The degradation of monoiodotyrosyl insulin isomers by insulin protease.

The four 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 four isomers (A14-, A19-, B16-, and B26-monoiodotyrosyl insulin) to degradation by purified insulin protease was examined using several different assay methods, including trichloroacetic acid precipitation, immunoprecipitation, and Sephadex G-50 chromatograpy. Using trichloroacetic 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 two B-chain-labeled isomers. Based upon the initial rate of isomer hydrolysis, the Michaelis Menten constant (Km) of insulin protease was higher for the B16 isomer (55 nM) than for the other three isomers, whose Km values were not different from one another (A14 = 24 nM; A19 = 35 nM; B26 = 29 nM). In addition, the values for maximum velocity (Vmax) were higher for the A14 and B26 isomers than for the A19 and B16 isomers. However, during incubation, the order of isomer susceptibility to insulin protease changed to B26 greater than A14 greater than A19 greater than 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 two 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 two 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 than with either of the other two assays.(ABSTRACT TRUNCATED AT 400 WORDS)