Purification to Homogeneity of an Insulin-Degrading Enzyme from Human Erythrocytes

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Abstract
The purification from human erythrocytes of an enzyme, a protease, is described, which degrades insulin with a high specificity at physiological hormone concentrations. Since the enzyme contains free sulfhydryl groups, affinity chromatography on organomercuri-Sepharose proved to be applicable as a valuable step in the isolation procedure. The purification factor amounted to .apprx. 6000, the yield to 8%. One mg of purified enzyme was capable of degrading 50 pmol of insulin/min into trichloroacetic acid-soluble split products. The purified insulin-degrading enzyme was homogeneous, as demonstrated by gel chromatography, gel electrophoresis and isoelectric focusing. The isoelectric point was at pH 5.8. The MW of native enzyme was estimated by gel chromatography and gel electrophoresis and found to be .apprx. 150,000-160,000, consisting of 4 subunits. Degradation products of insulin eluted from a Biogel P 30 column are smaller than the A-chain of the hormone, suggesting the activity of a protease. The enzyme appears to be specific for insulin in that it does not degrade other peptide hormones such as growth hormone, prolactin or thyroid-stimulating hormone [thyrotropin]. The enzyme does not inactivate enzyme such as lactate dehydrogenase, aldolase, fructose 1,6-bis-phosphatase, hexosephosphate isomerase or hexokinase.

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