Dinitrophenyl S-glutathione ATPase purified from human muscle catalyzes ATP hydrolysis in the presence of leukotrienes.
Dinitrophenyl S-glutathione (Dnp-SG) ATPase has been purified from human muscle to apparent homogeneity using Dnp-SG affinity chromatography and immunoaffinity chromatography using antibodies raised against human erythrocyte Dnp-SG ATPase. The enzyme purified from human muscle showed a subunit M(r) value of about 38 kDa in denaturing gels. The M(r) value of the native enzyme as determined by Sephadex G-200 gel filtration was found to be about 80 kDa, which indicates that it is a dimer. The N-terminus of the enzyme was blocked. Its immunological and kinetic properties were similar to Dnp-SG ATPase of human erythrocytes. Besides catalyzing the ATP hydrolysis in the presence of Dnp-SG, the muscle enzyme also catalyzed ATP hydrolysis in the presence of various leukotrienes, namely LTC4.LTD4, LTE4, and N-acetyl LTE4. The specific activity of the enzyme toward LTC4 was relatively higher than other GSH-xenobiotic conjugates. The muscle enzyme exhibits a low Km value for all leukotrienes as compared to Dnp-SG, indicating high affinity of the enzyme for leukotrienes as activators. The enzyme also catalyzed ATP hydrolysis in the presence of GSH conjugates of endogenously generated fatty acid epoxides. Our results might suggest that Dnp-SG ATPase is involved in the transport of GSH conjugates, leukotrienes, and other organic anions in muscle, erythrocytes, liver, and probably other tissues.
Saxena
M
M
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Singhal
S S
SS
Awasthi
S
S
Singh
S V
SV
Labelle
E F
EF
Zimniak
P
P
Awasthi
Y C
YC
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active; D002846:Chromatography, Affinity; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015289:Leukotrienes / Q000378:metabolism*; D008670:Metals; D009132:Muscles / Q000201:enzymology*; D011233:Precipitin Tests
D015289:Leukotrienes; D008670:Metals; C030190:S-(2,4-dinitrophenyl)glutathione; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C068387:S-(dinitrophenyl)glutathione ATPase; D005978:Glutathione