Conversion of proinsulin to insulin: involvement of a 31,500 molecular weight thiol protease.

Abstract

A lysed crude secretory granule fraction from rat islets of Langerhans was shown to process endogenous proinsulin to insulin with a pH optimum of 5.0-6.0. The converting activity in the lysed fraction was not inhibited by serine protease inhibitors (diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin) or metalloprotease inhibitors (EDTA and o-phenanthroline) but was inhibited by some thiol protease reagents (p-chloromercuribenzenesulfonic acid, antipain, and leupeptin) but not by others (N-ethylmaleimide and iodoacetamide). NG-p-Tosyl-L-lysyl chloromethyl ketone only mildly inhibited at higher concentrations, whereas L-alanyl-L-lysyl-L-arginyl chloromethyl ketone was a powerful inhibitor. L-Alanyl-L-lysyl-L-arginyl chloromethyl ketone was ['"I]iodotyrosylated and used as an affinity labeling agent for the converting activity. Because the crude granule preparation contained contaminating lysosomes the affinity labeling of the granule preparation proteins was compared with that in liver lysosomes purified from rats injected with Triton WR1339. In the crude granule fraction the affinity label bound in a cysteine-enhanced manner to a single 31,500 molecular weight protein, but in purified liver lysosomes the major affinity-labeled protein had a molecular weight of 25,000 and minor 31,500 and 35,000 molecular weight proteins were also labeled. Evidence suggests that these proteins are thiol proteases and that in islets the 31,500 molecular weight thiol protease is involved in the conversion of proinsulin to insulin.Insulin is synthesized as a larger precursor, preproinsulin (1), which is rapidly cleaved to proinsulin (2). The conversion of proinsulin to insulin is then a relatively slow process, which is thought to commence in the Golgi apparatus and continue into the maturing secretory granule (3). The unequivocal identification of the converting activities present in these organelles has proven to be an elusive problem. Conversion is thought to be mediated by two enzymes: one with trypsin-like specificity and the other similar to carboxypeptidase B. Evidence for this is based on the known structure of the cleavage products and intermediate forms (4), the ability of these exocrine pancreatic enzymes to correctly process proinsulin in vitro (5), and the detection ofsimilar activities in the appropriate subcellular fractions of rat islets (6). Previous studies have demonstrated that both activities have a slightly acidic pH optimum and that the trypsin-like activity is activated by thiols and inhibited by thiol reagents (6-8). This suggests that this enzyme may be related to the lysosomal cathepsins. Alternatively, serine proteases [kallikrein (9) and plasminogen activator (10)] have also been implicated in the conversion process (see ref. 11 for review).In the experiments reported here, we have further characterized the inhibitor profile of the trypsin-like converting activity associated with a crude secretory granule preparation from rat islets, and on the basis of this information have at...

The insulin factory: a tour of the plant surroundings and a visit to the assembly line

“…It is thus also of interest to consider the relationship between the presence of clathrin on the limiting membranes of the Golgi-derived coated compartment where conversion of proinsulin takes place and the putative role of a proinsulin-converting thiol-protease with acidic pH optimum [26]. The intragranular pH was estimated to be between 5 and 6 [27].…”

Cathepsin Z, a Novel Human Cysteine Proteinase with a Short Propeptide Domain and a Unique Chromosomal Location

“…All of them contain an essential cysteine residue in their active site but differ in tissue distribution and in some enzymatic properties, including substrate specificities and pH stability. Furthermore, several groups have reported the existence of additional cysteine proteinases including cathepsins M, N, P, and T, which were originally identified because of their degrading activity on specific substrates such as aldolase, collagen, proinsulin, or tyrosine aminotransferase, but whose characterization at the molecular level has not yet been reported (22)(23)(24)(25).…”

Proglobulin Processing Enzyme in Vacuoles Isolated from Developing Pumpkin Cotyledons

“…It has been reported that the proinsulin-and proglucagonconverting enzymes localized in the secretion vesicles of rat islets (2) or anglerfish islets of Langerhans (3) is a thiol protease and that the posttranslational processing enzymes present in chloroplasts (14) or mitochondria (10) are metalloproteases. The processing enzyme associated with the vacuoles presently studied is a thiol protease, clearly distinguishable from that in chloroplasts or mitochondria and rather similar to the proinsulin-converting enzyme.…”