Evidence of two forms of poly(A) polymerase in germinated wheat embryos and their regulation by a novel protein kinase.
Two forms of poly(A) polymerase (PAPI and PAPII) from germinated wheat embryos have been resolved on DEAE-cellulose ion-exchange chromatography by a linear gradient of 0-500 mM (NH(4))(2)SO(4). Further purification shows that both forms are monomeric in nature with an identical molecular weight, approximately 65 kDa. The phosphoprotein nature of PAPI and PAPII has been established by in vivo labelling with (32)P-orthophosphate. Acid hydrolysis of both (32)P-labelled purified PAPI and PAPII has revealed that phosphorylations generally take place in serine and threonine residues. PAPI and PAPII have also been characterised with respect to V(max) and K(m) for poly(A). The V(max) and K(m) values of PAPI are 28.57 and 11.37 microg, respectively, whereas 34.48 and 7.04 microg of PAPII. In vitro dephosphorylation of the purified enzyme by alkaline phosphatase leads to a significant loss of the enzyme activity, which is regained upon phosphorylation by a 65 kDa protein kinase (PK) purified from wheat embryos. The extent of phosphorylation by protein kinase shows that PK has similar affinity towards both PAPI and PAPII, whereas the phosphate incorporation in PAPII is twofold higher than PAPI suggesting their distinct chemical nature.
Sharma
Neelam
N
Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, CO 80523, USA. neelam26@hotmail.com
Dey
Madhusudan
M
Satpathy
Minati
M
Sachar
R C
RC
D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D011062:Polynucleotide Adenylyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology*; D014908:Triticum / Q000201:enzymology*
D007527:Isoenzymes; D010750:Phosphoproteins; D011494:Protein Kinases; D011062:Polynucleotide Adenylyltransferase