Interaction of human thiol-specific antioxidant protein 1 with erythrocyte plasma membrane.
During the purification from human erythrocytes, human thiol-specific antioxidant protein 1 (hTSA1), one human member of the TSA/alkyl hydroperoxide reductase subunit C (AhpC) family, was fragmented to a molecular mass of 20 323.9300. The fragmented form, in contrast to the intact form, did not bind to the C-terminal peptide (Gln-185-Gln-197) antibody. On the basis of the molecular mass of the fragmented form, the cleavage site was calculated to be between Val-186 and Asp-187. The C-terminal region of hTSA1 appeared to be unnecessary for the antioxidant reaction. In addition to hTSA1, two isoenzymes (hORF06 and hTSA2) were detected in the soluble fraction, whereas only hTSA1 was detected in the membrane fraction. A membrane binding study shows that the intact form binds to erythrocyte plasma membrane but the fragment does not, which suggests that the deleted C-terminal legion (Asp-187-Gln-197) is required for the membrane binding. A model membrane study using phospholipid vesicle showed a strong association of hTSA1 with the phospholipid. Human TSA1 exhibited high catalytic activity for the reduction of the fatty acid hydroperoxide as indicated by K(m) and V(max) (89.9 microM for linoleic acid hydroperoxide, 28.64 micromol(-1) min(-1) mg(-1), respectively). In this paper, we are making the first report of the involvement of the C-terminal region of hTSA1 in membrane binding as evidence supporting the existence of the membrane-associated forms in the erythrocyte. On the basis of our observations, we suggest that hTSA1 can act as a very effective antioxidant to remove oxidative stresses not only in matrix as a free form but also in the membrane surface of red blood cells (RBC) as a membrane-associated form.
Cha
M K
MK
National Creative Research Initiatives Center for Antioxidant Proteins, Department of Biochemistry, Paichai University, Taejon 302-735, Korea.
Yun
C H
CH
Kim
I H
IH
D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008054:Lipid Peroxides / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D009363:Neoplasm Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D061105:Peroxiredoxin III; D054464:Peroxiredoxins; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism
D000975:Antioxidants; D008054:Lipid Peroxides; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D020033:Protein Isoforms; D010544:Peroxidases; C516115:PRDX3 protein, human; D061105:Peroxiredoxin III; D054464:Peroxiredoxins