Insulin degradation. XV. Use of different assay methods for the study of mechanism of action of glutathione-insulin transhydrogenase.
Insulin degradation by glutathione-insulin transhydrogenase has been studied using three different assay procedures: the measurement of the change in insulin immunoreactivity; the formation of 5% trichloroacetic acid-soluble radioactivity from 125 I-labeled insulin and the formation of GSSG via coupling to the oxidation of NADPH with the use of glutathione reductase. The extent of reaction as measured by each assay was different, and the ratios between the assays were not constant with time. Kinetic experiments with the NADPH-coupled assay and the trichloroacetic acid assay yielded similar results: Line-weaver-Burke plots with insulin as variable and GSH as fixed substrate gave a set of straight, intersecting lines, and such plots with GSH as variable and insulin as fixed substrate were parabolic. Apparent Km values for insulin at 1 mM GSH were found to be quite similar by three assay techniques; however, the V values per unit of enzyme protein varied considerably with different procedures. The results are interpreted as indicating that immunoreactivity is lost after reduction of only one of the disulfide bonds of insulin whereas the two interchain disulfide linkages must be broken to produce the trichloroacetic acid-soluble A chain. The results of the NADPH-coupled assay suggest that all three disulfide bonds of insulin are possible substrates for the enzyme. The trichloroacetic acid precipitation assay seems to be the most practicable technique for general use because of the greater ease in performing large number of samples, precision and sensitivity.
D000818:Animals; D002417:Cattle; D005980:Glutathione Reductase; D007118:Immunoassay; D007328:Insulin / Q000378:metabolism*; D007330:Insulin Antibodies; D007700:Kinetics; D010088:Oxidoreductases / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D011490:Protein Disulfide Reductase (Glutathione) / Q000276:immunology / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods
D007328:Insulin; D007330:Insulin Antibodies; D010088:Oxidoreductases; D005980:Glutathione Reductase; D011490:Protein Disulfide Reductase (Glutathione)