Separation and Purification of both CK-I and CK-II Casein Kinases in Developing Maize Endosperm — Phosphorylation of Native HMG Proteins

Abstract

Summary Two enzymes possessing casein kinase activity were isolated from maize endosperm (3 weeks after anthesis). One of them met most of the type II (CK-II) casein kinase criteria and the other of type I (CK-I) and were named CK-2D and CK- 1E, respectively. These enzymes were further purified by ammonium sulfate precipitation, DEAE-cellulose, Shephadex G-75, Heparin-sepharose and ATP-agarose chromatography. CK- IE could bind on an ATP-agarose affinity column while CK-2D could not. The catalytic subunits of both enzymes were identified as a 36 kDa polypeptide by using the procedure of in situ phosphorylation after SDS/PAGE in an active gel polymerized in the presence of casein. CK-2D required 10 mmol · L−1 Mg2+ for maximal activity, could utilize equally well either ATP or GTP as phosphate donors, was slightly stimulated by low and inhibited by high concentrations of polyamines, was inhibited by low concentrations of heparin, was insensitive to polylysine, had an isoelectric point in the neutral region (pI 7.1), had Km values for ATP (11 µmol · L−1) and GTP (l5 mµol · L−1) and phosphorylated serine and threonine on casein. Also, it was in situ autophosphorylated in the presence of GTP while autophosphorylated by ATP/GTP under standard enzyme assay conditions. On the other hand, CK-IE required 15 mmol · L−1 Mg2+ for maximal activity, could utilize only ATP as phosphate donor with a Km value of 11µmol · L−1, was inhibited by polyamines, was insensitive to heparin and polylysine, specifically phosphorylated serine on casein and was not autophosphorylated. Both enzymes exclusively phosphorylated acid protein substrates (casein, phosvitin). Regarding the native substrates, non-histone chromosomal high mobility group (HMG) proteins were isolated from endosperm maize chromatin (3 weeks after anthesis) and used as substrates. The phosphorylation patterns of HMG-proteins of the two enzymes were different. Three HMG-proteins of Mrs about 14 kDa, 17kDa and 20kDa were phosphorylated by CK-2D while only one ofMr about 17kDa by CK-IE. This is the first report that HMG-proteins were phosphorylated by CK-I casein kinase.