Fresh peripheral blood lymphocytes from eight patients with congenital agammaglobulinemia demonstrate reduced ecto-5'-nucleotidase activity when compared to the mean activity of normal subjects and patients with other forms of immunoglobulin deficiency. A specific defect of ecto-5'-nucleotidase is further suggested by normal values for lymphocyte ecto-adenosinetriphosphatase and ecto-nonspecific phosphatase. The data provide evidence for an enzyme deficiency in this X-linked, B lymphocyte deficiency syndrome.
Deoxyadenosine metabolism was investigated in cultured human cells to elucidate the biochemical basis for the sensitivity of T lymphoblasts and the resistance of B lymphoblasts to deoxyadenosine toxicity. T lymphoblasts have a 20-to 45-fold greater capacity to synthesize deoxyadenosine nucleotides than B lymphoblasts at deoxyadenosine concentrations of 50-300 ,uM. During the synthesis of dATP, T lymphoblasts accumulate large quantities of dADP, whereas B lymphoblasts do not accumu ate dADP. Enzymes affecting deoxyadenosine nucleotide synthesis were assayed in these cells. No substantial differences were evident in activities of deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7. Deoxyadenosine and adenosine concentrations are increased in individuals with adenosine deaminase deficiency and severe combined immunodeficiency disease (1, 2). The accumulation of deoxyadenosine leads to the increased concentrations of dATP and dADP in erythrocytes, peripheral blood lymphocytes, and bone marrow cells of these patients (2-4). The elevated levels of deoxyadenosine nucleotides are believed to provide the biochemical basis for the immune dysfunction observed in the enzyme deficiency state.Although the precise mechanism for the immune dysfunction is unclear, several studies in vitro have attempted to determine the underlying molecular pathology. The addition of deoxyadenosine reduces the response of peripheral blood lymphocytes to mitogen stimulation when adenosine deaminase is inhibited (1, 5). The combination of deoxyadenosine and adenosine deaminase inhibition is also cytotoxic to T lymphoblasts but not B lymphoblasts (6, 7). Deoxyadenosine-mediated cytotoxicity in T lymphoblasts is accompanied by increased concentrations of dATP.Deoxyadenosine metabolism was investigated in cultured human cells to elucidate the biochemical basis for the sensitivity of T lymphoblasts and the resistance of B lymphoblasts to deoxyadenosine toxicity. These studies have revealed a major difference in the capacity of T lymphoblasts to accumulate deoxyadenosine nucleotides compared to B lymphoblasts, possibly because of a difference in activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5). MATERIALS AND METHODSReagents. Deoxyadenosine, deoxyinosine, ATP, dATP, dADP, dAMP, dIMP, Tris base, dithiothreitol, and EDTA were purchased from Sigma. Adenine and hypoxanthine were obtained from Calbiochem. Erythro-9-[3-(2-hydroxynonyl)]-adenine (EHNA) was a gift from G. B. Deoxyadenosine Metabolism. T and B lymphoblasts were removed from flasks in which they were grown in continuous culture. Cells were washed twice in normal saline and 100 mM Tris-HCl, pH 7.4. Cells were suspended in Eagle's minimal essential medium containing 10% dialyzed horse serum, 1.2 mM potassium phosphate at pH 7.4, 25 mM Tris-HCl at pH 7.4, and 0 or 5.0 ,uM EHNA. Cell counts were performed on the cell suspensions.Aliquots (50 ,l) of cell suspension (2.0-6.0 X 106 cells per ml) were incubated at 37°C for 20-35 min, removed and pl...
The contribution of plasma membrane 5'-nucleotidase (E.C. 3.1.3.5) to intracellular purine degradation and release was evaluated in cultured human lymphoblasts. B-lymphoblasts and T-lymphoblasts are characterized by high and low levels of plasma membrane 5'-nucleotidase activity, respectively. After radiolabeling of the cellular adenine nucleotide pools with [8-14C]adenine, deoxyglucose-induced purine nucleotide degradation resulted in a 2-2.5 times greater release of cellular radioactivity from the B-lymphoblasts than from the T-lymphoblasts. Specific inhibition of plasma membrane 5'-nucleotidase with 50 microM alpha, beta-methylene adenosine diphosphate (AMPCP) did not decrease purine release during deoxyglucose-induced nucleotide degradation. Similarly, the inhibition of B-lymphoblast membrane 5-nucleotidase did not alter the incorporation of [8-14C]adenine into the nucleotide pool. Therefore, to explain the relatively high release of purine nucleotide degradation products in B-lymphoblasts when compared with T-lymphoblasts, cytoplasmic 5'-nucleotidase activity was investigated in these cell lines. B-lymphoblasts have seven times more cytoplasmic 5'-nucleotidase activity for dAMP and two to three times more activity for other purine nucleoside 5'-monophosphates than do T-lymphoblasts at pH 7.4. Membrane and cytoplasmic nucleotidase activities are produced by different enzymes that can be distinguished by differences in pH optima, Michaelis constants for purine substrates, divalent cation requirements, and susceptibilities to AMPCP inhibition. The data suggest that plasma membrane 5'-nucleotidase hydrolyzes extracellular nucleoside 5'-monophosphates only. Cytoplasmic 5'-nucleotidase most likely regulates the degradation of intracellular nucleoside 5'-monophosphates and may be responsible for the increased purine release observed in B-lymphoblasts.
Three distinct 5'-phosphomonoesterase activities were isolated from soluble fractions of human placenta, cultured human T and B lymphoblasts, and rat liver using 5'-AMP-sepharose 4B affinity chromatography. We define these activities as "low-Km" 5'-nucleotidase, "high-Km" 5'-nucleotidase, and nonspecific phosphatase. High-Km 5'-nucleotidase was eluted with 0.5 M NaCl, low-Km 5'-nucleotidase was eluted with 10 mM ADP, and nonspecific phosphatase was not retained on the column. We have found significant variability in the relative content of high- to low-Km activities in the tissues studied with the ratios ranging from 5.5 to 264. The properties were studied after further purification. The molecular mass of the low-Km enzymes ranged from 72.5 to 209 kDa, optimum pH ranged from 7.4 to 9.0, Km for AMP ranged from 7 to 15 microM, and Km for IMP ranged from 10 to 26 microM. The molecular mass of the high-Km enzymes ranged from 182 to 210 kDa, pH optimum was at 6.5, Km for AMP ranged from 3.0 to 9.4 mM, and the Km for IMP ranged from 0.3 to 0.5 mM. The data indicate that the soluble low- and high-Km 5'-nucleotidase coexist in the mammalian cells and tissues studied. These observations suggest a complex system for the regulation of nucleoside 5'-monophosphate dephosphorylation.
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