Inactivation and the site of labeling of F1-ATPase from Mycobacterium phlei by dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and quinacrine mustard |
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Authors: | Neeraj Agarwal Vijay K Kalra |
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Institution: | Department of Biochemistry, University of Southern California, School of Medicine, Los Angeles, CA 90033 U.S.A. |
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Abstract: | The F1-ATPase from Mycobacterium phlei is inactivated by dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and quinacrine mustard. The inactivation is both time-and concentration-dependent and in the case of DCCD being more pronounced at acidic pH. The minimum inactivation half-time () for DCCD, NBD-Cl and quinacrine mustard was observed to be 14, 6 and 7 min, respectively. Inactivation of F1-ATPase resulted in the incorporation of 14C]DCCD as well as 14C]NBD-Cl into α and γ subunits. The incorporation of label into α and γ subunits, utilizing 14C]NBD-Cl, was reversible by dithiothreitol. Complete inactivation, by linear extrapolation to zero activity, revealed that 4 mol 14C]DCCD and 4 mol 14C]NBD-Cl bind per mol F1-ATPase. Kinetic and binding studies show that these probes bind to site(s) distinct from ATP-binding site in F1-ATPase from M. phlei. |
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Keywords: | Dicyclohexylcarbodiimide Quinacrine mustard Enzyme inactivation (M phlei) DCCD dicyclohexylcarbodiimide EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EEDQ Mes 4-morpholineethanesulphonic acid NBD-Cl 7-chloro-4-nitrobenzo-2-oxa-1 3-diazole TEMED To whom requests for reprints should be addressed |
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