Functional role of zinc in poly (A) synthesis catalyzed by nuclear poly (A) polymerase

Rose, K.M.; Allen, M.S.; Crawford, I.L.; Jacob, S.T.

European Journal of Biochemistry 88(1): 29-36

1978


ISSN/ISBN: 0014-2956
PMID: 208847
DOI: 10.1111/j.1432-1033.1978.tb12419.x
Accession: 068524142

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Abstract
The functional role of transition metals in poly(A) synthesis was elucidated by investigating the effect of the metal chelator o-phenanthroline on purified nuclear poly(A) polymerase (EC 2.7.7.19). This chelator inhibited the enzyme activity in a manner competitive with respect to the polynucleotide primer concentration. o-Phenanthroline was a non-competitive inhibitor with regard to ATP concentration and an 'uncompetitive' inhibitor with regard to dithiothreitol levels. The metal content of the purified enzyme preparations from rat liver and Morris hepatoma 3924A was determined using atomic absorption spectrometry. Of the transition metals measured, only Zn was present in detectable quantities, at levels less than 1 mol/mol of enzyme. Hepatoma enzyme contained 2-3 times as much Zn as the corresponding liver enzyme. Hepatoma poly(A) polymerase was also radioactively labeled in vivo by injection of tumor-bearing animals with 65Zn. Dialysis experiments with highly purified radiolabeled poly(A) polemerase showed that the enzyme .cntdot. Zn complex was labile and that a reduction in 65Zn content correlated with a loss in enzyme activity. These data indicate that poly(A) polymerase is a Zn-containing protein, this metal plays a role in the interaction between enzyme and its polynucleotide primer, relative to hepatoma poly(A) polymerase, the liver enzyme forms a less stable complex with Zn and the loss of Zn from poly(A) polymerase upon extensive purification may explain its low Zn content relative to that reported for other proteins.