Similarities and differences in the properties of multiple isoforms of maize endosperm casein kinase I (CK-I)

Abstract

Two novel type I casein kinases named CK-1B and CK-1C have been purified from maize endosperm (three weeks after anthesis) by a six step procedure involving ammonium sulfate precipitation, DEAE-cellulose, Sephadex G-75, Heparin-sepharose, and ATP-agarose chromatography. The catalytic subunits of both enzymes were identified as a 35-37 kDa polypeptide doublet by in situ phosphorylation after SDS/PAGE in active casein gel. Both enzymes required 5-10 mmol · L −1 Mg 2+ for maximal activity, could utilize only ATP as phosphate donor, were insensitive to heparin, were not autophosphorylated, had a pH optimum at pH 7 to 8.5, and exclusively phosphorylated acidic proteins (casein, phosvitin). Regarding the enzyme differences, their properties were as follows: a) CK-1B could bind on ATP-agarose affinity column, while CK-1C could not; b) the activity of CK-1C was strongly stimulated at low concentrations (1 mmol/L) of spermine, while that of CK-1B was inhibited; c) CK-1B and CK-1C Km values for ATP were 11 μmol · L −1 and 26 μmol · L −1 , respectively; d) Mg 2+ could substituted by Mn 2+ in the CK-1B catalytic activity (by about 80 percnt;); e) CK-1B phosphorylated serine, while CK-1C both serine and threonine on casein. The combination of these results with those from Babatsikos and Yupsanis (2000) brings the number of investigated maize endosperm CK-I isoforms to three (CK-1B, CK-1C, and CK-1E). This is the first biochemical approach demonstrating that multiple isoforms of CK-I casein kinases are present in the same plant tissue.