Factors controlling relative flux rates of the de novo and salvage pathways of purine nucleotide biosynthesis during animal cell growth are not fully understood. To examine the relative role of each pathway for cell growth, three cell lines including CHO K1 (a wild-type Chinese hamster ovary fibroblast cell line), CHO ade ؊ A (an auxotrophic cell line deficient of amidophosphoribosyltransferase (ATase), a presumed rate-limiting enzyme of the de novo pathway), and CHO ade ؊ A transfected with human ATase cDNA ( ؊ A؉hATase) resulting in 30 -350% of the ATase activity of CHO K1, were cultured in purine-rich or purine-free media. Based on the enzyme activities of ATase and hypoxanthine phosphoribosyltransferase, the metabolic rate of the de novo and salvage pathways, the rate of cell growth (growth rate) in three cell lines under various culture conditions, and the effect of hypoxanthine infusion on the metabolic rate of the de novo pathway in rat liver, we concluded the following. 1) In ؊ A؉hATase transfectants, ATase activity limits the rate of the de novo pathway, which is closely linked with the growth rate. 2) Purine nucleotides are synthesized preferentially by the salvage pathway as long as hypoxanthine, the most essential source of purine salvage, can be utilized, which was confirmed in rat liver in vivo by hypoxanthine infusion. The preferential usage of the salvage pathway results in sparing the energy expenditure required for de novo synthesis.3) The regulatory capacity of the de novo pathway (about 200%) was larger than that of the salvage pathway (about 20%) with constant hypoxanthine phosphoribosyltransferase activity.Purine nucleotides synthesized via de novo and salvage pathways are indispensable for cell growth through DNA and RNA syntheses and for the ATP energy supply. Interference of purine metabolism has thus been the target of antineoplastic drugs. However, it is unclear which of the two pathways is more important for the supply of purine nucleotides during cell growth. Purine nucleotide synthesis catalyzed by HPRT, 1 the key enzyme of the purine salvage pathway, was reported to be more active than that catalyzed by ATase, the presumed ratelimiting enzyme of the de novo pathway, in many tissues and malignant cells (1, 2). The increased metabolic rate via the de novo pathway and ATase activity are, however, more strongly linked with cell growth and malignant transformation than the metabolic rate via the salvage pathway and HPRT activity (1, 3-7). To interpret this enigma, we investigated the mutual regulation of purine biosynthesis between the de novo and salvage pathways. The cDNA cloning of rat and human ATase (hATase) in our laboratory (8, 9), an ATase-deficient auxotrophic Chinese hamster ovary fibroblast cell line of CHO ade Ϫ A, and CHO ade Ϫ A transfected with hATase cDNA driven by the cytomegalovirus promoter ( Ϫ AϩhATase) enabled us to explore the rate-limiting property of ATase and to study the relationship between the two pathways in three cell lines in two purine-free or two purine-...
We recently showed that an increased supply of purine nucleotides increased the growth rate of cultured fibroblasts. To understand the mechanism of the growth rate regulation, CHO K1 (a wild type of Chinese hamster ovary fibroblast cell line) and CHO ade (-)A (a cell line deficient in amidophosphoribosyltransferase, a rate-limiting enzyme of the de novo pathway) were cultured under various conditions. Moreover, a defective de novo pathway in CHO ade (-)A cells was exogenously restored by 5-amino-4-imidazole-carboxamide riboside, a precursor of the de novo pathway. The following parameters were determined: the growth rate of CHO fibroblasts, the metabolic rate of the de novo pathway, the enzyme activities of amidophosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, the content of intracellular nucleotides, and the duration of each cell-cycle phase. We concluded the following: (i) Purine de novo synthesis, rather than purine salvage synthesis or pyrimidine synthesis, limits the growth rate. (ii) Purine nucleotides are synthesized preferentially by the salvage pathway as long as hypoxanthine is available for energy conservation. (iii) The GTP content depends on the intracellular ATP content. (iv) Biosynthesis of purine nucleotides increases the growth rate mainly through ATP production and promotion of the G(1)/S transition.
The cDNA for a c-myc intron 1 binding protein 1 (MIBP1) in the rat was isolated from lambda gt11 and lambda ZAPII cDNA libraries. Sequencing of the cDNA clones revealed a long ORF which encoded a putative protein of 2437 amino acid residues. This protein has two widely separated zinc finger regions, each of which carries C2H2 motifs. When expressed in E. coli as a fusion protein, part of the MIBP1 showed sequence-specific binding to the target sequence, i.e., a 9-bp sequence in the rat c-myc intron 1. MIBP1 is most likely the rat counterpart of human MHC binding protein-2 (MBP-2/HIV-EP2), based on the 86% similarity in nucleotide sequence and 93% similarity in amno acid sequence. Northern blotting revealed a high level of MIBP1 mRNA in the brain.
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