Purification and properties of rat liver adenine phosphoribosyltransferase.
The adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) of rat liver was purified to a specific activity of 1.1 mumol of AMP formed per min per mg. The enzyme activity is associated with an apparently homogenous protein as shown by isoelectrofocusing, acrylamide gel electrophoresis, and N-terminal amino acids analysis (phenylalanine). The molecular weight of the enzyme was estimated to be approx. 20 000 by acrylamide gel electrophoresis in the presence of sodium dodecylsulfate and by sucrose density gradient zone sedimentation. The rat liver enzyme exhibited initial burst synthesis of AMP when 1-pyrophosphorylribose 5-phosphate was added. The 1-pyrophosphorylribose 5-phosphate initial-burst activity copurifies with the adenine phosphoribosyltransferase activity. A PH optimum of 10.0 was demonstrable for the adenine phosphoribosyltransferase. The initial-burst and steady-state phases of AMP synthesis catalyzed by highly purified rat liver adenine phosphoribosyltransferase have been partially characterized by the use of ligands which bind to sulfhydryl groups. Studies utilizing p-chloromercuribenzoate and HgCl2 as inhibitors of AMP sulfhydryl during the initial-burst and steady-state phases have revealed that sulfhydryl groups with different rates of ligand binding are present in the enzyme. The initial-burst phase was thereby delineated from the steady-state phase by use of these mercurial ligands. This delineation was also accomplished by titration with the Mg-2+ chelator, EDTA. The inhibitory effects of mercurials and EDTA were reversed by beta-mercaptoethanol and excess Mg-2+, respectively. Quantitative binding studies with 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate yielded values of 3.65 and 3.6 mol of sulfhydryl per mol of enzyme, respectively. 3.3 mol of cysteic acid per mol of performic acid-oxidized enzyme were found by amino acid analysis.
D000228:Adenine Phosphoribosyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000096:biosynthesis; D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008623:Mercaptoethanol / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000378:metabolism*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000032:analysis; D013997:Time Factors
D002729:Chloromercuribenzoates; D013438:Sulfhydryl Compounds; D000249:Adenosine Monophosphate; D008623:Mercaptoethanol; D004492:Edetic Acid; D010430:Pentosyltransferases; D000228:Adenine Phosphoribosyltransferase; D008628:Mercury