Simplified /sup 14/CO/sub 2/-trapping microassay for GTP cyclohydrolases I and II
GTP cyclohydrolases (GTP-CH) I and II catalyze the removal of the C/sub 8/ atom from GTP as formate. The reaction product of GTP-CH I is D-erythro-7,8-dihydroneopterin triphosphate, a key intermediate leading to the biosynthesis of folic acid in microorganisms and of tetrahydrobiopterin in mammals and microorganisms, and that of GTP-CH II is 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine 5'-phosphate, a key intermediate in the biosynthesis of riboflavin in microorganisms. They have simplified the /sup 14/CO/sub 2/-trapping assay of Burg and Brown for determining GTP-CH I and II activities. The assay consists of two consecutive steps which are carried out in a 2 ml tube. The first reaction yields formate from GTP (37/sup 0/C, 10 min). The reaction mixture contains 1 mM (8-/sup 14/C)-GTP (0.5 ..mu..Ci/..mu..mol), 50 mM Tris-HCl buffer (pH 8.2 for GTP-CH II and 7.7 for GTP-CH I), 0.2 M MgCl/sub 2/ for GTP-CH II or 0.3 M KCl and 1 mM EDTA for GTP-CH I, and enzyme in a final volume of 0.2 ml. The second reaction is the oxidation of /sup 14/C-formate to /sup 14/CO/sub 2/ (95/sup 0/C, 20 min) in the presence of 5% TCA and 1 mM formate (final volume 0.3 ml). Liberated /sup 14/CO/sub 2/ is trapped by the folded filter paper strip (1 x 4 cm), that has been placed inside the top of each tube and impregnated with 30 ..mu..l Protosol. This method is fast, comparable to the HPLC-fluorometric method for the assay of GTP-CH I activity, and ideal for performing a large number of determinations. Human and rat liver express both GTP-CH I and II activities. GTP-CH II is the predominant enzyme in both tissues and exists in multiple forms.
- Research Organization:
- Univ. of Texas Medical Branch, Galveston
- OSTI ID:
- 7171167
- Report Number(s):
- CONF-8606151-; TRN: 86-039081
- 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
HYDROLASES
BIOASSAY
ENZYME ACTIVITY
CARBON 14 COMPOUNDS
CARBON DIOXIDE
FORMIC ACID
GUANOSINE
LIVER
MAN
PHOSPHATES
RATS
TRACER TECHNIQUES
ANIMALS
AROMATICS
AZAARENES
BODY
CARBON COMPOUNDS
CARBON OXIDES
CARBOXYLIC ACIDS
CHALCOGENIDES
DIGESTIVE SYSTEM
ENZYMES
GLANDS
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MONOCARBOXYLIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
OXIDES
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
PRIMATES
PURINES
RIBOSIDES
RODENTS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques