The Degradation of Monoiodotyrosyl Insulin Isomers by Insulin Protease

Publisher Website
Google Scholar
Abstract
The 4 single-site monoiodotyrosyl insulin isomers were synthesized by lactoperoxidase-catalyzed iodination of porcine insulin and were separated from one another by high performance liquid chromatography. The susceptibility of the 4 isomers (A14-, A19-, B16- and B26-monoiodotyrosyl insulin) to degradation by purified insulin protease was examined using several different assay methods, including trichloracetic acid precipitation, immunoprecipitation, and Sephadex G-50 chromatography. Using trichloracetic acid precipitation, isomer susceptibility, determined from the initial rate of hydrolysis, was highest with the A14 isomer, lowest with the A19 isomer, and intermediate and roughly equal with the 2 B-chain-labeled isomers. Based upon the initial rate of isomer hydrolysis, the Km of insulin protease was higher for the B16 isomer (55 nM) than for the other 3 isomers, whose Km values were not different from one another (A14 = 24 nM; A19 = 35 nM; B26 = 29 nM). The values for maximum velocity (Vmax) were higher for the A14 and B26 isomers than for the A19 and B16 isomers. During incubation, the order of isomer susceptibility to insulin protease changed to B26 > A14 > A19 > B16. This change in apparent isomer susceptibility was prevented by including in the incubation mixture a rat renal peptidase, which did not degrade the intact isomers, suggesting that insulin protease converted the isomers to trichloroacetic acid-soluble products via trichloroacetic acid-precipitable intermediates. Using the immunoprecipitation assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the 2 B-chain-labeled isomers, of which the B16 isomer was degraded more rapidly. Each isomer was converted more rapidly to nonimmunoprecipitable products than to trichloroacetic acid-soluble products, implying that insulin protease converted the isomers to trichloroacetic acid-precipitable, nonimmunoprecipitable intermediates, which it then converted to trichloroacetic acid-soluble form. Using Sephadex G-50 chromatography (SGC) assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the 2 B-chain-labeled isomers, of which the B16 isomer was hydrolyzed more rapidly. With the exception of the A19 isomer, isomer hydrolysis appeared faster with SGC assay with either of the other 2 assays. Each of the isomers was converted to a complex mixture of intermediate and low MW products, some of which were not soluble in trichloracetic acid. Some of these products were detected with B-chain-labeled isomers, but were detected poorly or not at all with A-chain-labeled isomers. The rate isomer hydrolysis appears highest with SGC assay, lowest with trichloroacetic acid-precipitation assay, and intermediate with immunoprecipitation assay. Regardless of the assay used, the rate of hydrolysis by insulin by protease is highest for the A14 isomer, lowest for the A19 isomer, and intermediate for the 2 B-chain-labeled isomers, of which the B16 isomer is hydrolyzed more rapidly. The susceptibility of monoiodotyrosyl insulin to degradation by insulin protease is influenced by the location of the I atom.