EF-hands calcium binding regulates the thioredoxin reductase/thioredoxin electron transfer in human keratinocytes.
Thioredoxin reductases purified from Escherichia coli from human metastatic melanoma tissue and from human keratinocytes are subject to allosteric inhibition by calcium. 45Calcium has been used to show that this enzyme contains a single binding site. Bound calcium does not exchange from thioredoxin reductase upon dialysis for 48 hours or upon exposure to 10(-3) M EGTA. An intelligenetics computer analysis yielded a single EF-hands calcium binding site on E. coli thioredoxin reductase with homology to the first EF-hands site on calmodulin. Calcium exchange from the enzyme requires the addition of the natural electron acceptor oxidized thioredoxin which causes a concentration dependent slow exchange. Due to the large conformational change caused by calcium binding to thioredoxin reductase it has been possible to separate Calcium-free and Calcium-bound enzyme by FPLC chromatography. Human keratinocytes contain 5% thioredoxin reductase in their acidic protein cytosol fraction. The influence of extracellular calcium concentration on the intracellular equilibrium between calcium bound versus calcium free thioredoxin reductase has been assessed. This equilibrium was shown to determine the redox status of keratinocytes via the reduction of thioredoxin. Our results provide the first evidence for calcium dependent regulation of redox conditions in the human epidermis.
Schallreuter
K U
KU
Department of Dermatology, University of Hamburg, FRG.
Pittelkow
M R
MR
Wood
J M
JM
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007633:Keratins; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D012867:Skin / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000302:isolation & purification / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
D001426:Bacterial Proteins; D013879:Thioredoxins; D007633:Keratins; D009247:NADH, NADPH Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase; D002118:Calcium