Evidence for X-linkage of human phosphoribosylpyrophosphate synthetase

Abstract

The mode of genetic transmission of human phosphoribosylpyrophosphate synthetase (ribosephosphate pyrophosphokinase; ATP:D-ribose-5-phosphate pyrophosphotransferase; EC 2.7.6.1) was studied in fibroblasts cultured from members of a family with a structurally and electrophoretically altered phosphoribosylpyrophosphate synthetase that has increased activity per enzyme molecule. Enzyme activity in fibroblast lysates from the daughter of an affected male patient was intermediate to the activities in lysates from her father (and her affected paternal uncle) and from her mother and other normal individuals. Two bands of enzyme activity corresponding to normal and mutant phosphoribosylpyrophosphate synthetases were found in fibroblast lysates from the daughter after cellulose acetate strip electrophoresis. In contrast, only mutant enzyme was detectable in lysates derived from the male patients. Fibroblasts cloned from the daughter contained two phenotypically distinct (normal and mutant) populations of cells with respect to phosphoribosylpyrophosphate synthetase activity and electrophoretic mobility. These studies support assignment of the structural gene for human phosphoribosylpyrophosphate synthetase to the X-chromosome. No evidence for the presence of the normal enzyme was found in erythrocyte or lymphocyte lysates or in partially purified erythrocyte enzyme preparations from the heterozygous daughter, suggesting either nonrandom X-chromosome inactivation in precursors of these cells or selection against hematopoietic cells bearing the normal enzyme after random X-chromosome inactivation.5-Phosphoribosyl-l-pyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase; EC 2.7.6.1) catalyzes the reaction between ATP and ribose-5-P to form PRPP and AMP. PRPP is a rate-limiting substrate in purine synthesis de novo as well as an allosteric activator of amidophosphoribosyltransferase (EC 2.4.2.14) (1), the first committed enzyme in this pathway. In three families reported to date (2-4), affected male members with excessive PRPP synthetase activity due to apparently different structural alterations in the enzyme show increased intracellular PRPP synthesis, concomitant purine overproduction, and gout. Thus, increased PRPP synthetase activity is a prototype for the association of a hereditary disease state with a structurally aberrant protein of increased rather than decreased catalytic activity.The identification of an excessively active and electrophoretically distinct form of PRPP synthetase (5) provides an opportunity to study the mode of inheritance of the enzyme. Pedigree data from two of the families thus far reported (6, 7) are compatible with either autosomal dominant or X-linked transmission of the gene for PRPP synthetase. Father-to-son transmission of aberrant forms of the enzyme, a criterion essential for the exclusion of X-linkage, has not been observed. On the other hand, studies in erythrocytes from female memThe costs of publication of this article were defrayed in part by the p...

Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

“…Although the evidence for X-linkage of the gene for PP-ribose-P synthetase (23)(24)(25) is compelling, the use of erythrocyte lysates to identify heterozygous female carriers of superactive variants of the enzyme is unreliable. As previously described (15,18), enzyme activities intermediate to those of normal individuals and affected males, as predicted by the Lyon (46) hypothesis, seem to be the exception in erythrocyte lysates from heterozygous carriers of PP-ribose-P synthetase variants.…”

“…emphasizes this point. Measurement of enzyme activity in fibroblasts cultured from women suspected to carry variant forms of PPribose-P synthetase appears to provide an accurate basis for heterozygote detection (17,20,24), and the availability of cultured fibroblasts provides material for a thorough evaluation of the biochemical conse(luences of aberrations in this enzyme (17,18,20 …”

“…Mobilities of PP-ribose-P synthetase on cellulose acetate gel were examined with an activity stain (37) after electrophoresis of fibroblast extracts in several buffer systems (10,24,37).…”

“…Among these abnormal forms of the enzyme, superactive PP-ribose-P synthetases with either diminished sensitivity to feedback inhibition (16,17) or excessive reaction velocity per molecule of enzyme (10) have been described and extensively characterized. To date, the clinical manifestations of all the males bearing superactive forms of this enzyme, the structural gene for which has been mapped on the X-chromosome (23)(24)(25), have been restricted to gouty arthritis, uric acid urolithiasis, and/or renal insufficiency first manifested in early adulthood (15,18,20).…”

Selective expression of phosphoribosylpyrophosphate synthetase superactivity in human lymphoblast lines.

“…Studies of superactive enzymes in each of these classes (4-8, 14, 18, 19) have provided evidence for structural alteration underlying superactivity. Furthermore, enzyme superactivity is transmitted as an X chromosome-linked trait (20,21), and the structural gene for the single PRPP synthetase subunit (22) has been localized to the long arm of the X chromosome (23). Thus, even though demonstration of an altered primary structure of the enzyme protein has not, to date, been achieved for any superactive variant, structural gene mutation as the basis of enzyme superactivity is strongly suggested.…”