ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity

Biochemistry
S AwasthiS Pikula

Abstract

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that ...Continue Reading

Citations

Jan 30, 2007·Cancer Metastasis Reviews·Sushma YadavSanjay Awasthi
Jul 14, 1999·Toxicology Letters·P ZimniakY C Awasthi
Sep 6, 2000·Clinical and Experimental Pharmacology & Physiology·S Pepe
Feb 26, 2010·Journal of Toxicology and Environmental Health. Part B, Critical Reviews·Yogesh C AwasthiRajendra Sharma
May 7, 2009·Cancer Research·Sharad S SinghalSanjay Awasthi
Jul 17, 2008·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Sanjay AwasthiArthur E Frankel
Jul 5, 2001·Archives of Biochemistry and Biophysics·R SharmaY C Awasthi
Oct 4, 2006·Expert Opinion on Drug Metabolism & Toxicology·Aalok NadkarSanjay Awasthi
Jan 26, 2010·Biochemical Pharmacology·Rit VatsyayanSharad S Singhal
Nov 6, 2008·Biochemical Pharmacology·Sharad S SinghalJyotsana Singhal
Jul 17, 2003·International Journal of Cancer. Journal International Du Cancer·Sanjay AwasthiYogesh C Awasthi
Feb 15, 2005·Biochemical and Biophysical Research Communications·Sushma YadavSanjay Awasthi
Aug 21, 2007·Biochemical and Biophysical Research Communications·Mikael C Herlevsen, Dan Theodorescu
Mar 18, 1999·Toxicology and Applied Pharmacology·S AwasthiY C Awasthi
Nov 16, 2002·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Sanjay AwasthiYogesh C Awasthi
Dec 14, 2018·Oncotarget·Sanjay AwasthiSharda P Singh
Jun 6, 2020·Cancers·Chhanda BoseSanjay Awasthi
Mar 25, 2018·Proceedings of the National Academy of Sciences of the United States of America·Sanjay AwasthiSharad S Singhal
Feb 17, 2019·Biochimica Et Biophysica Acta. Reviews on Cancer·Sharad S SinghalSanjay Awasthi

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