Purification and Characterization of Bovine Brain 5′‐Nucleotidase

Montero, Josefa Mallol; Fes, Jorge Bozal

doi:10.1111/j.1471-4159.1982.tb11486.x

Cited by 57 publications

(31 citation statements)

References 34 publications

(28 reference statements)

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“…Thus it is tempting to speculate that an endogenous phospholipase C could release 5'-nucleotidase from its lipid anchor under controlled conditions. The presence of low-Km soluble forms of 5'-nucleotidase which bear similarities to the form we have studied has been described in a variety of tissues, including bovine brain (Montero & Fes, 1982;Mallol & Bozal, 1983) and rat brain (Lai & Wong, 1991;Orford et al, 1991). In rat liver (Fritzson et al, 1986) and kidney (Piec & Le Hir, 1991) the contribution to the total of the low Km soluble form is in the range of 1-7 % and similar to that observed for the electric-ray electric organ.…”

Section: Discussionsupporting

confidence: 77%

“…In rat liver (Fritzson et al, 1986) and kidney (Piec & Le Hir, 1991) the contribution to the total of the low Km soluble form is in the range of 1-7 % and similar to that observed for the electric-ray electric organ. In contrast, the contribution in human placenta is considerably higher (25 %; Klemens et al, 1990) and in brain has been estimated to be 30 % (Lai & Wong, 1991) or even 94.5 % (Montero & Fes, 1982). There may be tissue-specific differences in the degree of soluble low-Km 5'-nucleotidase, and this may correlate with the effectiveness of 5'-nucleotidase release by GPI-specific phospholipase C in vitro (Grondal & Zimmermann, 1987;Stochaj et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

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Soluble low-Km 5′-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage

Vogel

Kowalewski

Zimmermann

et al. 1992

Biochemical Journal

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Soluble and membrane-bound low-Km 5'-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6 %) than in bovine cerebral cortex (27.9 %). Membranebound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5'-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5'-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5'-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPIanchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

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Section: Discussionsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Soluble low-Km 5′-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage

Vogel

Kowalewski

Zimmermann

et al. 1992

Biochemical Journal

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“…Indeed, the K, value for AMP approached those observed in cytosol enzyme from bovine brain [34] and photoreceptor [35]. However, it was much lower than the K , observed in rat liver cytosol5'-nucleotidase [36,371.…”

Section: Discussionmentioning

confidence: 80%

Purification and properties of bovine liver plasma membrane 5′ nucleotidase

Harb

Méflah

Duflos

et al. 1983

European Journal of Biochemistry

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5′‐Nucleotidase from bovine liver plasma membranes has been extracted by the zwitterionic detergent sulfobetaine 14, and purified to apparent homogeneity. Two affinity chromatographies on concanavalin‐A‐Ultrogel and 5′AMP‐Sepharose 4B followed by AcA‐54‐Ultrogel filtration resulted in a purification of 16000 times relative to the homogenate. Sodium dodecyl sulphate gel electrophoresis indicates that the apparent molecular weight of the subunit is 70000. Cross‐linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows a band with an apparent molecular weight of 140000 indicating that the enzyme is a dimer. 5′‐Nucleotidase is a glycoprotein and its activity is inhibited to different degrees by various lectins, indicating a direct interaction with the enzyme. The purified enzyme shows a sevenfold greater affinity for AMP than the membrane‐bound enzyme. The optimum activity of the purified enzyme occurs at pH 7.5 while the membrane‐bound enzyme showed a wide range of pH optimum (7.5–8.3). An Arrhenius plot of the membrane‐bound enzyme shows a break at 28°C, which disappears in the purified enzyme. The enzyme was inhibited by EDTA, and this inhibition was reversed by divalent cations. This, as well as other evidence, indicates that the enzyme contains a highly bound metal cation, perhaps Mn2+ or Mg2+.

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“…In our study we found that the sequential elution of glycoproteins from the concanavalin-A -Sepharose column by two competing sugars (methyl a-D-glucoside and methyl a-D-mannoside) permitted a considerable increase in the degree of purification of the 5'-nucleotidase. Hence, the enrichment factor of our preparation is higher than that obtained from rat liver [13], rat heart [20], chicken liver [50], bovine brain [15] or human placenta [48] enzymes. Our results suggest that the bovine cytosolic 5'-nucleotidase is a heterodimeric protein of about 130 kDa and differs in its structure from the membrane 5'-nucleotidase [30] which is a homodimeric protein (140 kDa).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, structural heterogeneities seem to affect the cytosolic 5'-nucleotidase through its tissue origin. Thus, it has been reported a monomeric form (1 34-kDa) in a bovine brain [15], a homodimeric form in the human placenta [48] and a tetrameric form in the rat [50] and chicken [51] enzymes.…”

Section: Discussionmentioning

confidence: 99%

Purification of bovine liver cytosolic 5′‐nucleotidase

Zekri¹,

Harb²,

Bernard³

et al. 1988

European Journal of Biochemistry

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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 12000-fold purification. The sequential elution of glycoproteins from the concanavalin-A -Sepharose column with methyl a-D-glucoside and methyl a-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, [a,D-CH2]ADP and ATP were strong competitive inhibitors of the membrane enzyme, ADP and ATP activate the cytosolic form and [a,fi-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, MgC12 and MnC12, 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.5'-Nucleotidase is widely distributed in several mammalian tissues [I, 21 as well as in microorganisms [3, 41. The enzyme is a phosphomonoesterase which hydrolyzes all ribonucleoside 5'-monophosphates [S, 61. Commonly most tissues contain two isoenzymes of 5'-nucleotidase, the intrinsic membrane one [7 -101 and a soluble cytosolic form [ll -151. While the function of the membrane-bound enzyme remains obscure, cytosolic 5'-nucleotidase was shown to regulate the intracellular formation of adenosine resulting from nucleotide catabolism [16 -201. Thus, the enzyme contributes to the generation of adenosine in the heart during ischaemia through dephosphorylation of AMP [21-231. In the same way an increase of the enzyme activity occurs during regeneration of rat liver after partial hepatectomy [24]. The high activity of the cytosolic 5'-nucleotidase in chicken liver as compared with that of rat liver [25] suggests a role of this enzyme in elimination of amino acid nitrogen in uricotelic animals 1261. Though several studies have shown that the kinetic properties of both membrane-bound and cytosolic 5'-nuc...

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Purification and Characterization of Bovine Brain 5′‐Nucleotidase

Cited by 57 publications

References 34 publications

Soluble low-Km 5′-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage

Soluble low-Km 5′-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage

Purification and properties of bovine liver plasma membrane 5′ nucleotidase

Purification of bovine liver cytosolic 5′‐nucleotidase

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