Hypoxanthine phosphoribosyl transferase HPRT

Fig. 13.2. Synthesis of IMP. c = Hypoxanthine phosphoribosyl transferase (HPRT) GAR = glycinamide ribonucleotide FGAR = formyl glycinamide ribonucleotide PRPP = phosphoribosyl pyrophosphate AICAR = 5 aminoimidazole-4-carboxamide...

Some rare inherited deficiencies of the purine-salvage enzymes hypoxanthine-phosphoribosyl-transferase (HPRT) and adenine-PRT (APRT) lead to primary purine overproduction (Table 20.5). X-linked Lesch-Nyhan syndrome occurs in complete deficiency of HPRT. It is characterized by mental retardation, self-mutilation, choreoathetosis, gout. 

Fig. 14.2 Scheme of thiopurine drug metabolism. HPRT, hypoxanthine phosphoribosyl transferase 6-MMP, 6-methylmercaptopurine 6-TGN, 6-thioguanine nucleotides 6-TIMP, 6-thiosine monophosphate TPMT, thiopurine methyltransferase XO, xanthine oxidase... 

Fig. 13.3. Metabolic cooperation between BHK21/C3 cells. Growth of BHK-HPRT-and BHK-TK- cells separately and in mixed (1 1) culture, in medium containing hypoxanthine, aminopterin and thymidine (HAT medium). HPRT = hypoxanthine phosphoribosyl transferase TK = thymidine kinase. (Reproduced from Pitts, 1971, with kind permission of the author and publisher.)...

HAU HECS Hep cells HPRT HSV HTC cells IAA ITES haemagglutinin unit human endothelial cell supernatant human epithelial cells hypoxanthine phosphoribosyl transferase herpes simplex virus hepatoma tissue culture cells indole acetic acid medium supplement containing insulin, transferrin, ethanolamine and selenium... 

The assay was described by Clive and co-workers (Clive et al. 1972) as a mutational assay system using the TK locus in mouse lymphoma cells. In the following years, he and his collaborators undertook a large-scale of investigation of the potential and optimal conduct of the assay. This included the use of the above mentioned TFT to select tk mutants, a comparison of the hypoxanthine guanine phosphoribosyl transferase (hprt) and tk loci, an analysis of the best expression time for tk mutant selection and a description of distinct large and small colony tk mutants. 

CD-I mice were exposed to purified air or benzene by inhalation at 0.04, 0.1, or 1.0 ppm for 22 hours per day, 7 days per week for 6 weeks (Ward et al. 1992). The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period, lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The increased frequencies of mutant spleen lymphocytes were significant at the low and mid, but not the high dose, and the method does not take into account possible clonal expansion. Further evaluation of the induction of gene mutation at these dose levels seems warranted. 

Fig. 2 Scheme of thiopurine drug metabolism. 77Wthiopurine methyltransfer-ase, XO xanthine oxidase, HPRT hypoxanthine phosphoribosyl transferase, 6-TIMP 6-thiosine monophosphate, 6-MMP 6-methylmercaptopurine, 6-TGN 6-thioguanine nucleotides, AZA Azathioprine, 6-/WP6-Mercaptopurine... 

Fig. 2 Metabolism of 6-mercaptopurine (6-MP) via xanthine oxidase (XO) to the inactive metabolite 6-thiouric acid (6-TU), thiopurine S-methyltransferase (TPMT) to the inactive metabolite 6-methylmercaptopurine (6-MMP), and hypoxanthine guanine phosphoribosyl transferase (HPRT) to 6-thioinosine monophosphate (6-TIMP) which is then further metabolized to thioguanine nucleotides (6-TGN), 6-methylmercaptopurine ribonucleotides (6-MMPR) or 6-thio-inosine triphosphate (6-thio-ITP), these all being active metabolites...

DNA single-strand break frequency was measured by alkaline elution [16] and sister chromatid exchanges (SCEs) were assayed by the method described by Wolff [32]. Hypoxanthine-guanine phosphoribosyl transferase (HPRT 6-thioguanine) and Na/K ATPase (ouabain) resistant mutants of CHO were determined by the methods described by Cleaver [7]. Repair replication after MMS treatment was measured in isopycnic gradients [8]. 

Figure 1 The metabolism of azathioprine and mercaptopurine Key AO, aldehyde ojddase GMPS, guanine monophosphate synthetase HPRT, hypoxanthine phosphoribosyl transferase IMPDH, inosine monophosphate dehydrogenase ITPA, inosine triphosphate pyrophosphohydrolase TPMT, thiopurine methyltransfer-ase XO/XDH, xanthine oxidase/dehydrogenase.

An almost complete deficiency of the enzyme hypoxanthine guanine phosphoribosyl transferase (HPRT) is known to be the cause of the Lesch-Nyhan syndrome (1,2) The gene for HPRT is located on the X-chromosome, so that heterozygous females show two populations of cells, one HPRT and one HPRT ", as predicted by the hypothesis of Lyon (3,4). Such mosaicism has been demonstrated in populations of cultured fibroblasts and in hair root follicles of heterozygotes (4,5). 

In many cells, the capacity for de novo synthesis to supply purines and pyrimidines is insufficient, and the salvage pathway is essential for adequate nucleotide synthesis. In patients with Lesch-Nyhan disease, an enzyme for purine salvage (hypoxanthine guanine phosphoribosyl pyrophosphate transferase, HPRT) is absent. People with this genetic deficiency have CNS deterioration, mental retardation, and spastic cerebral palsy associated with compulsive self-mutilation, Cells in the basal ganglia of the brain (fine motor control) normally have very high HPRT activity. These patients also all have hyperuricemia because purines cannot be salvaged. 

HGPRT Hypoxanthine-guanine phosphoribosyl transferase (also called HPRT) an enzyme involved in the utiliza-... 

The loci that can typically be used for mutation assessment are ones that can be selected for, although PCR methods currently available and under development will allow for analysis of any gene for which the DNA sequence is known. The selection procedure requires that a locus be heterozygous or hemizygous such that only a single mutation is required for the selectable phenotype to be assessed. Examples of such loci are the hprt (hypoxanthine-guanine phosphoribosyl transferase) gene... 

FIGURE 52-2 Generation of monoclonal antibodies. Mice are immunized with the selected antigen, and spleen or lymph node is harvested and B cells separated. These B cells are fused to a suitable B-cell myeloma that has been selected for its inability to grow in medium supplemented with hypoxanthine, aminopterin, and thymidine (HAT). Only myelomas that fuse with B cells can survive in HAT-supplemented medium. The hybridomas expand in culture. Those of interest based upon a specific screening technique are then selected and cloned by limiting dilution. Monoclonal antibodies can be used directly as supernatants or ascites fluid experimentally but are purified for clinical use. HPRT, hypoxanthine-guanine phosphoribosyl transferase. 

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