Abstract
To verify a possible involvement of glutathione transferase π in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with K(d) values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase π was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The K(i) calculated was 4 μM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest that involvement of glutathione transferase π in the hemin transport.
| Original language | English |
|---|---|
| Pages (from-to) | 493-497 |
| Number of pages | 5 |
| Journal | European Journal of Biochemistry |
| Volume | 189 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 1990 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Interaction of hemin with placental glutathione transferase. / Caccuri, A. M.; Aceto, A.; Piemonte, F.; Di Ilio, C.; Rosato, N.; Federici, G.
In: European Journal of Biochemistry, Vol. 189, No. 3, 1990, p. 493-497.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Interaction of hemin with placental glutathione transferase
AU - Caccuri, A. M.
AU - Aceto, A.
AU - Piemonte, F.
AU - Di Ilio, C.
AU - Rosato, N.
AU - Federici, G.
PY - 1990
Y1 - 1990
N2 - To verify a possible involvement of glutathione transferase π in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with K(d) values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase π was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The K(i) calculated was 4 μM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest that involvement of glutathione transferase π in the hemin transport.
AB - To verify a possible involvement of glutathione transferase π in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with K(d) values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase π was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The K(i) calculated was 4 μM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest that involvement of glutathione transferase π in the hemin transport.
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UR - http://www.scopus.com/inward/citedby.url?scp=0025370743&partnerID=8YFLogxK
U2 - 10.1111/j.1432-1033.1990.tb15514.x
DO - 10.1111/j.1432-1033.1990.tb15514.x
M3 - Article
C2 - 2351133
AN - SCOPUS:0025370743
VL - 189
SP - 493
EP - 497
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
SN - 0014-2956
IS - 3
ER -