In this work the fate of A14-125 l-insulin inside human cells has been investigated by the complementary use of gel permeation and reversed-phase high performance liquid chromatography to obtain a better resolution of the cell processed radioactive material resulting from the internalization of labeled insulin. Mononuclear leukocytes from 12 normals were incubated with pure A14-125 l-insulin at 37°C and internalized radioactivity was characterized after 2, 15 and 60 min. Nearly 14% of intracellular radioactivity was associated to materials with a molecular weight of approximately 300,000. The remaining 86% had a molecular weight lower than 20,000. High molecular weight material showed an elution profile very similar to that obtained from purified human placental insulin receptor and was partially precipitable with antireceptor antibody. The reversed phase high performance liquid chromatography analysis of the low molecular weight material showed two main peaks corresponding to 125 l and A14-125 l-insulin and three intermediate peaks, a, b, c, accounting for about 8% of the recovered radioactivity. By increasing the incubation time of A14-125 l-insulin with monocytes a decrease of insulin peak (2 min: 38 ± 18%; 15 min: 25 ± 11%; 60 min: 6 ± 4%) and a corresponding increase of iodide peak was observed. Immunoprecipitability with anti-insulin antibody was 0% for iodide and a peaks, 60% for peak b, 78% for peak c and 90% for A14-insulin peak. Our results show that intracellular insulin degradation procedes rapidly and in a time-dependent manner and that this process produces insulin derivatives which partially retain the immunological properties of intact A14-125 l-insulin. Moreover a relevant percent of intracellular radioactivity is associated to high molecular weight products. The data presented strongly suggest, but definitely do not demonstrate, that the high molecular weight radioactive material is only the insulin receptor complex.

Insulin degradation into monocytes from normal subjects: a high performance liquid chromatographic analysis

MARCHETTI, PIERO;CICCARONE, ANNAMARIA;
1988

Abstract

In this work the fate of A14-125 l-insulin inside human cells has been investigated by the complementary use of gel permeation and reversed-phase high performance liquid chromatography to obtain a better resolution of the cell processed radioactive material resulting from the internalization of labeled insulin. Mononuclear leukocytes from 12 normals were incubated with pure A14-125 l-insulin at 37°C and internalized radioactivity was characterized after 2, 15 and 60 min. Nearly 14% of intracellular radioactivity was associated to materials with a molecular weight of approximately 300,000. The remaining 86% had a molecular weight lower than 20,000. High molecular weight material showed an elution profile very similar to that obtained from purified human placental insulin receptor and was partially precipitable with antireceptor antibody. The reversed phase high performance liquid chromatography analysis of the low molecular weight material showed two main peaks corresponding to 125 l and A14-125 l-insulin and three intermediate peaks, a, b, c, accounting for about 8% of the recovered radioactivity. By increasing the incubation time of A14-125 l-insulin with monocytes a decrease of insulin peak (2 min: 38 ± 18%; 15 min: 25 ± 11%; 60 min: 6 ± 4%) and a corresponding increase of iodide peak was observed. Immunoprecipitability with anti-insulin antibody was 0% for iodide and a peaks, 60% for peak b, 78% for peak c and 90% for A14-insulin peak. Our results show that intracellular insulin degradation procedes rapidly and in a time-dependent manner and that this process produces insulin derivatives which partially retain the immunological properties of intact A14-125 l-insulin. Moreover a relevant percent of intracellular radioactivity is associated to high molecular weight products. The data presented strongly suggest, but definitely do not demonstrate, that the high molecular weight radioactive material is only the insulin receptor complex.
Benzi, L; Marchetti, Piero; Brunetti, A; Ciccarone, Annamaria; Cecchetti, P; Trischitta, E; DI CIANNI, G; Squatrito, S; Navalesi, R.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11568/14674
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