Human hypoxanthine-guanine phosphoribosyltransferase. Demonstration of structural variants in lymphoblastoid cells derived from patients with a deficiency of the enzyme.

We have explored the possibility of using cultured lymphoblasts from patients with a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a source of cells for the isolation and characterization of mutant forms of the enzyme. HPRT from lymphoblasts derived from six male patients of five unrelated HPRT-deficient families was highly purified and characterized with regard to: (a) level of immunoreactive protein, (b) absolute specific activity, (c) isoelectric point, (d) migration during nondenaturing polyacrylamide gel electrophoresis, and (e) apparent subunit molecular weight. There experiments were performed on small quantities of lymphoblasts using several micromethods involving protein blot analysis of crude extracts as well as isolation and characterization of enzyme labeled in culture with radioactive amino acids. The lymphoblast enzymes from four of the patients exhibited structural and functional abnormalities that were similar to the recently described abnormalities found with the highly purified erythrocyte enzymes from these same patients. In addition, a previously undescribed HPRT variant was isolated and characterized from lymphoblasts derived from two male siblings. This unique variant has been called HPRT Ann Arbor. We conclude that lymphoblastoid cell lines can be used as a source of cells for the detection, isolation, and characterization of structural variants of human HPRT.

Human hypoxanthine-guanine phosphoribosyltransferase. Purification and characterization of mutant forms of the enzyme.

Erythrocyte hypoxanthine-guanine phosphoribosyltransferase has been highly purified from five unrelated patients with a deficiency of this enzyme. Affinity chromatography using either GMP-Sepharose or an immunoadsorbent was the most productive step in the purifications. The specific activity of the purified enzyme was unchanged for patients L. P. and G. S., and slightly decreased for patient R. H., as compared to control subjects. Enzyme from patient I. V. and from patient E. S. exhibited markedly reduced specific activities when purified to near homogeneity. The level of immunoreactive protein in patient I. V. appeared to be significantly higher than normal. The apparent subunit molecular weight of the enzyme from patient G. S. was decreased by approximately 1000 while it was increased by approximately 400 from patient I. V. The isoelectric points of the subunit isozymes were shifted to higher pH values from patients I. V. and E. S., and to lower pH values from patient L. P.; the subunit isozymes from patient G. S. were identical with normal. These studies provide direct evidence for the existence of at least four different mutations in the structural gene for hypoxanthine-guanine phosphoribosyltransferase. The four different mutant forms of human hypoxanthine-guanine phosphoribosyltransferase that have been identified are named as follows: patient L. P., HPRTToronto; patient G. S., HPRTLondon; patient E. S., HPRTKinston; and patient I. V., HPRTMunich.