Purification and characterization of Escherichia coli guanine-xanthine phosphoribosyltransferase produced by a high efficiency expression plasmid utilizing a lambda PL promoter and CI857 temperature-sensitive repressor.
The gene for Escherichia coli guanine-xanthine phosphoribosyltransferase was placed after the high efficiency lambda phage leftward promoter in plasmid pHEGPT also containing the lambda CI857 temperature-sensitive repressor. Guanine-xanthine phosphoribosyltransferase increases 780-fold when cells containing pHEGPT are shifted from 30 to 42 degrees C. Guanine-xanthine phosphoribosyltransferase represents approximately 5% of the protein in a crude extract of induced cells. Guanine-xanthine phosphoribosyltransferase may be purified to apparent homogeneity by ammonium sulfate fractionation, Sephadex G-100, and DEAE-cellulose column chromatography. The enzyme has a subunit molecular weight of 18,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a trimer during Sephadex G-100 column chromatography. Guanine-xanthine phosphoribosyltransferase is active from pH 7.5 to 10.5 with maximum activity at pH 9.5. The enzyme is protected from heat inactivation by phosphoribosylpyrophosphate (PRPP). At 65 degrees C, the enzyme has a half-life of 2 min in the absence of PRPP and 90 min in the presence of PRPP. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for guanine, xanthine, hypoxanthine, and PRPP of 2.6, 39, 167, and 95 microM, respectively. The activity of the enzyme with guanine is 2-fold greater than that with xanthine and 3-fold greater than that with hypoxanthine.
D004262:DNA Restriction Enzymes; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009876:Operon; D010430:Pentosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
D012097:Repressor Proteins; D014157:Transcription Factors; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D004262:DNA Restriction Enzymes