Isolation and characterization of the ecto-5'-nucleotidase from a rat glioblastoma cell line.
5'-Nucleotidase has been purified from rat glioblastoma cells (Rugli cells). The enzyme has been solubilized from plasma membranes by using Triton X-100 and CHAPS. Two affinity chromatographies on concanavalin A and 5'-AMP-Sepharose render the purified enzyme with a high specific activity (76.36 mumol AMP.min-1.mg-1). The purified enzyme gives a single polypeptide band on SDS-PAGE with an apparent molecular mass of 74 kDa. Active forms with an apparent molecular mass of 135 kDa and 268 kDa are observed when the purified enzyme is analyzed by gel filtration in the presence of either 0.6% sodium deoxycholate or 0.1% Triton X-100, respectively. The purified 5'-nucleotidase presents optimum activity at pH 7.8-8.1 either in the presence or in the absence of Mg2+. A linear Arrhenius plot is observed in the 25-46 degrees C temperature range and an activation energy of 33.7 KJ/mol is calculated. The enzyme is inhibited by EDTA; the activity is partially restored by different divalent cations as Zn2+, Mn2+, and Co2+. The hydrolysis of nucleosides 5'-monophosphate shows Michaelis kinetic. The enzyme is inhibited by nucleosides di- and triphosphate. 5'-Nucleotidase is a glycoprotein, being its activity inhibited at different extent by various lectins.
Turnay
J
J
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, Spain.
Olmo
N
N
Navarro
J M
JM
Gavilanes
J G
JG
Lizarbe
M A
MA
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D005910:Glioma / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D051381:Rats; D013379:Substrate Specificity; D013696:Temperature; D014407:Tumor Cells, Cultured
D002413:Cations, Divalent; D009711:Nucleotides; D015720:5'-Nucleotidase