An evaluation of purine nucleotide metabolism in human lymphocytes

Abstract

Purine nucleotide metabolism consists in three different sequences: 1) the purine de novo synthesis; 2) the "salvage pathway"; 3) the catabolic pathway. We have evaluated the behavior of purine nucleotide metabolism in human lymphocytes, in normal and leukemic cells, following the behavior of a) PRPP synthetase (PRPP syn) and PRPP amidotransferase (the main enzymes of the de novo synthesis), b) APRT (adenine phosphoribosyl transferase) and the HGPRT (hypoxanthine-guanine phosphoribosyl transferase), the enzymes of the salvage pathway, c) adenylic deaminase (AMP-D) , adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and 5'-nucleotidase (5'-N), which represent the catabolic pathway.As side by side examination of these three pathways under different conditions is very important, we evaluated the ratio between these enzymes in normal subjects and patients affected by B-chronic lymphocytic leukemia (B-CLL).Lymphocytes were isolated from peripheral blood using the Lymphocyte Separation Medium (L.S.M.) from Flow Laboratories (1) and were finally suspended in 0.29 M sucrose (containing 10 mM Tris, pH 7). Only for PRPP synthetase, an aliquot of the sediment was suspended in 25 mM K-phosphate, containing EDTA (pH 7.4). Final concentration of the cells was 140,000/ pl. Lymphocyte extracts for all enzymatic assays were prepared by sonication, spun down at 300 x g, treated with Norit A to eliminate the endogenous nucleotides.The determination of all enzymes was carried out radiochemically, using labelled substrates, separating either the substrates, or the labelled products, through HF'LC using a Beckman mod.332 instrument or DEAE-cellulose disk (for PRPP amidotransferase) .The assay mixtures were deproteinized with (final) 0.21 N HC104, and neutralized with 0.22 N KOH: supernatants were used for analysis. The indications of Welch and Rudolph (2). of Scholar and Calabresi (3), were followed to determinate the enzymes involved in nucleotide metabolism in lymphocyte extracts, those of Green and Smith (4), of Rylance et al. (5,6), to separate the nucleotides. We refer you to our previously published observations Protein was evaluated according to Lowry et al. (8) and was found to be less in the lymphocytes of B-CLL patients. Enzyme activity was expressed as I.U. and referred to the mg protein.Among the enzyme values of the salvage pathway, only APRT increased: PRPP synthetase and PRPP amidotransferase were elevated; among the catabolic enzymes, ADA specifically decreased.These data indicate a tendency of the leukemic cell to enhance purine nucleotide synthesis, both through de novo synthesis and the salvage pathway, which is selectively achieved not only through an increase in specific anabolic enzymes but also through a decrease in the catabolic enzymes. Activity values are not reported, but in Table 1 we show the ratios between the different activities and between the different pathways. This is clearly demonstrated by the ratio between the sums of activities, and specifically by the PRPP synthetase + PRPP amido...