Lead ion effect on creatine kinase: equilibrium and kinetic studies of inactivation and conformational changes.
The effects of lead ions on creatine kinase (CK) were studied by measuring activity changes, intrinsic fluorescence spectra and 8-anilo-1-naphthalenesulfonate (ANS)-binding fluorescence along with size-exclusion chromatography (SEC). Below 5 mM Pb(2+) concentration, there was nearly no change of the enzyme activity and a slight change of the ANS-binding fluorescence. The CK activity decreased significantly from 10 to 25 mM Pb(2+) concentrations. No residual activity was observed above 25 mM Pb(2+). The kinetic time courses of inactivity and unfolding were all mono-phase courses with the inactivation rate constants being greater than the unfolding rate constants for the same Pb(2+) concentration. The changes in fluorescence maximum and fluorescence intensity were relatively slow for 40-80 mM Pb(2+) as well as in the initial stage for less than 5 mM Pb(2+), showing that two transition states exist for Pb(2+) induced equilibrium-unfolding curves. The intrinsic fluorescence spectra and ANS-binding fluorescence measurements showed that even for high Pb(2+) concentrations, CK did not fully unfold. Additionally, the SEC results showed that the enzyme molecule still existed in an inactive dimeric state at 20 and 40 mM Pb(2+) solutions. All the results indicated the presence of at least one stable unfolding equilibrium intermediate of CK during Pb(2+) unfolding.
Zhou
Hong-Wei
HW
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, PR China. zhm-dba@mail.tsinghua.edu.cn
Park
Yong-Doo
YD
Zhou
Hai-Meng
HM
D000818:Animals; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D007854:Lead / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D007854:Lead; D003402:Creatine Kinase