Affiliations: Department of Medicinal Chemistry and Molecular
Pharmacology, HANS Life Sciences Research Building, Purdue University, West
Lafayette, IN, USA | Department of Foods and Nutrition, Purdue University,
West Lafayette, IN, USA
Note: [] Address for correspondence: D.J. Morré, Department of
Medicinal Chemistry and Molecular Pharmacology, HANS Life Sciences Research
Building, Purdue University, 201 South University Street, West Lafayette, IN
47907-2064, USA. Fax: +1 765 494 4007; E-mail: [email protected]
Abstract: 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.
DOI: 10.3233/BIO-2009-1078
Journal: BioFactors, vol. 34, no. 3, pp. 245-251, 2009
