Isolation and characterization of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina

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Abstract
Poly(A) polymerase was 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 .mu.mol AMP.cntdot.h-1.cntdot.(mg protein)-1. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis reveals the presence of only 2 proteins with MW 94,000 and 70,000. The MW 70,000 protein was identified as poly(A) polymerase. The enzyme is exclusively activated by Mn2+. Addition of Ca2+, Mg2+, Zn2+, NH4+, 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.