Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules.

Fletcher, Donald J.; Quigley, James P.; Bauer, G. Eric; Noe, Bryan D.

doi:10.1083/jcb.90.2.312

Cited by 116 publications

(45 citation statements)

References 49 publications

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“…Its activity was inhibited by leupeptin, p-chloromercuribenzoate, and pepstatin A, but not by serine or metalloprotease inhibitors. A thiol protease has also been implicated in the conversion ofproinsulin, proglucagon, and prosomatostatin in anglerfish islets (32,33). This acidic protease, which may be' bound to the secretory granule membrane, has an.inhibitor profile qualitatively similar to that reported here.…”

Section: Resultssupporting

confidence: 67%

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

Docherty¹,

Carroll²,

Steiner³

1982

Proc. Natl. Acad. Sci. U.S.A.

123

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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...

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Section: Resultssupporting

confidence: 67%

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

Docherty¹,

Carroll²,

Steiner³

1982

Proc. Natl. Acad. Sci. U.S.A.

123

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“…As expected for a non-receptor mediated process, kidney extraction varied conversely to liver extraction, being highest for human proinsulin and lowest for insulin. It is concluded that the kinetics of human proinsulin conversion intermediates depends upon the site of cleavage and deletion and is intermediate between those of insulin and intact human proinsulin.Key words: Biosynthetic human proinsulin, conversion intermediates, 123-I-labelling, scintillation scanning.Most of proinsulin is converted to insulin inside the B cells of the pancreas, a process resulting from the interplay of several endopeptidases, different from trypsin itself [1][2][3]. Recently, two distinct site-specific endopeptidases regulated by calcium concentration and pH were identified in a B granule fraction of rat insulinoma [4].…”

mentioning

confidence: 99%

“…Most of proinsulin is converted to insulin inside the B cells of the pancreas, a process resulting from the interplay of several endopeptidases, different from trypsin itself [1][2][3]. Recently, two distinct site-specific endopeptidases regulated by calcium concentration and pH were identified in a B granule fraction of rat insulinoma [4].…”

mentioning

confidence: 99%

Scintigraphic distribution of 123 I labelled proinsulin, split conversion intermediates and insulin in rats

et al. 1988

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Summary. Insulin, biosynthetic human proinsulin and 2 human proinsulin conversion intermediates, des (64, 65) human proinsulin and des (31, 32) human proinsulin, were labelled with 123 I and the derivatives monosubstituted on Tyr A14 were purified by reverse phase high performance liquid chromatography. The four tracers were injected into anaesthetized rats via a jugular or a portal vein and time activity curves were generated for the liver and kidneys using a gamma camera and an online computer. Liver extraction coefficients varied in the order insulin (38%), des (64, 65) human proinsulin (11.7%), des (31, 32) human proinsulin (3.2%), human proinsulin (1.6%); whereas half-life of hepatic activity varied in the reverse order, from 6 rain for insulin, to 45 min for human proinsulin. As expected for a non-receptor mediated process, kidney extraction varied conversely to liver extraction, being highest for human proinsulin and lowest for insulin. It is concluded that the kinetics of human proinsulin conversion intermediates depends upon the site of cleavage and deletion and is intermediate between those of insulin and intact human proinsulin.Key words: Biosynthetic human proinsulin, conversion intermediates, 123-I-labelling, scintillation scanning.Most of proinsulin is converted to insulin inside the B cells of the pancreas, a process resulting from the interplay of several endopeptidases, different from trypsin itself [1][2][3]. Recently, two distinct site-specific endopeptidases regulated by calcium concentration and pH were identified in a B granule fraction of rat insulinoma [4]. During proinsulin processing, conversion intermediates are formed, in particular the split products des (64, 65) human proinsulin (HPI) and des (31, 32) HPI. These intermediates and intact HPI have been found in human sera [5], in insulin producing islet cell tumours [6-81 and in familial hyperproinsulinaemia [9-111. Conversion to intermediates or insulin does not seem to occur after proinsulin enters the blood stream; partial conversion to intermediates might occur in the skin after subcutaneous injection [121.From the standpoint of biological activity, proinsulin has remarkable characteristics. Compared to insulin, HPI has a low biological potency, a low avidity for insulin receptors and a prolonged half-life [13][14][15][16][17]. Moreover, the proinsulin conversion intermediates have been shown to exhibit greater biological activity than proinsulin itself, and this is particularly true for the conversion intermediate in which the C peptide/A chain bond has been cleaved. Peavy et al. demonstrated that cleavage and/or dibasic amino acid deletions in the connecting peptide region of proinsulin enhance the receptor binding and hypoglycaemic activities of these compounds. In these studies, C peptide/A chain split products exhibited greater receptor affinity and hypoglycaemic activity than B chain C peptide split intermediates [18]. These observations prompted us to investigate the hepatic extraction and processing of HPI and its conver...

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“…This enzyme activity may be related or similar to other processing enzymes with acidic pH optima identified in pancreatic islet secretory granules of angler fish and rat pancreas (Fletcher et al, 1980(Fletcher et al, , 1981 and adrenal chromaffin granules (Mizuno et al, 1982;Evangelista et al, 1982;Troy and Musacchio, 1982). These enzymes have been defined on the basis of cleavage of pro-insulin, pro-glucagon, pro-somatostatin and/or enkephalin precursors.…”

Section: Processing Enzymes: Cleavage At Paired Basic Amino Acidsmentioning

confidence: 69%

Action of proteolytic enzymes on lipotropins and endorphins: Biosynthesis, biotransformation and fate

Burbach¹

1984

Pharmacology & Therapeutics

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Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules.

Cited by 116 publications

References 49 publications

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

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

Scintigraphic distribution of 123 I labelled proinsulin, split conversion intermediates and insulin in rats

Action of proteolytic enzymes on lipotropins and endorphins: Biosynthesis, biotransformation and fate

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