Levamisole inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes

1. The effect of levamisole (LMS) on alkaline phosphatase (EC 3.1.3.1) and 5'-nucleotidase (EC 3.1.3.5) activities of bovine milk fat globule membranes (MFGM) was examined. 2. LMS inhibited MFGM alkaline phosphatase activity in a concentration-dependent manner with 50% inhibition produced by 49 +/- 23 microM LMS. 3. 5'-Nucleotidase was resistant to LMS inhibition with 30.9% inhibition produced by 10 mM LMS, the highest concentration tested. 4. LMS was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 45 +/- 6 microM. 5. The extent of LMS inhibition of alkaline phosphatase was dependent on the substrate utilized in the assay. 6. The effect of LMS on bovine MFGM alkaline phosphatase was similar to LMS effects on other mammalian alkaline phosphatases of liver/kidney/bone/placental isoenzyme origin.     

Selective extraction of marker enzymes of bovine milk fat globule membrane by nonionic detergents.

Large amounts (66-97%) of marker enzymes such as alkaline phosphatase, 5'-nucleotidase, phosphodiesterase I, and gamma-glutamyl transpeptidase of bovine milk fat globule membrane (MFGM) were selectively solubilized by nonionic detergents such as Triton X-100, Tween 20, Nonidet P-40, Liponox NCK, and Emulgen 109-P. On the other hand, the extractability of MFGM protein with these detergents was less than 50%. Judging from the recovery of total activity, it is likely that alkaline phosphatase, phosphodiesterase I, and gamma-glutamyl transpeptidase are activated by nonionic detergents, whereas 5'-nucleotidase is somewhat inhibited by the detergents, except for Tween 20, and acid phosphatase is strongly inhibited by all detergents. In addition, solubilization of the protein with the nonionic detergents was found to be somewhat selective by SDS-polyacrylamide gel electrophoresis. There was no appreciable difference between the five brands of nonionic detergents used as regards the extractability of protein and the enzymatic activity of the extracted marker enzymes of MFGM, except that the solubilizing ability of Tween 20 was relatively low.     

Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells

The role of cyclic AMP on endothelial cell proliferation was investigated, since these cells can be exposed to high concentrations of physiological and pharmacological agents that alter cyclic AMP metabolism. Cloned bovine aortic endothelial cells were plated at 25,000 cells/35mm dish and grown for 5 days in the presence of phosphodiesterase (PDE) inhibitors, forskolin, or cyclic AMP analogs. The PDE inhibitors dipyridamole, ZK 62 711, isobutylmethylxanthine (IBMX) and theophylline inhibited cell growth in a concentration-dependent manner. Dipyridamole produced a 30% and a 50% inhibition at 5 microM and 12.5 microM, while higher concentrations were cytotoxic. At its therapeutic plasma concentration range (50-100 microM) theophylline inhibited cell proliferation by 15-25%, while IBMX and the highly specific cyclic AMP phosphodiesterase inhibitor, ZK 62 711 inhibited growth by 60-80% and 40-50%, respectively. Forskolin (5 microM) increased cyclic AMP levels and cyclic AMP-kinase activity ratios by 2.5-fold and 2-fold. In the absence of PDE inhibitors forskolin produced a 20% growth inhibition at 0.5 microM and a 60% inhibition at 10 microM. The forskolin dose-response curve was not altered by theophylline, but was shifted to the left by approximately 10-fold with dipyridamole and ZK 62 711 and 5-fold with IBMX. Forskolin (5 microM), by itself produced a 1.8-fold increase in cyclic AMP. In the presence of 5 microM theophylline, dipyridamole, IBMX, and ZK 62 711, cyclic AMP was increased by forskolin 2.0, 2.6, 3.5, and 6.6-fold, respectively. 8-Bromo cyclic AMP and dibutyryl cyclic AMP produced a 55% and 60% growth inhibition at 100 microM. The cyclic GMP analogs were less effective inhibitors of growth (15-30%). Our results demonstrate that cyclic AMP analogs and pharmacological agents that elevate intracellular cyclic AMP levels inhibit cell growth and suggest that cyclic AMP may be an important endogenous regulator of endothelial cell proliferation.     

Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: molecular identification, kinetic characterization and biochemical properties

In this study we describe the molecular identification, kinetic characterization and biochemical properties of an E-NTPDase and an 5'-nucleotidase in Walker 256 cells. For the ATP, ADP and AMP hydrolysis there were optimum pH in the range 6.5-8.0, and absolute requirement for divalent cations (Mg(2+)>Ca(2+)). A significant inhibition of ATP and ADP hydrolysis was observed in the presence of high concentrations of sodium azide and 0.5 mM of Gadolinium chloride. These activities were insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors. The K(m) values were 464.2+/-86.6 microM (mean+/-SEM, n=4), 137.0+/-31 microM (mean+/-SEM, n=5) and 44.8+/-10.2 microM (mean+/-SEM, n=4), and V(max) values were 655.0+/-94.6 (mean+/-SEM, n=4), 236.3+/-27.2 (mean+/-SEM, n=5) and 177.6+/-13.8 (mean+/-SEM, n=5) nmol of inorganic phosphate min(-1) mg of protein(-1) for ATP, ADP and AMP, respectively. Using RT-PCR analysis we identified the mRNA of two members of the ecto-nucleoside triphosphate diphosphohydrolase family (NTPDase 2 and 5) and a 5'-nucleotidase. The presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important to regulate the ratio adenine nucleotides/adenine nucleoside extracellularly, therefore motivating tumor growth.     

Alterations in hepatic 5'-nucleotidase in the tumor-bearing rat

The effect of the growth of the Walker 256 carcinoma on the level of 5'-nucleotidase and alkaline phosphatase in the whole liver and in an isolated hepatocyte membrane preparation of its host was investigated. Alkaline phosphatase activities of whole liver and plasma membrane were increased approximately 5-fold by tumor growth. A 50% decrease in whole liver 5'-nucleotidase activity was observed in tumor-bearing rats while the 5'-nucleotidase activity per milligram membrane protein was unaltered. Tumor growth would therefore appear to affect a pool of 5'-nucleotidase which is not associated with the plasma membrane.     

Induction of alkaline phosphatase activity in HeLa cells by sodium butyrate.

The induction of HeLa cell alkaline phosphatase activity by sodium butyrate could be inhibited by the coadministration of caffeine or theophylline. The inhibitions were dose dependent, and at any given concentration the potency was theophylline greater than caffeine. Although the induction by sodium butyrate was more sensitive to the inhibition by the xanthines than was that produced by 5-iodo-2'-deoxyuridine, the magnitudes of the increases in cyclic AMP concentrations after treatment with the xanthines were similar in the inhibition of both types of induction. The induction of alkaline phosphatase activity by sodium butyrate also produced a shift in the thermostability pattern of the enzyme, with a proportionately greater increase in the heat-labile, rather than heat-stable, form of the activity.     

Incorporation of urease into liposomes.

1. Plasma membranes were isolated from ascites hepatoma AH-130 and rat livers with or without partial hepatectomy or bile duct ligation. Reciprocal manifestations of two marker enzymes for plasma membranes were observed in these membrane preparations; alkaline phosphatase activity was found much higher in the hepatoma membrane than in any preparations of the liver membranes, whereas 5'-nucleotidase activity was much lower in the former than in the latter. 2. Effects of lectins and anti-plasma membrane antiserum on these two marker enzymes were examined. The results showed that about 50% of apparent activity of 5'-nucleotidase found in the hepatoma membrane was exhibited by alkaline phosphatase. 3. Localizations of alkaline phosphatase and 5'-nucleotidase in polyacrylamide gels after electrophoresis were demonstrated using 5'-AMP and 5-Br, 4-Cl-indoxylphosphate as substrate. There was a difference in electrophoretic mobility between the alkaline phosphatase of the hepatoma and that of the liver.     

Characterisation of an ATP-dependent Ca2+ transport system in a plasma membrane enriched fraction from rat parotid

A membrane fraction enriched in plasma membrane marker enzymes K+-dependent p-nitrophenyl phosphatase, 5'-nucleotidase and alkaline phosphatase was prepared from rat parotid glands using Percoll self-forming gradient. This fraction contained an ATP-dependent CA2+ transport system which was distinct from those located on the endoplasmic reticulum and mitochondria of parotid glands. The Km for ATP was 0.57 +/- 0.07 mM (n = 3). Nucleotides other than ATP such as ADP, AMP, GTP, CTP, UTP or ITP were unable to support significant Ca2+ uptake. ATP-dependent Ca2+ uptake displayed sigmoidal kinetics with respect to free Ca2+ concentration with a Hill coefficient of 2.02. The K0.5 for Ca2+ was 44 +/- 3.1 nM (n = 3) and the average Vmax was 13.5 +/- 1.1 nmol/min per mg of protein. The pH optimum was 7.2. Trifluorperazine inhibited Ca2+ transport with half maximal inhibition observed at 30.8 microM. Complete inhibition was observed at 70 microM trifluorperazine. Exogenous calmodulin however had no effect on the rate of transport. Na+ and K+ ions activated Ca2+ transport at 20 to 30 mM ion concentrations. Higher concentrations of Na+ or K+ were inhibitory.     

Ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio)

Adenosine, a well-known neuromodulator, may be formed intracellularly in the CNS from degradation of AMP and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. This study reports the enzymatic properties of an ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for AMP hydrolysis in a pH range of 7.0-7.5 in the presence of Mg(2+). The enzyme presented a maximal activity for AMP hydrolysis at 37 degrees C. The apparent K(m) and V(max) values for Mg(2+)-AMP were 135.3+/-16 microM and 29+/-4.2 nmol Pi.min(-1).mg(-1) protein, respectively. The enzyme was able to hydrolyze both purine and pyrimidine monophosphate nucleotides, such as UMP, GMP and CMP. Levamisole and tetramisole (1 mM), specific inhibitors of alkaline phosphatases, did not alter the enzymatic activity. However, a significant inhibition of AMP hydrolysis (42%) was observed in the presence of 100 microM alpha,beta-methylene-ADP, a known inhibitor of ecto-5'-nucleotidase. Since 5'-nucleotidase represents the major enzyme responsible for the formation of extracellular adenosine, the enzymatic characterization is important to understand its role in purinergic systems and the involvement of adenosine in the regulation of neurotransmitter release.     

X-Ray Crystal Structure of Pyrenocine A,a Phytotoxin from Pyrenochaeta terrestris

The milk fat globule membrane (MFGM) enclosing fat droplets in bovine milk was isolated, and its effects on hydrolysis of milk fat by lipases were investigated by using a gum arabic-stabilized milk fat emulsion as substrate. The addition of isolated MFGM to the reaction mixture markedly inhibited hydrolysis by pancreatic and microbial (Rhizopus delemer) lipases. The inhibition was completely lost on tryptic digestion of MFGM, suggesting that the protein moiety of MFGM played a role in the inhibition. Soluble glycoprotein (SGP) which was isolated from delipidated MFGM produced marked inhibitory activity. The inhibition by SGP was dependent on substrate concentration, suggesting that the inhibition was at least partly due to coverage and blockage of the substrate surface by SGP.     

Muscarinic Receptor Stimulated GTPase Activity in Synaptic Membranes from Bovine Retina

GTPase activity has been measured in synaptic membranes from bovine retina, with and without muscarinic receptor stimulation. Maximal stimulation above basal levels was achieved with 5 microM oxotremorine and 100 microM carbachol. (4-Hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride, which is selective for the M1 muscarinic receptor, failed to stimulate GTPase activity. 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) inhibition of oxotremorine stimulation demonstrated the presence of two populations of receptors, a low-affinity site (IC50 +/- SEM, 0.63 +/- 0.18 microM) which accounted for 63% of the inhibition and a high-affinity site (IC50 less than 1 nM) which accounted for the remaining 37%. When carbachol-stimulated GTPase activity was assayed, a single 4-DAMP inhibitory site was apparent (IC50 +/- SEM, 2.0 +/- 0.9 microM). Pirenzepine inhibited GTPase activity at a single site (IC50 values +/- SEM, 46.9 +/- 11 and 25.4 +/- 6.5 microM against oxotremorine and carbachol, respectively). Methoctramine was equipotent against carbachol and oxotremorine stimulation (IC50 values, 4.2 +/- 1.8 and 6.2 +/- 1.5 microM). Inhibition of maximal carbachol and oxotremorine stimulation by muscarinic antagonists at the major site had a rank order of potency of 4-DAMP = methoctramine greater than pirenzepine. Thus, the major site for muscarinic stimulation of GTPase activity in bovine retinal membranes is pharmacologically similar to M2 receptors.     

Purine catabolism in man: inhibition of 5'-phosphomonesterase activities from placental microsomes.

The 5'-phosphomonoesterase activity of 5'-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.5) participates in the catabolism of purine ribonucleotides to uric acid in humans. Initial velocity studies of 5'-nucleotidase suggest a sequential mechanism of interaction between AMP nad MgCl2, with a Km of 14 and 3 muM, respectively. With product inhibition studies the apparent Ki's for adenosine, inosine, cytidine, and inorganic phosphate were 0.4, 3.0, 5.0, and 42 mM, respectively. A large number of nucleoside mono-, di-, and tri-phosphate compounds were inhibitors of the enzyme. Allopurinol ribonucleotide, ADP, or ATP were competitive inhititors when AMP was the substrate, with a Ki slope of 120 muM. The phosphomonoesterase activity of human placental microsomal alkaline phosphatase had a pH optimum of 10.0 and had only 18% of maximum activity at pH 7.4. Substrates and inhibitors included almost any phosphorylated compound. The Km for AMP was 0.4 mM and the apparent Ki for Pi was 0.6 mM. Activity was increased only 19% by 5 mM MgCl2. These observations suggest that 5'-nucleotidase and alkaline phosphatase may be inhibited by ATP and Pi, respectively, under normal intracellular conditions, and that AMP may be preferentially hydrolyzed by 5'-nucleotidase.     

Selective Extraction of Alkaline Phosphatase and 51 -Nucleotidase from Milk Fat Globule Membranes by a Single Phase n-Butanol Procedure

ABSTRACT

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0°C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5¹- nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0°C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.     

Induction of alkaline phosphatase activity in HeLa cells by sodium butyrate

Summary The induction of HeLa cell alkaline phosphatase activity by sodium butyrate could be inhibited by the coadministration of caffeine or theophylline. The inhibitions were dose dependent, and at any given concentration the potency was theophylline > caffeine. Although the induction by sodium butyrate was more sensitive to the inhibition by the xanthines than was that produced by 5-iodo-2′-deoxyuridine, the magnitudes of the increases in cyclic AMP concentrations after treatment with the xanthines were similar in the inhibition of both types of induction. The induction of alkaline phosphatase activity by sodium butyrate also produced a shift in the thermostability pattern of the enzyme, with a proportionately greater increase in the heat-labile, rather than heat-stable, from of the activity. Supported by National Cancer Institute Grant CA16460.     

Reserpine attenuates interstitial adenosine-mediated activation of ecto-5'-nucleotidase in rat hearts in vivo

We examined whether reserpine-induced norepinephrine (NE) depletion attenuated the products of adenosine in rat heart. A flexibly mounted microdialysis technique was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase in rat hearts in situ. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP) at rate of 1.0 microliter/min. The baseline level of dialysate adenosine was 0.51 +/- 0.09 microM. The introduction of AMP (100 microM) through the probe increased markedly the dialysate adenosine to 8.95 +/- 0.86 microM, and this increase was inhibited by ecto-5'-nucleotidase inhibitor, alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP, 100 microM), to 0.66 +/- 0.38 microM. Thus, the level of dialysate adenosine is a measure of the ecto-5'-nucleotidase activity in the tissue in situ. AMP concentration for the half-maximal effect of adenosine release (EC(50)) was 107.3 microM. The maximum attainable concentration of dialysate adenosine (E(max)) by AMP was 21.1 microM. However, the EC(50) and E(max) values with reserpinized animals were 106.9 and 7.1 microM, respectively. Electrical stimulation of the left stellate ganglion increased significantly dialysate adenosine concentration, from the control level of 8.66 +/- 0.96 microM to 12.38 +/- 1.11 microM. After stimulation, dialysate adenosine returned to near the prestimulation level. When corresponding experiments were performed with reserpinized animals, the effect of electrical stimulation was abolished. Tyramine (endogenous catecholamine trigger) increased the adenosine concentration in a concentration-dependent manner. However, the elevation of adenosine concentration with reserpinized animals was not observed. These results suggest that reserpine attenuates NE-induced adenosine via stimulation of alpha(1)-adrenoceptor and protein kinase C mediated activation of ecto-5'-nucleotidase in rat heart.     

Effect of pH on the modulation of rat osseous plate alkaline phosphatase by metal ions.

1. Metal ions other than zinc and magnesium were effective in modulating the activity of rat osseous plate alkaline phosphatase. 2. Increasing pH had remarkable effects on the modulation of rat osseous plate alkaline phosphatase. 3. The modulation of enzyme activity by zinc, manganese and cobalt ions was slightly affected by pH variations. 4. Zinc ions were stimulatory for the enzyme at very low concentrations (50 nM). Above 50 nM zinc ions inhibited the enzyme by displacing magnesium ions. 5. Calcium ions were inhibitors of alkaline phosphatase (Kd = 10 microM) whereas manganese (Kd = 1.3 microM) and cobalt (Kd = 0.2 microM) ions were stimulatory in the pH range 8.0-10.0.     

Properties of 5'-nucleotidase in rat heart sarcolemma

The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.     

Stimulation of melanotic expression in a melanoma cell line by theophylline

Theophylline, a phosphodiesterase inhibitor, was found to be a potent stimulator of melanogenesis in the RPMI 3460 hamster melanoma cell line. This stimulation was greater than that caused by either dibutyryl cyclic AMP (db-cAMP) or another phosphodiesterase inhibitor, papaverine. Theophylline and db-cAMP treatments also produced strikingly different morphologies in the monolayered cells. The theophylline effect on melanogenesis was diminished by db-cAMP, whereas simultaneous treatment of cells with db-cAMP and papaverine produced greater stimulation of melanotic activity than either agent acting alone. Theophylline, therefore, may have phenotypic effects that are at least partially independent of phosphodiesterase inhibition. Theophylline stimulated melanin biosynthesis, as measured by rates of 2- [2-¹⁴C] thiouracil incorporation, and also caused an increase in the level of tyrosinase (EC 1.10.3.1) activity. This melanotic stimulation was prevented by the presence of cordycepin or cycloheximide. Theophylline inhibited DNA synthesis and mitosis in the melanoma cell cultures but stimulated protein synthesis. However, inhibition of proliferation and the first appearance of induced melanotic activity did not bear an immediate direct relationship to one another.     

Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics.

The inhibitory effect of a marine-sponge toxin, okadaic acid, was examined on type 1, type 2A, type 2B and type 2C protein phosphatases as well as on a polycation-modulated (PCM) phosphatase. Of the protein phosphatases examined, the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited. For the phosphorylated myosin light-chain (PMLC) phosphatase activity of the enzyme, the concentration of okadaic acid required to obtain 50% inhibition (ID50) was about 1 nM. The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited (ID50 0.1-0.5 microM). The PMCL phosphatase activity of type 2B phosphatase (calcineurin) was inhibited to a lesser extent (ID50 4-5 microM). Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p-nitrophenyl phosphate phosphatase activity of calcineurin. The following phosphatases were not affected by up to 10 microM-okadaic acid: type 2C phosphatase, phosphotyrosyl phosphatase, inositol 1,4,5-trisphosphate phosphatase, acid phosphatases and alkaline phosphatases. Thus okadaic acid had a relatively high specificity for type 2A, type 1 and PCM phosphatases. Kinetic studies showed that okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes.     

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.