Open AccessIsolation and characterization of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina
Author(s) -
ROGGEN Erwin,
SLEGERS Herman
Publication year - 1985
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1985.tb08740.x
Subject(s) - sepharose , enzyme , size exclusion chromatography , agarose , chemistry , affinity chromatography , biochemistry , microbiology and biotechnology , gel electrophoresis , cytoplasm , artemia salina , polyacrylamide gel electrophoresis , specific activity , chromatography , biology , organic chemistry , toxicity
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 M r 94000 and 70000. The M r ‐70000 protein has been identified as poly(A) polymerase. The enzyme is exclusively activated by Mn 2+ . Addition of Ca 2+ , Mg 2+ , Zn 2+ , 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.