Analysis of HGPRT- CRM+ human lymphoblast mutants.

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.     

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).     

Differences between rat liver epithelial and fibroblast cells in metabolism of purines

Epithelial and fibroblast cells from adult rat liver were found to differ markedly in their metabolism of the purine hypoxanthine. Both cell types took up hypoxanthine and possessed hypoxanthine-guanine phosphoribosyl transferase for phosphoribosylating the purine. However, in the transferase assay, lysates from epithelial cells converted hypoxanthine predominantly to inosine monophosphate, with small amounts of the nucleoside inosine as product, whereas fibroblast cell lysates converted hypoxanthine predominantly to inosine. The inosine appeared not to be produced by direct ribosylation of the base, since fibroblast cell lysates had less purine nucleoside phosphorylase activity than epithelial cell lysates. Rather, the inosine produced by fibroblast lysates appeared to be derived from inosine monophosphate through catabolism of the mononucleotide by 5' nucleotidase. An inhibitor of 5' nucleotidase, thymidine triphosphate, reduced the amount of inosine formed.     

Hypoxanthine-guanine phosphoribosyltransferase in human erythroid cells: Properties of the isozymes

We have previously given evidence that the hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) isozymes in human erythroid cells result from posttranslational modifications of a single gene product [Johnson, G. G., et al. (1982). Biochemistry 21:960]. In the present work we compare the properties of the unmodified and two major modified isozymes, which collectively account for 90% of the HGPRT enzyme activity in cell lysates. The modified isozymes differ from the parent molecule in the pH dependence of activity and in the relative utilization of the two purine base substrates, hypoxanthine and guanine. In contrast to the changes in the catalytic properties of the enzyme, the modifications have no detectable effects on the heat stability or on the equilibrium between enzyme dimers and enzyme tetramers.This work was supported by United States Public Health Service Grant 5 RO1 CA 16754-03 and by the San Diego State University Foundation.     

Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase.

N-Acetylglucosamine-1-phosphotransferase (GlcNAcPTase) was solubilized with 2% Tergitol NP-10 from cultured human lymphoblast cells and purified 3840-fold with 14% recovery using lentil lectin-Sepharose 4B, DEAE-Sephacel and Sephacryl S-400 chromatographies. The partially purified enzyme requires the non-ionic detergent Tergitol NP-10 and a divalent cation, Mn2+ or Mg2+, for its activity and exhibits an optimal pH at 7.2-7.5 in Tris-maleate buffer. Kinetic studies demonstrated an apparent Km of 24 microM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor alpha-methylmannoside. The GlcNAcPTase is inhibited by UDP and UDP-glucose, and by negatively charged phospholipids including phosphatidylserine, phosphatidylglycerol and phosphatidic acid. The apparent mol. wt of the human lymphoblast GlcNAcPTase is approximately 1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheed et al., 1982).     

Mutagen-induced diploid human lymphoblast variants containing altered hypoxanthine guanine phosphoribosyl transferase

The human lymphoblast line MGL8 was treated with HAT and subsequently "mutagenized" with EMS (200 microgram/ml) to give 15% survival, and 6-thioguanine-resistant cells were selected by cloning in soft agarose containing the drug (1 microgram/ml). Eighteen sublines of independently derived resistant clones were isolated and studied in detail. One subline had a low residual HGPRT activity of about 1% of the parental cells. The HGPRT of this subline had a higher Km for PRPP, was more sensitive to heat, and was degraded faster by trypsin than the enzyme in extracts of MGL8 cells. This resistant subline and three others contained CRM levels of 1--38%, compared to the wild-type, so they probably represent true structural mutants of the HGPRT gene. All the variants maintained the karyotype of the parental line (46, XY, 6p-).     

Molecular and biochemical analyses of spontaneous and X-ray-induced mutants in human lymphoblastoid cells

We have isolated a series of 14 spontaneously arising and 28 X-ray-induced mutants at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in human lymphoblastoid cells. Among the spontaneous mutants, 5/14 (36%) had detectable alterations in their restriction fragment pattern after hybridization with a human cDNA probe for hgprt. Of the 10 remaining mutants, 4 had partial HGPRT enzyme activity, which suggested that they contained point mutations. Among the 28 mutants induced by 150 rad of X-rays, 15 (54%) had deletions of part or all of the hgprt gene. 5 of the remaining 13 (18% overall) had partial HGPRT enzyme activity, which suggested that they contained point mutations. These data imply that in this human cell system, X-rays induce both point mutants which have residual enzyme activity as well as mutations involving relatively large deletions of DNA.     

Bacterial expression and characterization of a novel,soluble, calcium-binding,and calcium-activated human nucleotidase

A newly discovered human analogue of a bed bug apyrase, which we named hSCAN-1 for human soluble calcium-activated nucleotidase-1, was expressed in bacteria, refolded from inclusion bodies, purified, and characterized. This apyrase, which is distinct from the eNTPDases exemplified by the endothelial CD39 (NTPDase1) apyrase, is a 38 kDa monomeric enzyme capable of hydrolyzing a variety of nucleoside di- and triphosphates, but not monophosphates. Preferred substrates include GDP, UDP, and IDP, with a pH optimum for activity between 6 and 7. The specific activity and substrate preference of the bacterially expressed enzyme closely mimic those of the enzyme expressed in mammalian COS cells, as well as the enzyme synthesized in an in vitro bacterial expression system. This suggests that glycosylation and other posttranslational modifications that do not occur in bacteria are not necessary for nucleotidase activity or proper folding of this human apyrase. hSCAN-1 absolutely requires Ca(2+), but not Mg(2+) or other divalent cations analyzed, for enzymatic activity. Surprisingly, the activity does not increase in a quasi-linear fashion at sub-millimolar Ca(2+) concentrations, as would be expected if Ca(2+) were only used as a cosubstrate for the nucleotide substrate, but rather follows a sigmoidal curve. The intrinsic fluorescence and difference absorption studies of hSCAN-1 in the absence of nucleotides revealed Ca(2+)-induced changes in the environment of tryptophan and tyrosine residues with half-saturation at about 90 microM Ca(2+). NaCl increased the half-saturating Ca(2+) concentration needed for both structural changes detected by optical spectroscopy and enzymatic activation of hSCAN-1 detected by nucleotidase assay. These results suggest that Ca(2+) triggers a conformational change in hSCAN-1, converting the enzymatically inactive protein to the active enzyme, in addition to forming the metal-nucleotide substrate complex necessary for nucleotidase activity.     

Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase with reduced affinity for PP-ribose-P in four related males with gout

Summary A family is described in which four affected males, spanning two generations, have hyperuricemia and gout accompanied by hematuria but are without severe neurologic involvement. The affected males were found to have markedly reduced levels of erythrocytic hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity; these were 5–12% with hypoxanthine and 0.5–3% with guanine as compared to controls. Erythrocytic adenine phosphoribosyltransferase (APRT) was approximately three-fold elevated in the affected individuals.The residual HGPRT activity in affected males enabled characterization of some of the properties of this mutation. The apparent Michaelis constants (km) for both hypoxanthine and guanine were essentially unchanged, whereas the km for PP-ribose-P was approximately 10–20-fold elevated for all four affected males. The enzyme was more sensitive to product inhibition by IMP and GMP than controls, and exhibited greater thermal lability at 65°C than found with control lysates.     

Mevalonate kinase in lysates of cultured human fibroblasts and lymphoblasts: kinetic properties, assay conditions, carrier detection and measurement of residual activity in a patient with mevalonic aciduria

An assay has been developed for the measurement of mevalonate kinase activity in extracts of cultured human fibroblasts and lymphoblasts. Individual elements of the assay were investigated in order to achieve optimum conditions. Apparent Michaelis constants (KMapp) for the substrates mevalonic acid and adenosine-5'-triphosphate were 22 +/- 10 mumol/l and 0.42-0.53 mmol/l, respectively, in lysates of control fibroblast lines. The same values in lysates of a control lymphoblast line were 17 mumol/l and 0.23 mmol/l, respectively. Mevalonate kinase activity in extracts of cultured fibroblasts derived from 6 control individuals was 3.24 +/- (SD) 0.91 nmol/min/mg protein. The activity in extracts of fibroblasts derived from a patient with mevalonic aciduria was 0.15 +/- 0.10 nmol/min/mg protein, approximately 5% of the control mean. The parents and brother of the patient displayed mevalonate kinase activities in fibroblast extracts approximating 38-42% of the control mean. Substantially higher mevalonate kinase activity was documented in extracts of cultured lymphoblasts. When assayed on various occasions, the mean activity of mevalonate kinase in extracts of lymphoblasts derived from the parents, brother and maternal grandmother of the patient ranged from 27 to 32% of the mean activity of 9.8 +/- (SD) 3.4 nmol/min/mg protein measured in a parallel control lymphoblast line, while the mean activity in a maternal and paternal uncle approximated 65-89% of the same control mean. The mean activity in extracts of lymphoblasts derived from the patient approximated 2% of the control mean. The data suggest that the parents, brother and maternal grandmother are carriers of the defective gene responsible for mevalonate kinase deficiency, consistent with an autosomal recessive mode of inheritance.     

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency with full expression of the Lesch-Nyhan syndrome

Summary A patient with the full clinical expression of the classical Lesch-Nyhan syndrome is presented with a residual hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity of 5–10% in erythrocyte lysate and about 30% in fibroblast lysate. The activities of other erythrocyte enzymes of purine metabolism were typical for a classical Lesch-Nyhan patient. The effects of allopurinol therapy on the excretion of urinary purine metabolites were studied by a newly developed isotachophoretic technique.The unusually high residual activity of HGPRT in erythrodytes and fibroblasts of the patient enabled the enzymologic characterization of the mutant enzyme: in fibroblasts the affinities for the substrates hypoxanthine and guanine were normal. However, there was an increased apparent K _m for phosphoribosylpyrophosphate (PRPP), a complete absence of product inhibition by IMP and GMP, and a decreased heat stability. Addition of PRPP did not stabilize the mutant enzyme. In addition to the altered properties of the fibroblast enzyme, the K _m of the erythrocyte enzyme for hypoxanthine was also increased.Immunoprecipitation experiments revealed the presence of an approximately normal amount of material cross-reacting with anti-human HGPRT antiserum. However, it appeared that this cross-reacting material had a decreased stability. When intact erythrocytes were incubated with radiolabeled purine bases, no formation of IMP or GMP could be detected, despite the relatively high residual activity of HGPRT in the hemolysate. The results fit the following hypothesis: as a consequence of a structural mutation affecting the PRPP-site of the enzyme and a decreased heat stability, the activity of the mutant enzyme under in vivo conditions is virtually zero.In the erythrocytes of the patient's mother a normal HGPRT-activity was found. However, the activity in her fibroblasts was lower than normal, while a decreased heat stability and an intermediate behavior towards IMP could be shown.Hair root analysis of several members of the patient's family confirmed the heterozygosity of the mother, whereas no other heterozygotes could be detected. The family anamnesis did not show other cases of Lesch-Nyhan syndrome. These findings were taken as evidence that the patient described in this paper might represent a mutation orginating from the gametes in either of the maternal grandparents.     

Hypoxanthine transport in normal and hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient diploid human lymphoblasts

We have examined the possible relation between hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.7., HGPRT) activity and hypoxanthine transport in the normal human lymphoblast line MGL8 and two HGPRT- mutant lines derived from it. The mutant line MGL8A29 (L8A29) had considerable amounts of material cross-reacting immunologically to HGPRT, while mutant MGL8A18 (L8A18) had none. In the normal cells, hypoxanthine is taken up by both a saturable and non-saturable process. Kinetic studies show that the velocity of transport is much lower than HGPRT activity, while both have similar values of K_m. In the two mutant lines, we failed to demonstrate saturable transport, and the rates of hypoxanthine uptake by these cells were directly proportional to its concentration in the medium. Active HGPRT molecules appear to be related to the saturable transport process.     

Properties of N-acetylglucosamine 1-phosphotransferase from human lymphoblasts.

Human lymphoblast and fibroblast cell lines from a patient with I-cell disease and normal individuals were characterized with respect to certain properties of UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine phosphotransferase. The enzyme isolated from normal lymphoblast and fibroblast cell lines expressed similar kinetic properties, substrate specificities and subcellular localizations. Coincident with the severe reduction of N-acetylglucosamine phosphotransferase activity in both I-cell fibroblast and lymphoblast cell lines, there was an increased secretion of several lysosomal enzymes compared to normal controls. Subsequent examination of N-acetyl-beta-D-hexosaminidase secreted by the I-cell lymphoblasts demonstrated a significant increase in adsorption of the I-cell enzyme to Ricinus communis agglutinin, a galactose-specific lectin. However, the I-cell lymphoblasts did not exhibit the significant decrease in intracellular lysosomal activities seen in I-cell fibroblasts. Our results suggest that lymphoblasts not only represent an excellent source for the purification of N-acetylglucosamine phosphotransferase, but in addition, represent a unique system for studying alternate mechanisms involved in the targeting of lysosomal enzymes.     

5′-Nucleotidase

Summary The distribution of 5-nucleotidase in several tissues of rat and mouse has been investigated. The enzyme is greatly specific in dephosphorylating 5-nucleotides. Two 5-nucleotidases seem to exist: one showing greatest activity at pH 5.0, while the other is most active at pH 7.0–7.5. The localization of these two enzymes is not identical. At the acid pH the deoxyribonucleotides are dephosphorylated faster than the ribonucleotides, while at the neutral pH the ribonucleotides are hydrolysed more rapidly. In contrast to the non-specific phosphatases the nucleotidases can be stimulated by magnesium and manganese ions. The acid nucleotidase can be considered as a lysosomal enzyme. The neutral nucleotidase can be involved in transport processes in capillaries and sinusoids. Other localizations of the enzyme suggest a role of the enzyme in the catabolism of nucleic acids.     

Elucidation of aberrant purine metabolism: application to hypoxanthine-guanine phosphoribosylstransferase- and adenosine kinase-deficient mutants, and IMP dehydrogenase- and adenosine deaminase-inhibited human lymphoblasts

We propose that the ratio of [14C]formate-labelled purine nucleosides and bases (both intra and extracellular) to nucleic acid purines provides, in exponentially growing cultures, a sensitive index for comparative studies of purine metabolism. This ratio was 4-fold greater for an HGPRT- mutant than for the parental HGPRT+ human lymphoblast line. The major components of the labelled nucleoside and base fraction were hypoxanthine and inosine. By blocking adenosine deaminase activity with coformycin we found that approx. 90% of inosine was formed directly from IMP rather than the route IMP leads to AMP leads to adenosine leads to inosine. The ratio of labelled base + nucleosides to nucleic acids was essentially unchagned for an AK- lymphoblast line and 2-fold greater than control for an HGPRT(-)-KAK- line, demonstrating that a deficiency of adenosine kinase alone has little effect on the accumulation of purine nucleosides and bases. Although adenosine was a minor component of the nucleoside and base fraction, the adenosine fraction increased from 3 to 13% with the addition of coformycin to the HGPRT(-)-AK- line. In the parental and HGPRT- lines, adenosine was shown to be primarily phosphorylated rather than deaminated at concentrations less than 5 microM. Inhibition of IMP dehydrogenase activity by mycophenolic acid caused a 12- and 3-fold increase in the rate of production of labelled base and nucleoside in the parent and HGPRT- cells respectively. These results suggest that a mutationally induced partial deficiency in the activities converting IMP to guanine nucleotides may result in an increased catabolism of IMP.     

Regulation of purine nucleotide synthesis in human B lymphoblasts with both hypoxanthine-guanine phosphoribosyltransferase deficiency and phosphoribosylpyrophosphate synthetase superactivity.

Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal and phosphoribosylpyrophosphate (PP-Rib-P) synthetase-superactive cell lines and were compared with their respective parental cell lines with regard to growth and PP-Rib-P and purine nucleotide metabolism. During blockade of purine synthesis de novo with 6-methylthioinosine or aminopterin, inhibition of growth of all HGPRT-deficient cell lines was refractory to addition of Ade at concentrations which restored substantial growth to parental cell lines. Ade-resistant inhibition of growth of parental lines by 6-methylthioinosine, however, occurred during Ado deaminase inhibition. Insufficient generation of IMP (and ultimately guanylates) to support growth of lymphoblasts lacking HGPRT activity and blocked in purine synthesis de novo best explained these findings, implying that a major route of interconversion of AMP to IMP involves the reaction sequence: AMP----Ado----Ino----Hyp----IMP. PP-Rib-P generation and purine nucleoside triphosphate pools were unchanged by introduction of HGPRT deficiency into normal lymphoblast lines, in agreement with the view that accelerated purine synthesis de novo in this deficiency results from increased availability of PP-Rib-P for the pathway. Cell lines with dual enzyme defects did not differ from PP-Rib-P synthetase-superactive parental lines in rates of PP-Rib-P and purine synthesis despite 5-6-fold increases in PP-Rib-P concentrations, excretion of nearly 50% of newly synthesized purines, and diminished GTP concentrations. Fixed rates of purine synthesis de novo in PP-Rib-P synthetase-superactive cells appeared to reflect saturation of the rate-limiting amidophosphoribosyltransferase reaction for PP-Rib-P. In combination with accelerated purine excretion, increased channeling of newly formed purines into adenylates, and impaired conversion of AMP to IMP, fixed rates of purine synthesis de novo may condition cell lines with defects in HGPRT and PP-Rib-P synthetase to depletion of GTP with consequent growth retardation.     

Rapid determination of hypoxanthine-guanine-phosphoribosyl transferase in human fibroblasts and amniotic cells

Summary A micromodification of the method of HGPRT and APRT assay is described, which measures the incorporation of ¹⁴C hypoxanthine and ¹⁴C adenine into cultured skin fibroblasts and amniotic cells grown on microtiter plates. Only about 10 000 cells are needed per assay. By this method HGPRT deficient cells can be easily distinguished from normal cells. Investigations with respect to the effect of substrate concentrations and time of incubation have been carried out on some normal fibroblast cell lines, amniotic cell lines and 3 Lesch-Nyhan cell lines.Another modified method is described for quantitative determination of HGPRT activity by means of radio thin-layer chromatography.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Single-strand-specific nuclease of pea seeds: glycoprotein nature and associated nucleotidase activity

A single-strand-specific nuclease from germinating pea seeds has been purified to homogeneity. The purification procedure includes affinity chromatography on concanavalin A-Sepharose and gel filtration. The nuclease exhibits its activity at neutral pH and does not have an absolute requirement for a divalent cation. The purified nuclease also possesses a 3'-nucleotidase activity and is a glycoprotein containing about 20% carbohydrate. On native polyacrylamide gels the nuclease activity comigrates with the nucleotidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits in the native enzyme. The nuclease and nucleotidase activities show differential rates of thermal inactivation, the latter following simple first order kinetics and the former exhibiting a more complex reaction. The nucleotidase was also found to be stimulated by DNA, the increase being greater with native DNA than with denatured DNA. These properties are possibly accounted for by the dimeric structure of the enzyme where the nucleotidase catalytic site resides in one subunit while the nuclease site is formed by interaction of both subunits. The enzyme also hydrolyzes double-stranded alkylated DNA and depurinated DNA at a higher rate than native DNA. Experimental evidence suggests that depurinated DNA is hydrolyzed in the region of apurinic sites.     

Characterization of a bacteriophage-induced, host-specific lytic enzyme from lysates of Bacillus stearothermophilus infected with bacteriophage TP-8

Phage TP-8 lysates of Bacillus stearothermophilus 4S or 4S(8) contain lytic activity exhibiting two pH optima, one at pH 6.5 and the other at pH 7.5. Using a variety of fractionation procedures, the two lytic activities could not be separated. At pH 7.5 the lytic enzyme is an endopeptidase which hydrolyzes the l-alanyl-d-glutamyl linkage in the peptide subunits of the cell wall peptidoglycan and at pH 6.5 it exhibits N-acetylmuramidase activity. Endopeptidase activity is inhibited by NaCl and neither lytic activity was significantly affected by divalent cations or ethylenediaminetetraacetic acid. Crude lysates contain 2.5 to 3.0 times more endopeptidase activity than N-acetylmuramidase activity. The ratio of the two lytic activities (endopeptidase/N-acetylmuramidase) changes to 1.3 to 1.7 during the course of purification, to 1.0 after isoelectric focusing, and 3.9 and 6.00 after exposure for 2 h at 60 and 65 C, respectively. We conclude that the two lytic activities may be associated with a single protein or a lytic enzyme complex composed of two enzymes. Lytic activity at pH 7.5 is more effective in solubilizing cells or cell walls than the lytic activity at pH 6.5. LiCl extracts of 4S and 4S(8) cells contain lytic activity exhibiting endopeptidase activity at pH 7.5 and N-acetylmuramidase activity at pH 6.5. Lytic activity in these LiCl extracts also has a number of other properties in common with those in lysates of phage TP-8. We proposed that the lytic enzyme(s) are not coded for by the phage genome but are part of the host autolytic system.     

Different timing of increases in activities of four X-chromosome linked enzymes during the cell cycle of synchronized human lymphoblasts

A permanent human lymphoblast culture was synchronized with repetitive thymidine blocks, and the changes in the levels of activity of four X-chromosome-linked enzymes were followed during the cell cycle. The four enzymes studied were phosphoglycerate kinase (PGK), α-galactosidase (α-Gal), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and glucose-6-phosphate dehydrogenase (G6PD). The levels of PGK and α-Gal activities increased simultaneously in G1 and in S, while HGPRT and G6PD increased close together in middle and late S. Therefore, different control mechanisms may be involved in the increases of the activities of these two sets of enzymes.