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

Stanley, Keith K.; Edwards, Michael R.; Luzio, J. Paul

doi:10.1042/bj1860059

Cited by 151 publications

(66 citation statements)

References 58 publications

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“…Enzyme was preincubated in buffer A containing 10 mM MgC12 at 37°C according to the method of Zachowski et al [36]. 20mM sodium 2-glycerophosphate was present to saturate any non-specific phosphatase [37]. The reaction was started by addition of 10 mM CMP.…”

Section: Assays Of 5'-nucleotidasementioning

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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Section: Assays Of 5'-nucleotidasementioning

confidence: 99%

Purification of bovine liver cytosolic 5′‐nucleotidase

Zekri¹,

Harb²,

Bernard³

et al. 1988

European Journal of Biochemistry

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“…20mM sodium glycerol 2-phosphate was present to saturate non-specific phosphatases [22]. The reaction was started by addition of 1 mM AMP.…”

Section: Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

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 contrast, rabbit antisera inhibit the rat enzyme almost completely [2]. There are however many precedents for partial inhibition of enzyme activity by antibodies [20].…”

Section: Discussionmentioning

confidence: 99%

“…Inhibitory antisera have been used in defining it as an ectoenzyme [I], to investigate its rno~~en~ent during endocytosis and membrane re-cycling [2], and to assess its physiological function [3]_ The enzyme has been extensively purified [4,5], but not to homogen~it~~. Antisera raised to S'nucleotidase preparations therefore contain antibodies to other prateins and this has hindered their use for immunological localisation and other studies.…”

Section: Introductionmentioning

confidence: 99%