ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity.
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 the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.
Awasthi
S
S
Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas, USA. sawasthi@utmb.edu
Singhal
S S
SS
Srivastava
S K
SK
Torman
R T
RT
Zimniak
P
P
Bandorowicz-Pikula
J
J
Singh
S V
SV
Piper
J T
JT
Awasthi
Y C
YC
Pikula
S
S
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004317:Doxorubicin / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins; D019920:Photoaffinity Labels; D013050:Spectrometry, Fluorescence
D001386:Azides; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D019920:Photoaffinity Labels; C086860:glutathione transporter; C030190:S-(2,4-dinitrophenyl)glutathione; C017040:8-azidoadenosine 5'-triphosphate; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C068387:S-(dinitrophenyl)glutathione ATPase; D005978:Glutathione