The dose-response relationship for ultraviolet-light-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster ovary cells.

Exposure of Chinese hamster ovary (CHO) cells clone K1BH4 to ultraviolet (UV) light at doses up to 86 ergs/mm2 did not significantly reduce cell survival, but UV doses of 86-648 ergs/mm2 produced an exponential cell killing. Observed mutation frequency ro 6-thioguannine resistance induced by UV increases approximately in proportion to increasing doses up to 260 ergs/mm2 in a range of 5-648 ergs/mm2 examined. The pooled data of mutation frequency f(X) as a function of dose X from 0-260 ergs/mm2 is adequately described by f(X)=10(-6) (13.6 + 2.04 X). That the UV-induced mutations to 6-thioguanine resistance affects the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus is supported by the observation that all randomly isolated drug-resistant colonies contained highly reduced or undetectable HGPRT activity.     

Optimal conditions for uptake of exogenous DNA by Chinese hamster lung cells deficient in hypoxanthine-guanine phosphoribosyltransferase.

Conditions were characterized for maximizing the uptake of exogenous mammalian cell DNA by hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster lung cells. Recipient cell cultures in an exponential growth phase were found to be more competent in taking up DNA than stationary cultures. Polyornithine enhanced the uptake of exogenous DNA more reproducibly and to a greater extent than did any of the other facilitators tested (DEAE-dextran, CaCl2, latex spheres, spermine, polylysine and polyarginine). Maximal DNA incorporation occurred when polyornithine and DNA were mixed together prior to inoculation. About 25-30% of the DNA inoculum became deoxyribonuclease-resistant in a typical experiment utilizing polyornithine as the facilitator. Both homologous and heterologous exogenous DNAs rapidly became associated with recipient cell nuclei: approximately 95% of the deoxyribonuclease-resistant donor DNA was nuclear-associated 15 min after inoculation.     

3-Deazaguanosine is metabolized to the triphosphate derivative in Chinese hamster cells deficient in hypoxanthine-guanine phosphoribosyltransferase

3-Deazaguanosine containing a 14C label in the ribose moiety was prepared using [U-14C]inosine as the [14C] ribose donor and commercial purine-nucleoside phosphorylase (EC 2.4.2.1) both to degrade the inosine, in the presence of phosphate, and to synthesize [14C-ribosyl]3-deazaguanosine in reduced phosphate and an excess of 3-deazaguanine. Purification was by high-pressure liquid chromatography (HPLC). [14C-ribosyl]3-Deazaguanosine was metabolized by Chinese hamster ovary cells to two metabolites, one major and one minor, eluting in the triphosphate region after HPLC analysis, and appeared to be incorporated into perchloric acid-insoluble material. Cell line TGR-3, deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and resistant to 3-deazaguanine, also formed both metabolites. Line TGR-1/DGRR-9, deficient in hypoxanthine-guanine phosphoribosyltransferase and resistant to both 3-deazaguanine and 3-deazaguanosine, formed greatly reduced levels of the major metabolite. 3-Deazaguanosine 5'-triphosphate, prepared enzymically from authentic 3-deazaguanosine 5'-monophosphate, co-eluted with the major metabolite peak during HPLC analysis. Treatment of a metabolite-containing extract with bacterial alkaline phosphatase (EC 3.1.3.1) resulted in the formation of 3-deazaguanosine. These observations indicate that 3-deazaguanosine can be metabolized, in Chinese hamster ovary cells, to the triphosphate derivative in lieu of the action of hypoxanthine-guanine phosphoribosyltransferase.     

Chinese hamster cells mutant at the hypoxanthine-guanine phosphoribosyltransferase (GPRT) locus. V. Complementation analysis of ts mutants

Four temperature-sensitive HPRT clones were used for hybridological analysis, which led to increase in complementation rate about 5 times. The probability of complementation, in respect of the HPRT locus proved to be rather high: 14 of 45 hybridization-tested mutants had complementation ability (including 3 ts mutants). Analysis of the complementation rate among mutants revealed clear-cut dependence on the selection conditions: clones grown in a medium with 8-azaguanine showed most frequent complementation. The use of mutants with a new phenotype in hybridization analysis revealed four additional complementation groups, three of which are made of temperature-sensitive clones. Biochemical analysis revealed the presence of hybrid forms of the HPRT enzyme in all hybrids tested. This confirms the intragenic character of complementation. At present, the functional map of the HPRT locus is represented by 9 groups, including a group of mutants with no complementation ability.     

Elevated intracellular glycine associated with hypoxanthine-guanine phosphoribosyltransferase deficiency in glioma cells

Abstract— The intracellular concentrations of a number of amino acids were measured in a normal clone of rat glioma cells, and in several independently derived clones selected for gross deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). A significant, approx 2-fold increase in the concentration of free glycine was observed in both mutagenized and non-mutagenized HGPRT deficient clones. The increase in glycine was independent of the phase of cell growth. A similar increase did not occur in HGPRT deficient lymphoblasts.     

Converting the guanine phosphoribosyltransferase from Giardia lamblia to a hypoxanthine-guanine phosphoribosyltransferase

Guanine phosphoribosyltransferase from Giardia lamblia, a key enzyme in the purine salvage pathway, is a potential target for anti-giardiasis chemotherapy. Recent structural determination of GPRTase (Shi, W., Munagala, N. R., Wang, C. C., Li, C. M., Tyler, P. C., Furneaux, R. H., Grubmeyer, C., Schramm, V. L., and Almo, S. C. (2000) Biochemistry 39, 6781-6790) showed distinctive features, which could be responsible for its singular guanine specificity. Through characterizing specifically designed site-specific mutants of GPRTase, we identified essential moieties in the active site for substrate binding. Mutating the unusual Tyr-127 of GPRTase to the highly conserved Ile results in 6-fold lower K(m) for guanine. A L186F mutation in GPRTase increased the affinity toward guanine by 3. 3-fold, whereas the corresponding human HGPRTase mutant L192F showed a 33-fold increase in K(m) for guanine. A double mutant (Y127I/K152R) of GPRTase retained the improved binding of guanine and also enabled the enzyme to utilize hypoxanthine as a substrate with a K(m) of 54 +/- 15.5 microm. A triple mutant (Y127I/K152R/L186F) resulted in further increased binding affinity with both guanine and hypoxanthine with the latter showing a lowered K(m) of 29.8 +/- 4.1 microm. Dissociation constants measured by fluorescence quenching showed 6-fold tighter binding of GMP with the triple mutant compared with wild type. Thus, by increasing the binding affinity of 6-oxopurine, we were able to convert the GPRTase to a HGPRTase.     

Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells.

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.     

Human hypoxanthine-guanine phosphoribosyltransferase. Evidence for tetrameric structure

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) has been purified 23,000-fold from normal human erythrocytes. The purification includes affinity chromatography on a GMP column. The subunit molecular weight of the enzyme obtained from this purification is 24,000. The finding of four protein species after cross-linkage of the highly purified enzyme with dimethylsuberimidate, dimethyladipimidate, and glutaraldehyde suggests that the enzyme may exist in the native state as a tetramer.     

Purification and characterization of mouse hypoxanthine-guanine phosphoribosyltransferase.

Hypoxanthine-guanine phosphoribosyltransferase (HGPR transferase) (EC 2.4.2.8) has been purified approximately 4500-fold to apparent homogeneity from mouse liver. The procedure involves the use of affinity chromatography and was designed to be readily adaptable to small scale isolations. The enzyme appears to be composed of 3 subunits of identical molecular weight (27,000 per subunit). The subunit molecular weight has also been determined by the analysis of radioactively labeled HGPR transferase immunoprecipitated from wild type and mutant (HGPR transferase) mouse tissue culture cell lines.     

Expression of human and Chinese hamster hypoxanthine-guanine phosphoribosyltransferase cDNA recombinants in cultured Lesch-Nyhan and Chinese hamster fibroblasts

The results presented in this communication demonstrate that hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA can be expressed in both Chinese hamster and human fibroblasts deficient in the endogenous gene product at levels permitting normal growth of the transformants. All the elements necessary for this expression are present in a pBR322-derived plasmid containing HPRT cDNA coding sequence and a retroviral long terminal repeat. These molecules function in both species investigated and, at least in the case of the Chinese hamster transformants, are efficient at the single copy level. Although the effects of the presence of intron sequences and a polyadenylation signal within the plasmids have yet to be evaluated, these studies demonstrate that neither is an absolute requirement for expression of HPRT cDNA sequences in cultured mammalian cells. We describe the construction of recombinant plasmids containing wild type human or Chinese hamster HPRT cDNA sequences in tandem with a retroviral LTR which confer the HPRT+ phenotype in HPRT-deficient V79 and Lesch-Nyhan fibroblasts. Both stable and unstable transformants, that expressed HPRT mRNA and protein, were isolated at high frequency.     

Steady-state kinetics of the schistosomal hypoxanthine-guanine phosphoribosyltransferase.

Schistosomiasis is a trematode infection of some 200 million people. The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of the major etiologic agent, Schistosoma mansoni, has been proposed as a potential target for antischistosomal chemotherapy [Dovey, H. F., McKerrow, J. H., & Wang, C. C. (1984) Mol. Biochem. Parasitol, 11, 157-167]. The steady-state kinetic mechanism for the schistosomal HGPRTase has been determined by including both hypoxanthine and guanine in the forward and reverse reactions under identical conditions. Double-reciprocal plots of initial velocity versus the concentration of one substrate, at a series of fixed concentrations of the other, give groups of intersecting straight lines indicating a sequential mechanism for the schistosomal HGPRTase-catalyzed reactions. In product inhibition studies, the results show that magnesium pyrophosphate (MgPPi) is a noncompetitive inhibitor with respect to dimagnesium phosphoribose pyrophosphate (Mg2PRPP), hypoxanthine, and guanine. Also, magnesium inosine monophosphate (MgIMP) and magnesium guanosine monophosphate (MgGMP) are noncompetitive inhibitors with respect to hypoxanthine or guanine, respectively, but are competitive inhibitors to Mg2PRPP. Furthermore, Mg2PRPP is a competitive inhibitor with respect to MgIMP and MgGMP but is a non-competitive inhibitor to MgPPi. The minimum kinetic model which fits the experimental data is an ordered bi-bi mechanism, where the substrates bind to the enzyme in a defined order (first Mg2PRPP followed by the purine bases), while products are released in sequence (first MgPPi followed by MgIMP or MgGMP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic properties of an azaguanine-resistant variant of Chinese hamster ovary cells (azarts) with normal levels of hypoxanthine-guanine phosphoribosyltransferase activity

Azarts Chinese hamster ovary cells were 20 to 50 times more resistant to 8-azaguanine and 50 to 10 times more resistant to both 6-thioguanine and 6-mercaptopurine than wild-type cells. Resistance correlated with a failure of azarts cells to incorporate 8-azaguanine into the nucleotide pool and into nucleic acids. The uptake of hypoxanthine and guanine, on the other hand, was about the same in both types of cells and the hypoxanthine-guanine phosphoribosyltransferase of the azarts cells as measured in cell lysates was unaltered both in concentration and kinetic properties with hypoxanthine as well as 8-azaguanine as substrate. Plasma membrane permeability to 8-azaguanine and the regulation of intracellular pH were also not altered in azarts cells and there was no significant degradation of 8-azaguanine or azaguanine nucleotides. We conclude therefore that in azarts cells the phosphoribosylation of 8-azaguanine per se is specifically blocked but that this effect is abolished upon cell lysis.     

Substrate inhibition in a human variant of hypoxanthine-guanine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase from a young man with purine overproduction and decreased purine salvage in fibroblast cultures was found to have low activity at concentrations of purine substrates at which the enzyme from normal individuals showed near maximal activity. The low enzyme activity was not associated with changes in the values of the Km(app) and Vmax(app) for any of the enzyme substrates. However, the enzyme activity was susceptible to substrate inhibition by hypoxanthine and guanine. The values obtained for the true Km, true Vmax, and true Ki for hypoxanthine were 26 +/- 10 microM, 1761 +/- 382 microunits/mg of protein, and 80 +/- 20 microM, respectively. The pattern of the substrate inhibition, as seen on a plot of 1/v versus hypoxanthine concentration, was characteristic of that associated with the formation of a dead-end complex between the inhibitory substrate and an enzyme form with which it normally does not react. The nature of this enzyme form and that of the dead-end complex was determined from double inhibition experiments, which indicated that hypoxanthine interacted with an enzyme-PPi intermediate to form an enzyme-hypoxanthine-PPi dead-end complex. The trapping of the enzyme in this inactive form explains the low activity at high purine base concentrations. Further information as to the nature of the reaction mechanism was obtained from plots of the reciprocal of enzyme activity versus the reciprocal of PP-ribose-P concentration at different fixed hypoxanthine concentrations. A pattern characteristic of uncompetitive substrate inhibition was obtained. This is indicative of an ordered sequential binding of substrates on the enzyme; PP-ribose-P binding before hypoxanthine. Thus, the variant enzyme showed an ordered sequential reaction mechanism, with the inhibitory substrate forming a dead-end complex with an enzyme-PPi intermediate.     

The incorporation of homologous and heterologous hypoxanthine-guanine phosphoribosyltransferase into mutant cells

Experiments are described leading to partial compensation of a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase in mutant cells by supplying the cells with exogenous purified enzymes. DEAE-dextran is an effective helper agent, whereas poly(L-lysine, lysolecithin and amphotericin B seem to inhibit the entry of the enzymes or their activity. Enzyme preparation from Chinese hamster was found to have different effects in different mutant cell lines. In mutant Chinese hamster cells, the electrophoretic activity pattern remains unchanged for the Chinese hamster enzyme, but changes progressively to faster-moving activity peaks for the human enzyme after several hours. The metabolic effect of the incorporated enzyme is in the range between 3 and 4% of the normal cellular enzyme activity which corresponds to a 10–20 fold increase of hypoxanthine-guanine phosphoribosyltransferase activity in the mutant cells.     

Adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase immunoprecipitation reactions in human-mouse and human-hamster cell hybrids.

Male New Zealand White rabbits were immunized with human adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which were purified about 2000-fold and 800-fold, respectively, from erythrocytes by DEAE-cellulose chromatography, ammonium sulfate precipitation and preparative polyacrylamide gel electrophoresis. Specific immunoprecipitations of APRT and HGPRT were achieved with the antisera that were obtained and by using polyethylene glycol as a substitute for goat anti-(rabbit) gamma globulin. The activities of the human forms of these enzymes, whether from red blood cells or from cultured cells, were almost completely eliminated under the conditions of immunoprecipitation used. Little or no reduction of APRT and HGPRT activities from mouse and Chinese hamster cells was observed. This discriminatory capacity of the antisera was successfully used for the identification of human APRT and HGPRT in human-mouse and human-hamster cell hybrids using the immunoprecipitation reaction.     

A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key enzyme in purine base salvage in humans and in purine auxotrophs, including Plasmodium falciparum, the leading cause of malaria. Hydrogen/deuterium (H/D) exchange into amide bonds, quantitated by on-line HPLC and mass spectrometry, has been used to compare the dynamic and conformational properties of human HGPRT alone, the HGPRT-GMP-Mg(2+) complex, the HGPRT-IMP-MgPPi <==> HGPRT-Hx-MgPRPP equilibrating mixture, and the transition-state analogue complex HGPRT-ImmGP-MgPPi. The rate and extent of H/D exchange of 26 peptic peptides, spanning 91% of the primary structure, have been monitored. Human HGPRT has 207 amide H/D exchange sites. After 1 h in D2O, HGPRT alone exchanges 160, HGPRT-GMP-Mg(2+) exchanges 154, the equilibrium complex exchanges 139, and the transition-state analogue complex exchanges 126 of these amide protons. H/D exchange rates are correlated with structure for peptides in (1) catalytic site loops, (2) a connected peptide of the subunit interface of the tetramer, and (3) a loop buried in the catalytic site. Structural properties related to H/D exchange are defined from crystallographic studies of the HGPRT-GMP-Mg(2+) and HGPRT-ImmGP-MgPPi complexes. Transition-state analogue binding strengthens the interaction between subunits and tightens the catalytic site loops. The solvent exchange dynamics in specific peptides correlates with hydrogen bond patterns, solvent access, crystallographic B-factors, and ligand exchange rates. Solvent exchange reveals loop dynamics in the free enzyme, Michaelis complexes, and the complex with the bound transition-state analogue. Proton transfer paths, rather than dynamic motion, are required to explain exchange into a buried catalytic site peptide in the complex with the bound transition-state analogue.