Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase.
The course of refolding and reactivation of urea-denatured creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) has been studied in the absence and presence of molecular chaperonin GroEL. The enzyme was denatured in Tris--HCl buffer containing 6 M urea for 1 h. In the refolding studies, the denatured enzyme was diluted 60-fold into the same buffer containing GroEL or not for activity, turbidity, fluorescence measurements and polyacrylamide gel electrophoresis. The results show that the reactivation process is dependent of creatine kinase concentration in the concentration range 2.5--4 microM. The levels of activity recovery decrease with increasing enzyme concentration because of the formation of wrong aggregates. The molecular chaperonin GroEL can bind the refolding intermediate of creatine kinase and thus prevent the formation of wrong aggregates. This intermediate is an inactive dimeric form that is in a conformation resembling the 'molten globule' state.
Li
S
S
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 10084, People's Republic of China.
Bai
J H
JH
Park
Y D
YD
Zhou
H M
HM
D000818:Animals; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism*; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D009132:Muscles / Q000201:enzymology; D011485:Protein Binding / Q000502:physiology; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D014508:Urea / Q000737:chemistry
D018834:Chaperonin 60; D018832:Molecular Chaperones; D014508:Urea; D003402:Creatine Kinase