Creatine kinase is modified by 2-chloromercuri-4-nitrophenol at the active site thiols with complete inactivation.
Creatine kinase modified by mercurials has been reported to be fully reactive as the native enzyme. This was ascribed to the modification of a second class of thiol groups instead of the reactive thiols at the active site (Laue, M.C. and Quiocho, F.A. (1977) Biochemistry 16, 3838-3845). It has now been shown by spectrophotometric titration and fluorescence studies that 2-chloromercuri-4-nitrophenol (MNP) reacts preferentially with the active-site thiol. Moreover, if the activity of the modified enzyme is determined in the absence of added bovine serum albumin or other enzymes, as usually employed in coupled activity assay systems for creatine kinase, the modified enzyme is completely inactive. Addition of an excess of bovine serum albumin or rabbit muscle glyceraldehyde-3-phosphate dehydrogenase restores the activity of the enzyme to over 80% of its original level. It appears that the active thiol groups at the active site of creatine kinase are after all modified by MNP with complete inactivation.
Wu
H
H
Institute of Biophysics, Academia Sinica, Beijing, People's Republic of China.
Yao
Q Z
QZ
Tsou
C L
CL
D000818:Animals; D002730:Chloromercurinitrophenols / Q000494:pharmacology*; D003402:Creatine Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D066298:In Vitro Techniques; D010663:Phenylmercury Compounds / Q000494:pharmacology*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000502:physiology; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D002730:Chloromercurinitrophenols; D010663:Phenylmercury Compounds; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C026149:2-chloromercuri-4-nitrophenol; D003402:Creatine Kinase