Identification of S100b protein as copper-binding protein and its suppression of copper-induced cell damage.
We have isolated from bovine brain a protein with a high capacity to inhibit the copper ion-catalyzed oxidation of L-ascorbate and identified it as S100b protein, an EF-hand calcium-binding protein, by sequencing its proteolytic peptides. Copper binding studies showed that this protein has four copper-binding sites per dimeric protein molecule with a dissociation constant of 0.46 microM and that in the presence of L-ascorbate, copper ions bind to a total of six binding sites with a great increase in affinity. Furthermore, we examined whether S100b protein can prevent copper-induced cell damage. Bovine S100b protein was found to suppress dose-dependently the hemolysis of mouse erythrocytes induced by CuCl2. We transformed Escherichia coli cells with pGEX-5X-3 vector containing a cDNA for rat S100b protein, so that this protein could be expressed as a fusion protein with glutathione S-transferase. The transformed cells were demonstrated to be markedly resistant to a treatment with CuCl2 plus H2O2 as compared with the control cells expressing glutathione S-transferase alone. These results indicate that S100b protein does suppress oxidative cell damage by sequestering copper ions.
Nishikawa
T
T
Department of Biochemistry, Wakayama Medical College, Wakayama 640, Japan.
Lee
I S
IS
Shiraishi
N
N
Ishikawa
T
T
Ohta
Y
Y
Nishikimi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D003300:Copper / Q000378:metabolism* / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D006461:Hemolysis / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000633:toxicity; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins
D002135:Calcium-Binding Proteins; D009414:Nerve Growth Factors; D011993:Recombinant Fusion Proteins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579159:S100b protein, mouse; C579158:S100b protein, rat; D003300:Copper; D006861:Hydrogen Peroxide; D001205:Ascorbic Acid