5'-Nucleotidase in rat liver lysosomes

5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (Mr = 72,000). 5'-Nucleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.     

Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum.

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.     

Purification of 5'-nucleotidase from human placenta after release from plasma membranes by phosphatidylinositol-specific phospholipase C

5'-Nucleotidase was purified greater than 1000-fold from human placenta by treatment of plasma membranes with S. aureus phosphatidylinositol-specific phospholipase C and affinity chromatography on Con A Sepharose and AMP-Sepharose. The resulting enzyme had a specific activity of greater than 5000 mumol/hr/mg protein and a subunit molecular weight of 73,000. Goat antibodies against 5'-nucleotidase inhibited enzyme activity and detected 5'-nucleotidase after Western blotting. These antibodies also recognized a soluble form of 5'-nucleotidase and residual membrane-bound 5'-nucleotidase which could not be released by phosphatidylinositol-specific phospholipase C treatment, suggesting that the three forms of the enzyme are structurally related. The soluble 5'-nucleotidase may be derived from the membrane-bound form by the action of an endogenous phospholipase C. The structural basis for the inability of some of the membrane-bound 5'-nucleotidase to be released by phosphatidylinositol-specific phospholipase C is unknown.     

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.     

Light and electron microscopical immunocytochemistry of 5'-nucleotidase in rat cerebellum

5'-Nucleotidase in nervous tissue has so far not been localised at the ultrastructural level using immunocytochemical techniques. We have now applied monoclonal antibodies and a polyclonal antiserum raised against this ecto-enzyme and describe the distribution of 5'-nucleotidase antigenicity in rat cerebellum both at the light and electron microscopic levels. Within all cerebellar layers, 5'-nucleotidase immunoreactivity was found on plasma membranes of glial elements, i.e. Bergmann glial cell processes crossing the molecular layer, astrocytic end-feet around blood vessels and glial cell extensions surrounding single Purkinje cells. In the granular layer, 5'-nucleotidase immunoreactivity was present on glial membranes interposed between granule cells. Neuronal cells or processes were devoid of immunoreactivity. The immunocytochemical results were compared with conventional 5'-nucleotidase histochemistry. Both techniques showed the same ecto-localisation of the enzyme and favour the view of 5'-nucleotidase being predominantly situated at glial plasma membranes.     

Inhibition of lymphocyte 5'-nucleotidase by lectins: effects of lectin specificity and cross-linking ability

5'-Nucleotidase, an integral glycoprotein enzyme of the lymphocyte plasma membrane, is inhibited cooperatively by the lectin concanavalin A. Because divalent succinyl-concanavalin A is a poor enzyme inhibitor, both binding and lectin-induced cross-linking of 5'-nucleotidase may be necessary for inhibition. Succinyl-concanavalin A does not compete with concanavalin A for binding to the enzyme; however, maleyl-concanavalin A, another poor inhibitor, competes effectively with the parent lectin. Thus, maleyl-concanavalin A binds to the same site as concanavalin A but causes little inhibition, whereas succinyl-concanavalin A does not bind to this site. The monovalent lectin from Ricinus communis (RCA-60) is a more effective enzyme inhibitor than the related divalent lectin (RCA-120), and inactivation of the second low-affinity sugar binding site on RCA-60 does not abolish inhibition, suggesting that multivalent cross-linking is not required for 5'-nucleotidase inhibition. Peanut and wheat germ agglutinins do not inhibit the enzyme, whereas lectins from lentil, pea, soybean, Griffonia simplicifolia, and Phaseolus vulgaris inhibit 5'-nucleotidase with various degrees of effectiveness. The only lectin showing strong positive cooperativity in its interaction with 5'-nucleotidase is concanavalin A.     

Solubilization, characterization, and detergent interactions of lymphocyte 5'-nucleotidase

5'-Nucleotidase is a member of a recently identified class of membrane proteins that is anchored via a phosphatidylinositol-containing glycolipid. The enzyme was readily solubilized with full retention of catalytic activity by nonionic and anionic detergents such as alkylthioglucosides, deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), while the cationic detergent dodecyltrimethylammonium bromide (DTAB) caused loss of activity. 5'-Nucleotidase was released only at high detergent concentrations, suggesting that it is tightly associated with the membrane. DTAB and deoxycholate caused a loss of heat stability, while alkylthioglucosides had no effect. CHAPS produced a remarkable increase in the heat stability of the partially purified (glycoprotein fraction) and purified enzyme. Arrhenius plots of solubilized 5'-nucleotidase showed "break points" for all detergents in the temperature range 30-37 degrees C. SDS-PAGE of pure 5'-nucleotidase showed a single subunit of molecular mass 70 kilodaltons (kDa), while sucrose density gradient sedimentation gave a peak of activity corresponding to 132 kDa, indicating that the enzyme exists as a dimer. Gel filtration of the solubilized enzyme in several detergents showed apparent molecular masses between 200-630 kDa, suggesting that lymphocyte 5'-nucleotidase may be present in high molecular mass aggregates in its native state.     

An improved procedure for purifying 5'-nucleotidase from various sources. Evidence for tissue and species differences in their molecular mass and affinity for F-actin

5'-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5'-nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E.M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J.P. (1984) Biochem. J. 221, 369-377). Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5'-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5'-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5'-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.     

Reconstitution of purified chicken gizzard 5'-nucleotidase in phospholipid vesicles. Evidence for its transmembraneous character and the existence of functional domains on both sides of the phospholipid bilayer

5'-Nucleotidase, purified to homogeneity from chicken gizzard using published procedures [Dieckhoff, J., Knebel, H., Heidemann, M. and Mannherz, H. G. (1985) Eur. J. Biochem. 151, 377-383] was incorporated into artificial phospholipid vesicles after prolonged dialysis against detergent-free buffer or by a gel filtration procedure. After dialysis the obtained liposomes exhibit a mean diameter of 80 nm and contain 5'-nucleotidase at random orientation, demonstrated by finding up to 50% of the total liposome-incorporated AMPase activity to be cryptic, i.e. could only be measured after their permeabilization by addition of detergent. By affinity chromatography a phospholipid vesicle fraction could be obtained containing almost exclusively cryptic AMPase activity, thus representing the inside-out orientation of 5'-nucleotidase. Comparative analysis of physiochemical and enzymatic properties of 5'-nucleotidase reveals differences between the detergent-solubilized and the liposome-incorporated 5'-nucleotidase including a changed accessibility of the enzyme to polyclonal and monoclonal antibodies. Binding and AMPase inhibition studies with different polyclonal antibodies strongly indicate to the existence of a cytoplasmic domain of chicken gizzard 5'-nucleotidase. F-actin appears preferentially to interact with the cytoplasmic domain of liposome-incorporated 5'-nucleotidase.     

Purification of 5'-nucleotidase from human seminal plasma.

5'-Nucleotidase from human seminal plasma was purified to electrophoretic homogeneity and some of its kinetic and molecular properties compared with those of 5'-nucleotidase from bull seminal plasma. The purification of the enzyme was achieved by using the same affinity chromatography media (Con A-Sepharose and AMP-Agarose or ADP-Agarose) previously used for the purification of bull seminal plasma 5'-nucleotidase (Fini, C., Ipata, P.L., Palmerini, C.A. and Floridi, A. (1983) Biochim. Biophys. Acta 748, 405-412). However, in the present purification procedure no detergent was used as it had been necessary for the purification of the bovine enzyme. The experimental data reveal some main differences between these two enzymes; first, the human enzyme seems to be constituted of a single polypeptide chain of about 71 kDa, while the 5'-nucleotidase of bull seminal plasma, in non denaturing detergent solutions, is a homodimer of about 160 kDa. Another most remarkable difference is that the human enzyme does not seem to contain a phosphatidylinositol anchoring system like the one present in the bovine enzyme and in 5'-nucleotidase of different sources (Low, M.G. (1987) Biochem. J. 244, 1-13). Finally, the AMPase activity of 5'-nucleotidase from human seminal plasma is not affected by dithiothreitol which, on the contrary, is a powerful inhibitor of the bovine enzyme causing the dissociation of its subunits which are held together by disulphide bridges (Fini, C., Minelli, A., Camici, M. and Floridi, A. (1985) Biochem. Biophys. Acta 827, 403-409).     

Purification of bovine liver cytosolic 5'-nucleotidase. Kinetic and structural studies as compared to the membrane isoenzyme

Cytosolic 5'-nucleotidase from bovine liver has been purified to homogeneity. Two affinity chromatographies on concanavalin A and 5'AMP-Sepharose columns result in a 12,000-fold purification. The sequential elution of glycoproteins from the concanavalin-A-Sepharose column with methyl alpha-D-glucoside and methyl alpha-D-mannoside greatly increases the degree of purification of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows two subunits having apparent molecular masses of 65 kDa and 57 kDa respectively, while only one band at 70 kDa is observed in the case of the membrane-bound 5'-nucleotidase. Both the Stokes radii, measured by gel exclusion HPLC, and the sedimentation coefficient, determined by density gradient ultracentrifugation, indicate that the cytosolic enzyme is a heterodimer of about 130 kDa. This contrasts with the membrane-bound 5'-nucleotidase which is a homodimer of 140 kDa. Moreover, the antibodies raised against the membrane 5'-nucleotidase inhibited the cytosolic form indicating that a common antigenic determinant(s) exists between the two isoenzymes. However, structural differences are revealed by immunoblotting. In the same way, the effect of lectins suggests that differences in the structure of the carbohydrate chains exist between the two isoenzymes. The purified cytosolic enzyme has lower affinity for the nucleotides than does the membrane enzyme. In addition, while ADP, [alpha,beta-CH2]ADP and ATP were strong competitive inhibitors of the membrane enzyme, ADP and ATP activate the cytosolic form and [alpha,beta-CH2]ADP has no effect. Moreover, two pH optima at 7.5 and 9.5 are observed in the cytosolic enzyme while only one at 7.5 occurred in the membrane form. Finally the exogenous cations, MgCl2 and MnCl2, are necessary for the maximal activity of the cytosolic but not of the membrane 5'-nucleotidase. All these observations indicate that the two isoenzymes are different.     

Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from salmon liver

Salmon liver was chosen for the isolation of 6-pyruvoyl tetrahydropterin synthase, one of the enzymes involved in tetrahydrobiopterin biosynthesis. A 9500-fold purification was obtained and the purified enzyme showed two single bands of 16 and 17 kDa on SDS/PAGE. The native enzyme (68 kDa) consists of four subunits and needs free thiol groups for enzymatic activity as was shown by reacting the enzyme with the fluorescent thiol reagent N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The enzyme is heat-stable up to 80 degrees C, has an isoelectric point of 6.0-6.3, and a pH optimum at 7.5. The enzyme is Mg2+ -dependent and has a Michaelis constant for its substrate dihydroneopterin triphosphate of 2.2 microM. The turnover number of the purified salmon liver enzyme is about 50 times as high as that of the enzyme purified from human liver. It does not bind to the lectin concanavalin A, indicating that it is free of mannose and glucose residues. Polyclonal antibodies raised against the purified enzyme in Balb/c mice were able to immunoprecipitate enzyme activity. The same polyclonal serum was not able to immunoprecipitate enzyme activity of human liver 6-pyruvoyl tetrahydropterin synthase, nor was any cross-reaction in ELISA tests seen.     

Polyclonal and monoclonal antibodies against chicken gizzard 5′-nucleotidase inhibit the spreading process of chicken embryonic fibroblasts on laminin substratum

Polyclonal and monoclonal antibodies raised against chicken gizzard 5'-nucleotidase were tested in adhesion assays of embryonic chicken fibroblasts (CEF) for their ability to interfere with the adhesion process of these cells on either laminin or fibronectin substrata. The initial attachment process of CEF on fibronectin and laminin substrata was not influenced by preincubating these cells with antibodies against chicken gizzard 5'-nucleotidase. However, the subsequent spreading process of these cells was found to be inhibited for at least 2 h on a laminin substratum. This effect was obtained with a polyclonal antibody as well as with one from 12 monoclonal antibodies raised against the native enzyme purified from chicken gizzard. In vitro assays demonstrated a competition of laminin and this monoclonal antibody for the binding site on purified 5'-nucleotidase. Spreading-arrested and rounded CEF do not develop prominent intracellular stress-fibers like control cells, instead they seem to concentrate their available actin in areas of presumptive initial contact with the laminin substratum.     

Isolation and characterization of Neurospora crassa plasma membranes.

The isolation and characterization of plasma membranes from a cell wall-less mutant of Neurospora crassa are described. The plasma membranes are stabilized against fragmentation and vesiculation by treatment of intact cells with concanavalin A just prior to lysis. After lysis, the concanavalin A-stabilized plasma membrane ghosts are isolated by low speed centrifugation techniques and the purified ghosts subsequently converted to vesicles by removal of the bulk of the concanavalin A. The yield of ghosts is about 50% whereas the yield of vesicles is about 20%. The isolated plasma membrane vesicles have a characteristically high sterol to phospholipid ratio, Mg2+-dependent ATPase activity and (Na+ plus K+)-stimulated Mg2+ATPase activity. Only traces of succinate dehydrogenase and 5'-nucleotidase are present in the plasma membrane preparations.     

A family of phosphohydrolases from bovine intestinal mucosa: 5'-nucleotidase

Bovine intestinal 5'-nucleotidase has been partially purified and characterized for comparison with two other phosphohydrolases from the same tissue, alkaline phosphatase and 5'-nucleotide phosphodiesterase, which are closely related structurally and mechanistically. Kinetic studies with a variety of nucleotides and phosphonate analogs show that, although 5'-nucleotidase is a monoesterase like alkaline phosphatase, it more closely resembles 5'-nucleotide phosphodiesterase in its high affinity and specificity for nucleotide binding. 5'-Nucleotidase is bound very strongly by an affinity column containing a bound phosphonate analog of ADP but is not bound by an affinity column containing a non nucleotide phosphonate which selectively binds alkaline phosphatase. 5'-Nucleotidase is strongly bound by immobilized antibodies prepared against 5'-nucleotide phosphodiesterase, and is less strongly bound by immobilized antibodies prepared against alkaline phosphatase. We conclude that 5'-nucleotidase is structurally more similar to 5'-nucleotide phosphodiesterase than to another monoesterase, alkaline phosphatase.     

Absence of 5'-nucleotidase in porcine polymorphonuclear leucocyte membranes

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess p-nitrophenyl phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5'-nucleotidase inhibitor alpha, beta-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5'-nucleotidase. An antibody against mouse liver 5'-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5'-nucleotidase and the necessity for distinguishing between true 5'-nucleotidase and non-specific phosphatase activity is discussed.     

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.     

5'-Nucleotidase I from rabbit heart

5'-Nucleotidase I (N-I) from rabbit heart was purified to homogeneity. After ammonium sulfate precipitation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose, AMP-agarose, and ADP-agarose. The pure enzyme has a specific activity of 318 mumol (mg of protein)-1 min-1. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a subunit molecular weight of 40,000. N-I is activated by ADP but not by ATP, in contrast to the 5'-nucleotidase (N-II) purified by Itoh et al. (1986), which is activated by ATP and, less well, by ADP. N-I displays sigmoidal saturation kinetics in the absence of ADP and hyperbolic kinetics in the presence of ADP. Partially purified N-I was previously shown to prefer AMP over IMP as substrate (Truong et al., 1988); this has been confirmed for pure N-I. Comparison of AMP and ADP concentrations reported to occur in heart with the kinetic behavior of N-I implicates N-I as the enzyme responsible for producing adenosine under conditions leading to a rise in ADP and AMP, such as hypoxia or increased workload. N-I is not activated by the ADP analogue adenosine 5'-methylenediphosphonate (AOPCP) and is only weakly inhibited by relatively high concentrations of AOPCP, in contrast to 5'-nucleotidase from plasma membrane, which is powerfully inhibited by this analogue. N-I shows an absolute dependence on Mg2+ ions. Mn2+ and Co2+ ions can replace Mg2+ ions as activator; Ni2+ and Fe2+ are much less effective, while Ca2+, Ba2+, Zn2+, and Cu2+ fail to activate the enzyme.     

Concanavalin A induced inhibition of 5'-nucleotidase from guinea pig skeletal muscle and bull seminal plasma: a comparative study

Both purified and membrane-bound 5'-nucleotidases (EC 3.1,3.5) from guinea pig skeletal muscle and bull seminal plasma are inhibited by Concanavalin A (Con A). 5'-Nucleotidase purified from skeletal muscle is inhibited by Con A by an apparent uncompetitive process (K'i = 160 nM), while the lectin inhibits the particulate enzyme by an apparent non-competitive process (Ki = K'i = 50 nM). 5'-Nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process (K'i = Ki = 270 nM), while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin (K'i greater than Ki; 30 and 14 nM, respectively). The enzyme purified from skeletal muscle exhibits a significant cooperativity in the interaction with Con A. The inhibition of bull seminal plasma particulate 5'-nucleotidase brought about by Con A is not completely reversed by addition of alpha-methyl-D-mannoside.