Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: Activity in Normal, Mutant, and Heterozygote- Cultured Human Skin Fibroblasts Wilfred Y

Proceedings of the National Academy of Sciences Vol. 65, No. 3, pp. 577-584, March 1970 Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: Activity in Normal, Mutant, and Heterozygote- Cultured Human Skin Fibroblasts Wilfred Y. Fujimoto* and J. Edwin Seegmillert SECTION ON HUMAN BIOCHEMICAL GENETICS, NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND Communicated by E. R. Stadtman, June 27, 1969 Abstract. Cultured skin fibroblasts from patients deficient for the enzyme hypoxanthine-guanine phosphoribosyltransferase (PRT) activity show very low but nevertheless significant levels of apparent PRT enzyme despite absence of detectable activity (<0.004% of normal) in erythrocytes of the same patients. In fibroblasts this mutant enzyme is more heat labile than the normal enzyme. These findings indicate that PRT deficiency in this disorder is not due to a dele- tion mutation of the PRT locus. Individual cultured skin fibroblasts from heterozygote females for PRT deficiency show normal, intermediate, or very low levels of PRT activity. The mosaicism demonstrated in the heterozygotes for this X-linked disorder ac- counts for the cells with normal and very low activities of PRT. Intermediate activity can best be explained by the phenomenon of metabolic cooperation pre- sumably from the transfer of either PRT enzyme or messenger RNA, from normal to mutant cells. The enzyme hypoxanthine-guanine phosphoribosyltransferase (PRT) (E.C. 2.4.2.8.) is not detectable, (<0.004% of normal activity), in erythrocyte hemolysates from patients affected with the Lesch-Nyhan syndrome, an X-linked reces- sive familial disorder characterized by spasticity, choreoathetosis, psychomotor retardation, a compulsive automutilation by biting of the lips and fingers, and accelerated purine synthesis de novo.1. 2 Since this enzyme normally converts the free purine bases hypoxanthine and guanine to their respective ribonucleotides by reaction with 5-phosphoribosyl-1-pyrophosphate, the deficiency of this enzyme may in some manner affect the feedback control of purine synthesis.3 Incom- plete deficiency of PRT activity (0.007-17% of normal) which is also X-linked and associated with excessive purine synthesis de novo, has been reported in erythrocytes of some gouty patients.4' 5 A subgroup of these patients with the least amount of enzyme (<0.5% of normal) have relatively mild neurological abnor- malities while patients with larger quantities of enzyme are completely spared the neurological dysfunction.5 The enzyme PRT is normally detectable in a number of different human tissues as well as in cultured skin fibroblasts and amniotic fluid cells." 4. 64 In the present studies we found that fibroblasts cultured from skin biopsies of patients 577 Downloaded by guest on October 1, 2021 578 GENETICS: FUJIMOTO AND SEEGMILLER PROC. N. A. S. with the complete Lesch-Nyhan syndrome show very low but definite levels of PRT activity despite the absence of detectable activity in erythrocytes. Fibro- blasts from patients whose erythrocytes show the incomplete deficiency of enzyme show slightly higher levels of activity, and those from heterozygous females show normal, intermediate, or low levels of activity. The mutant enzyme has a greater heat lability than the normal PRT. Materials and Methods. Hypoxanthine-8-"4C was obtained from Schwarz Bio- Research, Inc., Orangeburg, N. Y.; 5-phosphoribosyl-1-pyrophosphate from Sigma Chemical Co., St. Louis, Mo., or from Calbiochem, Los Angeles, Calif.; and hypoxanthine-3H from New England Nuclear, Boston, Mass. Cell cultures: Human fibroblasts were established from minced skin biopsies as reported previouslyI Preparation of cell extracts: Cells were harvested for studies by brief incubation at 370C in 0.25% trypsin in Dulbecco's phosphate-buffered saline. Cells were col- lected by centrifugation, washed at least twice with 0.154 M sodium chloride, and stored at -780C if not used immediately. Disrupted cells were prepared with the micro- tip of a Branson Sonifier after suspension of the cells in ice-cold 0.01 M Tris buffer, pH 7.4. Samples of the supernatant fluid after centrifugation at 19,000 g for 45 min were used for assays of protein content9 and PRT activity. Assay of PRT: Cell extract (50 Ml) was added to 50 ul of a substrate mixture containing 10 ,dl of 0.5 M Tris buffer, pH 7.4, 5 ,ul of 0.1 M MgCl2; 10 ,ul of 9 mM 5-phosphoribosyl-1-pyrophosphate solution; 15 Ml of 4.56 mM hypoxanthine-8-'4C solution (3.8 mCi/mmole); and 10 Ml of water. The mixture was incubated with shaking at 250C for 60 min and the reaction terminated by the addition of 20 ,l of 0.1 M EDTA, followed by freezing in a dry ice-alcohol bath. The reaction mixture was thawed and 20-MA ali- quots were spotted at 1-inch intervals on a Whatman 3 MM paper overlying previously spotted and dried carrier inosine (20 1l of 1 mg/ml solution) and inosinic acid (20 Jl of 1 mg/ml solution). Reaction products were separated from substrates by high voltage electrophoresis in 0.05 M borate buffer, pH 9.0, containing 0.001 M EDTA at 4000 volts for 20 to 30 min. The areas corresponding to inosinic acid and inosine were identified by inspection of the paper under ultraviolet light, cut out, and counted in a liquid scin- tillation counter at about 50% efficiency. The sum of counts in the inosinic acid and inosine spots was used to determine activity of the enzyme and showed a linear relation- ship to incubation time and to concentration of protein. Radioautography: Cells were plated onto coverslips in Petri dishes at low density. After at least 24 hr of growth, hypoxanthine-3H (3.14 Ci/mmole) was added at a concentration of 10 Mc/ml. After another 24 hr of growth, coverslips were removed, washed with 0.154 M sodium chloride, and fixed with absolute methanol for 5 min. The coverslips were then treated with ice cold 5% trichloroacetic acid for 25 min. After a quick wash with water, the coverslips were dried, coated with Kodak NTB 3 emulsion, and after exposure for 3 to 5 days, were developed and treated with Giemsa stain. Results and Discussion. Table 1 summarizes the apparent PRT activity in cultured skin fibroblasts from individuals who are normal, partially deficient, or virtually completely deficient for PRT activity, and from heterozygous females. The specific activity of normal-cultured skin fibroblasts is about three- fourths of the level found in normal erythrocyte hemolysates.4 On the other hand, the level of activity in fibroblasts cultured from the fully affected patients, although markedly decreased, is still detectable. This is in contrast to the absence of PRT activity in erythrocytes from such patients.' Of note is the finding that, whereas in normal erythrocyte hemolysates the ratio of inosinic acid: inosine formed from hypoxanthine-8-14C is about 9: 1, virtually none of the radio- Downloaded by guest on October 1, 2021 VOL. 65, 1970 GENETICS: FUJIMOTO AND SEEGMILLER 579 TABLE 1. PRT activity in cultured human skin fibroblasts. Diagnosis Specific activity* Diagnosis Specific activity* Normal: (Sex) Partial deficiency 1. Female 66.3 16. 7.7 2. Male 61.2 17. 10.9 3. Male 88.7 18. 5.0 4. Male 68.4 19. 4.2 5. Female 87.6 Heterozygote for Lesch-Nyhan 6. Female 83.1 20. 22.0 7. Male 54.6 21. 21.5 8. Female 67.2 22. 9.2 9. Female 62.6 23. 57.8 10. Female 132.3 24. 5.5 11. Male 119.1 25. 10.7 Average + SD 81.0 + 24.9 26. 12.2 Lesch-Nyhan 27. 76.3 12. 2.1 Heterozygote for partial deficiency 13. 3.7 28. 46.7 14. 2.8 15. 2.7 * Specific activity of PRT represents the mjsmoles of hypoxanthine-S-14C converted to inosinic acid and inosine per milligram cell extract protein per hr at 250C. activity is found in inosinic acid in normal fibroblast extracts. This is explained by the presence of nucleotidase activity in fibroblast extracts which, in one assay as measured by the conversion of inosinic acid-8-14C to inosine, was found to be about five times the specific activity of PRT in normal cells. Hence, as soon as inosinic acid is formed from hypoxanthine, it is split into inosine and orthophos- phate, and virtually all of the inosinic acid formed from hypoxanthine is rapidly converted to inosine. Furthermore, hypoxanthine may be directly converted to inosine by a ribosyltransferase (nucleoside phosphorylase) reaction. This could result in apparent PRT activity in fibroblast extracts from the affected patients, since PRT activity is also related to the conversion of hypoxanthine-8-14C to inosine, but by way of inosinic acid. Under the PRT assay conditions, when ribose-l-phosphate was substituted for 5-phosphoribosyl-1-pyrophosphate, the specific activity of this ribosyltransferase as measured by the conversion of hypoxanthine-8- 14C to inosine, was found to be about five times that of PRT in normal cells. Further studies showed that the heat stability curves for PRT in normal and mutant fibroblasts were clearly different, with the latter showing greater heat lability (Fig. 1A), whereas the heat stability curves for ribosyltransferase were virtually identical in normal and mutant fibroblasts (Fig. 1B). Furthermore, although the heat stability curve for ribosyltransferase more closely resembles that of mutant PRT, these are still clearly not identical. These findings indicate that although ribosyltransferase activity may be contributing to the apparent PRT activity in the method of assay used, difference in heat stability of the PRT activity of normal and mutant cells from that of ribosyltransferase suggests that a low but nevertheless significant amount of PRT activity may be present in the mutant fibroblasts.

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