2-deoxyuridine as permeant

Journal Article · · Anal. Biochem.; (United States)
;

5(/sup 125/I)Iodo-2-deoxyuridine (IdUrd) has been shown to serve as a permeant for the nucleoside transport system of human erythrocytes and to be metabolically inert in these cells. Linear initial velocities were obtained at 20/sup 0/C for /sup 125/IdUrd transport, yielding a K/sub m/ of 73 +/- 18 ..mu..M (n = 6). Low-affinity inhibitors of /sup 125/IdUrd transport, such as adenosine (K/sub i/ = 32 +/- 2 ..mu..M, n = 2), could be characterized by Michaelis-Menten kinetics. However, high-affinity inhibitors, such as 6-(4-nitrobenzyl)thio)9-..beta..-D-ribofuranosylpurine, caused nonlinear initial velocities when added to the cells simultaneously with /sup 125/IdUrd. Conditions were defined (viz., 20-min pretreatment of cells with test compound followed by 5.0-min incubation with 1.0 ..mu..M /sup 125/IdUrd, all at 20/sup 0/C) whereby high-affinity inhibitors of IdUrd transport can be identified and evaluated according to their 50% inhibitory concentrations. The use of /sup 125/IdUrd as permeant greatly expedites the testing of compounds as inhibitors of nucleoside transport by allowing the cell pellets generated in these assays to be monitored directly in a gamma spectrometer, thereby circumventing the solubilization and decolorization of cell pellets required by assays that use /sup 3/H- or /sup 14/C-labeled nucleoside permeants.

Research Organization:
Burroughs Wellcome Co., Research Triangle Park, NC
OSTI ID:
5578871
Journal Information:
Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 154:1; ISSN ANBCA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
550501 -- Metabolism-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADENOSINE
ANTIMETABOLITES
AROMATICS
AZAARENES
AZINES
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ERYTHROCYTES
GAMMA SPECTROMETERS
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
INHIBITION
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
IODODEOXYURIDINE
IODOURACILS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
MATERIALS
MEASURING INSTRUMENTS
MEMBRANE TRANSPORT
METABOLITES
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC IODINE COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PURINES
PYRIMIDINES
RADIOISOTOPES
REACTION KINETICS
RIBOSIDES
SPECTROMETERS
TRACER TECHNIQUES
URACILS