R. Kapoor et al., PURIFICATION AND CHARACTERIZATION OF POLY(A) POLYMERASE FROM GERMINATED WHEAT EMBRYOS - ENZYME GLYCOSYLATION, PLANT SCI, 89(2), 1993, pp. 167-176
Wheat embryo poly(A) polymerase was purified (1348-fold) to electropho
retic homogeneity by adenosine triphosphate(ATP)-Sepharose and concana
valin A(Con A)-agarose affinity chromatography. The purified polymeras
e was devoid of cryptic nuclease activity after Con A-agarose affinity
chromatography. Thus the Con A-agarose fraction showed a linear incre
ase in poly(A) polymerase activity as a function of time up to 90 min.
Fractionation of purified enzyme on native PAGE showed a single prote
in stained band that coincided with the activity peak of poly(A) polym
erase. The molecular weight of poly(A) polymerase was 65 000- 70 000 a
s determined by molecular sieve chromatography. A single subunit of pu
rified 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 mannop
yranoside suggested its glycoprotein nature. The apparent K(m) of poly
(A) polymerase for ATP was 86 muM. The H-3-labelled reaction product o
f purified enzyme binds to oligo(dT)-cellulose affinity matrix. In add
ition, 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.