Insulin degrading activity (IDA) was studied in subcellular fractions from homogenized human mononuclear cells. More than 90% (mean ± SD >> 94.1% ± 4.0) of total IDA was found in the soluble fraction (100,000 x g supernatant) of monocytes and it was completely inhibited by protease inhibitors such as Bacitracin (1 mg/ml) and NEM (1 mM). IDA was time-, temperature- and pH-dependent, being completely inhibited at 4°C and being maximal at pH 7.0-7.5; its Km for insulin was 1.9 x 10-7 M. The partially purified insulin degrading enzyme had a molecular weight of 120,000. When A14-125I-insulin was incubated with purified IDA a variety of degradative products were identified by HPLC, in addition to 125I and a small percentage of intact insulin. Different elution patterns were observed after incubating with IDE monoiodinated A19-, B16, and B26-insulin. In conclusion, IDA from human mononuclear cells has characteristics similar to those of other IDA previously reported in typical insulin target tissues. Mononuclear cells therefore represent a suitable model for studying insulin degradation in humans.
Characterization of insulin degrading activity in subcellular fractions of human monocytes
Brunetti A.;
1988-01-01
Abstract
Insulin degrading activity (IDA) was studied in subcellular fractions from homogenized human mononuclear cells. More than 90% (mean ± SD >> 94.1% ± 4.0) of total IDA was found in the soluble fraction (100,000 x g supernatant) of monocytes and it was completely inhibited by protease inhibitors such as Bacitracin (1 mg/ml) and NEM (1 mM). IDA was time-, temperature- and pH-dependent, being completely inhibited at 4°C and being maximal at pH 7.0-7.5; its Km for insulin was 1.9 x 10-7 M. The partially purified insulin degrading enzyme had a molecular weight of 120,000. When A14-125I-insulin was incubated with purified IDA a variety of degradative products were identified by HPLC, in addition to 125I and a small percentage of intact insulin. Different elution patterns were observed after incubating with IDE monoiodinated A19-, B16, and B26-insulin. In conclusion, IDA from human mononuclear cells has characteristics similar to those of other IDA previously reported in typical insulin target tissues. Mononuclear cells therefore represent a suitable model for studying insulin degradation in humans.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.