Rapid prenatal diagnosis of HG-PRT deficiency using ultra-microchemical methods

Summary A previously developed simple ultramicromethod has been used for the rapid prenatal diagnosis of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) deficiency. The method is based on the incubation of small numbers of visually selected, lyophilized fibroblasts (in the present study five cells per incubation) with radioactive substrate in an end volume of 0.3 l. Fibroblasts derived from the amniotic fluid of a 15-week male fetus in a heterozygote for the X-linked Lesch-Nyhan syndrome showed a severe degree of HG-PRT deficiency. In total 50 fibroblasts were used. The diagnosis was confirmed upon termination of the pregnancy by the demonstration of HG-PRT deficiency in fetal erythrocytes and cultured skin fibroblasts.HG-PRT: hypoxanthine-guanine phosphoribosyl transferase     

Hypoxanthine-guanine phosphoribosyl transferase deficiency

Summary In man congenital lack of an enzyme of the purine salvage system, hypoxanthineguanine phosphoribosyl transferase (HG-PRT E.C. 2.4.2.8), is mostly accompanied by a picture known as the Lesch-Nyhan syndrome. The degree of deficiency may vary from zero to a few percent of normal activity but a correlation between the severity of HG-PRT deficiency and the clinical picture has not been observed, no more than a correlation between HG-PRT deficiency and neurological dysfunction. But individuals with undetectable HG-PRT activity but without the Lesch-Nyhan syndrome have been described. Patients with partial HG-PRT deficiency have clinically distinctive findings. Sometimes mild neurological abnormalities are observed. Because of marked overproduction of uric acid severe gouty arthritis and renal dysfunction are often encountered in both complete and partial deficiency.There is considerable molecular heterogeneity in HG-PRT deficiency in man. Mutant enzymes may exhibit different kinetic and electrophoretic properties, indicating that there might be a mutation on the structural gene coding for HG-PRT.Lack of HG-PRT disturbs purine interconversions profoundly. In addition to an important function of HG-PRT in the uptake of the purine bases hypoxanthine and guanine into the cell, the effective uptake of inosine, guanosine and adenosine also seems to be dependent on HG-PRT. Uptake of purine bases into intact red blood cells occurs according to a two component mechanism, one component probably involving a phosphoribosyl transferase system.The inheritance of HG-PRT deficiency is X-linked recessive and it is transmitted by asymptomatic carrier females. Several methods have been introduced for carrier detection. As a consequence of X chromosome inactivation, in these females a mosaicism of HG-PRT positive and HG-PRT negative fibroblasts can be demonstrated after cloning or after selection of HG-PRT negative cells in a selective medium. A more rapid method involves direct measurements of HG-PRT activities in single hair roots from the scalp. Because hair roots develop more or less clonally, in heterozygote females HG-PRT positive and negative hair roots are encountered. HG-PRT deficiency can be detected antenatally by demonstrating the presence or absence of enzyme activity in ammiotic fluid derived fibroblasts qualitatively by autoradiography and quantitatively by ultramicrochemical measurements of enzyme activities in single or small numbers of cells.In studies with isolated cells the metabolic defect can be corrected in several ways. Metabolic cooperation between HG-PRT positive and HG-PRT negative cells leads to apparently normal phenotype of all cells, provided there is cell to cell contact. There is evidence that a missing enzyme product or a derivative might be transferred from the normal to the mutant cells. Apparent correction of the enzyme defect is also observed when HG-PRT deficient lymphocytes are stimulated with phytohaemagglutinin.The first data suggestive of genetic complementation between two human HG-PRT deficient cell strains by which hybrid cells can synthesize a functionally active HG-PRT, are consistent with the view that HG-PRT deficiency in man is due to a structural gene mutation. Recent results show that other interesting findings might come from experiments in which HG-PRT deficient cells are treated with exogenous genetic material (isolated DNA or metaphase chromosomes) to reactivate or induce HG-PRT activity.Supported by grants from FUNGO (Foundation for Medical Scientific Research in the Netherlands) and the Medical Prevention Fund.     

Metabolic cooperation studied by a quantitative enzyme assay of single cells

A new method making use of a radiochemical enzyme assay at the single cell level is presented to investigate metabolic cooperation, a widely studied form of cellular communication. In this case metabolic cooperation between normal human fibroblasts and fibroblasts derived from a patient deficient for the enzyme hypoxanthine-guanine phosphoribosyl transferase has been studied.A mixture of an equal number of both cell types was cultured in close physical contact and after trypsinisation, replating and culturing the cells for several hours in a high dilution, quantitative enzyme measurements with individual cells isolated from the mixture were carried out. From the distribution curve of the enzyme activities of the individual cells the conclusion could be drawn that a macromolecule, either the enzyme itself or DNA or mRNA, coding for that enzyme, is transferred from normal to mutant cells.     

Characterization of the biochemical basis of a complete deficiency of the adenine phosphoribosyl transferase (APRT)

Summary In order to study the biochemical basis of a complete deficiency of adenine phosphoribosyl transferase (APRT) the enzyme was purified to homogeneity, its properties were characterized, and antibodies raised. The enzyme is indirectly involved in adenine uptake. Apparently, by forming AMP the internal concentration of adenine is kept low allowing its diffusion.The same APRT is present in various tissues as was revealed by antibody inactivations employing anti-erythrocyte APRT as well as by direct enzyme assays in cells from the APRT deficient patient. In vitro cultured fibroblasts derived from this patient had less than 0.02% enzyme activity. No cross-reacting material was found in erythrocytes obtained from an APRT deficient child.     

X-chromosome inactivation and enzymatic activities in diploid and triploid fibroblasts

Diploid and triploid rabbit embryos obtained by artificial fertilization were cultured. No Barr's body in XXY and only one Barr's body in XXX triploid fibroblasts was observed. Six enzymatic activities were also determined. Two X-chromosome-bound enzymatic activities, glucose-6-phosphate dehydrogenase and phosphoglycerate kinase, were significantly increased in triploid fibroblasts, while another X-chromosome-bound activity (hypoxanthine phosphoribosyl transferase) was not modified. Among the autosome-bound activities studied, pyruvate kinase and adenine phosphoribosyl transferase were not modified, whereas in the triploid cells the 6-phosphogluconate dehydrogenase activity was decreased. The relationship between these modifications of enzymatic activities in triploids and the X-chromosome inactivation is discussed.     

Transient assay, by [3H]guanine incorporation of Escherichia coli xanthine-guanine phosphoribosyl transferase (GPT) in transfected human fibroblasts

We have used [3H]guanine incorporation as a rapid and sensitive assay of xanthine-guanine phosphoribosyl transferase (GPT) activity in SV40 transformed human fibroblasts. The SV40 early promoter is more efficient than the Rous sarcoma virus long terminal repeat for transient expression of the gpt gene. The assay works well in a derivative of AT5BIVA which lacks hypoxanthine-guanine phosphoribosyl transferase (hprt-) and we show here how the assay has been adapted to work in the hprt+ AT5BIVA parent.     

Microscale isoelectric focusing studies of mouse and human hypoxanthine-guanine phosphoribosyl transferases

A microscale isoelectric focusing technique has been developed and used to study hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C. 2.4.2.8, inosinate-guanylate:pyrophosphate phosphoribosyl transferase) activities in mouse and human cell lines. The enzymes of both mouse and human origin are shown to exhibit considerable heterogeneity, but each type has a unique range of isoelectric pH. The enzyme of a mouse × human hybrid cell line, derived by fusion of HGPRT^– parental cells, gives a homogeneous peak of activity, unlike the wild-type enzyme of either parent. The possibility is suggested that this enzyme activity is due to intra-allelic complementation.Centennial Fellow of the Medical Research Council of Canada, 1967–1970.     

Characterization of hypoxanthine-guanine phosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electrophoretic method

The hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in a group of man-mouse somatic cell hybrids, produced by Sendai virus-mediated cell fusion and HAT selection, has been analyzed by a new electrophoretic technique. Evidence is presented which shows that the hybrid lines derived from fusion of a mouse fibroblast deficient in HGPRT with various human cell strains have an HGPRT activity that is characteristic of the human enzyme, whereas a hybrid line derived from a mouse fibroblast which is deficient in thymidine kinase has an HGPRT activity characteristic of the mouse. This new technique involves electrophoresis of cell extracts on cellulose acetate gel, followed by the localization of the enzyme activity by autoradiography.This research was supported in part by a research grant from the U.S. National Institutes of Health (No. GM-13415).     

Introduction of a Fluorine Atom at C3 of 3-Deazauridine Shifts Its Antimetabolic Activity from Inhibition of CTP Synthetase to Inhibition of Orotidylate Decarboxylase, an Early Event in the de Novo Pyrimidine Nucleotide Biosynthesis Pathway

The antimetabolite prodrug 3-deazauridine (3DUrd) inhibits CTP synthetase upon intracellular conversion to its triphosphate, which selectively depletes the intracellular CTP pools. Introduction of a fluorine atom at C3 of 3DUrd shifts its antimetabolic action to inhibition of the orotidylate decarboxylase (ODC) activity of the UMP synthase enzyme complex that catalyzes an early event in pyrimidine nucleotide biosynthesis. This results in concomitant depletion of the intracellular UTP and CTP pools. The new prodrug (designated 3F-3DUrd) exerts its inhibitory activity because its monophosphate is not further converted intracellularly to its triphosphate derivative to a detectable extent. Combinations with hypoxanthine and adenine markedly potentiate the cytostatic activity of 3F-3DUrd. This is likely because of depletion of 5-phosphoribosyl-1-pyrophosphate (consumed in the hypoxanthine phosphoribosyl transferase/adenine phosphoribosyl transferase reaction) and subsequent slowing of the 5-phosphoribosyl-1-pyrophosphate-dependent orotate phosphoribosyl transferase reaction, which depletes orotidylate, the substrate for ODC. Further efficient anabolism by nucleotide kinases is compromised apparently because of the decrease in pK(a) brought about by the fluorine atom, which affects the ionization state of the new prodrug. The 3F-3DUrd monophosphate exhibits new inhibitory properties against a different enzyme of the pyrimidine nucleotide metabolism, namely the ODC activity of UMP synthase.     

X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: Detection of heterozygotes by selective medium

Skin fibroblasts from five unrelated males with X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency and from their families have been exposed to medium containing 6-thioguanine. This purine analogue selects against cells with normal hypoxanthine-guanine phosphoribosyl transferase activity and therefore permits detection of mutant cells in heterozygous populations. The results of these studies are compared to those obtained by autoradiography of single-cell clones of skin fibroblasts from the same subjects. In each case, the results of the selective method are similar to those obtained by clonal analysis. The use of selective medium therefore provides a sensitive means to detect heterozygosity at this locus and may provide a general method to select cells with X-linked markers from heterozygous populations.This work was supported by grants from the U.S.P.H.S. (#HD 00486), The Joseph P. Kennedy, Jr., Memorial Fluid Research Fund, and the National Foundation for Neuromuscular Diseases, Inc.     

Hypoxanthine-guanine phosphoribosyl transferase with altered substrate affinity in mutant mouse lymphoma cells

Cells with altered hypoxanthine-guanine phosphoribosyl transferase (HPRT) (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) have been selected. Compared to wild type, mutant enzyme has a reduced affinity for the substrate phosphoribosyl pyrophosphate and is more labile to heat inactivation. Mutant cells are resistant to 6-thioguanine at 33-39 degrees C and sensitive to hypoxanthine-aminopterin-thymidine at 37-39 degrees C, but not at 33 degrees C. We hypothesize that a single structural mutation of HPRT can explain these results.     

Partial Characterization of Phosphoribosyl Transferase, Phosphoribosyl Anthranilate Isomerase, and Indole Glycerol Phosphate Synthase from Serratia marcescens

The molecular organization of the enzymes phosphoribosyl (PR) transferase, phosphoribosyl anthranilate (PRA) isomerase, and indole glycerol phosphate (InGP) synthase of the tryptophan biosynthetic pathway of Serratia marcescens was investigated and compared with that reported in other enteric bacteria. PRA isomerase and InGP synthase activities were found to reside in a single polypeptide chain, a situation analogous to that in Escherichia coli, Salmonella typhimurium, and Aerobacter aerogenes. This bifunctional enzyme was purified to near homogeneity. Its molecular weight was estimated to be 48,000. PR transferase was found unassociated with PRA isomerase and InGP synthase after gel filtration and ion-exchange chromatography. Whereas in other enteric organisms PR transferase has been reported to form an aggregate with anthranilate synthase, it is a distinct entity in S. marcescens.     

A Simple and Rapid Method for the Separation and Characterization of Hypoxanthine-Guanine Phosphoribosyltransferase Isoenzymes

Purinephosphoribosyltransferases catalyze the conversion of purine bases to their nucleotides in the presence of 5-phosphoribosyl-l- pyrophosphate (PRPP) (1). This salvage pathway plays an important role in the regulation of de novo purine synthesis (2). In mammalian cells two distinct phosphoribosyltransferases were demonstrated: the enzyme adenine phosphoribosyltransferase (AMP: pyrophosphate phosphoribosyl- transferase; A-PRT; E.C. 2.4.2.7) and the enzyme hypoxanthine-guanine phosphoribosyltransferase (PIP: pyrophosphate phosphoribosyltranferase; HG-PRT; E.C. 2.4.2.8). There has been a great interest in this latter enzyme as the complete absence of this enzyme activity in patients with Lesch-Nyhan syndrome (3) and a partial deficiency in some patients with gout (4) has been demonstrated.     

Characterization of mutants with single and multiple defects in the tryptophan biosynthetic pathway in Bacillus subtilis

Sixty-five tryptophan auxotrophs which map in a cluster on the genome of Bacillus subtilis were characterized on the basis of (i) growth response, (ii) accumulation of intermediate compounds, and (iii) determination of enzymatic defects. They could be placed into six phenotypic classes. Certain of the mutants exhibited pleiotropic effects on more than one enzymatic activity in a manner different from those effects reported for the tryptophan pathway in other organisms. Invariably, mutations in the second gene, that coding for phosphoribosyl transferase activity, were found to lack the indoleglycerol phosphate synthase activity specified by the third gene in the cluster; however, this polarity did not extend to genes more distal in the cluster. Furthermore, mutations in the gene which codes for phosphoribosyl-anthranilate isomerase not only led to a loss of this enzyme but also to a loss of phosphoribosyl transferase and indoleglycerol phosphate synthase. In contrast, mutations in either of the loci coding for these latter functions had no apparent effect on isomerase activity. No polarity of the conventional type was found, e.g., none of the mutations in any gene led to polarized effects on the levels of the enzymes specified by the other genes of the cluster. These observations indicated a possible in vivo aggregation involving the transferase, isomerase, and synthase enzymes, with the isomerase acting as the "key" enzyme in the aggregate.     

An approach to the production of helminth antigens in vitro: The formation of hybrid cells between Fasciola hepatic a and a rat fibroblast cell line

Using polyethylene glycol, hybrid cells were formed between rat fibroblasts lacking the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT, B.C. 2.4.2.8) and cells of the liver fluke Fasciola hepatica. The hybrid cells survived in a medium containing hypoxanthine, aminopterin and thymidine (HAT) indicating that the enzyme deficiency of the parental rat cells had been corrected. Isoelectric focusing in agarose gels showed that the HGPRT activity in the hybrids was of F. hepatica rather than rat origin. F. hepatica chromosomes could not be identified with certainty in hybrids; and fluke antigens, other than HGPRT, could not be detected in them or in culture medium in which they had grown.     

Nucleotide sequence of the Escherichia coli xanthine-guanine phosphoribosyl transferase gene.

The Escherichia coli gene coding for the enzyme xanthine-guanine phosphoribosyl transferase (gpt) has been widely used as a dominant selectable marker in a variety of mammalian cells. We have determined the complete nucleotide sequence of the 1057 base pair (bp) segment of DNA containing this gene. The coding sequence for the enzyme is 456 nucleotides long and can code for a 152 amino acid (16.9 Kd) polypeptide. A comparison of the amino acid sequence of the bacterial enzyme with that of the mammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) reveals no significant homology between the two polypeptides.     

Stopped flow kinetic studies of adenosine triphosphate phosphoribosyl transferase, the first enzyme in the histidine biosynthesis of Escherichia coli

1. Stopped flow kinetic studies of ATP phosphoribosyl transferase (EC 2.4.2.17) from Escherichia coli showed that high protein concentration and elevated temperature do not give a drastic reduction of the transferase activity when measured before inhibitory amounts of PRibATP (product) has accumulated. 2. A small and slow increase in activity follows a reduction of the protein concentration showing a slow dissociation of the enzyme from an inactive to an active species. 3. By lowering the concentration of the inhibitor histidine, a fairly slow increase in activity is observed indicating a dissociation of the enzyme. 4. AMP and histidine together give a strong inhibition of the activity, while AMP alone stimulates the enzyme activity.     

Metabolic investigations of fibroblasts from horses, Equus caballus, with hereditary severe combined immunodeficiency

In an attempt to determine the metabolic defect causing severe combined immunodeficiency (SCID) in horses in which altered purine metabolism has been observed, various parameters of purine and pyrimidine metabolism were evaluated. The activities of nine purine enzymes (adenosine kinase, purine nucleoside phosphorylase, deoxyadenosine kinase, deoxycytidine kinase, 5'-nucleotidase, AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase were measured in fibroblasts. All activities determined for SCID horses were normal. Uptake of 10 microM adenosine or 2'-deoxyadenosine (a growth inhibitory concentration for SCID fibroblasts) by SCID fibroblasts was identical to that found for normal fibroblasts in the presence of both 1 and 50 microM phosphate. The Km determined for the transport of both adenosine and 2'-deoxyadenosine was 35 microM. In the presence of p-nitrobenzylthioguanosine (a nucleoside transport inhibitor), 2'-deoxyadenosine uptake was inhibited to the same extent in all fibroblast lines tested. To determine if the last step in pyrimidine biosynthesis might be altered in SCID fibroblasts, UMP synthase activities were evaluated but found to be normal (0.5 nmol UMP formed/min/mg protein).     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

Model involving gene inactivation in the generation of autosomal recessive mutants in mammalian cells in culture.

We present evidence for a two-step model for expression of the recessive phenotype at the diploid adenine phosphoribosyl transferase (aprt) locus in Chinese hamster ovary cells. This model proposes a high-frequency event leading to allelic inactivation and a low-frequency event leading to a structural alteration of the APRT protein. Either event can occur first, resulting in two types of heterozygous cells. The proposed model is based on analysis of Chinese hamster ovary presumptive aprt heterozygotes and APRT- mutants, derived by two different laboratories. The major class of heterozygotes (class 1) had approximately 50% parental APRT activity, 50% immunologically precipitable APRT protein, and only wild-type enzyme as based on two-dimensional gel electrophoresis and thermal inactivation studies. We propose that one allele at the aprt locus has been inactivated in these heterozygotes. APRT- mutants derived from any single class 1 heterozygote arose at a low frequency and contained either no immunologically detectable APRT protein or an APRT enzyme which was, in most cases, demonstrably altered. The second class of heterozygotes, consisting of two independent isolates, gave rise to APRT- cells at a high frequency (10(-3) to 10(-5). These heterozygous cell lines had 50% of parental APRT activity and only wild-type spot, or wild-type and an electrophoretic variant spot, on two-dimensional gels. These aprt heterozygotes appear to have arisen by mutation at one allele. APRT- mutants derived from either heterozygote of this class had all lost the wild-type activity, consistent with the proposed model.