Tissue culture fibroblasts derived from patients with Lesch-Nyhan disease (congenital hyperuricosuria) have a reduced IMP:pyrophosphate phosphoribosyltransferase (EC 2.4.2.8) activity and therefore incorporate, as detected by radioautography, much smaller amounts of tritiated hypoxanthine or guanine into cell nuclei and cytoplasm than do normal cells. However, Lesch-Nyhan cells grown in close contact with normal fibroblasts incorporate these purines. This phenomenon, which requires cell to cell contact for correction of the mutant phenotype, has been called metabolic cooperation. After separation of Lesch-Nyhan cells from normal cells, there is a prompt reversion to the mutant phenotype although the transferase is stable under these conditions for many hours.These results are most compatible with the transfer from normal to mutant fibroblasts of the product of the normal enzyme, a nucleotide or a nucleotide derivative, rather than the transfer of the transferase or informational macromolecules leading to the synthesis of the enzyme. Metabolic cooperation may provide a mechanism for maintaining normal cell function in the heterozygote in vivo. Evidence has been presented previously that selection of normal cells, presumably during embryogenesis, also provides a means for achieving normal function in the heterozygote.

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IMP:pyrophosphate phosphoribosyltransferase (IPPase) (EC 2.4.2.8) has been purified over 7000-fold from human erythrocytes. The purified enzyme moved as a single band on disc electrophoresis. Antisera prepared in rabbits and rats against the purified enzyme precipitated and neutralized the enzyme, but had no effect on AMP-pyrophosphate phosphoribosyltransferase (EC 2.4 In Lesch-Nyhan (LN) disease (10), the activity of the enzyme IMP:pyrophosphate phosphoribosyltransferase [recommended trivial name, IMP pyrophosphorylase, here abbreviated IPPase; identical with hypoxanthine (guanine) phosphoribosyltransferase, EC 2.4.2.8] is operationally absent (1). It is not known whether phenotypic expression is due to lack of synthesis of enzyme, or whether the enzyme is synthesized but is structurally ineffective. In view of the importance of the enzyme in human neurochemistry, as evidenced by the severity of symptoms when it is absent, it would be desirable to have purified enzyme available, and to determine whether a modified form is produced in patients.We have approached this question using immunological techniques. A highly purified IPPase has been isolated from normal human erythrocytes, and antibodies to it have been prepared in the rat and rabbit. The antisera have been used to test for the presence of an altered IPPase in extracts of erythrocytes of five patients with LN disease. Salt Fractionation. Solid ammonium sulfate (Schwarz BioResearch, special enzyme grade) was added to the DEAEcellulose eluate to a final concentration of 27.4 g/100 ml (47% saturation). 6 ml of concentrated NH40H was added per liter of eluate to maintain the pH in the range of 7-8. Precipitation was allowed to proceed overnight. The protein that was collected by centrifugation at 10,000 X g for 40 min was discarded. The supernatant was then brought to 70% saturation by the addition of further ammonium sulfate (14.5 g/100 ml). Precipitation was complete within 5 hr and the protein was collected by centrifugation at 10,000 X g for 40 min. The supernatant was discarded and the precipitate was dissolved in a minimal volume of 0.01 M K-PO4 (pH 6.4)-0.001 M 2-mercaptoethanol.Removal of Heat-Labile Proteins. The 47-70% fraction was concentrated in dialysis tubing by packing in aquacide II (MW 250,000, Calbiochem) to a final concentration of 20-30 mg/ml. This solution was placed in an Erlenmeyer flask and immersed in a 68°C water-bath. The sample was allowed to remain in the bath for 3 min after it reached 60°C. Precipitated protein was removed by centrifugation at 15,000 X g and