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Increased 5-Phospho--D-ribose-1-diphosphate Synthetase (Ribosephosphate Pyrophosphokinase, EC 2.7.6.1) Activity in Rat Hepatomas

Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46223

The behavior of the activity of 5-phosphoribosyl 1-pyrophosphate (PRPP) synthetase (ribosephosphate pyrophosphokinase, EC 2.7.6.1) was elucidated in normal rat liver, in 11 hepatomas of different growth rates, and in rapidly growing differentiating and regenerating liver. Tissue extracts were prepared by centrifugation of 10% homogenates at 100,000 x g for 30 min, and enzyme activity was measured in the protein fractions obtained by 40 and 47% ammonium sulfate saturation of the supernatant fluids from livers and hepatomas, respectively. In the tissue extracts, there was no interfering enzyme activity that utilized PRPP under the standard assay conditions.

The affinity of PRPP synthetase for its substrates, ribose 5-phosphate and adenosine triphosphate (ATP), and to Mg²⁺ was similar in liver and hepatoma extracts. The K_m for ribose 5-phosphate was 0.3 mM; for ATP, it was 0.1 mM in the presence of excess Mg²⁺. The K_m for Mg²⁺ATP was 1.2 mM in the presence of excess ATP. There was no difference in the affinity of the enzyme for its activators, Mg²⁺ and inorganic phosphate, in liver and hepatoma preparations; the K_m for Mg²⁺ was 0.6 mM in the presence of excess ATP; the K_m for inorganic phosphate was 14.0 mM. The requirement of hepatoma extracts for full phosphate saturation was higher than that of liver extracts (85 versus 65 mM).

A standard assay was worked out for the liver and hepatoma systems; in liver, the enzyme activity was linear for 30 min incubation, and in hepatoma it was linear for 15 min incubation. PRPP synthetase activity was proportionate with amounts of protein added over a range of 0.4 to 3.0 mg in both liver and hepatoma extracts.

In the liver of normal adult Wistar rats, PRPP synthetase activity was 108 ±10 nmol/hr/mg protein. In rat tissues of high cell renewal activity, thymus, testis, spleen, and small intestine, synthetase specific activity was 3.7-, 3.6-, 1.2-, and 1.3-fold higher than that of normal liver.

The synthetase specific activity in hepatomas of slow growth rate increased 1.2- to 1.5-fold, and in intermediate and rapidly growing hepatomas it was elevated 1.9- to 4.1-fold higher than that of normal liver. In the 24-, 36-, 48-, and 72-hr regenerating liver, the synthetase specific activity was increased 1.5-, 1.4-, 1.4-, and 1.3-fold, respectively; activity returned to normal range by 96 hr after operation. In the differentiating liver in postnatal days 1, 6, and 23, the synthetase activity in the average cell was 31, 38, and 69% of that of the adult liver, and it was in normal range at 32 days after birth.

The markedly increased activity in the rapidly proliferating hepatomas appears to be more characteristic of neoplastic growth than of normal liver proliferation. The increased activity of PRPP synthetase should provide an elevated capacity to produce the key metabolite, PRPP, and this should confer selective advantages to cancer cells. The increased synthetase activity marks this enzyme as a potentially important target in the design of chemotherapy.

¹ Visiting Assistant Professor at Indiana University School of Medicine. Pemanent address: Department of Dermatology, Semmelweis University Medical School, Budapest, Hungary.

² Recipient of USPHS Grants CA-13526 and CA-05034. To whom requests for reprints should be addressed, at the Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, IN 46223.

Received 4/23/84. Accepted 8/13/84.