William J. Arnold

Human adenine phosphoribosyltransferase. Purification, subunit structure, and substrate specificity.

Adenine phosphoribosyltransferase (APRT) catalyzes the magnesium dependent conversion of adenine to adenosine 5’-monophosphate (AMP) utilizing the high energy compound, 5-phosphoribosyl-lpyrophosphate (PP-ribose-P) as a cosubstrate. In humans this enzyme provides the only apparent pathway for conversion of dietary adenine into utilizable nucleotides. The finding of elevated levels of APRT activity in patients with the Lesch-Nyhan syndrome (Seegmiller, Rosenbloom and Kelley, 1967; Kelley, 1968) and the discovery of at least three families with a genetically determined partial deficiency of APRI activity in circulating erythrocytes (Kelley, et al., 1968; Kelley, Fox and Wyngaarden, 1970; Emmerson, et al., present symposium) stimulated our study of a highly purified preparation of human adenine phosphoribosyltransferase.

Hypoxanthine-guanine phosphoribosyltransferase: characteristics of the mutant enzyme in erythrocytes from patients with the Lesch-Nyhan syndrome

The Lesch-Nyhan syndrome is characterized clinically by choreoathetosis, spasticity, selfmutilation, and mental and growth retardation. Biochemically, there is a striking reduction of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in affected individuals. We have examined erythrocytes from 14 patients with the Lesch-Nyhan syndrome for the presence of hypoxanthine-guanine phosphoribosyltransferase activity and enzyme protein. In contrast to the usual finding of no detectable hypoxanthine-guanine phosphoribosyltransferase activity, we have found low levels (0.002-0.79 nmoles/mg protein per hr) of hypoxanthine-guanine phosphoribosyltransferase activity in erythrocyte lysates from five of these patients. In three of the five patients, hypoxanthine-guanine phosphoribosyltransferase activity appeared to be substantially more labile in vivo than normal using erythrocytes which had been separated according to their density (age). Immunochemical studies using a monospecific antiserum prepared from a homogeneous preparation of normal human erythrocyte hypoxanthine-guanine phosphoribosyltransferase revealed immunoreactive protein (CRM) in hemolysate from all 14 patients with the Lesch-Nyhan syndrome. The immunoreactive protein from each patient gave a reaction of complete identity with normal erythrocyte hypoxanthine-guanine phosphoribosyltransferase and was present in quantities equal to those observed in normal erythrocytes. In addition, a constant amount of CRM was found in erythrocytes of increasing density (age) from patients with the Lesch-Nyhan syndrome despite the decreasing hypoxanthine-guanine phosphoribosyltransferase activity. These studies confirm previous data which indicate that the mutations leading to the Lesch-Nyhan syndrome are usually, if not always on the structural gene coding for hypoxanthine-guanine phosphoribosyltransferase. In addition, although the mutant proteins appear to be present in normal amounts, they are often very labile in vivo with respect to enzymatic activity. These observations suggest that therapy directed at stabilization or activation of enzyme activity in vivo may be of potential benefit.

Dietary-Induced Variation of Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Patients with the Lesch-Nyhan Syndrome

We have studied three patients with the Lesch-Nyhan syndrome to assess the effect of dietary purines on erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity. During dietary purine restriction HGPRT activity rose in all three patients; resumption of normal dietary purine intake or the addition of adenine (10 mg/kg per day) to a purinefree diet resulted in a fall in HGPRT activity. These changes in enzyme activity appeared to be due to an activation or inactivation of the mutant enzyme without a change in the half-life or absolute amount of HGPRT enzyme protein.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency: immunologic studies on the mutant enzyme.

The Lesch-Nyhan syndrome is a bizarre, X-linked disease characterized by spasticity, choreoathetosis, self-mutilation, mental and growth retardation as well as hyperuricemia and hyperuricaciduria (Lesch and Nyhan,1964) which is due to a striking reduction of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity in all tissues of affected individuals (Seegmiller, Rosenbloom and Kelley, 1967; Rosenbloom, et al., 1967). We have examined hemolysates from 14 patients and autopsy tissue from one patient with the Lesch-Nyhan syndrome for their content of HGPRT activity and immunologically detectable HGPRT enzyme protein by using a monospecific rabbit antiserum prepared against a homogeneous preparation of normal human HGPRT (Arnold, Meade and Kelley, 1972).

STUDIES ON THE ELECTROPHORETIC VARIANTS OF HUMAN HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE

ABSTRACT. During the purification of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) from normal human male erythrocytes, three distinct peaks of HGPRT activity (I-pI 5.65; II-pI 5.80; III-pI 6.01) have been reproducibly distinguished by preparative isoelectric focusing and subsequently purified to homogeneity. With these highly purified preparations of variants I, II, and III we have found that: (1) the three variants are interconvertible as determined by polyaery1amide gel electrophoresis; (2) the three variants are immunologically identical; (3) the three variants have similar substrate utilization and end-product inhibition; (4) the three variants have the same native molecular weight (68,000) and Stokes radius (36a) and each is composed of two non-covalently bound subunits of equal molecular weight (34,000) and net charge; (5) the amino acid composition of variants II and III is nearly identical. We conclude that the electrophoretic variants of human erythrocyte HGPRT most likely result from a non-genetic post-transcriptional alteration of one or both subunits of the HGPRT enzyme molecule. Although the exact nature of the post-transcriptional alteration is not known, differential sialiation, differential binding of ribose-5-phosphate or ampholytes and association of the subunits into trimers, tetramers, etc. have been excluded.

Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT) Deficiency: Effect of Dietary Purines on Enzyme Activity

The X-linked, virtually complete deficiency of hypoxanthineguanine phosphoribosyltransferase (HGPRT) activity results in a bizarre neurologic disorder, the Lesch-Nyhan syndrome (Seegmiller, Rosenbloom and Kelley, 1967). In initial reports HGPRT activity was noted as undetectable in erythrocytes from patients with the Lesch-Nyhan syndrome. However, more recently several investigators have noted low but detectable levels of HGPRT activity in erythrocytes from these patients (Mizuno, et al., 1970; Sorenson, 1970). In addition, it is now known that despite this striking reduction in HGPRT activity there is a normal amount of immunologically detectable HGPRT protein present (Rubin, et al., 1971; Arnold, Meade and Kelley 1972). We subsequently found that a given patient would also exhibit a wide variability in HGPRT activity even when assay conditions were standardized. This suggested that envir- onmental factors might be responsible for changes in enzyme actiivty and that one potential approach to therapy of patients with this disease would be activation of the structurally abnormal HGPRT protein. The present report describes the influence of alterations in dietary purine content on the erythrocyte HGPRT activity from three patients with the Lesch-Nyhan syndrome.

Human Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT): Purification and Properties

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (E.C. 2.4.2.8.) catalyzes the formation of guanosine-5’-monophosphate and inosine-5’-monophosphate from 5’-phosphoribosyl-l-pyrophosphate (PPribose-P) and the purine bases guanine and hypoxanthine, respectively. Although originally assigned simply a “salvage” function in purine metabolism, the discovery of a virtually complete, X-linked deficiency of HGPRT associated with hyperuricemia, hyperuricaciduria and a bizarre neurologic syndrome (Lesch-Nyhan syndrome) has led to a reevaluation of the importance of HGPRT in the regulation of purine metabolism and central nervous system function (Seegmiller, Rosenbloom and Kelley, 1967). Therefore, an analysis of the structure of the normal enzyme is necessary to provide a basis for understanding the genetic lesion(s) producing altered or absent enzyme function. We have purified HGPRT to homogeneity from non-pooled, human male erythrocytes and have attempted to elucidate the nature of the electrophoretic heterogeneity of HGPRT activity observed during the purification (Arnold and Kelley, 1971; Kelley and Arnold, 1973).

Immunoadsorbent Chromatography of Hypoxanthine-Guanine Phosphoribosyltransferase

Recent evidence indicates that the virtual absence of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in patients with the Lesch-Nyhan syndrome is due in most if not all instances to a mutation(s) on the gene coding for the HGPRT protein (Kelley and Meade, 1971; Rubin, et al., 1971; Arnold, Meade and Kelley, 1972). This mutation(s) results in the synthesis of a normal amount of a catalytically defective yet immunoreactive HGPRT enzyme protein. We have purified normal human HGPRT from erythrocytes and have prepared a highly specific antiserum in rabbits (anti-HGPRT) which displays a single precipitin line on immunodiffusion and immunoelectrophoresis with both normal hemolysate and hemolysate from patients with the Lesch-Nyhan syndrome. We have succeeded in coupling a partially-purified preparation of anti-HGPRT to cyanogen bromide activated sepharose and will present data which indicates that this technique is a promising method for the rapid isolation of both normal and mutant HGPRT.