Inactivation of creatine kinase by high pressure may precede dimer dissociation.
The effects of hydrostatic pressure on creatine kinase activity and conformation were investigated using either the high-pressure stopped-flow method in the pressure range 0.1-200 MPa for the activity determination, or the conventional activity measurement and fluorescence spectroscopy up to 650 MPa. The changes in creatine kinase activity and intrinsic fluorescence show a total or partial reversibility after releasing pressure, depending on both the initial value of the high pressure applied and on the presence or absence of guanidine hydrochloride. The study on 8-anilinonaphthalene-1-sulfonate binding to creatine kinase under high pressure indicates that the hydrophobic core of creatine kinase was progressively exposed to the solvent at pressures above 300 MPa. This data shows that creatine kinase is inactivated at low pressure, preceding both the enzyme dissociation and the unfolding of the hydrophobic core occurring at higher pressure. Moreover, in agreement with the recently published structure of the dimer, it can be postulated that the multistate transitions of creatine kinase induced both by pressure and guanidine denaturation are in direct relationship with the existence of hydrogen bonds which maintain the dimeric structure of the enzyme.
Zhou
J M
JM
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Zhu
L
L
Balny
C
C
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004789:Enzyme Activation; D019791:Guanidine / Q000494:pharmacology; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation; D011817:Rabbits; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D012997:Solvents; D003402:Creatine Kinase; D019791:Guanidine