Isolation and characterization of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina
In: European Journal of Biochemistry. Springer: Berlin. ISSN 0014-2956; e-ISSN 1432-1033, more
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| Abstract |
Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina cryptobiotic gastrulae by ion-exchange chromatography on DEAE-cellulose, DEAE-Sepharose CL-6B and phosphocellulose P11, gel filtration on CL-Sepharose 6B, affinity chromatography on poly(A)-Sepharose 4B and ATP-agarose. The enzyme is fully dependent on exogeneous oligo(riboadenylic acid) and is free of any nuclease or other enzyme activities. In standard assay conditions the enzyme preparation has a specific activity of 5.6 μmol AMP · h−1· (mg protein)−1.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals the presence of only two proteins with Mr 94000 and 70000. The Mr-70000 protein has been identified as poly(A) polymerase.The enzyme is exclusively activated by Mn2+. Addition of Ca2+, Mg2+, Zn2+, NH+4, K+ or Na+ inhibits the enzymatic reaction. The activity is specific for ATP and competitive inhibition is observed in the presence of other ribonucleoside 5′-triphosphates. AMP incorporation is time-dependent and is increased non-linearly with protein and primer concentration. |
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