Synthesis and affinity purification of. beta. -/sup 32/P-labeled (. gamma. -S)GTP
Journal Article
·
· Anal. Biochem.; (United States)
A method for the synthesis and purification of guanosine 5'-(..gamma..-S)triphosphate labeled with /sup 32/P in the ..beta..-position is described. The first step in the synthesis involves the quantitative transfer of /sup 32/P/sub i/ from (..gamma..-/sup 32/P)dATP to 5'-GMP catalyzed by GMP kinase. Further incubation of the (..beta..-/sup 32/P)GDP product with (..gamma..-S)GTP and nucleoside diphosphate kinase results in the synthesis of (..beta..-/sup 32/P)(..gamma..-S)GTP with a yield of 10 to 18%. The /sup 32/P-labeled (..gamma..-S)nucleotide is purified by binding to mercury-agarose and eluting with buffer containing ..beta..-mercaptoethanol. Specific incorporation of /sup 32/P into the ..beta..-position was demonstrated by treating (..beta..-/sup 32/P)(..gamma..-S)GTP with 7% formic acid to remove the ..gamma..-thiophosphate and digesting the remaining (..beta..-/sup 32/P)GDP with nucleotide pyrophosphatase. Although 5'-GMP was released after pyrophosphatase digestion, the only /sup 32/P radioactivity detected was as inorganic phosphate.
- Research Organization:
- The Johns Hopkins Univ., Baltimore, MD
- OSTI ID:
- 5724271
- Journal Information:
- Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 130:1; ISSN ANBCA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEAR CHEMISTRY
400702 -- Radiochemistry & Nuclear Chemistry-- Properties of Radioactive Materials
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CATALYSIS
CHEMICAL PREPARATION
CHEMISTRY
DAYS LIVING RADIOISOTOPES
ENZYMES
GUANOSINE
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PURIFICATION
PURINES
RADIOCHEMISTRY
RADIOISOTOPES
RIBOSIDES
SYNTHESIS
TRACER TECHNIQUES
TRANSFERASES
YIELDS
400702 -- Radiochemistry & Nuclear Chemistry-- Properties of Radioactive Materials
550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CATALYSIS
CHEMICAL PREPARATION
CHEMISTRY
DAYS LIVING RADIOISOTOPES
ENZYMES
GUANOSINE
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHATES
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PURIFICATION
PURINES
RADIOCHEMISTRY
RADIOISOTOPES
RIBOSIDES
SYNTHESIS
TRACER TECHNIQUES
TRANSFERASES
YIELDS