Regulation of Purine Biosynthesis and Interconversion in the Chick*

In ureotelic species, such as the rat, adaptive changes in metabolic flux and enzyme levels occur in the purine metabolic pathway when cells are rapidly growing. This is observed in both regenerating liver and in ma-lignant tissues. The enzymes P-Rib-PP amidotransfer- ase and IMP dehydrogenase increase in activity in both situations. The level of purine biosynthesis is much highr in uricotelic species, such as the chick, when compared to ureotelic animals. By treating immature roosters with the hormone 8-estradiol, it is possible to induce rapid liver growth, allowing comparison of the regulation of purine biosynthesis and interconversion in high metabolic rate cells with different roles for purine metabolism. The tissue activities of P-Rib-PP amidotransferase, xanthine dehydrogenase, adenylosuccinate synthetase and lyase, AMP deaminase, IMP dehydrogenase, and GMP synthetase did not rise in livers from estradiol-treated chicks, as compared to controls. However, the rate of de novo purine synthesis triples and the intracellular level of P-Rib-PP doubles within 24 h of treatment. The biosynthesis of GMP is elevated at 12 and 24 h, but the levels of soluble nucleotide pools do not change. These data indicate that regulation of the de novo purine pathway in uricotelic species in a high metabolic situation is at the level of substrate availability (P-Rib-PP ) and not due to changes in enzyme level or to feedback inhibition. Uricotelic

Uricotelic animals use synthesis of the purine ring system as a pathway for the excretion of excess nitrogen. In such animals, the domestic chicken for example, the level of de novo purine synthesis is 15-fold higher than that found in the rat (1). When birds are subjected to stress situations such as starvation or diets high in proteins (both conditions that lead to higher nitrogen excretion), the rate of purine synthesis is increased (2). Burns and Buttery (3) have demonstrated that a number of amino acids stimulate both total and de novo urate biosynthesis. They also found that both nitrogenous precursors and a readily available energy source were necessary for optimal urate biosynthesis in isolated chicken hepatocytes. Brand and Lowenstein (4) have shown that the activity of two enzymes (adenolosuccinate lyase and xanthine dehydrogenase) involved in the synthesis of urate, the end product of purine biosynthesis from small molecule precursors, increases in the livers of chickens starved for 4 days. In addition, the level of the first committed enzyme in purine * TO whom reprints should be addressed at, Department of Biochemistry, Rice University, P. 0. Box 1892, Houston, TX 77001. biosynthesis, amidophosphoribosyltransferase, is elevated in livers from chicks fed diets high in protein (2). These studies show that, though normal levels of both purines and purine synthetic enzymes are high in the chick, the flux through the de novo pathway and the level of the enzymes can be increased by high levels of the amino acid substrates.
Livers from chickens treated with &estradiol can provide a rapidly growing normal cell as a model system. Treatment with this hormone causes a rapid increase in liver weight and in protein, RNA and DNA content (a 60% increase in 48 h) in preparation for the production of egg proteins (5). Jackson et at. (6,7) and Katonuma and Weber (8) have shown that, in regenerating rat liver, another system that is used as a model for normal rapid growth, the level of certain purine biosynthetic enzymes is increased. This is also true in certain rat liver hepatomas (6)(7)(8). In livers from chicks treated with pestradiol, a larger than normal portion of newly synthesized purines will be incorporated into RNA and DNA during the rapid liver growth instead of being excreted as uric acid. Thus, this tissue can be used as a model for study of regulation of purine biosynthesis as requirements for nucleotides vary.
The results of this study indicate that purine metabolism in rapidly growing uricotelic liver is regulated in a manner differnt than observed in mammalian ureotelic liver or in high amino acid feeding in the chick where nitrogen excretion is elevated. Enzyme levels in the chick do not change in response to the increased metabolic flux, in contrast to rat liver, Regulation appears to be primarily at the level of P-Rib-PP' concentration which serves as the precursor for de novo purine biosynthesis. (v:v), wlth a wick of Whatman 3MU paper stapled on top (10). Sheets Of 3*II tillatlon system with an efflciency Of 8540%. OIganlc Scintillation fluld c o n t a m i n g 14.7 9 PPO and 0.2 g POeOP in 3.8 llters Of toluene was used for counting thin layer plates. The radioactlvity of DEAE-cellulo~e disks "015 measured using scintlllatlon fluld containing 2100 a1 toluene, 1230 nl 100% ethanol, 12 9 PPO. and 0 . 3 g POPOP. All re'eaqents were sclntillatlon arade.

AND DISCUSSION
The liver from chicks treated with the hormone /3-estradiol is a fast growing nonmalignant tissue. In an analogous mammalian system, regenerating rat liver, increases in the levels of several purine biosynthetic enzymes have been reported (6)(7)(8). The goal of this study was to determine how the avian system adapts to high growth rates. In addition to determining the activities of purine biosynthetic and interconversion enzymes, the rate of purine biosynthesis, P-Rib-PP levels, and adenine and guanine pool sizes were determined in order to gain a better understanding of the regulation of purine biosynthesis cle nouo.
The activities of seven enzymes involved in purine biosynthesis, interconversion, and catabolism were measured in liver extracts obtained from normal and /3-estradiol-treated chickens using assays specially developed for the chicken system. The enzyme levels present in the chick homogenates do not change after hormonal treatment (Table I). This is in direct contrast to the regenerating rat liver system, in which amidophosphoribosyltransferase activity doubles and IMP dehydrogenase activity is elevated 5-fold on a similar time scale (6)(7)(8). The observed activity levels for amidotransferase, adenylosuccinate lyase, and xanthine dehydrogenase indicate the high capacity of chick liver for IMP biosynthesis relative to the branch pathways to AMP and GMP. It should be noted that the lyase is a dual function protein catalyzing a reaction in both IMP and AMP biosynthesis.
Its level is clearly regulated for IMP production and is not likely to be rate-limiting for AMP formation from IMP.
The overall rate of purine biosynthesis was determined by the incorporation of formate into cellular purines (both acidsoluble and those incorporated into RNA and DNA). The rate of de nouo purine synthesis (Table II) is increased at 12 h after hormone treatment and remains at a high level for at least 48 h. At 12 h, though, much more of the newly synthesized IMP is being channelled into the GMP branch of the interconversion pathway, as shown by the adenineguanine ratios, than at any other time. By 48 h, the ratio of GMP and   AMP synthesis has returned almost to normal, even though the overall rate of synthesis remains high. Thus, it appears that the rapid growth of the liver induced by the treatment with /3-estradiol is supported by an increase in the rate of purine synthesis de nouo. The branches of the purine interconversion pathways are more equally expressed during the initial 24 h of this increase in de nouo synthesis than under normal growth conditons. P-Rib-PP, which is a substrate of the first committed step in purine biosynthesis, has often been implicated in the regulation of the rate of de novo synthesis (24,25). Amidophosphoribosyltransferase, which forms phosphoribosylamine from P-Rib-PP, purified from pigeon liver shows sigmoidal kinetics at P-Rib-PP concentrations below the K,,, so that small changes in cellular levels of P-Rib-PP could greatly influence the rate of purine biosynthesis (25,26). Determination of the level of P-Rib-PP in livers of chickens treated with ,L?-estradiol shows that, after 24 h, the amount of P-Rib-PP in the tissues has doubled (Table III).
Numerous suggestions have been made that changes in purine nucleotide pools regulate de nouo synthesis by feedback inhibition.
To determine if the concentration of these compounds changes during the increase in purine biosynthesis that follows P-estradiol treatment, soluble pool sizes of adenine, guanine, there was no significant change in the levels of adenine, guanine, and hypoxanthine after stimulation with ,@estradiol, compared to the control.
The results of this study suggest that the major response of purine metabolism in chick liver to rapid growth is to increase the level of P-Rib-PP increasing the flux through the de novo pathway. Some regulation changes also occur as the IMP branch of GMP synthesis increases. Enzyme levels do not respond and soluble pool sizes do not change. Chick liver purine metabolism in a rapid growth situation is regulated in a manner different than observed in ureotelic species.