On the Biosynthesis, Intracellular Transport and Mechanism of Conversion of Proinsulin to Insulin and C-Peptide

Abstract

The detailed mechanism of the enzymic transformation of proinsulin has not yet been elucidated, but it appears to be a complex process requiring the participation of several proteolytic activities. A useful model system is described in which ordinary pancreatic trypsin arid carboxypeptidase B are shown to rapidly and quantitatively convert proinsulin to insulin. This reaction is accompanied by the liberation of the C-peptide and four basic amino acids. Studies on the biosynthesis and conversion of proinsulin in isolated islets of Langerhans suggest that the conversion to insulin begins in the Golgi apparatus and continues within newly formed secretory granules. Evidence bearing on this conclusion is summarized, and experiments are described that demonstrate conversion of proinsulin to insulin in a crude secretory granule fraction prepared from islets of Langerhans. Lysates of this subcellular fraction have been shown to contain low levels of trypsin-like activity. With lightly trypsin-treated proinsulin labeled with [3H]-arginine in the regions of specific cleavage as a substrate, a carboxypeptidase B-like activity has also been detected. Further studies will be required to elucidate the nature of these enzymes and their possible relationship to the pancreatic acinar trypsins and carboxypeptidases. Structural studies on C-peptides from several mammalian species are briefly discussed. Comparisons of these amino acid sequences indicate that considerably greater interspecies variation occurs in this region of the molecule. Extension of these comparative studies of C-peptide structure should shed light on the role of this region in facilitating the correct folding of the proinsulin polypeptide chain, and these also may disclose new aspects of insulin evolution.

Characterization of PC2, a mammalian Kex2 homologue, following expression of the cDNA in microinjected Xenopus oocytes

“…Cleavage usually occurs at sites marked by pairs of basic amino acids, although peptides can also be generated by cleavage at single basic amino acids [2]. An endopeptidase is thought to cleave on the COOH-terminal side of the pairs of basic amino acids and a carboxypeptidase then removes the COOH-terminal basic residues [3].…”

Golgi/granule processing of peptide hormone and neuropeptide precursors: A minireview

“…In neurosecretory cells a similar sequence of cellular events may occur during the long period of axonal transport, providing ample time for the proteolytic maturation of precursors [30]. Only low levels of proteolytic enzymes are probably required, eg, for the @-cell if it is assumed that the converting enzyme is as active as pancreatic trypsin a molar ratio of about one molecule protease per lo4-lo5 substrate molecules should suffice to achieve the observed in vivo rates of conversion [31]. This fact, and the inherent technical difficulties of efficiently isolating and recovering intact Golgi and secretion granule fractions free of other contaminating cellular organelles (such as lysosomes) from small amounts of endocrine tissues complicates the task of identifying converting protease(s) 1321.…”

Isoelectric focusing of proinsulin and intermediate in polyacrylamide gel

“…ly, suggesting that in these intermediate forms only the residues Arg,, or Arg,, are cleaved out from the proinsulin molecule [8]. Further evidence supporting the existence of such closely related intermediates is the precipitation with insulin antibodies (fig.…”