Ribonucleotide Phosphohydrolase Research Articles

Suppressing a phosphohydrolase of cytokinin nucleotide enhances grain yield in rice.

One-step and two-step pathways are proposed to synthesize cytokinin in plants. The one-step pathway is mediated by LONELY GUY (LOG) proteins. However, the enzyme for the two-step pathway remains to be identified. Here, we show that quantitative trait locus GY3 may boost grain yield by more than 20% through manipulating a two-step pathway. Locus GY3 encodes a LOG protein that acts as a 5'-ribonucleotide phosphohydrolase by excessively consuming the cytokinin precursors, which contrasts with the activity of canonical LOG members as phosphoribohydrolases in a one-step pathway. The residue S41 of GY3 is crucial for the dephosphorylation of iPRMP to produce iPR. A solo-LTR insertion within the promoter of GY3 suppressed its expression and resulted in a higher content of active cytokinins in young panicles. Introgression of GY302428 increased grain yield per plot by 7.4% to 16.3% in all investigated indica backgrounds, which demonstrates the great value of GY302428 in indica rice production.

5′-Nucleotidase in the Detection of Increased Activity of the Liver Form of Alkaline Phosphatase in Serum

5′-Nucleotidase (5′-ribonucleotide phosphohydrolase; EC 3.1.3.5; 5NT), an intrinsic membrane glycoprotein present as an ectoenzyme in a wide variety of mammalian cells, hydrolyzes 5′-nucleotides to their corresponding nucleosides (1). Despite its ubiquitous distribution, serum concentrations of 5NT appear to reflect hepatobiliary disease with considerable specificity (2). Dixon and Purdom (3) first observed that serum 5NT was clinically useful for differential diagnosis of hepatobiliary and osseous diseases, the enzyme activity being increased only in hepatobiliary diseases. This finding has been confirmed by several authors (4)(5)(6)(7). Assay of 5NT activity may have value as an addition to measurement of nonspecific total alkaline phosphatase (AP) in patients with suspected hepatobiliary disease (8)(9)(10)(11)(12), and abnormal 5NT activity is routinely interpreted by some clinicians as evidence of a hepatic origin of increased AP activity in serum. However, a direct comparison of 5NT and the liver form of AP (L-AP) in a clinical setting has not been reported (13). The aim of this …

Alterations of gene expression in endoderm differentiation of F9 teratocarcinoma cells.

During the endoderm differentiation of F9 mouse embryonal carcinoma cells, as induced by sodium butyrate (NaBu) or retinoic acid (RA), gene expressions of alkaline phosphatase (ALPase), pyruvate kinase (PKase) and 5' ribonucleotide phosphohydrolase (5'-Nase) were examined. The specific activity of ALPase was found to increase by 3.5-fold after 48 hr treatment with NaBu. In contrast, specific activity of PKase were decreased by 63%. Northern blot analysis revealed that the elevation of ALPase activity resulted from an increase in the level of liver/bone/kidney (L/B/K)-type ALPase mRNA and that the decrease of PKase activity was dependent on a reduction in the level of M(2)-PKase mRNA. Interestingly, when NaBu was removed from the culture medium, the levels of these mRNAs reverted to their original levels after 16 h. During these processes, the specific activity of 5'-Nase and the level of its mRNA remained unchanged. In contrast, when F9 cells were treated with RA, only the level of L/B/K-type ALPase mRNA increased. Lastly, we examined the issue of whether an increase in the level of ALPase mRNA is dependent on the transcriptional activation of the mouse L/B/K-type ALPase gene. Transient transfection assays using luciferase reporter constructs showed that the promoter activity increased as the result of treatment with RA but not with NaBu.

Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.

Substrate cycling between 5-amino-4-imidazolecarboxamide riboside and its monophosphate in isolated rat hepatocytes

AICA (5-amino-4-imidazolecarboxamide)-riboside is taken up by isolated rat hepatocytes and converted by adenosine kinase (ATP:adenosine 5′-phosphotransferase, EC 2.7.1.20) into AICAR (ZMP), an intermediate of the de novo synthesis of purine nucleotides. We investigated if, in these cells, a cycle analogous to the adenosine-AMP substrate cycle operates between AICAriboside and ZMP. When 50 μM ITu, an inhibitor of adenosine kinase, was added to hepatocytes that had metabolized AICAriboside for 30 min, the concentration of ZMP decreased immediately. This was mirrored by a reincrease of AICAriboside. Rates of the ITu-induced decrease of ZMP and the increase of AICAriboside, calculated at different concentrations of ZMP, were first order, up to the highest concentration of ZMP (approx. 5 μmol/g of cells). Dephosphorylation of ZMP added to crude cytosolic extracts of rat liver displayed hyperbolic kinetics, with a V max of 0.65 μmol/min per g protein and an apparent K m of 5 mM, and was markedly inhibited by Pi, an inhibitor of IMP-GMP 5′nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5). We conclude that hepatocyte ZMP is continuously dephosphorylated, most likely by IMP-GMP 5′-nucleotidase, into AICAriboside, which is rephosphorylated into ZMP by adenosine kinase. Substrate cycling was also shown to occur between other nucleoside analogs and their phosphorylated counterparts.

Human placental cytosolic purine 5'-nucleotidase is effectively reactive with an anti-chicken liver enzyme antibody.

Dephosphorylation of nucleoside 5’-monophosphates by 5’-nucleotidase (5’-ribonucleotide phosphohydrolase, EC 3.1.3.5.) is the first irreversible step in the nucleotide degradation pathway. Its activity has been found in different subcellular fractions such as the plasma membrane (1), the cytoplasm (2–7), the mitochondria (8), and the lysosome (9, 10) (Fig. 1). While ecto-5’-nucleotidase localized on the plasma membrane degrades extracellular purine/pyrimidine 5’-nucleotides with micromolar Km values, intracellular degradation of 5’-nucleotides is catalyzed possibly by several distinct cytosolic 5’-nucleotidases coexistent in the cytoplasm (11, 12). Namely, purine 5’-nucleotidase (2, 3), pyrimidine 5’-nucleotidase (4), deoxyribonucleotidase (5’(3’)-nucleotidase (EC 3.1.3.31.)) (5), and AMP-selective 5’-nucleotidase (6, 7) are cytosolic enzymes with millimolar Km values for various nucleotides, and may have a critical role in the maintenance of a constant composition of intracellular purine/pyrimidine 5’-nucleotides (4, 13).KeywordsHuman PlacentaPurine NucleosidePurine Nucleoside PhosphorylaseChicken LiverNucleotidase ActivityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Effect of dopamine on ecto-5'-nucleotidase expression in human glomerular mesangial cells.

Ecto-5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) of mesangial cells may be the main source of adenosine within the glomerulus, and thus essential in the regulation of glomerular microcirculation. c-AMP and c-AMP-stimulating agents were found to induce ecto-5'-nucleotidase of mesangial cells. Dopamine is a catecholamine known to increase c-AMP levels in mesangial cells. We have studied the effect of dopamine on ecto-5'-nucleotidase expression and DNA synthesis of glomerular mesangial cells in culture. Human mesangial cells were exposed to dopamine in the concentration range from 0.1 microM- to 1 mM, for 6-72 h. Ecto-5'-nucleotidase activity of human mesangial cells increased from 118.6 +/- 7.7 to 171 +/- 12 nmol/min/mg in a 72 h culture. This effect was time- and dose-dependent. Cycloheximide, an inhibitor of protein synthesis did not modify basal 5'-nucleotidase activity but it suppressed the stimulatory effect of 10 microM dopamine. DNA synthesis of human mesangial cells, studied after exposure of these cells to the same concentrations of dopamine used in the 5'-nucleotidase stimulation, was inhibited, being also dose dependent. These results indicate that dopamine induces ecto-5'-nucleotidase and inhibits DNA synthesis of cultured human mesangial cells. This action of dopamine on glomerular mesangial cells may be important in the regulation of glomerular hemodynamics.

Characterization of a filtrable 3′-nucleotidase of Leishmania donovani

A plasma membrane 3′-nucleotidase (3′-ribonucleotide phosphohydrolase, EC 3.1.3.6) having a apparent subunit molecular mass of 38 kDa but filtrable through a Centriprep-10 microconcentration (Amicon) membrane was purified from Leishmania donovani promastigotes. The enzyme activity has an optimum at pH around 7.5. EDTA strongly inhibited the enzyme activity which was fully restored only by Co 2, from various metal ions added. Citrate ions, Zn 2+ or dithiothreitol were also strongly inhibitory. The enzyme is apparently located on the inner side of the parasite plasma membrane. The substrates specificity and kinetics of the filtrable enzyme are similar to those of the non-filtrable outer-surface-membrane-bound 3′-nucleotidase reported by Gbenle and Dwyer (Gbenle, G.O. and Dwyer, D.M. (1992) Biochem. J. 285, 41–46). Therefore, it is suggested that both enzymes are implicated in the supply of nucleosides to the parasite.

Application of solid-phase extraction on anion-exchange cartridges to quantify 5'-nucleotidase activity.

The enzyme 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) catalyzes a critical reaction in intermediary metabolism, the phosphohydrolysis of nucleoside 5'-monophosphates to their corresponding nucleosides. We have evaluated solid-phase extraction on pre-packed anion-exchange cartridges as a chromatographic technique with which 5'-nucleotidase activity may be detected and quantified. Chromatographic conditions were established whereby substrate nucleotide was rapidly and completely separated from its corresponding nucleoside by solid-phase extraction. Both analytes were recovered quantitatively, without loss or degradation. This chromatographic system was integrated into a discontinuous radiochemical assay for 5'-nucleotidase which enabled both substrate utilization and product formation to be assessed simultaneously. Enzyme reaction samples could be analyzed directly for 5'-nucleotidase activity without any pre-chromatography preparation. The high capacity of the solid-phase cartridges and the inability of 5'-nucleotidase to enter the packing bed during analyte elution facilitated termination of the enzyme reaction by applying the entire reaction mixture to the cartridge. Loaded cartridges could then be stored at 4 degrees C prior to chromatography and subsequently batch-eluted. The excellent resolution between substrate and product in solid-phase extraction and the sensitivity of radioisotopic counting enabled detection/quantification of low tissue levels of 5'-nucleotidase in conjunction with ancillary assays for secondary enzyme reactions with the potential to elicit the artifactual loss of 5'-nucleotidase substrate/product when crude biological preparations are examined for 5'-nucleotidase activity. Our results demonstrate that solid-phase extraction on anion-exchange cartridges with elution solvents of appropriate pH offers several unique advantages for 5'-nucleotidase determination.

Recommended screening test for pyrimidine 5′-nucleotidase deficiency

Pyrimidine 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, E.C. 3.1.3.5) (P5N) deficiency is one of the more common causes of hereditary non-spherocytic haemolytic anaemia. This syndrome is characterized by haemolytic anaemia, pronounced basophilic stippling of red cells, and marked increase in red cell pyrimidine-containing nucleotides (Valentine et al. 1974). P5N activity is quantitatively measured by a colorimetric method (Valentine et al. 1974) and a radiometric method (Torrance, West & Beutler 1977). For screening of P5N deficiency, the recommended method is to determine the u.v. absorption of blood extract (Beutler 1984).

Topography, purification and characterization of thyroidal 5'-nucleotidase

1. 1. Subcellular studies of bovine thyroid indicate that 5'-nucleotidase is predominantly associated with plasma membranes, although a considerable part of this ectoenzyme is also found internalized. 2. 2. The enzyme displaying the features of a glycoprotein has been purified 1400 times by detergent solubilization and two subsequent affinity Chromatographic steps. 3. 3. Thyroidal 5'-nucleotidase can be classified as an unspecific metallo-dependent 5'-ribonucleotide phosphohydrolase. The native enzyme exists as a dimer (MW 150 kDalton), composed of two similar or identical subunits.

Inhibition of cAMP-phosphodiesterase by molybdate.

Inhibition of 3':5'-cyclic-AMP-5'-nucleotidohydrolase (EC 3.1.4.17) type II (cAMP-phosphodiesterase) by sodium molybdate was studied: While determination of inorganic phosphate and 5'-ribonucleotide phosphohydrolase (5'-nucleotidase) (EC 3.1.3.5) activity was not disturbed by sodium molybdate in concentrations up to 10 mM, cAMP-phosphodiesterase was inhibited by millimolar concentrations of molybdate. The half maximal effect was observed at about 2 mM sodium molybdate (0.75 mM cAMP in the assay).

Chemical modification of essential carboxyl group and histidine residue in the plasma-membrane 5′-nucleotidase

An investigation, using specific chemical reagents, of the amino acids involved in the catalytic activity of the purified 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) from bovine liver plasma membranes, was carried out. The enzyme was irreversibly inactivated by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The inhibition kinetics were of the first-order type and decreased partially in the presence of nucleotides and divalent cations. These results indicate for the first time that a carboxyl group is essential for the catalytic process of 5′-nucleotidase. Moreover, chemical modification by diethylpyrocarbonate also produced inactivation of the enzyme and showed a differential spectrum with a peak at 240 nm characteristic of N-carbethoxyhistidine residues. This inactivation was efficiently released upon decarbethoxylation by hydroxylamine only when the extent of inactivation, due to low concentration of diethylpyrocarbonate, was limited. The time-dependent inactivation followed first-order kinetics and nucleotides afforded significant protection against diethylpyrocarbonate modification. The results indicate the involvement of the histidine residue in catalysis.

Human placental cytoplasmic 5'-nucleotidase: kinetics and molecular properties.

Dephosphorylation of nucleoside 5′-monophosphates is the first and irreversible reaction of purine nucleotide catabolism.1 This reaction is catalyzed by 5′-nucleotidase 5′-ribonucleotide phosphohydrolase, EC 3.1.3.5. Most of this activity is present on the external surface of the cell. This activity has no clear role in intracellular nucleotide catabolism. However, recent studies indicate that cytoplasmic 5′-nucleotidase may have an important role in intracellular nucleotide degradation.2 KeywordsHuman PlacentaPyridine NucleotidePurine NucleosideChicken LiverNucleotidase ActivityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Inhibition of smooth muscle 5′-nucleotidase by imidazole and its reversal by magnesium

Imidazole, commonly used as an effective pH-buffering reagent in aqueous media maintained at pH 7–8, was found to depress the 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity of microsomal membrane fraction isolated from rat vas deferens smooth muscle in a dose-dependent manner in the absence of added Mg 2+. Such an inhibitory effect of imidazole on the smooth muscle 5′-nucleotidase was not dependent upon the purity or integrity of the membrane fractions used and could be fully reversed by the inclusoin of 5–10 mM Mg 2+ in the assay medium. Of the five different pH-buffering reagents tested, imidazole was specific in exerting inhibitory effect on the 5′-nucleotidase in the absence of Mg 2+ and this inhibition could not be accounted for by the impurities present in the imidazole. Differential effects of chelating reagents and other divalent metal ions on the 5′-nucleotidase activity were also observed in imidazole and Tris buffer solutions. The 5′-nucleotidase activity was not affected if the membranes were preincubated and washed with a large volume of 50 mM imidazole and subsequently assayed in 50 mM Tris in the absence of Mg 2+. Similar findings were obtained with EDTA treated membrane. These results suggest that imidazole does not act by removal of the activating metal ion but rather interacts directly with 5′-nucleotidase and alters the metal-enzyme interactions.

The interaction of 5′-nucleotidase purified from chicken gizzard and actin, and the reversible loss of the inhibitory capacity of actin on deoxyribonuclease I

Evidence is presented for a direct interaction of the intrinsic membrane protein 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) purified from avian smooth muscle (chicken gizzard) and the cytoskeletal component actin. Two different modes of interaction can be discerned: firstly, an immediate inhibitory effect of preferentially filamentous actin (F-actin) on the enzymic (i.e., AMPase) activity of 5′-nucleotidase and a direct binding of this enzyme to immobilized F-actin. Since these effects are suppressed by the addition of myosin subfragment 1, binding of 5′-nucleotidase appears to occur along the F-actin filament axis. Secondly, a time- and 5′-nucleotidase concentration-dependent transformation of also preferentially F-actin into a form unable to inhibit the enzymic activity of deoxyribonuclease I (DNAase I). This desensitization of actin versus DNAase I is not due to a denaturation process and was found to be reversible after addition of ATP. Furthermore, it does not seem to effect the ability of actin to bind to DNAase I. The transformation is accompanied by the hydrolysis of actin-bound nucleotide into adenosine, which remains bound to actin. Therefore, the desensitization of actin versus DNAase I appears to be due to a nucleotide-dependent conformational change of actin. An unidentified contamination of the 5′-nucleotidase preparations to a varying degree with ADPase and ATPase activities appears to be responsible for the desensitization process, although a synergistic role of these activities and 5′-nucleotidase cannot be excluded.

Continuous fluorimetric assay of 5′-nucleotidase with formycin 5′-phosphate as substrate, and its application to properties of substrates and inhibitors

The modifications in ultraviolet absorption and fluorescence emission accompanying dephosphorylation of formycin 5′-phosphate may be employed for the continuous assay of 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity. The sensitivity of the fluorimetric method is additionally enhanced by coupling the reaction with adenosine deaminase, which deaminates formycin more rapidly than adenosine. The final product is then formycin B, which is non-fluorescent at neutral pH and only slightly so at alkaline pH. The fluorescence procedure permits the use of substrate concentrations as low as 1 μM in a 10 mm curvette. Details are described for the use of the foregoing systems to follow continuously the kinetics as well as the properties of a number of substrate and inhibitor analogues of the enzyme from snake venom. Kinetic parameters are presented and compared with reported values for the enzyme from other sources. In particular, the pH dependence of the inhibitory properties of nucleoside 5′-diphosphates (NDP) points to the non-dissociated form, NDP 2−, as the potent inhibitory species. An especially useful inhibitor is adenosine α,β-methylene-5′-pyrophosphate, because of its higher p K value for the β-phosphate secondary hydroxyl ionization so that it is the most suitable inhibitor for kinetic and in vivo investigations over a broad pH range. The spectral properties of formycin analogues are tabulated, and reference made to their potential applications to other enzyme systems.

5′-Nucleotidase from bull seminal plasma

5′-Nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) occurs in bull seminal plasma in multiple forms. The heterogeneity does not reflect the existence of true isoenzymes, but is due to the association of the enzyme with particulate material and to molecular aggregation phenomena. Addition of detergents to native bull seminal plasma prevents molecular aggregation, solubilizes the particulate form of the enzyme, and results in the appearance of a single molecular form of the enzyme. Enzyme purification can be achieved after three chromatographic steps which involve negative adsorption of 5′-nucleotidase activity on DEAE-Sephadex A-50 followed by two affinity chromatographies on concanavalin A-Sepharose 4B and ADP-agarose. The enzyme appears to be a dimeric glycoprotein. Some properties of the enzyme, including substrate specificity and the effects of hydrogen ion concentration and of various divalent cations, are reported.

Evidence for a substrate cycle between AMP and adenosine in isolated hepatocytes.

The effect of adenosine on the metabolism of prelabeled adenine nucleotides was investigated in isolated hepatocytes. Adenosine caused an approximately equal to 2-fold increase in the ATP content of the cells. This effect was in part counteracted by an increased rate of adenine nucleotide catabolism that could be explained by a stimulation of both AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) and the cytoplasmic 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) because of the increased concentration of ATP. The unexpected finding that labeled adenosine was formed immediately after the addition of the unlabeled nucleoside could be explained by the trapping effect of adenosine. An accumulation of labeled adenosine was observed also in the presence of 5-iodotubercidin, a potent inhibitor of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). Under these conditions, there was a decrease in the concentration of ATP in the cell and a 2- to 3-fold increase in the rate of formation of allantoin. This formation of adenosine was only slightly decreased by inhibition of the membranous 5'-nucleotidase; it led to the accumulation of S-adenosylhomocysteine in the presence of coformycin and an excess of L-homocysteine. It was concluded that, under basal conditions, the cytoplasmic 5'-nucleotidase present in the liver cell continuously produces adenosine, which is immediately reconverted into AMP by adenosine kinase, without giving rise to allantoin. This futile cycle between AMP and adenosine amounts to at least 20 nmol/min per g of liver and, thus, exceeds the basic rate of allantoin formation.

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 ( K m 35 μM), and ATP was a potent competitive inhibitor. In contrast, the 5′-nucleotidase displayed by fraction S showed a low substrate affinity ( K m 130 μM) 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 μM relative to 50 μM 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 μM, 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.