Localization, purification, and characterization of the rabbit sarcoplasmic reticulum associated calmodulin-dependent protein kinase.
The Ca2+/calmodulin dependent protein kinase associated with the sarcoplasmic reticulum membranes (SR CaM kinase) plays a specific and important role in the modulation of both Ca2+ uptake and release functions of the sarcoplasmic reticulum itself. In this work we have localized a 60 kD SR CaM kinase in slow and fast twitch rabbit skeletal muscle fractions; the kinase was present in both the longitudinal and the junctional sarcoplasmic reticulum. We then developed a procedure for the purification of the active kinase from the longitudinal sarcoplasmic reticulum and performed biochemical and functional characterization of the enzyme. Differently from what was previously suggested, our analysis shows that the biochemical properties of the purified SR CaM kinase (Ca2+ sensitivity, K0.5 for calmodulin, Km for ATP, IC50 for the specific inhibitory peptide (290-309), autophosphorylation properties) are not significantly different from those of the soluble multifunctional CaM kinase II. Moreover, we show that the purified SR CaM kinase retains the ability to autophosphorylate in a Ca2+/calmodulin-dependent manner, becoming a Ca2+-independent enzyme. In the light of the knowledge of the rabbit SR CaM kinase biochemical properties, we propose and discuss the possibility that, under physiological conditions, the activity of the autophosphorylated kinase persists when the Ca2+ transient is over.
Pelosi
M
M
Department of Biological Chemistry, University of Padova, Padova, 35121, Italy. mpelosi@civ.bio.unipd.it.
Donella-Deana
A
A
D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium