Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The N-linked carbohydrate chain of the PZP3 glycoprotein family of pig zona pellucida is shown by competition assay to possess sperm receptor activity. Structural analysis reveals that the N-linked chains comprise neutral and acidic complex-type chains. The tri- or tetra-antennary fraction in the neutral chains possesses the sperm receptor activity, but the activity is markedly reduced compared with that of PZP3, suggesting that the protein moiety participates in the maintenance of proper orientation of the active chain. Two components in the PZP3 family can be separated by reverse-phase HPLC after endo-beta-galactosidase (E beta G) digestion and one of the digests, E beta G-PZP3 alpha, possesses the sperm receptor activity. E beta G-PZP3 alpha deprived of O-linked chains by treatment with alkali retains the activity comparable with E beta G-PZP3 alpha, while E beta G-PZP3 alpha deprived of N-linked chains by N-glycanase digestion loses the activity. Furthermore, competition assay of the digests of E beta G-PZP3 alpha with lysyl-endopeptidase indicates that the active chain is linked to Asn67 or Asn84 of PZP3 alpha or to both residues. We conclude that sperm-egg binding in pigs is mainly mediated by tri- or tetra-antennary, neutral, complex-type N-linked carbohydrate chain(s) localized in the N-terminal region of PZP3 alpha protein.
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PMID:Structure and function of the N-linked carbohydrate chains of pig zona pellucida glycoproteins. 898 85

The major N-linked carbohydrate structures were determined for recombinant human plasma lecithin:cholesterol acyltransferase (LCAT). The analysis of the structure of oligosaccharides by fast atom bombardment mass spectrometry (FAB-MS) and linkage analysis was preceded by reduction and carboxymethylation of the intact glycoproteins and digestion with trypsin and proline specific endopeptidase. The N-glycans were subsequently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak cartridge. The data from the combination of FAB spectrometry and linkage analysis show that the N-linked glycans present on recombinant LCAT (rLCAT) were composed primarily of triantennary and tetraantennary structures with and without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or more antennae. The LCAT activities of both recombinant and circulating forms of plasma LCAT were determined using low molecular weight and lipoprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical. These findings validate the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundation for subsequent structural studies.
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PMID:Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties. 955 45

We have investigated the proteolytic mechanisms of glucagon degradation within hepatic endosomes at neutral pH before lumen acidification. Hepatic endosomes incubated at neutral pH rapidly degraded native glucagon into 13 intermediate products, one of which corresponded to the bioactive fragment glucagon-(19-29) (miniglucagon). The serine protease inhibitor phenylmethylsulfonyl fluoride as well as the nonspecific protease inhibitor bacitracin inhibited the endosomal degradation of glucagon at pH 7. In purified endosomal fractions, miniglucagon endopeptidase was undetectable as evaluated by immunoblotting, and immunoprecipitation with antibodies to insulin-degrading enzyme, cathepsins B and D, or furin failed to remove the endosomal neutral glucagonase activity. Incubation of endosomal fractions and [125I]iodoglucagon with the zero-length bifunctional cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in specific labeling of a 170-kDa polypeptide. The labeling was completely inhibited by unlabeled glucagon (IC50 value, 5 x 10-7 m) and bacitracin (IC50 value, 1 microg/ml), suggesting that it may correspond to a bacitracin-sensitive glucagon-degrading enzyme. Treatment of the 125I-labeled 170-kDa cross-linked polypeptide with N-glycanase demonstrated that the cross-linked complex contained approximately 30 kDa of N-linked oligosaccharides. Specific cross-linking of the 170-kDa polypeptide was also observed using [125I]Tyr12-miniglucagon as the radioligand. Together, these data suggest that the 170-kDa glycoprotein represents a novel glucagon-degrading activity that could mediate glucagon proteolysis within endosomes before the acidification step and generate the bioactive (19-29) miniglucagon peptide.
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PMID:Endosomal proteolysis of glucagon at neutral pH generates the bioactive degradation product miniglucagon-(19-29). 1295 81

Vasoactive intestinal peptide (VIP) exerts many biological functions through interaction with the VPAC1 receptor, a class II G protein-coupled receptor. Photoaffinity labeling studies associated with receptor mapping and three-dimensional molecular modeling demonstrated that the central part of VIP (6-24) interacts with the N-terminal ectodomain of VPAC1 receptor. However, the domain of the VPAC1 receptor interacting with the C-terminus of VIP is still unknown. A photoaffinity probe, Bpa28-VIP, was synthetized by substitution of amidated Asn28 of VIP by amidated photoreactive para-benzoyl-L-Phe (Bpa). Bpa28-VIP was shown to be a hVPAC1 receptor agonist in CHO cells expressing the recombinant VPAC1 receptor. After obtaining a covalent 125I-[Bpa28-VIP]/hVPAC1 complex, it was cleaved by CNBr, PNGase F, and endopeptidase Glu-C and the cleavage products were analyzed by electrophoresis. The data demonstrated that 125I-[Bpa28-VIP] was covalently bonded to the 121-133 fragment within the N-terminal ectodomain of the receptor. This fragment is adjacent to those covalently attached to the central part (6-24) of VIP.
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PMID:Spatial approximation between the C-terminus of VIP and the N-terminal ectodomain of the VPAC1 receptor. 1688 62