Purification and characterization of adenine phosphoribosyltransferase from mouse mammary carcinoma FM3A cells in culture.
Adenine phosphoribosyltransferase has been purified to apparent homogeneity from mouse mammary tumor FM3A cells. The purified enzyme, with a specific activity of 20.6 X 10(6) units/g protein at 30 degrees C, was homogeneous as judged by polyacrylamide gel electrophoresis and Ouchterlony double immunodiffusion analysis. The native enzyme had a molecular weight of 44,000 and a subunit composition of 23,000. Apparent Km values for adenine and 5-phosphoribosyl-1-pyrophosphate (PRib-PP) were 6.6 microM and 1.2 microM, respectively. Free Mg2+ was an essential activator with a half-maximal effect at 0.4 mM. AMP was an inhibitor, competitive with PRib-PP, and the Ki value was estimated to be 24 microM. The enzyme activity was not significantly affected by 2,6-diaminopurine, 4-carbamoylimidazolium 5-olate, 8-azaadenine, and 2-fluoro-6-aminopurine. An antibody against the purified mouse adenine phosphoribosyltransferase was raised in a rabbit. The enzyme derived from either mouse, Chinese hamster, or human cells was completely neutralized and precipitated by this antibody, indicating that these enzymes share a common antigenic determinant.
D000228:Adenine Phosphoribosyltransferase / Q000302:isolation & purification*; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000302:isolation & purification*
D010430:Pentosyltransferases; D000228:Adenine Phosphoribosyltransferase