Hypoxanthine phosphoribosyltransferase: radiochemical assay procedures for the forward and reverse reactions

Journal Article · Fri Feb 15 04:00:00 EST 1985 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(85)90320-3· OSTI ID:5079767

Smithers, G W; O'Sullivan, W J

Simple and rapid radiochemical assay procedures for the forward (IMP synthesis) and reverse (IMP pyrophosphorolysis) reactions catalyzed by hypoxanthine phosphoribosyltransferase have been developed. Enzyme activity in the forward direction was assessed by measuring the amount of (8-/sup 14/C)IMP formed from (8-/sup 14/C)hypoxanthine following their separation by polyethyleneimine-cellulose TLC in methanol:water (1:1, v/v). (8-/sup 14/C)IMP has been synthesized from (8-/sup 14/C)hypoxanthine, using hypoxanthine phosphoribosyltransferase derived from human brain, with subsequent purification by elution from phenyl boronate-agarose. Enzyme activity in the reverse direction was assessed by measuring the amount of (8-/sup 14/C)uric acid formed from the labeled IMP following their separation by polyethyleneimine-cellulose TLC in 0.2 M LiCl saturated with boric acid (pH 4.5):95% ethanol (1:1, v/v), the transferase reaction being coupled with excess xanthine oxidase and catalase to overcome the unfavorable equilibrium.

• OSTI ID: 5079767
• Journal Information: Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 1; ISSN ANBCA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AROMATICS
AZAARENES
BODY
BRAIN
CARBON 14 COMPOUNDS
CATALYSIS
CENTRAL NERVOUS SYSTEM
ENZYME ACTIVITY
ENZYMES
GLYCOSYL TRANSFERASES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
HYPOXANTHINE
HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
NERVOUS SYSTEM
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PENTOSYL TRANSFERASES
PURINES
RADIOASSAY
TRACER TECHNIQUES
TRANSFERASES