Degradation of Insulin and Proinsulin by Various Organ Homogenates of Rat

Abstract

Degradation of biologically active and immunoreactive insulin and proinsulin was studied in the particulate and soluble fractions of homogenates of liver, brain, fat, lung, kidney, spleen, heart, muscle, pancreas, and testis of four groups of male rats at various time intervals. In addition, the above studies were also conducted in the ovaries, muscles, and adrenals of one group of female rats. Approximately 90 per cent of the degradative activity for immunoreactive insulin and immunoreactive proinsulin was located in the supernatant fraction with the remaining in the particulate fraction. The rate of degradation of immunoreactive insulin in the supernatant fraction expressed as picomoles destroyed per minute per gram of tissue was 53.8, 27.2, 26.6, 26.6, 25.0, 24.3, 14.1, 13.0, 11.5, 8.0, 5.9, and 1.7 for liver, pancreas, kidney, testis, adrenal, spleen, ovary, lung, heart, muscle, brain, and fat, respectively. With the exception of pancreas and kidney, no organ extract exhibited greater than 10 per cent of immunoreactive proinsulin degradation as compared to immunoreactive insulin. The rate of immunoreactive proinsulin degradation (picomoles per minute per gram of tissue) in the supernatant fraction was 5.0, 5.0, 5.0, 2.4, 2.0, 2.0, 1.6, 1.6, 1.0, 1.0, 0.6, and 0.2 for liver, pancreas, kidney, adrenal, testis, spleen, ovary, lung, heart, muscle, brain, and fat, respectively. Studies on partially purified insulin protease of kidney indicated (a) an approximately tenfold greater rate for degradation of insulin than proinsulin and no appreciable degradation of intermediate II; (b) degradation of intermediate I was approximately one-third as much as that of insulin; (c) addition of other hormones such as ACTH, glucagon, and growth hormone did not affect the degradation rate of insulin; (d) an inhibition of the enzyme by the sulfhydryl group inhibitor and by heating at 60° for five minutes; (e) a lack of effect of glutathione or trasylol; (f) a pH optimum of 7.4 to 7.6; and (g) a Km for insulin of 2 × 10−8 M. These properties all agree with similar findings of enzyme systems in liver and muscle, and suggest a similar degree of selectivity for degradation of insulin.