Effects of pH and KCl on the conformations of creatine kinase from rabbit muscle. Infrared, circular dichroic and fluorescence studies.
The activity loss of creatine kinase (CK), observed at low pH (midpoint was 4.8) corresponded to the monomerization of the dimeric protein and was correlated with structural changes. The acid-induced unfolding was not complete at this pH, as probed by circular dichroic (CD) and fluorescence methods. Further decrease of pH, led to a second transition (midpoint was pH 3.5). The loss of activity was irreversible at pH 4.8 (< 20% native activity was recovered) while it was almost fully reversible (> 90% of native activity was recovered) for the enzyme incubated at pH 0.9-2.5. The amount of intermolecular beta-sheets (monitored with the 1620 cm-1 infrared component band) was maximal when the enzyme was incubated at pH 4.8, as a consequence of protein aggregation, while it was minimal at extremes of pH and at low ionic strength. Acid-induced and alkaline-induced denaturations promoted different structural changes, leading to distinct partially unfolded conformational states. The addition of KCl (from 0.05 M to 0.5 M) to an acidic solution of monomeric creatine kinase (pH 1.6) resulted in a highly cooperative transition from the partially unfolded conformation (UA) to the more compact conformation (A) with the properties of a molten globule, as probed by CD spectra and by fluorescence. The formation of intermolecular beta-sheets in the compact conformation was observed by infrared spectroscopy, indicating formation of unstable aggregates.
Raimbault
C
C
Laboratoire de Physico-Chimie Biologique, Université Claude Bernard, Lyon 1, CNRS URA, France.
Couthon
F
F
Vial
C
C
Buchet
R
R
D000818:Animals; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry*; D004789:Enzyme Activation; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D009132:Muscles / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D011817:Rabbits; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared
D011189:Potassium Chloride; D003402:Creatine Kinase