Detection of the substance immunologically cross-reactive with insulin in insulin RIA is an artifact caused by insulin tracer degradation: involvement of the insulin-degrading enzyme.
In previous literature, the existence of a new insulin-like substance found in tumor tissues, termed substance immunologically cross-reactive with insulin (SICRI), has been proposed. In these studies, insulin-specific radioimmunoassay (RIA) was the only detection method for SICRI. The mouse melanoma B16BL6 cell line was found to be a rich source of SICRI. In this paper, we show that SICRI is not expressed in B16BL6 cells. Previous RIA measurements were wrongly ascribed to SICRI. What was really measured was a positive artifact caused by insulin tracer degradation in RIA. Several lines of evidence indicate that protease responsible for insulin degradation in B16BL6 cells in insulin-degrading enzyme (IDE; EC 3.4.22.11). First, SICRI activity of B16BL6 cytosol measured by insulin RIA was inhibited by thiol protease inhibitor N-ethylmaleimide (NEM). Thiol active agents as well as metal chelators, both potent IDE blockers, inhibited also the insulin-degrading activity of the same sample. Second, cross-linking to 125I-labeled insulin of partially purified sample with highest insulin RIA activity specifically labeled only a single protein with molecular mass similar to IDE (110 kDa). Labeling was blocked by 'cold' insulin in excess. Third, kinetic studies of insulin degradation by RIA active chromatographic fractions revealed an apparent Kd of 90 nM which is very similar to the reported affinity of insulin for IDE (Kd = 100 nM). Additionally, in B16BL6 as well as in mouse myeloid leukemia cells, IDE gene is actively transcribed and this expression was found to be much stronger than in normal mouse tissues. In conclusion, our results strongly question the real existence of SICRI.(ABSTRACT TRUNCATED AT 250 WORDS)
Saric
T
T
Rudjer Boskovic Institute, Department of Molecular Medicine, Zagreb, Croatia.
Seitz
H J
HJ
Pavelic
K
K
D000818:Animals; D015152:Blotting, Northern; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000378:metabolism; D005189:False Positive Reactions; D007328:Insulin / Q000032:analysis* / Q000378:metabolism*; D007339:Insulysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008546:Melanoma, Experimental / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011863:Radioimmunoassay / Q000706:statistics & numerical data*; D012680:Sensitivity and Specificity; D013696:Temperature; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D007328:Insulin; D010446:Peptide Fragments; D007339:Insulysin