PURIFICATION AND CHARACTERIZATION OF 2 CASEIN KINASE TYPE-II ISOZYMESFROM BOVINE BRAIN GRAY-MATTER

Highly purified casein kinase II (CK II) isozymes from bovine brain gr ay matter (BBGM) were obtained by means of a new purification procedur e consisting of one phosphocellulose and three Mono-Q steps. The phosp hocellulose eluate showed two BBGM-CK II activities. The first minor c omponent (BBGM-CK IIa) was eluted with 0.9 M NaCl and the major compon ent was eluted at 1.1 M NaCl (BBGM-CK IIb). The protein complexes resp onsible for these two activities were comprised of three subunits, i.e ., alpha (40 kDa), alpha' (38 kDa), and beta (28 kDa), with various su bunit ratios. The two isozymes displayed the same behavior on Superose 12 fast protein liquid chromatographic gel filtration and sucrose den sity centrifugation. BBGM-CK IIa and b showed chromatographic and bioc hemical differences including differing K-m for ATP and GTP and K-i fo r heparin and 2,3-bisphosphoglycerate. The properties of the main peak (BBGM-CK IIb) were studied in detail. The stimulatory effect of Mg2+, Mn2+, and Co2+ was highly dependent both on the nature of the substra te and on ionic type and concentration. It is surprising that with pho svitin as substrate, BBGM-CK IIb was fully active even in the absence of Mg2+ and NaCl. The inhibitory effect of heparin and the stimulatory effects of NaCl, KCl, spermine, and polylysine were highly dependent on the ionic strength, buffer type, and substrate. BBGM-CK II isozymes phosphorylated stathmine in the presence of polylysine, but the requi rement for polybasic compounds was not absolute, as is the case with c almodulin and clathrin beta-light chain. The unusual chromatographic b ehavior and biochemical properties of these BBGM-CK II isozymes, compa red with the classical CK II, could be explained at least in part by t heir subunit ratios.