Assay of covalent intermediate of 5'-nucleotide phosphodiesterase.
We report a new procedure for isolating a covalent phosphoryl enzyme (diester) intermediate of bovine intestinal 5'-nucleotide phosphodiesterase. The convenience of the procedure makes it possible to determine effects of reaction conditions on the yield of covalent intermediate. Under optimum conditions, using [methyl-3H]deoxythymidine 5'-triphosphate as substrate, more than 50% of the enzyme is recovered as thymidylyl enzyme, a 10-fold increase in yield over the previous procedure (M. Landt and L. G. Butler, 1978, Biochemistry 17, 4130-4135). Yields of thymidylyl enzyme were maximal at pH 4, whereas optimum catalytic activity is observed at pH greater than 9.
Blytt
H J
HJ
Brotherton
J E
JE
Butler
L
L
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000032:analysis*; D013938:Thymidine Monophosphate / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism; D014316:Tritium
D013942:Thymine Nucleotides; D014316:Tritium; D013938:Thymidine Monophosphate; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C024157:thymidine 5'-triphosphate