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Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.

Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively.

Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.

Mammalian mitochondrial DNA (mtDNA) is a highly polymorphic, high-copy-number genome that is maternally inherited. New mutations in mtDNA segregate rapidly in the female germline due to a genetic bottleneck in early oogenesis and as a result most individuals are homoplasmic for a single species of mtDNA. Sequence variants thus accumulate along maternal lineages without genetic recombination. Most of the extant variation in mtDNA in mammalian populations has been assumed to be neutral with respect to selection; however, comparisons of the ratio of replacement to silent nucleotide substitutions between species suggest that the evolution of mammalian mtDNA is not strictly neutral. To test directly whether polymorphic mtDNAs behave as neutral variants, we examined the segregation of two different mtDNA genotypes in the tissues of heteroplasmic mice. We find evidence for random genetic drift in some tissues, but in others strong, tissue-specific and age-related, directional selection for different mtDNA genotypes in the same animal. These surprising data suggest that the coding sequence of mtDNA may represent a compromise between the competing demands of different tissues and point to the existence of unknown, tissue-specific nuclear genes important in the interaction between the nuclear and mitochondrial genomes.

Point mutations at the purine nucleoside phosphorylase locus impair thymocyte differentiation in the mouse.

Three point mutations on the Np(b) allele of the purine nucleoside phosphorylase locus in the mouse have been recovered by male germ cell mutagenesis. The mutants were backcrossed, 12-14 generations, and are designated in increasing order of severity of enzyme deficiency and phenotype: B6-NPE, Met-87 --> Lys; B6-NPF, Ala-228 --> Thr; and B6-NPG, Trp-16 --> Arg. A marked decline in total cell numbers per thymus occurs between 2 and 3 months for the more severe B6-NPF and B6-NPG mutants (35% and 52%, respectively) and by 8 months for the less severe B6-NPE mutation. The thymocyte population is thereafter characterized by a 3- or 8-fold expanded precursor, CD4-CD8- double-negative population and 15% or 55% reduced CD4+CD8+ double-positive cells for the B6-NPF and B6-NPG strains, respectively. Spleen lymphocyte Thy-1+ cells are reduced by 50% and spleen lymphocyte response to T cell mitogen and interleukin 2 is reduced by 80%. Increases of thymocyte dGTP pools of 5- and 2.5-fold for B6-NPF and B6-NPG mutants, respectively, are observed. The purine nucleoside phosphorylase-deficient mouse exhibits age-dependent progressive perturbations in thymocyte differentiation, reduced numbers of thymocytes, and reduced splenic T cell numbers and response. The progressive T cell deficit is similar to the human disorder.

Random genetic drift in the female germline explains the rapid segregation of mammalian mitochondrial DNA.

Mitochondrial DNA (mtDNA) is maternally inherited in mammals. Despite the high genome copy number in mature oocytes (10(5)) and the relatively small number of cell divisions in the female germline, mtDNA sequence variants segregate rapidly between generations. To investigate the molecular basis for this apparent paradox we created lines of heteroplasmic mice carrying two mtDNA genotypes. We show that the pattern of segregation can be explained by random genetic drift occurring in early oogenesis, and that the effective number of segregating units for mtDNA is approximately 200 in mice. These results provide the basis for estimating recurrence risks for mitochondrial disease due to pathogenic mtDNA mutations and for predicting the rate of fixation of neutral mtDNA mutations in maternal lineages.

Modelling of purine nucleoside metabolism during mouse embryonic development: relative routes of adenosine, deoxyadenosine, and deoxyguanosine metabolism.

The individual activities for adenosine kinase, deoxyadenosine kinase, adenosine deaminase, deoxyguanosine kinase, and purine nucleoside phosphorylase were determined during days 7 to 13 of mouse embryonic development. Adenosine deaminase increased 74-fold between days 7 and 9; deoxyadenosine kinase increased 5.4-fold during the same interval. Adenosine kinase, deoxyguanosine kinase, and purine nucleoside phosphorylase exhibited less than 2-fold changes in activity between days 7 and 13. Using Michaelis constants for each enzyme and the maximal velocities determined from enzyme assay, the relative routes of adenosine and deoxyadenosine metabolism via phosphorylation or deamination were modeled as a function of nucleoside concentration for days 7 through 13. For days 7 and 8, phosphorylation of adenosine is the principle route of metabolism at physiological concentrations. A switch occurred at day 9 and following where deamination is at least 5-fold greater than phosphorylation at all substrate concentrations. Deoxyadenosine phosphorylation was at most 10% of deamination at day 7 and then declined to less than 1% for days 9 to 13. Phosphorolysis was the principle route of deoxyguanosine metabolism through the 7 to 13 day period. Thus catabolism rather than phosphorylation was the principle pathway for purine deoxynucleoside metabolism during this period.

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.

Assignment of a gene that determines erythrocytic guanosine-5'-triphosphate concentration (Gtpc) to mouse chromosome 9.

Nine inbred mouse strains surveyed for erythrocytic guanosine-5'-triphosphate (GTP) concentration were found to segregate into two discrete groups. Strains having low GTP levels between 1.4 and 3.4 nmol/10(9) cells were C3H/HeJ, C3H/HeHa, A/J, and WB/ReJ. Strains having high GTP levels between 11.0 and 14.8 nmol/10(9) cells were AKR/J, DBA/2J, CBA/J, C57BL/6J, and C57L/J. Erythrocytic ATP levels did not vary significantly among these groups. Crosses between low and high GTP strains gave F1 progeny having intermediate levels of GTP, and the progeny of F1's backcrossed to parental strains segregated in a 1:1 ratio for GTP concentration. We designated the GTP concentration determining trait, Gtpc. Typing the C57BL/6J x C3H/HeJ (B x H) recombinant inbred strains for GTP levels revealed 0/12 strain distribution pattern differences for loci on both chromosomes 5 and 9. Backcross analysis did not provide evidence for linkage of Gtpc to W (dominant white spotting) on chromosome 5 with 15/45 recombinants. A test for linkage of Gtpc to transferrin (Trf) on chromosome 9 gave evidence of linkage with an observed recombination frequency of 14.6 +/- 5.5 and a 99% confidence interval of 3.9-33.9 cM.

cDNA sequence of four purine nucleoside phosphorylase (Np) alleles in the mouse.

The molecular basis of four electrophoretic and activity variants of purine nucleoside phosphorylase in the mouse was examined by amplification and sequence analysis of cDNA. Compared with the cDNA coding sequence for C3H/HeHa designated Npa, there were five nucleotide changes for C57BL/6J, Npb; three for MOLF/Ei, Npc; and five for SPRET-1, Npd. There was only a single codon change between Npa and Npb, the deduced substitution of threonine 176 by serine. Similarly, there was only a single codon change between Npa and Npc, resulting in substitution of methionine 258 by lysine. There were three codon changes between Npa and Npd, resulting in substitution of glutamate 22 by lysine, threonine 39 by alanine, and aspartate 152 by glutamate. These amino acid substitutions-neutral to neutral, neutral to basic, and acidic to basic--are in agreement with the electrophoretic properties of the gene products of Npa relative to Npb, Npc, and Npd previously described by isoelectric focusing. Codon differences were confirmed by PCR-RFLP or single nucleotide primer extension analysis and extended to include the assignment of other strains as Npa: C3H/HeHa, DBA/2J, CLA, Posch-2; or Npb: C57BL/6J, C57L/J, C58/J. Both RFLP analysis of amplified genomic DNA and Southern analysis are consistent with single but unique Np alleles present in the C3H/HeHa and C57BL/6J genomes. As these data do not support the previous two-loci, Np-1 and Np-2, classification, we propose and employ a new single locus multiple allele classification for Np on the basis of the sequence analysis.