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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A study was made of the enzymes of the islands of Langerhans which could participate in the transformation of proinsulin into insulin. The homogenate of the islands of Langerhans of rat and man catalized the hyppuril-L-arginine splitting at pH 5.4-5.8 and 6.8-7.2 which was completely blocked with N-ethyl maleimide. The enzyme of the islands with the optimum action pH of 5.4-5.8 was similar to the enzyme of the exocrine tissue and was possibly a catheptic carboxypeptidase. The second enzyme of the islands differing from the exocrine carboxypeptidase could apparently participate in the insulin formation from the intermediate forms of proinsulin. In the formation of these proinsulin fomrs the participation of the enzyme of the endopeptidase character with a more acid optimum of the action pH is supposed.
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PMID:[Nature of the enzymes participating in the transformation of proinsulin into insulin]. 1 42

To determine the cellular localization of glandular kallikrein in the human pancreas, immunohistochemical studies were performed with a monospecific antibody against the antigenically identical urinary kallikrein (urokallikrein). The localization of glandular pancreatic kallikrein to the beta cells of the islets was the same as that of insulin in normal human pancreas and in two islet-cell tumors. When beta cells were lacking in islet-cell tumors or in the pancreas of a patient with juvenile-onset diabetes, kallikrein antigen was not detectable. Anti-urokallikrein absorbed with purified urinary or pancreatic kallikrein no longer identified a pancreatic antigen, whereas absorption with insulin had no effect. The beta-cell localization of human pancreatic kallikrein, an endopeptidase that, in concert with carboxypeptidase B, converts bovine proinsulin to a polypeptide with the electrophoretic mobility of insulin, suggests that pancreatic kallikrein may be involved in the physiologic activation of proinsulin.
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PMID:Identification of human glandular kallikrein in the beta cell of the pancreas. 22 May 34

A pancreatic endopeptidase localized to the beta-cells of the pancreas by immunohistochemical techniques has been purified to homogeneity by following its functional and antigenic characteristics as a glandular kallikrein (EC 3.4.21.8). The enzyme gave a single stained band on alkaline disc gel electrophoresis which corresponded in location with the kinin-generating activity eluted from a replicate gel, was of 54,000 molecular weight by gel filtration, was devoid of caseinolytic activity, elicited a monospecific antiserum in a rabbit, and gave a line of complete identity with a single constituent in pancreatic extract, crude urine, and purified urokallikrein when analyzed with monospecific antibody to urokallikrein. The pancreatic glandular kallikrein generated three cleavage products of increasing anodal mobility from bovine and porcine proinsulin, and the presence of pancreatic kininase or bovine carboxypeptidase B increased the quantity of these products. Although the conversion products did not correspond to diarginyl- and monoarginylinsulin, the product of intermediate mobility was also obtained when proinsulin was treated with a low concentration of trypsin in the presence of kininase. The most rapidly migrating product did correspond to desalanylinsulin in the reference standard. Kininase alone had no action on proinsulin, and aprotinin prevented cleavage by kallikrein alone or in combination with kininase. Although the chemical structure of the proinsulin cleavage products has not been established, human pancreatic kallikrein is considered a putative activator of proinsulin because of its location in the beta-cell, its preferential action on proinsulin and kininogen as compared to azocasein, and its capacity to generate insulin intermediate products that are further modified by human pancreatic kininase or bovine carboxypeptidase B.
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PMID:Sequential cleavage of proinsulin by human pancreatic kallikrein and a human pancreatic kininase. 38 42

Two Ca(2+)-dependent endopeptidase activities are involved in proinsulin to insulin conversion: type I cleaves COOH-terminal to proinsulin Arg31-Arg32 (B-chain/C-peptide junction); and type II preferentially cleaves at the Lys64-Arg65 site (C-peptide/A-chain junction). To further understand the mechanism of proinsulin processing, we have investigated types I and II endopeptidase processing of intact proinsulin in parallel to that of the conversion intermediates, des-31,32-proinsulin and des-64,65-proinsulin. The type I processed des-64,65-proinsulin and proinsulin at the same rate. In contrast, the type II endopeptidase processed des-31,32-proinsulin at a much faster rate (> 19-fold; p < 0.001) than it did intact proinsulin. Furthermore, unlabeled proinsulin concentrations required for competitive inhibition of 125I-labeled des-64,65-proinsulin and 125I-proinsulin processing by a purified insulin secretory granule lysate were similar (ID50 = 14-16 microM), whereas inhibition of 125I-labeled des-31,32-proinsulin processing required a higher nonradiolabeled proinsulin concentration (ID50 = 197 microM). Synthetic peptides corresponding to the sequences surrounding Lys64-Arg65 (AC-peptide/substrate) and Arg31-Arg32 (BC-peptide/substrate) of human proinsulin were synthesized for use as specific substrates or competitive inhibitors. Cleavage of the BC-substrate by type I and AC-substrate by type II was COOH-terminal of the dibasic sequence, with similar Ca(2+)-and pH requirements previously observed for proinsulin cleavage. Apparent Km and Vmax for type I processing of the BC-substrate was Km = 20 microM; Vmax = 22.8 pmol/min, and for type II processing of the AC-substrate was Km = 68 microM; Vmax = 97 pmol/min. In competitive inhibition assays, the BC-peptide similarly blocked insulin secretory granule lysate processing of des-64,65-proinsulin and proinsulin (ID50 = 45-55 microM), but did not inhibit des-31,32-proinsulin processing. However, the AC-peptide preferentially inhibited insulin secretory granule lysate processing of des-31,32-proinsulin (ID50 = microM) compared to proinsulin (ID50 = 330 microM), and not des-64,65-proinsulin. We conclude that the type I endopeptidase recognized des-64,65-proinsulin and proinsulin as similar substrates, whereas the type II endopeptidase has a stronger preference for des-31,32-proinsulin compared to intact proinsulin. Furthermore, we suggest that in intact proinsulin there exists a constraint to efficient processing that is relieved following type I processing. Structural flexibility, in addition to the presence of Lys64-Arg65, therefore appears to be important for type II endopeptidase specificity and may provide a molecular basis for a preferential route of proinsulin conversion via des-31,32-proinsulin.
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PMID:Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing. 142 23

A simple, rapid and sensitive assay for the type-1 endopeptidase (Arg-Arg cleaving) was developed by using an antiproinsulin monoclonal immunoadsorbent to separate reaction products from the substrate. The values obtained by this assay were identical with those obtained by an h.p.l.c.-based procedure and yielded similar values for the pH optimum (5.6) and Ca2+ activation (K0.5 = 2 mM). It was shown that the type-1 endopeptidase was readily solubilized by Triton X-114 (87 +/- 3%, n = 12) and partitioned principally into the aqueous phase at 30 degrees C (90.1 +/- 2.6%, n = 12). Activity was lost on gel filtration, but could be restored by adenosine 5'-[gamma-thio]triphosphate (K0.5 = 6 microM), 50 microM-dithiothreitol or 50 microM-Ca(2+)-trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid (CDTA), indicating that the enzyme was particularly sensitive to heavy metal ions. The Km obtained with proinsulin as substrate (13 +/- 1.7 microM) indicated that the enzyme works at close to its Vmax. in the nascent secretory granule. The Vmax. of the enzyme prepared from insulin granules (0.6% proinsulin converted/min) corresponded closely to the rate measured in vivo in rat islets. The type-1 endopeptidase also appears to be capable of binding to proinsulin in the region of the C-peptide/A-chain junction, since a peptide spanning this region was found to inhibit the 125I-proinsulin processing measured by this assay.
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PMID:Kinetic analysis of the type-1 proinsulin endopeptidase by a monoclonal antibody-based immunoadsorbent assay. 152 Feb 72

A novel fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC (RSKR-AMC) was used to characterize Ca(++)-activated proteolytic activity present in purified insulinoma secretory granules. Secretory granules efficiently cleaved this substrate in a time- and protein-dependent manner; the hydrolysis rate was between 2 and 4 pmol/min/ug of protein, with an apparent Km of 55 microM. Greater than 90% of the activity against this substrate was dependent on the presence of Ca++, with half-maximal stimulation obtained at 100 microM Ca++. The pH optimum of enzymatic activity was 5.5-6, and the profile of inhibition by various proteinase inhibitors was similar to that previously described for the type I and II proinsulin processing enzymes. These biochemical characteristics and co-elution of the RSKR-AMC processing activity with the type II endopeptidase activity on anion-exchange chromatography suggest that the new assay selectively detects the Lys-Arg-directed, or type II, proinsulin processing endopeptidase. This fluorogenic assay is more quantitative, sensitive and rapid than methods previously used, and therefore presents a significant improvement for the study of similar Ca(++)-activated processing endopeptidases.
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PMID:Fluorometric assay of a calcium-dependent, paired-basic processing endopeptidase present in insulinoma granules. 154 79

Enzymological studies have implicated two Ca(2+)-dependent endopeptidases in the conversion of proinsulin to insulin; a type 1 activity which cleaves on the C-terminal side of Arg31-Arg32 and a type 2 activity which cleaves C-terminally to Lys64-Arg65 in the proinsulin sequence. The possibility that these enzymes are related to the recently discovered family of mammalian subtilisin-like gene products (furin, PC2, and PC3) and the yeast propheromone-converting enzyme (KEX-2), was investigated. Degenerate oligonucleotide primers flanking the putative catalytic domain within this gene family were used in a polymerase chain reaction to amplify related sequences from rat insulinoma cDNA. One major product of 700 base pairs was obtained which was greater than 99% identical to the corresponding rat PC2 sequence. This cDNA was subcloned into the bacterial expression vector pGEX-3X to generate a recombinant protein for antibody production. Western blot analysis showed the immunoreactivity was prominent in neuroendocrine tissues as a 65-kDa protein. It was concentrated in secretory granule-enriched fractions of insulinoma tissue, where it was present as a readily solubilized monomeric protein. Deglycosylation studies using endoglycosidase H and N-glycanase showed that the 65-kDa protein was comprised of approximately 9% carbohydrate, consistent with the presence of three consensus sequences for N-linked glycosylation in rat PC2. The immunoreactivity co-eluted with the type 2 proinsulin endopeptidase on gel filtration and ion-exchange chromatography and the antisera specifically immunoprecipitated type 2 activity from insulin granule extracts. N-terminal sequence analysis of the immunoreactive protein gave two sequences which corresponded to residues 109-112 and 112-119 of rat PC2. This indicated that posttranslational processing of PC2 itself occurs C-terminally to basic amino acids to produce the mature enzyme. It is concluded that PC2 is the type 2 endopeptidase involved in proinsulin conversion. Localization of PC2 immunoreactivity to other tissues of the diffuse neuroendocrine system suggests that the type 2 endopeptidase also functions in the processing of precursor forms of other prohormones and polypeptide neurotransmitters.
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PMID:Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. 163 53

PC3, a mammalian homologue of the yeast subtilisin-like proteinase Kex2, was expressed in Xenopus oocytes and its activity was characterized. PC3 cleaved human proinsulin at one of the two dibasic sites (KTRR32 but not LQKR65). The specificity, inhibitor profile, pH optimum (5.5) and Ca(2+)-dependence (K0.5 = 2.5-3 mM) paralleled those of the insulin-granule type 1 endopeptidase activity, suggesting a role for PC3 in the conversion of prohormones.
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PMID:A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase. 163 32

Enzymological studies have implicated two Ca2+ dependent endopeptidases in the conversion of proinsulin to insulin: a type 1 activity and a type 2 activity which cleave on the C-terminal side of R31R32 and K64R65 in proinsulin, respectively. These activities were further characterized and their relationship to the mammalian family of subtilisin-like proteases was investigated. PC2 was expressed in neuroendocrine tissues and in insulinoma secretory granule fractions predominantly as a 65kDa protein. On anion-exchange chromatography of solubilized granules, PC1/3 immunoreactivity comigrated with a peak of type 1 activity whereas PC2 immunoreactivity coeluted with the peak of type 2 endopeptidase activity. PC2 antiserum gave a specific immunoprecipitation of type 2 activity from insulin granule extracts. It was concluded that the PC2 gene-product has type 2 endopeptidase activity.
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PMID:Proprotein-processing endopeptidases of the insulin secretory granule. 184 83

The proinsulin-insulin system provides a general model for the proteolytic processing of polypeptide hormones. Two proinsulin-specific endopeptidases have been defined, a type I activity that cleaves the B-chain/C-peptide junction (Arg31-Arg32) and a type II activity that cleaves the C-peptide/A-chain junction (Lys64-Arg65). These endopeptidases are specific for their respective dibasic target sites; not all such dibasic sites are cleaved, however, and studies of mutant proinsulins have demonstrated that additional sequence or structural features are involved in determining substrate specificity. To define structural elements required for endopeptidase recognition, we have undertaken comparative 1H NMR and photochemical dynamic nuclear polarization (photo-CIDNP) studies of human proinsulin, insulin, and split proinsulin analogues as models of prohormone processing intermediates. The overall conformation of proinsulin is observed to be similar to that of insulin, and the connecting peptide is largely unstructured. In the 1H NMR spectrum of proinsulin significant variation is observed in the line widths of insulin-specific amide resonances, reflecting exchange among conformational substates; similar exchange is observed in insulin and is not damped by the connecting peptide. The aromatic 1H NMR resonances of proinsulin are assigned by analogy to the spectrum of insulin, and assignments are verified by chemical modification. Unexpectedly, nonlocal perturbations are observed in the insulin moiety of proinsulin, as monitored by the resonances of internal aromatic groups. Remarkably, these perturbations are reverted by site-specific cleavage of the connecting peptide at the CA junction but not the BC junction. These results suggest that a stable local structure is formed at the CA junction, which influences insulin-specific packing interactions. We propose that this structure (designated the "CA knuckle") provides a recognition element for type II proinsulin endopeptidase.
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PMID:NMR and photo-CIDNP studies of human proinsulin and prohormone processing intermediates with application to endopeptidase recognition. 225 1


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