Trevor Lukey

Kinetic considerations for the regulation of adenosine and deoxyadenosine metabolism in mouse and human tissues based on a thymocyte model

Metabolic regulation at a branch point may be determined primarily by relative enzyme activities and affinity for common substrate. Adenosine and deoxyadenosine are both phosphorylated and deaminated and their metabolism was studied in intact mouse thymocytes. From kinetic considerations of two activities competing for a common substrate, the deamination:phosphorylation ratio, vd/vk, at high nucleoside concentration, [S] congruent to infinity, is equal to Vd/Vk, or 34 and 1090 for adenosine and deoxyadenosine, respectively. At low substrate concentrations, [S] congruent to o, vd/vk is equal to VdKkm/VkKdm, or 0.7 and 285 for adenosine and deoxyadenosine, respectively. The analysis was extended to other mouse and human tissues by measurement of adenosine kinase, deoxyadenosine kinase and adenosine deaminase activities. All tissues were found to preferentially deaminate deoxyadenosine. Three tissue types were apparent with respect to adenosine metabolism: those which preferentially phosphorylate adenosine at all concentrations, those which switch from phosphorylation to deamination between low and high adenosine concentration and those for which deamination is quantatively important at all concentrations. Lymphoid tissues are representative of the latter category. The kinetic approach we describe offers a means of predicting nucleoside metabolism over a range of concentration which may be technically difficult to otherwise measure. The phosphorylation of adenosine and deoxyadenosine was also studied in intact thymocytes in the presence of adenosine deaminase inhibitors. The rate of deoxyadenosine phosphorylation was unaffected by coformycin or EHNA, whereas adenosine phosphorylation decreased with increasing substrate concentrations to 18% the rate in the absence of adenosine deaminase inhibitors.

Genetic variability of purine nucleoside phosphorylase activity in the mouse: Relationship to Np-1 and Np-2

A survey of 37 inbred strains for erythrocyte purine nucleoside phosphorylase activity showed a greater than threefold range. Six of these strains had significantly greater activity than the others, and all of the high-activity strains had the Np-2 electrophoretic band. The high-purine nucleoside phosphorylase activity trait corresponding to Np-2 was inherited in an autosomal codominant manner and minor differences were apparent in thermal and kinetic properties between low- and high-activity strains. This work provides further support for there being either two structural loci for purine nucleoside phosphorylase, Np-1 and Np-2, or a regulatory-modifier locus.

S-adenosylhomocysteine hydrolase activity, deoxyadenosine triphosphate accumulation, and competence of thymocyte and spleen leucocyte response to mitogens in coformycin-treated mice

The inhibition of S-adenosylhomocysteine hydrolase and accumulation of dATP in thymus, spleen and other tissues of mice treated with the adenosine deaminase inhibitor coformycin were studied in parallel with the competence of thymocytes and spleen leucocytes to undergo mitogen-induced transformation. Newborn mice were lethally sensitive to daily injections of coformycin, 0.2 mg/kg, whereas adult mice were not. Developmental profiles of enzymes of nucleoside metabolism showed adenosine deaminase and purine nucleoside phosphorylase to be greatest in thymus around day 20 and to decrease for animals older than 60 days. The most notable change was a 3-fold increase in spleen leucocyte adenosine deaminase activity between days 10 and 30. Adenosine deaminase activity was reduced to less than 10% of normal in tissues of newborns treated with coformycin for 12-14 days. S-Adenosylhomocysteine hydrolase was also reduced to 5-40% of normal with no evidence of tissue specificity. Both thymocytes and erythrocytes of coformycin-treated mice accumulated dATP whereas spleen leucocytes did not. For coformycin-treated mice, spleen leucocyte and thymocyte response to concanavalin A (Con A) was reduced to 20 and 60% of controls respectively. Coformycin, 3.6 microM, also potentiated the in vitro toxicity of adenosine and deoxyadenosine toward thymocytes or spleen leucocytes by approximately an order of magnitude. Our observations are consistent with dATP being involved in impairment of thymocyte responsiveness; however, it appears unlikely that either dATP elevation or S-adenosylhomocysteine hydrolase inhibition is involved in the mechanism of impairment of spleen leucocyte response by coformycin.

Purine nucleoside phosphorylase (Np) in the mouse: Linkage relationship of Np-2 to esterase-10 (Es-10) and Np-1 on chromosome 14

An improved method for detecting four Np-1 (purine nucleoside phosphorylase) alleles in mouse erythrocytes by cellulose acetate electrophoresis is described. The previous linkage of Np-1 and Es-10 (esterase-10) was confirmed, with a map distance of 13.0±2.6 cM. Np-2 was detected by either specific activity assay or starch gel electrophoresis and shown to be linked to Es-10, 15.9 ± 3.1 cM, on chromosome 14. No recombinants between Np-1 and Np-2 were observed in 52 offspring, indicating either that these loci are either closely associated or that Np-2 represents simply a property of existing allelic products of the Np-1 locus.

Implementation of a debugging aid for logic errors in Pascal programs

This paper describes a debugging utility with some tutorial capabilities. It is designed to assist novice programmers in finding logic errors in syntactically correct programs. Flow and use analysis techniques are employed to aid in the recognition of some instances of incorrect code sequence, improper variable use and improper nesting of constructs. The utility is menu driven with built in facilities for displaying user source programs and debugging information in a multiple window format.

Adenosine and deoxyadenosine metabolism in horse erythrocytes and lymphocytes

Abstract 1. 1. Horse erythrocytes are essentially devoid of adenosine deaminase activity, but phosphorylate both adenosine and deoxyadenosine. 2. 2. In horse lymphocytes, deamination of adenosine and deoxyadenosine is equal or greater than the rate of phosphorylation. 3. 3. Horse lymphocyte adenosine deaminase activity is 5-fold more labile than the human activity at 60°C. 4. 4. On sucrose density gradients, there are distinct adenosine and deoxyguanosine kinase activities and a co-sedimenting deoxyadenosine-deoxycytidine kinase activity.

Purine Ribonucleoside and Deoxyribonucleoside Metabolism in Thymocytes

The human deficiencies of adenosine deaminase and purine nucleoside phosphorylase activities are associated with an accumulation of the purine deoxyribonucleoside triphosphates, dATP (1–4) and dGTP (4) respectively. In the absence of adenosine deaminase or purine nucleoside phosphorylase activities, deoxyadenosine or deoxyguanosine are presumed to be rephosphorylated, thereby producing increased dATP or dGTP concentrations which may in turn inhibit ribonucleotide reductase. We have therefore examined the specificity and optimal assay conditions for the principal purine ribonucleoside and deoxyribonucleoside kinase activities in mouse thymocytes. The role of these activities was evaluated by analyzing the competition for common substrates between the nucleoside kinases and adenosine deaminase or purine nucleoside phosphorylase.

Physiological amino acid data management: Quantitation, assessment, reporting and storage

A series of routines, written in BASIC, have been developed to aid in the analysis, reporting and storage of physiological amino acid data used in the diagnosis and management of inherited metabolic disorders. The concentrations of 44 compounds are determined for three types of physiological samples: plasma, urine or cerebral spinal fluid. The programs facilitate the editing of numerical data, the creation of a patient and sample information file to be merged with the results, the flagging of abnormal results, the addition of diagnostic or interpretive comments and the generation of hard copy reports. Files containing the foregoing information provide records which may be manipulated using data base programs for further analysis.

Genetic Variability of Purine Nucleoside Phosphorylase in the Mouse

The inherited human deficiency of purine nucleoside phosphorylase is associated primarily with cellular immune dysfunction1. We have begun to screen for quantitative activity variants of purine nucleoside phosphorylase in Mus musculus in attempts to establish an animal model for the enzyme deficiencies associated with immunodeficiency disease. A preliminary survey of feral and inbred mouse strains revealed a 4-fold range in erythrocyte purine nucleoside phosphorylase activity. We have examined some of the biochemical and genetic aspects of this variability.

Biochemical and Immunological Evaluation of Long Term Coformycin Administration in the Mouse

The inherited human deficiency of adenosine deaminase is associated with severe combined immunodeficiency disease1,2. Coformycin is a potent inhibitor of adenosine deaminase and markedly potentiates the in vitro toxicity of adenosine and deoxyadenosine toward lymphoid cells. We wished to evaluate some of the biochemical and immunological events associated with coformycin administration in vivo. Increased dATP concentrations observed in adenosine deaminase deficiency3 are thought to inhibit ribonucleotide reductase and thereby restrict DNA synthesis. Another secondary consequence of a deficiency of adenosine deaminase may be inhibition of S-adenosylhomocysteine hydrolase by de oxyadenosine4.