V.V. Risnik

Some properties of the nucleotide-binding site of troponin T kinase-casein kinase type II from skeletal muscle

Investigation of properties of skeletal muscle troponin T kinase (EC 2.7.1.37) has revealed that the enzyme belongs to the group of casein kinases of the second type. The enzyme consists of two subunits with apparent molecular weights of 44 000 and 26 000 and contains a protein with molecular weight of 39 000, which is probably the proteolytic fragment of the 44 000 subunit. The substrate specificity of troponin T kinase was tested, using 20 analogs of the nucleotide. The enzyme has a low substrate specificity toward the purine base and uses both ATP and GTP as substrates. Modification of the ribose ring does not influence the enzyme interaction with the nucleotide; however, the cleavage of ribose leads to a decrease of the enzyme-nucleotide interaction. Elimination of the gamma-terminal phosphate or its modification by bulky hydrophobic radicals do not affect this interaction. A comparison of the Ki values for different analogs suggests that the interaction of troponin T kinase with the nucleotide occurs via the binding of the purine base and the beta-phosphate group of the analog.

Casein kinases I and II bound to pig brain microtubules

Summary1.Microtubules prepared from pig brain by two cycles of assembly-disassembly comprise cyclic nucleotide-independent protein kinase activity with phosvitin and troponin T as substrates.2.Phosphocellulose chromatography resolved two phosvitin kinase activity peaks, one of which coincided with the troponin T kinase peak.3.The activity peak corresponding to troponin T kinase was inhibited by heparin (I50 = 0.06µg/ml), whereas the other phosvitin kinase peak was unaffected.4.Both kinase fractions phosphorylated tubulin and microtubule-associated protein (MAP-2).5.It is concluded that pig brain microtubules contain bound casein kinases I and II. The association may target the action of these kinases toward microtubular proteinsin vivo.