An analysis of the reassembly of denatured creatine kinase from monkey brain.
Creatine kinase isolated from monkey brain was characterized with respect to denaturation/inactivation and renaturation/reactivation/reassociation in order to determine the mechanism of reassembly. Enzyme unfolded in 8 M urea exhibits several characteristics of denatured protein: complete loss of enzymatic activity, decrease in intrinsic fluorescence with a red shift in the emission maximum and loss of circular dichroism at 220 nm. The renatured protein reassembles to its apparently native condition as judged by these criteria, but small differences of uncertain origin persist. Dependence of activity and fluorescence on denaturant concentration indicate that inactivation is more sensitive to urea than is unfolding; spectral changes at the intermediate urea concentrations suggest formation of aggregated protein. Upon dilution, enzyme previously exposed to 8 M urea for 40 min regains 70-80% native activity, independent of protein concentration over the range of 0.56-160 nM. Reactivation kinetics, measured using the assay mixture with and without trypsin, are independent of protein concentration, and are adequately described by a single rate constant, 3.2 X 10(-3) s-1 and 4.2 X 10(-3) s-1, respectively. Reactivation is completed 20-30 min after initiation of renaturation. Fluorescence changes during refolding are at least biphasic, exhibiting a rapid increase, then a slow decrease completed at approximately 15-20 min after initiating refolding. Reassociation is measured by competitive hybridization between dimerizing B subunits and M subunits to form MB heterodimer. The time dependent decay in heterodimer formation during competitive dimerization shows that reassociation is completed between 60 and 90 min after initiation of reassembly. These results indicate that the brain isozyme of creatine kinase, like the muscle form, is composed of subunits which do not require association for expression of catalytic activity. Furthermore, a comparison of spectral data and susceptibility to trypsin inactivation between the muscle and brain isozymes supports previous suggestions that in the native state, the brain isozyme is a conformationally looser, more open protein.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008253:Macaca mulatta; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D055503:Protein Multimerization; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D007527:Isoenzymes; D014508:Urea; D003402:Creatine Kinase; D014357:Trypsin