ASSAY OF HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE IN HUMAN FIBROBLAST LYSATES - INACTIVATION OF NUCLEOTIDASE

Assays of the hypoxanthine-guanine phosphoribosyltransferase enzyme (HGPRT: EC 2.4.2.8) in human fibroblast lysates are affected by the presence of a nucleotidase enzyme which converts the reaction product nucleotide to nucleoside. These enzymes, HGPRT and nucleotidase, have substantially different thermostabilities and pH optima, and the nucleotidase activity can be selectively eliminated. The conditions include preheating the cell lysates at 60°C for 10 min, a temperature at which the HGPRT enzyme is relatively stable, and using HGPRT assays buffered at pH 10. Also, we provide evidence that there is no nucleotidase activity in human lymphoblast lysates. Thus, human lymphoblast and preheated fibroblast lysates can be assayed for HGPRT activity by the DEAE-filter disk method. © 1979.