ECTO-NOX (ENOX) proteins of the cell surface lack thioredoxin reductase activity.
This study was to determine if ENOX proteins of the cell surface act as cell surface thioredoxin reductases. To measure formation of thiols a turbimetric insulin assay was used. No turbidity was observed with insulin alone or with insulin plus DTT. However, the combination of insulin +DTT + recombinant his-tagged ENOX2 (tNOX) did result in increased turbidity. An ENOX1 (CNOX) preparation also resulted in turbidity changes. In contrast, we were unable to demonstrate ENOX2-dependent insulin reduction by high density SDS-PAGE. Inclusion of reduced serum albumin as a source of free thiols for the protein disulfide interchange activity catalyzed by ENOX2 failed to result in insulin reduction in the presence of ENOX2. A direct effect of ENOX2 on thioredoxin reduction in the presence of NADPH also was not observed. The DTNB assay for thioredoxin reductase activity also failed to reveal activity. Thus, ENOX proteins appear not to function as thioredoxin reductases at the cell surface nor do they appear to recognize reduced insulin as a substrate for protein disulfide-thiol interchange. The enhanced turbidity of insulin solutions resulting from ENOX presence was traced to ENOX-catalyzed insulin fibrillation either through nucleation enhancement or some other mechanism. Fibrillation was determined using Thioflavin T fluorescence which paralleled the turbimetric results and the formation of multimers (polymerization) observed on SDS-PAGE.
Bosneaga
Elena
E
Department of Medicinal Chemistry and Molecular Pharmacology, HANS Life Sciences Research Building, Purdue University, West Lafayette, IN 47907-2064, USA.
Kim
Chinpal
C
Shen
Bernard
B
Watanabe
Takahiro
T
Morre
Dorothy M
DM
Morré
D James
DJ
D004229:Dithiothreitol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism
D007328:Insulin; D013879:Thioredoxins; D009247:NADH, NADPH Oxidoreductases; C431909:tumor-associated NADH oxidase; D013880:Thioredoxin-Disulfide Reductase; D004229:Dithiothreitol