High performance liquid chromatographic analysis of insulin degradation by rat skeletal muscle insulin protease

Frederick G. Hamel, Daniel E. Peavy, Michael P. Ryan, William C. Duckworth

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The degradation of [125I]iodoinsulin (A14) by insulin protease (EC 3.4.22.11) was studied using HPLC. A reverse phase HPLC method is presented which allows the separation and quantitation of insulin degradation products. After incubation of [125I]iodoinsulin (A14) with insulin protease, there was an initial rapid loss of radioactivity from the [125I] iodoinsulin (A14) peak, which was quantitatively accounted for by the appearance of radioactivity in 11 different peaks, but was not accompanied by a proportional increase in the solubility of the sample in trichloroacetic acid. Two of the peaks showed appreciable accumulation before the others, and all but the firsteluted peak plateaued by 20 min. After 20 min of incubation, the amount of radioactivity present as the first-eluted peak, solubility in trichloroacetic acid, and insulin loss continued to increase at a steady, but slowed, rate. The order of appearance suggests that insulin protease acts on insulin in an ordered sequence of steps to generate a number of intermediates that are precipitable by trichloroacetic acid, but are subsequently degraded to material that is soluble in trichloroacetic acid. Sulfitolysis of 5 major peaks and subsequent HPLC analysis of the fragments showed none of the peaks to possess intact A chains. Peptide sequencing of 2 of the peaks indicates that the A-chain is cleaved in at least 2 positions, one beyond the 14th position, and one between the 13th and 14th amino acids (leucine and tyrosine).

Original languageEnglish (US)
Pages (from-to)328-333
Number of pages6
JournalEndocrinology
Volume118
Issue number1
DOIs
StatePublished - Jan 1986

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Insulysin
Trichloroacetic Acid
Skeletal Muscle
High Pressure Liquid Chromatography
Insulin
Radioactivity
Solubility
Leucine
Tyrosine
Amino Acids
Peptides
iodo-insulin

ASJC Scopus subject areas

  • Endocrinology

Cite this

High performance liquid chromatographic analysis of insulin degradation by rat skeletal muscle insulin protease. / Hamel, Frederick G.; Peavy, Daniel E.; Ryan, Michael P.; Duckworth, William C.

In: Endocrinology, Vol. 118, No. 1, 01.1986, p. 328-333.

Research output: Contribution to journalArticle

Hamel, Frederick G. ; Peavy, Daniel E. ; Ryan, Michael P. ; Duckworth, William C. / High performance liquid chromatographic analysis of insulin degradation by rat skeletal muscle insulin protease. In: Endocrinology. 1986 ; Vol. 118, No. 1. pp. 328-333.
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