Calcium regulates thioredoxin reductase in human metastatic melanoma.
Thioredoxin reductase has been purified from human metastatic melanotic melanoma and amelanotic melanoma tissues. Enzyme from the melanotic melanoma tissue contains bound calcium showing classical sigmoidal allosteric kinetics, whereas enzyme from the amelanotic melanoma yielded normal Michaelis-Menten saturation with substrate. Calcium inhibition can be partially reversed by oxidized thioredoxin. 45Ca has been used to label the amelanotic melanoma enzyme in order to determine the number of calcium-binding sites. These isotope experiments yielded only one calcium-binding site per enzyme molecule. Enzyme labeled with 45Ca was dialyzed for 24 h without loss of radioactivity, but the addition of oxidized thioredoxin to this labeled enzyme caused 60% calcium exchange in 24 h. Comparative studies with Escherichia coli thioredoxin reductase showed similar calcium inhibition as well as partial reactivation with oxidized thioredoxin. The enzyme from E. coli previously sequenced by others, showed considerable homology with the first EF-hands calcium-binding site of calmodulin. Detailed calcium-binding studies indicated that 10(-5) M of this fast exchange ion was sufficient to cause allosteric regulation in 10 min. This strong calcium-binding property could explain the allosteric nature of the thioredoxin reductase purified from human metastatic melanotic melanoma and its role in the regulation of melanin biosynthesis.
Department of Dermatology, University of Hamburg, F.R.G.
K U
KU
Schallreuter
J M
JM
Wood
D002118:Calcium / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008545:Melanoma / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry; D013880:Thioredoxin-Disulfide Reductase / Q000302:isolation & purification / Q000378:metabolism*
D004228:Dithionitrobenzoic Acid; D009247:NADH, NADPH Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase; D002118:Calcium