Altered turnover of allelic variants of hypoxanthine phosphoribosyltransferase is associated with N-terminal amino acid sequence variation

The results of our previous studies suggested that differences in the primary structures of the hypoxanthine phosphoribosyltransferase (HPRT) A and B proteins (EC 2.4.2.8) of mice are associated with altered turnover of these proteins in reticulocytes. On the basis of nucleotide sequence comparisons of their corresponding cDNAs, we show here that the HPRT A and B proteins differ at two positions; there is an alanine/proline substitution at amino acid position 2 and a valine/alanine substitution at amino acid position 29 (HPRT A/B proteins, respectively; total protein length, 218 amino acids). On the basis of results obtained from sequencing of the N termini of the purified HPRT A and B proteins, we also show that these amino acid substitutions are associated with differences in processing of the proteins; HPRT B, which is encoded as N-terminal Met-Pro, has a free N-terminal proline residue; HPRT A, which is encoded as N-terminal Met-Ala, lacks a free N-terminal alpha-amino group and is presumed to be acetylated following removal of the N-terminal methionine (i.e. AcO-Ala). These observations are discussed in reference to the idea that the N terminus of a protein plays a role in determining the rate at which the protein is degraded in erythroid cells.     

Human brain hypoxanthine guanine phosphoribosyltransferase: structural and functional comparison with erythrocyte hypoxanthine guanine phosphoribosyltransferase

A rapid and simple method, based on GMP Sepharose affinity chromatography, was used for the purification of human brain hypoxanthine guanine phosphoribosyltransferase. A single protein band was detected by polyacrylamide gel electrophoresis of the native purified enzyme. A subunit molecular weight of 25,000 was estimated by SDS gel electrophoresis. The Km values for hypoxanthine and phosphoribosyl pyrophosphate were 50 and 111 microM, respectively. The Ki values for GMP and IMP with phosphoribosyl pyrophosphate were 21 and 37 microM, respectively. The purified enzyme from human brain did not differ significantly from the human erythrocyte one in amino acid composition. The brain and erythrocyte hypoxanthine guanine phosphoribosyltransferases showed complete immunochemical identity on Ouchterlony double diffusion.     

Elevated erythrocyte CDP-choline levels associated with beta-thalassaemia in patients with transfusion independent anaemia

The accumulation of CDP-ethanolamine as well as CDP-choline in a small cohort of patients with normal UMPH1 and no defined cause for their anaemia suggested a defect in both phosphotransferases. Here we report 10 patients with transfusion independent beta-thalassaemia; 8 being pure heterozygotes and 2 heterozygotes also for Hb E. Mean CDP-choline (86.xxx +/- 48 microM) and CDP-ethanolamine (34.6 microM +/- 34.5 microM), mean control <3 microM. Elevated CDP-choline in patients with no defined cause for their haemolytic anaemia was previously suggested as a possible indicator of CDP-choline phosphotransferase deficiency. Here we associate it with transfusion independent beta-thalassaemia.     

Elevated aspartate transcarbamylase and dihydroorotase activities in erythrocytes from patients with hypoxanthine guanine phosphoribosyltransferase deficiency.

Activities of aspartate transcarbamylase and dihydroorotase are increased 6 to 8-fold in erythrocytes from individuals with hypoxanthine guanine phosphoribosyltransferase deficiency. The increased enzyme activities do not appear to be due to enzyme activation.     

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.     

Natural allelic variation of duck erythrocyte histone H1b

In our previous work (J. Palyga, Genetic polymorphisms of histone H1. b in duck erythrocytes. Hereditas 114, 85-89, 1991) we reported a genetic polymorphism of duck erythrocyte histone H1.b. Here, we screened H1 preparations in a two-dimensional polyacrylamide gel to refine the distribution of allelic forms of H1.b in fifteen duck populations. We have revealed that the frequency of H1.b allelic variants was significantly different among many conservative and breeding duck groups. While b(1) and b(3) were common in all populations screened, the allele b(2), with a slightly lower apparent molecular weight, was confined mainly to brown-feathered ducks (Khaki Campbell and Orpington) and descendent lines.The C- and N-terminal peptides released upon cleavage with N-bromosuccinimide and Staphylococcus aureus protease V8 from duck allelic histones H1. b2 and H1.b3, respectively, migrated differently in the gel, probably as a result of potential amino acid variation in a C-terminal domain.     

Evidence for tetrameric structure of mammalian hypoxanthine phosphoribosyltransferase

A fast electrophoretic variant of hypoxanthine phosphoribosyltransferase (HPRT) has been detected in Mus musculus bactrianus, a mouse subspecies from Middle Asia (USSR). The electrophoretic HPRT pattern yielded by hybrids between the somatic cell of LMTK^– (deficient in thymidine kinase) and the splenocytes of a male of M. m. bactrianus was five-banded. The pattern obtained from the germ cells of the ovaries from 14.5-day-old embryos from laboratory CBA mice × M. m. bactrianus crosses was also composed of five bands. The hybrids between the somatic cells of human fibroblasts × LMTK^– cells gave a three-banded electrophoretic HPRT pattern because the asymmetrical heteropolymeric isozymes are probably unstable. Taken together, all the evidence is in favor of a tetrameric structure of mammalian HPRT.     

Transformation of the gene for hypoxanthine phosphoribosyltransferase.

Purified DNA from wild-type Chinese ovary (CHO) cells has been used to transform three hypoxanthine phosphoribosyltransferase (HPRT) deficient murine cell mutants to the enzyme positive state. Transformants appeared at an overall frequency of 5 x 10(-8) colonies/treated cell and expressed CHO HPRT activity as determined by electrophoresis. One gene recipient, B21, was a newly isolated mutant of LMTK- deficient in both HPRT and thymidine kinase (TK) activities. Transformation of B21 to HPRT+ occurred at 1/5 the frequency of transformation to TK+; the latter was, in turn, an order of magnitude lower than that found in the parental LMTK- cells, 3 x 10(-6). Thus both clonal and marker-specific factors play a role in determining transformability. The specific activity of HPRT in transformant extracts ranged from 0.5 to 5 times the CHO level. The rate of loss of the transformant HPRT+ phenotype, as measured by fluctuation analysis, was 10(-4)/cell/generation. While this value indicates stability compared to many gene transferents, it is much greater than the spontaneous mutation rate at the indigenous locus. The ability to transfer the gene for HPRT into cultured mammalian cells may prove useful for mutational and genetic mapping studies in this well-studied system.     

Levels of hypoxanthine phosphoribosyltransferase RNA in human cells

The gene for the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) is expressed at a low level in many cells. As is the case with several other “housekeeping genes,” thorough studies of hprt gene regulation have been hampered by the low levels of its mRNA. We have used RNA/RNA hybridization in solution to determine the concentration of hprt-RNA in human cells. The sensitivity and specificity of the method have been validated, and it is shown that hprt-RNA can be accurately determined at a level of a few mRNA molecules per cell. As expected for a housekeeping gene, low and relatively constant hprt-RNA levels (0.3–0.8 pg/μg DNA) were found in primary cultures of normal amnion cells and fibroblasts, EBV-transformed lymphoblastoid cell lines, neuroblastoma, glioblastoma, and melanoma cell cultures. While resting lymphocytes were found to contain very low amounts of hprt-RNA, lymphocytes stimulated with phytohemagglutinin (PHA) showed a 10-fold increase to about 0.8–1.2 pg/μg DNA, which corresponds to 6–10 hprt-RNA molecules per cell. The level started to increase about 20 h after PHA stimulation, 5–10 h before the onset of DNA synthesis, and a steady-state level was reached after 2–3 days in culture. In PHA-stimulated lymphocytes from two brothers with inherited HPRT deficiency (LeschNyhans syndrome), the hprt-RNA level in PHA-stimulated lymphocytes was only about 25% of that in normal subjects. In T-cells selected for HPRT deficiency by growth in 6-thioguanine medium, the levels of hprt-RNA were either normal or very low, which probably reflects the different nature of the mutations involved. These results demonstrate the sensitivity of this method for determinations of low levels of RNA and clearly show induction of hprt-RNA after mitogenic stimulation of human lymphocytes.     

Steady-state kinetics of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi

The kinetic mechanism for the reaction catalyzed by the hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi was analyzed to determine the feasibility of designing a parasite-specific mechanism-based inhibitor of this enzyme. The results show that the HPRT from T. cruzi follows an essentially ordered bi-bi reaction, and like its human counterpart also likely forms a dead end complex with purine substrates and the product pyrophosphate. Computational fitting of the kinetics data to multiple initial velocity equations gave results that are consistent with the dead end complex arising when the hypoxanthine- or guanine-bound form of the enzyme binds pyrophosphate rather than the phosphoribosylpyrophosphate substrate of the productive forward reaction. Limited proteolytic digestion was employed to provide additional support for formation of the dead end complex and to estimate the K(d) values for substrates of both the forward and reverse reactions. Due to similarities with the kinetic mechanism of the human HPRT, the results reported here for the HPRT from T. cruzi indicate that the design of a mechanism-based inhibitor of the trypanosomal HPRT, that would not also inhibit the human enzyme, may be difficult. However, the results also show that a potent selective inhibitor of the trypanosomal HPRT might be achieved via the design of a bi-substrate type inhibitor that incorporates analogs of moieties for a purine base and pyrophosphate.     

Orotate phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase from yeast: kinetic analysis with chromium (III) pyrophosphate

The reactions catalyzed by orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) from yeast differ in the kinetic mechanisms by which they are activated by divalent metal ions. Moreover, whereas OPRTase is activated specifically by Mg(II) or Mn(II), the reactions catalyzed by HGPRTase can utilize a wider range of divalent metal ions, including Mg(II), Mn(II), Co(II), and Zn(II). In this report we describe the results of a kinetic analysis of the effects of the addition of Cr(III) pyrophosphate (Cr-PPi) to the OPRTase and HGPRTase assay solutions, which delineates further the differences between these enzyme activations by metal ions. (1) Cr-PPi is an effective competitive inhibitor of the OPRTase catalysis, when the steady-state forward velocity of orotidine monophosphate (OMP) formation is examined over a range of phosphoribosyl alpha-pyrophosphate (PRibPP) concentrations, whereas pyrophosphate (PPi) has been reaffirmed to be a noncompetitive product inhibitor under the same conditions. (2) Cr-PPi itself serves as a substrate for the OPRTase-catalyzed reverse pyrophosphorolysis of OMP and does not inhibit the utilization of PPi as substrate during this reaction. (3) In contrast, Cr-PPi, at concentrations as high as 6 mM, has no effect on the HGPRTase-catalyzed formation of inosine monophosphate, whereas the inhibition exhibited by PPi during this reaction is noncompetitive but defined by two sets of lines in the double reciprocal plot of the initial velocity versus 1/PRibPP. (4) Cr-PPi is not a substrate for the HGPRTase-catalyzed pyrophosphorolysis of IMP under the conditions of these assay procedures.     

Irreversible inactivation of hypoxanthine phosphoribosyltransferase by periodate oxidized nucleotides.

Hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) from rat brain or human erytherocytes can be irreversibly inactivated by incubation with periodate-oxidized analogues of the enzyme products GMP or IMP. This inhibition is specific and directed against the product binding site of the enzyme. Inactivation is not produced by periodate-oxidized AMP or other aldehydes, for example periodate-oxidized glycerol. The inactivation is concomitant with the binding of the inhibitor to the enzyme protein. The bound inhibitor cannot be removed from the protein by dialysis, Sephadex chromatography or polyacrylamide-gel electrophoresis. Adenine phosphoribosyltransferase (EC 2.4.2.7), on the other hand, is not influenced by any of the inhibitors mentioned above.     

Modified GMP-affinity chromatography for the purification of mutant hypoxanthine phosphoribosyltransferase

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the conversion of hypoxanthine and guanine to IMP and GMP, respectively, in the presence of 5-phosphoribosyl-1-pyrophosphate. Deficiencies of HPRT are associated with neurological abnormalities and gout. A human HPRT variant enzyme failed to bind to a GMP-affinity column under standard purification conditions. We developed a series of predictive tests for designing the affinity chromatography protocol which enabled purification of both normal and variant HPRT. The primary variable for the present variant was a difference in toleration of salt; other aspects recommended for evaluation are assessment of ligand-enzyme affinity, pH optimum, and tolerance of nonspecific ligands for washes. In addition, a method for determining the amount of GMP linked to the column material was developed and consisted of acid hydrolysis and HPLC quantitation of guanine.     

Reaction of antibody to normal human hypoxanthine phosphoribosyltransferase with products of mutant genes.

Immunoglobulin produced in rabbits against normal human red cell hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8) was used to study cell lysates of individuals with deficient enzyme activity. The reaction of immunoglobulin with HPRT formed partially active insoluble and fully active soluble complexes. The insoluble complexes were separated from soluble complexes and the free enzyme by centrifugation. The soluble complexes and free enzyme were separated by electrophoresis. Hemolysates from 13 patients with the Lesch-Nyhan syndrome who have virtually total deficiency of HPRT activity and 2 patients with hyperuricemia and 2–5% of normal activity were unable to neutralize immunoglobulin and showed no evidence of cross-reacting material (CRM). In contrast, 2 other partially deficient males with 4.5 and 50% of normal actvity, and a partially deficient heterozygous female with 34% of normal activity, were CRM⁺ in this assay. The amount of CRM present in the cells of these 2 males appeared to be disproportionate to their HPRT activity. The heterozygous female contained about 30% of normal CRM which was consistent with the estimated activity provided by her normal cell population. This indicated that her abnormal cells were CRM^?. Absence of CRM in her abnormal cells was consistent with the observed lack of CRM in hemolysates of her hyperuricemic half-brother. These data indicate the presence of considerable heterogeneity in human mutation at the HPRT locus.     

Elevated erythrocyte glutathione associated with elevated substrate in high- and low-glutathione sheep.

Erythrocyte glutathione concentration increases dramatically in sheep when they become anemic. To determine the mechanism of this change in glutathione control, we measured the enzymes and substrates necessary for glutathione control, we measured the enzymes and substrates necessary for glutathione synthesis after acute blood loss in both low- (gamma-glutamylcysteine synthetase deficient) and high-glutathione sheep. Erythrocyte glutamate, ATP, and glycine increased dramatically in all sheep. Erythrocyte gamma-glutamylcysteine synthetase increased slowly and seemed unrelated to changes in glutathione. Erythrocyte glutathione synthetase and cysteine and plasma cysteine, glutamate and glycine did not change significantly. Apparently substrate concentrations may be important in regulating erythrocyte glutathione levels.     

Elevated in vivo frequencies of mutant T cells with altered functional expression of the T-cell receptor or hypoxanthine phosphoribosyltransferase genes in p53-deficient mice

We have studied the effects of a defect in the p53 gene on spontaneous and radiation-induced somatic mutation frequencies in vivo by measuring T-cell receptor (TCR) and hypoxanthine phosphoribosyltransferase (HPRT) mutant frequencies (MFs) in p53 deficient mice both before and after exposure to X-irradiation. In the absence of irradiation, the TCR and HPRT mutant frequencies were roughly two-fold higher in p53 null (-/-) mice than in wild-type (+/+) mice. Unexpectedly, the TCR and HPRT MFs were slightly lower in heterozygote p53 (+/-) than in wild-type (+/+) mice, however. After 2 weeks 2Gy whole body irradiation the TCR and HPRT MFs were about two-fold higher in the p53 null (-/-) and p53 (+/-) mice than in the wild-type. Taken together, these findings suggest that a defect in the p53 gene may lead to TCR and HPRT mutants being recovered at higher frequencies in both irradiated and unirradiated mice, but it should be emphasized that the effects we have observed are not particularly strong, albeit that they are statistically significant. Interestingly, several of the highest TCR MF values that we observed in the course of our experiments were recorded in p53 (-/-) animals that had developed thymomas and hence appeared to be cancer prone.