Regulation of purine metabolism by plasma membrane and cytoplasmic 5'-nucleotidases

Abstract

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.

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes

“…Other mechanisms that limit the accumulation of dGTP may include reduced dGuo transport or high ecto-5Ј-NT activity. In fact, B lymphoblasts have been reported to contain higher levels of ecto-5Ј-NT than T lymphoblasts (23). In one case, a patient was found to have variable immunodeficiency related to partial deficiencies of both PNP and ecto-5Ј-NT activity (24), implicating a role of ecto-5Ј-NT in modulating the impact of PNP deficiency.…”

Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B- and T-lymphocyte cell lines, and monocyte-macrophages.

“…Low activities of plasma membrane ecto-5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) (16,17), of a cytosol nucleotidase (18,19), and of an ecto-ATPase (20) in T lymphoblasts have been invoked to explain their propensity to accumulate deoxyribonucleotides. However, neither impaired degradation nor enhanced synthesis from deoxyadenosine has been convincingly implicated in the disparate ability of various lymphocyte cell lines to accumulate adenine deoxyribonucleotides (18,(21)(22)(23)(24).…”

Azathioprine-related bone marrow toxicity and low activities of purine enzymes in patients with rheumatoid arthritis

“…Two major forms of 5NT are recognized: ecto-5NT and cytosolic 5NT (13). Ecto-5NT is located on outer membranes, which are not permeable for thionucleotides, and most likely plays no role in the intracellular thiopurine metabolism and its subsequent cytotoxicity (14,15). This is supported by the observation that in vitro drug resistance to 6-thioguanine in childhood lymphoblastic leukemia is not related to ecto-5NT activity and is associated with high cytosolic 5NT activity (16).…”