Kinetic characterisation of the enzymatic activity of the eEF-2-specific Ca2(+)-and calmodulin-dependent protein kinase III purified from rabbit reticulocytes.
The Ca2(+)-and calmodulin-dependent protein kinase III, which specifically phosphorylates the eukaryotic elongation factor 2 (eEF-2), has been purified to apparent homogeneity from the post-ribosomal fraction of rabbit reticulocytes by an efficient four-step method. The method results in a more than 4000-fold purification of the enzyme. SDS-gel electrophoresis showed that the purified kinase contained only one polypeptide with the apparent molecular mass of 90 kDa. The kinase activity was associated with the 90-kDa protein as shown by analyzing the phosphorylating activity of SDS gel electrophoretically purified protein electroblotted to nitrocellulose membranes. The purified kinase was dependent on Ca2+, Mg2+ and calmodulin for activity. Kinetic analysis of the phosphorylation reaction indicates that the turnover number of the kinase was approximately 1 s-1. The Km for the two substrates ATP and eEF-2 was calculated to be approximately 100 microM and 10 microM, respectively. The activity of the kinase was competitively inhibited by cAMP. The inhibition constant Ki (0.5 mM) was found to be in the same order of magnitude as that calculated for the competitive product inhibition caused by ADP. GTP was ten-times less efficient as competitor, indicating that the kinase had a preference for adenosine nucleotides. Phosphorylation of eEF-2 did not interfere with the diphtheria-toxin-catalysed ADP-ribosylation of the factor nor did ADP-ribosylation inhibit phosphorylation.
Nilsson
A
A
Department of Cell Biology, Stockholm University, Sweden.
Carlberg
U
U
Nygård
O
O
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects / Q000201:enzymology*; D013379:Substrate Specificity; D019892:eIF-2 Kinase
D000246:Adenosine Diphosphate Ribose; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D019892:eIF-2 Kinase; D002118:Calcium