Properties of lymphocyte 5′nucleotidase in normal subjects and patients with chronic lymphocytic leukemia

Heterogeneity for 5′nucleotidase has been demonstrated in chronic lymphocytic leukemia [12]. The enzyme is not detectable in the lymphocytes from the majority of patients with this disorder, but normal and even supranormal levels are found in some cases [17]. In the present studies, the properties of this ecto-nucleotidase were investigated in unhomogenized lymphocytes isolated from the blood of normal subjects and patients with chronic lymphocytic leukemia. The activity was found to have a pH optimum of 8.0–8.5 and a preference of 5′ribo- over 5′deoxyribonucleotides. The enzyme was inactive towards 2′,3′AMP. The Km for AMP showed a broad range from 19 to 210 μM in unhomogenized lymphocytes. An unexpected relationship was observed between sp. act. and this Km in that higher Km values were noted with cells from subjects with high lymphocyte 5′nucleotidase sp. act. and low Km values in lymphocyte preparations with low sp. act. When plasma membranes were prepared, a narrow range of low Km values unrelated to sp. act. was noted. The change in Km was not due to Pi concentration or nucleotide effects. The ecto-enzyme was inhibited by α,β-methylene adenosine diphosphate, while cytosol phosphatase activity was not inhibited by this compound. Heat stability studies revealed a 45% loss in 5′nucleotidase activity after incubation for 20 min at 65°C. A comparison of the substrate preference. Km values, effect of inhibitor and subcellular localization revealed no differences between the enzyme from patients with chronic lymphocytic leukemia and from normal subjects. This finding is consistent with the suggestion that the undetectable or supranormal levels observed in the lymphocytes of patients with this disorder stems from variations in the percentage of cells having 5′nucleotidase rather than changes in the enzyme proper.