Ellen R. Mably

Secondary loss of deoxyguanosine kinase activity in purine nucleoside phosphorylase deficient mice

The T-cell immunodeficiency associated with purine nucleoside phosphorylase (PNP) deficiency in man is believed to be due to the accumulation of dGTP which may be preferentially formed from deoxyguanosine in T-lymphocytes or their precursor cells. We found no evidence for dGTP accumulation in thymocytes or spleen leucocytes, < 1 nmol/10(9) cells, nor in erythrocytes, < 0.05 nmol/10(9) cells, of the B6-NPE- or B6-NPF PNP-deficient mice strains. There were no changes in purine or pyrimidine ribonucleotide pools. As these mice had been previously shown to excrete PNP nucleoside substrates, we examined the metabolism of deoxyguanosine. Deoxyguanosine kinase activity as compared to control mice was 6 to 52% for the B6-NPE mutant, 2 to 22% for the B6-NPF mutant. Fractionation of erythrocyte and liver lysates from the F mutation and the background strain, C57BL/6J, by anion exchange chromatography confirmed the secondary deficiency of deoxyguanosine kinase and demonstrated that this activity was distinct from adenosine kinase and two major peaks of deoxycytidine kinase activity. Mouse PNP, expressed and purified as a fusion protein, did not show evidence of being bifunctional and having deoxyguanosine kinase activity. Metabolic modelling revealed that the ratio of deoxyguanosine phosphorylation versus phosphorolysis was < 0.06 in control mice, and < or = 0.3 in lymphocytes of PNP-deficient mice. Were deoxyguanosine kinase not reduced in the PNP-deficient mice, all tissues of the B6-NPF mutant would preferentially phosphorylate deoxyguanosine at low substrate concentrations.

Purine nucleoside phosphorylase heterogeneity in the mouse as analyzed by isoelectric focusing and specific activity

1. Isoelectric focusing in polyacrylamide gels identified three erythrocytic electrophoretic patterns of purine nucleoside phosphorylase in a survey of 16 mouse strains. 2. Three strain-specific electrophoretic types were also evident in liver, kidney and spleen leukocytes. 3. There are 3-fold differences in purine nucleoside phosphorylase activities between strains for several tissues; C57BL/6J and Mus spretus having the greatest and the least activity, respectively. 4. Within strains there were up to 8-fold tissue-specific differences in activity with the order from greatest to least being: liver, kidney, spleen leukocyte, erythrocyte, heart.

Glucose transport and oxidation in adipose tissue of patients with myotonic dystrophy

The effect of insulin on the transport of 2-deoxyglucose and the oxidation of glucose in chopped adipose tissue was investigated in 14 myotonic dystrophy (MyD) patients and 28 age and size-matched control subjects. The transport of 0.55 mM 2-deoxyglucose was measured over 3 min at 37 degrees C both with and without 32 ng/ml of insulin. Oxidation was determined at 37 degrees C for 90 min by the measurement of 14CO2 released from a system containing 0.55 mM glucose with and without 50 ng/ml of insulin. Basal 2-deoxyglucose transport was not reduced in MyD subjects but insulin-stimulated 2-deoxyglucose transport in MyD was significantly less at 0.512 +/- 0.220 nmole compared to control subjects with 0.906 +/- 0.160 nmole/100 mg tissue/3 min (P less than 0.02). Both the basal and insulin-stimulated glucose oxidation were significantly less in the MyD group. Insulin-stimulated oxidation was 2.92 +/- 0.21 nmole in the control subjects compared to 2.20 +/- 0.27 nmole/100 mg tissue/90 min in the MyD cases (P less than 0.02). Similar findings were obtained when calculations were based on nmoles of 2-deoxyglucose transport and glucose oxidation/100 mg lipid. The findings indicate that both glucose transport and oxidation are impaired in MyD.

Absence of dGTP accumulation and compensatory loss of deoxyguanosine kinase in purine nucleoside phosphorylase deficient mice.

The first reported cases of purine nucleoside phosphorylase (PNP) deficiency in man were characterized by selective cellular immune dysfunction1. Patients have a pronounced decrease in T-cell numbers with normal or exaggerated B-cell function2–4. They excrete PNP substrates and have elevated plasma levels of these metabolites. Intracellular nucleotide pools of erythrocytes show an increase in deoxyguanosine triphosphate (dGTP), nicotinamide adenine dinucleotide (NAD) and a decrease in guanosine triphosphate (GTP)4–7. The metabolite thought to be responsible for the T-cell dysfunction is dGTP5.

Purine Nucleoside Phosphorylase Deficient Mice Exhibit Both an Age Dependent Attrition of Thymocytes and Impaired Thymocyte Differentiation

Purine nucleoside phosphorylase deficiency is characterized by T-cell immunodeficiency with a majority of patients also showing signs of neurological disorders ranging from spasticity to mental retardation (1,2). The accumulation of dGTP in T lymphocytes is thought to be of importance in inhibiting ribonucleotide reductase and thereby potentially affecting T cell differentiation or function (2). In addition the observations of reduced GTP levels in erythrocytes have suggested the possibility that reduced levels of GTP in the brain might correlate with neurologic deficits (3).

Rescue of a Lethal Purine Nucleoside Phosphorylase Mutation in the Mouse Via a Second Locus Interaction

We have recovered five independent mutations at the purine nucleoside phosphorylase (NP) locus in the mouse among the offspring of mutagenized male micel,2. The biochemical and metabolic features of two of these mutations, the NP-1E and NP-1F alleles, have been described2. One of the five mutations, NP-1G, is lethal on the C57BL/6J background, but viable homozygous PNP deficient mice have been recovered in crosses with a second inbred strain, DBA/2J.

146 MOUSE MODELS OF PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY. CHARACTERIZATION OF PARTIALLY AND SEVERELY ENZYME DEFICIENT MUTANTS

Two mutations of purine nucleoside phosphorylase (PNP) were identified in the carrier state of the first generation progeny of male mice treated with ethylnitrosourea mated to untreated females. The variants are assigned the gene symbols Np-1e and Np-1f. Both carriers have approximately half normal PNP activity in erythrocytes and Np-1a/Np-1a was distinct from the inbred strain background, Np-1a/Np-1a on isoelectric focusing, whereas Np-1a/Np-1a was not distinguishable. In the homozygous state, Np-1a and Np-1a differ and determine a more basic pattern of PNP activity than the Np-1a allele. NP-1A is stable in the presence of phosphate at 55° C whereas the half lives for the mutants were 30 and 7.5 min for NP-1E and NP-1F respectively. The substrate Michaelis constants for the variants were unchanged from controls and the maximal velocities for erythrocytes were: 16.8 ± 1.1, NP-1A; 2.16 ± 0.12, NP-1E; and 0.50 ± 0.03, NP-1F (nmole/min/mg protein). Brain, heart, kidney, liver, spleen leukocytes and thymocytes also showed reductions in activity, 4-27% of normal for NP-1E and 0.1-3.9% for NP-1F. Purine nucleoside excretion correlated with the severity of the enzyme deficiency. The substrates of PNP are not found normally ingurine < 10 uM, but inosine and guanosine were present for Np-1e/Np-1e mice, total 150 ± 84 uM; as were inosine, guanosine, deoxyinosine, and deoxyguanosine for Np-1f/Np-1f mice, total 1490 ± 190 uM. Supported by the Medical Research Council grant MT-6376.

Synthesis and turnover of purine nucleoside phosphorylase in human lymphocytes.

The inherited deficiency of purine nucleoside Phosphorylase results in severe T cell immunodeficiency disease1. We have studied the synthesis of purine nucleoside Phosphorylase during phytohumagglutinin induced T cell transformation and have examined the turnover of purine nucleoside Phosphorylase in the human lymphoblast WI-L2.