[51] Nonlysosomal insulin-degrading proteinases in mammalian cells

The purification of the metalloproteinase from human erythrocytes is described in this chapter. The insulin-degrading serine proteinase co-elutes from DEAE-cellulose with the metalloproteinase, but the enzymes are separated during subsequent purification steps. The serine proteinase may be purified 8000-fold using the same procedures. The procedures steps involves: preparation of lysate; DEAE-cellulose chromatogarphy; ammonium sulfate precipitation; pentyl-agaarose chromatography; hydroxyapatite chromatography; and gel filtration. The hydrolysis of radiochemically labeled proteins is measured as an increase in radioactive fragments soluble in trichloracetic acid. Insulin can be used in assays for several reasons. A single endoproteolytic cleavage of small proteins, such as the chains of insulin or glucagon, generally produces acid-soluble fragments. In contrast, the products of the limited proteolysis of larger proteins are generally not acid soluble. Because the iodinated tyrosines are not located at the termini of the insulin peptides, the assay is relatively insensitive to exoproteases. Furthermore, insulin is hydrolyzed very slowly or not at all by two major, soluble intracellular proteinases, the ATP-stimulated proteinase and the calcium-activated proteinase, and thus these enzymes does not complicate the measurement of insulin-degrading proteinases in extracts and in purified fractions.