Abstract
This chapter describes two purification procedures related to the enzyme orotate phosphoribosyltransferase. Orotate phosphoribosyltransferase and orotidylate decarboxylase are purified together from mammalian sources and exist as a complex. The assay and preparation of these enzymes from beef and human erythrocytes are described in the chapter. Orotate phosphoribosyltransferase activity may be measured spectrophotometrically by following the change in absorbance that results upon the the conversion of a pyrimidine or purine base to the corresponding ribonucleotide. In the procedure, the activity of erythrocyte orotate phosphoribosyltransferase may be measured spectrophotometrically after the removal of hemoglobin. Reaction mixtures are incubated at 37 ° for 5 minutes before adding enzyme. The spectrophotometric assay of the transferase activity with orotic acid as the base substrate depends upon the conversion of the corresponding product––orotidylic acid––to uridylic acid by the decarboxylase. In the other procedure, orotate phosphoribosyltransferase activity may be measured prior to the removal of hemoglobin. Pyrophosphorolysis of orotidylic acid could be readily demonstrated in transferase preparations that contained low decarboxylase activity.
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