APRT stabilization

As a result of an extensive screening program, a family was found that had a genetically regulated deficit in APRT (K3). The enzyme produced did not appear to be abnormal in electrophoretic mobility, pH optimum, Michaelis constants, or heat stability (H6). The four subjects studied did not suffer any apparent physical disability attributable to the lack of the enzyme. 

These facts showed that the increased amount of APRT is not due to an increased rate of synthesis, but to posttranscriptional stabilization of the enzyme. Since earlier workers had shown that PRibPP stabilizes... 

Despite the fact that APRT is coded for by a gene on chromosome 16, there seemed to be differences between APRT from normal and I.esch-Nyhan subjects in crude lysates. As the enzymes are purified, these differences lessen both in terms of heat stability, chromatographic and antigenic activity (R3, R4). Any differences must be reflective of secondary changes in the cell milieu and could be lost on purification. 

A large range of abnormalities has been observed. In some cases no detectable enzyme is found in the red cells (D3). In other cases the amount of activity is about 30 of normal (Y3). Families have been reported with 1-3 of normal enzyme levels. No simple correlation between behavior and enzyme level is apparent. Some patients have concurrently elevated APRT, some do not. In some, but not in all, there is increased heat stability of the APRT. In one study (E2) eight patients from five kindreds were examined in detail. No correlation was found between clinical manifestations and the level of HPRT in red cell lysates. These authors seemed to find the line between partial deficiency gout and the symptoms of Lesch-Nyhan disease a question of degree, not of kind. 

We measured APRT-activity from erythrocytes lysates of 524 unselected patients of the Medical Policlinic Munich micro-radiochemically using the method of Kelley (1967). Moreover the activity in 127 patients with renal failure on dialysis, and of 41 patients with nephrolithiasis was determined. The family of a patient with low APRT-activity was further investigated. The enzyme of patients with diminished actwity was investigated by measuring heat and cold stability at 55°C and 4 C respectively. 

On the other hand, PRPP in concentrations as low as 0.05 mM partially stabilized both enzymes against the inactivation in the presence of 1 mM PRPP the decrease in the specific activities of HGPRT and APRT by the above 3-fold dilution was 30% and 14% respectively, causing the ratio of the specific activities of APRT and HGPRT to increase from 1.6 to 2.0 only. 

PRPP has been previously shown to protect HGPRT and APRT against thermal inactivation in-vitro (7). In addition an increased concentration of PRPP in LNS erythrocytes has been implicated in the stabilization of the APRT in such cells against degradation in vivo (7) ... 

A major problem in the APRT purification was stabilization of the enzyme. While APRT activity in crude hemolysate is relatively stable, enzyme activity rapidly deteriorates at 4 in more purified preparations, a finding noted by previous investigators (Hori and Henderson, 1966 Srivastava and Beutler, 1971). Stability studies on... 

Partial deficiency of HGPRT, a salvage enzyme of purine metabolism, has been demonstrated to be the primary abnormality causing purine overproduction in a small proportion of patients with gout (l-4). The quantitative deviation in the activity of this enzyme has been shown by Kelley et al. to be associated with decreased stability to thermal inactivation (2). These authors suggest that in the affected subjects HGPRT is structurally altered. Furthermore, in some of these patients erythrocyte adenine phosphoribosyltransferase (APRT) activity was found to be increased and relatively thermostable (2). 

Regarding the APRT of these patients with partial HGPRT deficiency, all properties examined - Km for substrates, electrophoretic mobility on starch gel, pH profile and sensitivity to product and feedback inhibition, were found normal. In agreement to the findings of other investigators (2), the APRT exhibited increased activity and relative thermostability and PRPP stabilized the enzyme against thermal inabtivation. 

The Lesch-Nyhan Syndrome (LNS) is a rare x-linked neurological disease of children characterized by choreoathetosis, spasticity, mental retardation and compulsive self mutilation accompanied by excessive purine production and hyperuricemia (l). The virtually complete deficiency of activity of a purine salvage enzyme, hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) (EC 2.4.2.8.) (2), due to structural gene mutation (3 4) has been shown to be the basic abnormality in this disease. In erythrocytes of LNS patients, HGPRT deficiency has been found to be associated with increased activity and relative thermal stability of adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7 ) (5 6) an autosomally determined enzyme (7) ... 

In the dialyzed hemolysates of the LNS children, HGPRT activity could not be detected (< 0,05% of normal activity) and the activity of APRT was 22k% and 213% of normal respectively. In addition APRT in the dialyzed LNS hemolysates exhibited relative stability to thermal inactivation. Following k min. at 96 C, the residual activity of the enzyme in hemolysates from patients H.B. and T.N. was 7 % and 8l respectively (averages of 3 and 5 experiments) in comparison to the range of 13.6 - 6B,k% (m + 1SD 44.2 + ll.l9i) obtained for hemolysates of 59 control subjects. 

Addition of 5-phosphoribosyl-1-pyrophosphate (PRPP) to normal and LNS dialyzed hemolysates resulted in stabilization of APRT to heat Inactivation. Under these conditions APRT in both normal and LNS hemolysates attained the same degree of stability (approximately 95 residual activity). 

A significant difference between the APRT in the LNS and normal hemolysates could be demonstrated by exposing them to destabilization against thermal inactivation. Hypoxanthine caused a significant decrease in the thermal stability of the enzyme in normal hemolysates but affected only slightly the LNS enzyme. In the presence of adenine APRT of both normal and INS hemolysates was equally destabilized to thermal inactivation (Table l). Similar findings were recently reported also by Rubin and Balis (lO). 

The mechanism by which PRPP stabilizes APRT against heat inactivation is not yet clarified, but it would be reasonable to assume that PRPP is inducing a conformational change in the APRT molecule resulting in thermal stability, and that the increased heat stability of the enzyme in the UlS hemolysate is due to the increased level of PRPP in such cells. Indeed, both increased specific activity and thermal stability of the APRT in LNS hemolysates have been attributed to stabilization of the enzyme by the elevated concentration of PRPP accumulation in the HGPRT deficient erythrocytes (ll,12). In view of the fact however, that the increased stability of APRT is observed in dialyzed hemolysates, the postulated conformational change would have to be irreversible, unless the stabilizing PRPP is bound to the enzyme. 

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