PURIFICATION AND CHARACTERIZATION OF POLY(A) POLYMERASE FROM GERMINATED WHEAT EMBRYOS - ENZYME GLYCOSYLATION

Wheat embryo poly(A) polymerase was purified (1348-fold) to electrophoretic homogeneity by adenosine triphosphate(ATP)-Sepharose and concanavalin A(Con A)-agarose affinity chromatography. The purified polymerase was devoid of cryptic nuclease activity after Con A-agarose affinity chromatography. Thus the Con A-agarose fraction showed a linear increase in poly(A) polymerase activity as a function of time up to 90 min. Fractionation of purified enzyme on native PAGE showed a single protein stained band that coincided with the activity peak of poly(A) polymerase. The molecular weight of poly(A) polymerase was 65 000- 70 000 as determined by molecular sieve chromatography. A single subunit of purified poly(A) polymerase (mol. wt., 64 000) was observed on SDS-PAGE. This proved the monomeric nature of the enzyme. The binding of poly(A) polymerase to Con A-agarose and its elution with alpha-methyl mannopyranoside suggested its glycoprotein nature. The apparent K(m) of poly(A) polymerase for ATP was 86 muM. The H-3-labelled reaction product of purified enzyme binds to oligo(dT)-cellulose affinity matrix. In addition, the putative poly(A) product was not hydrolysed by RNase A and RNase T(i). Thus, it was proved that poly(A) polymerase catalyzes the synthesis of poly(A) sequences.