Implications of a 5′-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer

5′-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56°C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37°C for 1 h and it was destroyed completely by heating at 100°C for 30 min. When the heated (56°C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-glucosaminidase}$ or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5′-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35 000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggests that the previously reported undetectability of 5′-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5′-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5′-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.     

Enhancement of 5'-nucleotidase activity of human leukemic cells after fractionation: implications for cancer and aging

Previous studies reported that 5'-nucleotidase activity was undetectable or at much lower levels in the homogenate of human chronic lymphocytic leukemic (CCL) cells than in normal lymphocytes. In the present study, 5'-nucleotidase specific activity in acute myelocytic leukemia (AML), which varied in a range from undetectable to 1.4 (nmoles/min.mg protein), was enhanced by cell fractionation, from undetectable in the homogenate, up to 18.8 +/- 1.2, 6.4 +/- 0.7 and 0.68 +/- 0.12 in plasma membranes, microsomes, and cytosol fraction, respectively. In a further fractionation of the cytosol of various leukemic cells with ammonium sulfate, 5'-nucleotidase specific activity increased up to 14-fold in the 60% (NH4)2SO4 fraction, with a recovery of 1266 +/- 115%. These data suggest that 5'-nucleotidase activity in fractionated leukemic cells is higher than reported previously and that the sum of 5'-nucleotidase activity in subcellular compartments is higher than that detected in the homogenate. Furthermore, even when 5'-nucleotidase was undetectable in a homogenate, it became detectable in the plasma membranes, suggesting that its ecto-enzyme function is still active in leukemic cells. The undetectable or low 5'-nucleotidase in the homogenate is indicative of (1) the enzyme itself being in an inactive form but becoming active after the fractionations, or (2) the presence of a factor(s) that prevents the enzyme from being detected but that is separated from the enzyme by the fractionations. In both cases, the rate of nucleotide catabolism by inactive 5'-nucleotidase in rapidly proliferating leukemic cells should be slower than when the enzyme is active. The present finding is consistent with our previous findings that during normal cell aging the high 5'-nucleotidase activity is associated with senescent non-proliferating cells but low or undetectable activity with rapidly proliferating immortal cells. The implications of 5'-nucleotidase for DNA synthesis in aging and cancer are discussed.     

5′-nucleotidase activity in permanent human lymphoid cell lines Implication for cell proliferation and aging in vitro

5′-Nucleotidase of 11 human lymphoid cell lines was measured. These cell ines were homogeneous B, T and Null cells, had an unlimited lifespan in vitro, and were subcultivated from leukemic cells of patients with Burkitt lymphoma, the blastic phase of chronic myelocytic leukemia, or acute lymphoblastic leukemia. 5′-Nucleotidase activities in normal human lymphocytes and in human fibroblasts (VA-13 and IMR-90) could be determined at a cellular protein concentration as low as 0.025 and 0.007 mg/ml, respectively. In all the eleven lymphoid cell lines, including 8 B-cell lines (RPMI 8422, B46M, RPMI 1788, DAUDI, HRIK, B411-4, B85 and DND-3-9A), 2 T-cell lines (MOLT 3 and RPMI 8402) and 1 Null cell line (NALM-1) 5′-nucleotidase was undetectable with the protein concentration range from 0.033 to 8.543 mg/ml. Previously 5′-nucleotidase activity was found to increase 10-, 6- and 20-fold in normal human embryonic lung (WI-38 and IMR-90 cells) and chick embryo fibroblasts, respectively, from a young rapidly proliferative to a senescent non-proliferative stage (Sun, A.S., Aggarwal, B.B. and Packer, L. (1975) Arch. Biochem. Biophys. 170, 1 and Sun, A.S., Alvarez, L.J. Reinach, P.S. and Rubin, E. (1979) Lab. Invest. 41, 1). These data demonstrate that the large increase in 5′-nucleotidase activity occurs concomitantly with the in vitro senescence of these normal cell lines. The present study suggests that this large increase in 5′-nucleotidase activity during cell aging is absent in these permanently lymphoid cell lines. The undetectable 5′-nucleotidase activity may be a biochemical characteristic of these homogeneous B, T and Null cells originating from the aforesaid leukemias. The implications of these results for cell proliferation and aging are discussed.     

The isolation of endosome-derived vesicles from rat hepatocytes.

Intracellular 5'-nucleotidase involved in membrane circulation in rat hepatocytes is latent, and is protected from inhibition when whole cells are incubated with inhibiting antiserum at 2 degrees C [Stanley, Edwards & Luzio (1980) Biochem. J. 186, 59-69]. These two criteria were used to identify intracellular membrane vesicles containing 5'-nucleotidase on Ficoll density gradients. A sharply defined turbid band containing intracellular 5'-nucleotidase isolated on density gradients was further fractionated by immunoadsorption of plasma-membrane fragments derived from the cell surface of surface-inhibited cells on to an anti-(immunoglobulin G) immunoadsorbent. The resulting non-adsorbed membrane fraction consisted of vesicles of uniform size (approx. 65 nm diam.), but was not identifiable as any known organelle. This fraction could account for approx. 5% of the total cell 5'-nucleotidase activity, and the enzyme activity measured was 55% latent. The fraction had a restricted polypeptide composition but similar phospholipid composition compared with plasma membrane. We suggest that the vesicles observed in this fraction were derived from the endocytic pathway.     

Disappearance of terminal deoxynucleotidyltransferase in human malignant T-lymphoblasts treated with a human alpha interferon preparation

Human T-lymphoblastoid cell lines RPMI 8402, MOLT-3, and CCRF-CEM were treated with interferon (IFN) to determine if the treatment would result in the disappearance of cellular terminaldeoxynucleotidyltransferase (TdT), a possible differentiation marker for T-lymphocytes. Incubation of RPMI 8402 cells in the presence of IFN preparation caused a decrease in the number of TdT-positive cells and in TdT activity of the cell extract. The inhibition of cell multiplication was dose dependent. The anticellular effect of IFN preparation was cytostatic, not cytocidal. The IFN preparation modified neither the TdT content nor proliferation of MOLT-3 and CCRF-CEM cell lines. The effects of IFN preparation thus varied with the cell line.     

Subcellular distribution and movement of 5''-nucleotidase in rat cells.

1. Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme, but over 75% inhibition of the cell-surface enzyme. 7. When surface-inhibited hepatocytes were washed and resuspended in buffer at 37 degrees C, 5'-nucleotidase was observed to redistribute from the intracellular pool to the cell surface.     

Recycling of 5''-nucleotidase in a rat hepatoma cell line.

Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine.     

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5''-nucleotidase.

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.     

The autorelease of alkaline phosphatase from the plasma membrane during the incubation of cultured liver cell homogenates

When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 degrees C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 degrees C. The optimum pH value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

The proposed 5'-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5'-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577-584) mentioned that this phenomenon resulted from the presence of a 5'-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5'-[3H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [3H]Adenosine derived from 5'-[3H]AMP hydrolysis was further transformed into [3H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [3H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5'-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5'-nucleotidase inhibitor. We did not observe 5'-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.     

Comparison of 5'-nucleotidase activities among cultured murine melanoma cell lines

The activity of 5'-nucleotidase and ouabain-sensitive Na/K ATPase was determined in seven different mouse melanoma cell lines. Ouabain-sensitive Na/K ATPase activity was found in NP40-treated cell homogenates of all cell lines. However, 5'-nucleotidase activity was found in only one mouse melanoma cell line--JB/RH. The absence of expression of 5'-nucleotidase activity in the other six cell lines is not associated with pigmentation in melanoma cells, nor is the gene switched off in all transformed melanocytes of C57BL/6 origin.     

Production of cytotoxic factor(s) in human T cell lines transformed by a human retrovirus

Human T cell lines, MT-2, TCL-Ter, TCL-Haz, and TCL-Kan which were transformed by a human retrovirus, constitutively produced cytotoxic factor(s) (CF) in the culture supernatants. In these cell lines, MT-2 produced the largest amount of CF. The amount of CF produced by MT-2 was 9-10 or 3-4 times larger than that produced by a human B cell line, RPMI 1788, or normal peripheral blood leukocytes stimulated with mitogens and phorbol ester. The kinetics of the production by MT-2 was similar in media with and without serum. The activity was stable at 56 degrees C for 30 min but was lost at 80 degrees C for 30 min and at pH 2 for 20 hr. On gel filtration, the molecular weight of the factor produced by MT-2 was approximately 90,000. On isoelectric focusing, the activity was recovered in the fraction at pH 6.5-7.0.     

Interferon production in human T lymphoblastoid cells

Human T lymphoblastoid cell (RPMI 8402 cell) produced interferon (IFN) through the induction by Sendai virus. The priming effect on the interferon production in the RPMI 8402 cell could be found by the pretreatment of human leukocyte IFN (Hu IFN-alpha), but not by that of the IFN produced in the RPMI 8402 cell (T-IFN). The superinduction by the irradiation of ultraviolet rays or the treatment of antimetabolites (actinomycin D and cycloheximide) or 5-bromodeoxyuridine was not found. The T-IFN was completely neutralized by the anti-Hu IFN-beta serum, but not by the anti-Hu IFN-alpha serum at all. In conclusion, it was confirmed that the IFN produced in the RPMI 8402 cell through the induction by Sendai virus was antigenically identical to Hu IFN-beta.     

Membrane-bound 5'-nucleotidase inhibitor in Ehrlich ascites tumour cells and newborn mouse liver

5'-Nucleotidase activity in Ehrlich ascites tumour cells was undetectable. The cell homogenate, when mixed with adult mouse liver homogenate, inhibited the 5'-nucleotidase activity of the latter, without affecting its p-nitrophenyl phosphate-hydrolysing activity. The inhibitor activity was enriched (6.8-fold) in a membrane fraction which was enriched in (Na+ + K+)-ATPase (14-fold) and alkaline phosphatase (8-fold). 5'-Nucleotidase activity in this membrane fraction could be detected only after separating the inhibitor activity from the enzyme on Sephadex G-50. The inhibitor activity was decreased by 27% when heat-treated, 33% when treated with 6 M urea and was almost completely lost when treated with trypsin. It was dialysable from a tubing with a molecular exclusion limit of 10,000, but was retained in a tubing with an exclusion limit of 3000. From these results we conclude that a small molecular weight protein inhibitor(s) of 5'-nucleotidase is present in the plasma membrane of Ehrlich ascites tumour cells. Also, the presence of such an inhibitor in the newborn mouse liver but not in the adult liver suggests that it may have some role in cellular ageing and cancer.     

Cytolytic activity of Naegleria fowleri cell-free extract

The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30-60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by 51Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50 degrees C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing 51Cr release from target cells by the soluble fraction of N. fowleri extracts.     

Localization of the human angiogenin gene to chromosome band 14q11, proximal to the T cell receptor alpha/delta locus.

The gene encoding angiogenin, a potent inducer of blood vessel formation, has been localized within the human genome. It is present as a single copy per haploid genome and is located on chromosome 14, on the basis of discordancy analysis of human-rodent hybrid cell lines. This localization was refined to 14q11 by in situ hybridization of an angiogenin probe to metaphase chromosomes prepared from both normal human lymphocytes and RPMI 8402 cells. The results from the RPMI 8402 cells also establish that the angiogenin gene resides proximal to a translocation breakpoint within the T cell receptor alpha/delta locus and therefore upstream from that locus. An AvaII RFLP, present at a frequency of 29% in an unselected collection of human placental DNAs, was identified in the coding region of the gene and results from a single silent transversion.     

Activity of cell-detached 5'-nucleotidase in thymocytes of various densities

The rat thymocytes submitted to heating at 45 degrees C for 1 hr liberate plasma membrane fragments containing 5'-nucleotidase activity in the supernatant. The thymocytes were separated by ficoll density gradient centrifugation. High activity of 5'-nucleotidase per 10(6) cells was found in the supernatant of low density (1.069) subset of thymocytes. Thymocyte supernatant of rats treated with hydrocortisone demonstrated higher 5'-nucleotidase activity per 10(6) cells than in intact animals. This is due to an increase of the low density population of thymocytes in treated rats since the 5'-nucleotidase activity per 10(6) cells of the supernatant obtained from this density fraction is the same both in treated with hydrocortisone and intact rats. Hydrocortisone seems to induce a selection of the thymocytes with high 5'-nucleotidase activity.     

Acquisition of deoxyguanosine resistance by TPA-induced T lymphoid lines

The human leukemic T cell line 8402, which contains terminal deoxynucleotidyl transferase (TdT) and phenotypically resembles precursor thymocytes, when exposed to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) undergoes in vitro T maturation. TdT disappears from virtually all the cells and a fraction of TdT- -cells express specific T surface markers, such as the T3 determinant. Like T lymphocytes from the thymus, 8402 cells are extremely sensitive to the cytotoxic effect of deoxyguanosine (dGuo). As a consequence of TPA treatment, resistance to dGuo is observed in 8402, as well as in two other TdT+ lymphoid T cell lines, Molt-4 and CEM-10. These results suggest the occurrence of changes in deoxynucleoside metabolism in TPA-treated cells related to the in vitro maturation process. Maturation of 8402 cells, once started, progresses in the presence of additional physiologic stimuli provided by conditioned medium from lymphocyte culture, because a portion of cells display the T8+/T4- phenotype characteristic of cytotoxic/suppressor T cells. This in vitro lymphoid system may thus be used to study the relationships of molecular differentiation between precursor thymocytes and cytotoxic/suppressor T cells.     

Variations in 5'-nucleotidase activity in established mesenchymal cell lines from syngeneic A/J mice

5'-Nucleotidase activity was analyzed in four different mesenchymal cell lines (F, m, e and SP) established from syngeneic A/J mice. The 5'-nucleotidase activity of fibroblasts was lower in transformed cells (F and m) than in nontransformed cells (e). An increase in cell contact during confluence or during high cell density increased 5'-nucleotidase activity, and a decrease in cell contact caused a decrease in 5'-nucleotidase activity in both fibroblastic (F, m and e) and reticulum (SP) cell lines. These results are evidence that 5'-nucleotidase activity in mesenchymal cells is influenced by intercellular contact as well as transformation.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.