Purification and characterization of glutathione transferases with an activity toward nitroglycerin from human aorta and heart. Multiplicity of the human class Mu forms.
Although recent studies suggest involvement of glutathione transferase (GST) of blood vessels in vasodilation by nitroglycerin, GST forms in blood vessels remain to be studied. In this study, three GST forms (pI values 8.3, 6.6, and 4.8) were purified from human aorta and four (pI values 6.0, 5.6, 5.3, and 4.6) from the heart by affinity chromatography followed by chromatofocusing. The major form of both aorta (pI 4.8) and heart (pI 4.6) was identified as GST-pi, and the other five forms were immunologically related to GST-mu, suggesting that the five belong to the Mu class. Among nine human GST forms, including three in the Alpha class purified from the liver, GST-mu, aorta pI 8.3 form, and GST-I (a form of the Alpha class, corresponding to GST-epsilon (B1B1)) showed high activities toward nitroglycerin, 1.08, 0.85, and 0.78 units/mg protein, respectively. GST-pi did not exhibit the activity. The Km values of the aorta form (pI 8.3) for glutathione (GSH) and nitroglycerin were calculated as 0.12 and 1.1 mM, respectively. The Km values of GST-mu and GST-I for GSH were 0.29 and 0.09 mM, and those for nitroglycerin were 2.5 and 0.3 mM, respectively. The activity of the pI 8.3 form as well as GST-mu toward nitroglycerin was inhibited by bromosulfophthalein, which is known to inhibit the relaxation of rabbit aorta induced by nitroglycerin, at the lower concentration (IC50, 2 microM) than was GST-I (IC50, 32 microM). Two-dimensional gel electrophoresis and N-terminal amino acid sequence analysis revealed that five forms in the Mu class are homo- or heterodimers of five different subunits named M1 (pI 7.0/Mr 27,000), M2 (6.6/27,000), M3 (6.0/27,000), N1 (6.5/26,500), and N2 (5.9/26,500). The subunit structures of the five forms are as follows: pI 8.3 form, M1M2; 6.6 form, M2N1; 6.0 form, M3M3; 5.6 form, M3N2; and 5.3 form, N2N2. M3 and N2 seem to correspond to the subunits of GST-mu, and -4 (Board, P. G., Suzuki, T., and Shaw, D. C. (1988) Biochim. Biophys. Acta 953, 214-217), respectively. These subunits except N1 are different from each other at two or three positions in the first 20 residues of N-terminal amino acid sequence. These results indicate the presence of five different subunits in the human Mu class and also suggest that GST-M1M2 and -M2N1 found in the aorta are involved in the expression of the pharmacologic effect of nitroglycerin.
Tsuchida
S
S
Second Department of Biochemistry, Hirosaki University School of Medicine, Japan.
Maki
T
T
Sato
K
K
D000595:Amino Acid Sequence; D001011:Aorta / Q000201:enzymology*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005982:Glutathione Transferase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009206:Myocardium / Q000378:metabolism*; D005996:Nitroglycerin / Q000378:metabolism*; D013379:Substrate Specificity
D007527:Isoenzymes; D046911:Macromolecular Substances; D005982:Glutathione Transferase; D005996:Nitroglycerin