Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The uptake by rat yolk sacs of native invertase and invertase which was deglycosylated by treatment with endo-beta-N-acetylglucosaminidase was compared. The initial rate of uptake of the deglycosylated enzyme was severalfold greater and its accumulation leveled off much earlier than that of the native enzyme. Uptake rates of the deglycosylated and native forms of the enzyme were proportional to their concentration in the medium in the range employed and were inhibited about 85% by 10(-6) M glucagon in both cases. After preloading of yolk sacs with native invertase, the tissue level of activity remained relatively constant over a subsequent 6-h time period, while with the deglycosylated form, activity declined substantially. Since this difference appears not to be attributable to differences in thermal stability, it is suggested that the deglycosylated form of the protein is more susceptible to intracellular proteolytic digestion. In vitro studies on the digestion of these two forms of invertase by trypsin are consistent with this suggestion.
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PMID:Effect of deglycosylation of yeast invertase on its uptake and digestion in rat yolk sacs. 37 2

Recombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-prorenin by employing a simple two-step procedure which consisted of ammonium sulfate precipitation and immunoaffinity chromatography using a monoclonal antibody specific for the profragment of human prorenin. About 100-fold purification with 35% recovery was achieved after the two steps. Purified rh-prorenin migrated as a single protein band with apparent molecular weights of 46,000-47,000 and about 50,000 on SDS-PAGE and gel filtration (HPLC), respectively, although it consisted of multiple components (pI values, 5.6-6.4) that could be resolved by isoelectric focusing (IEF). The treatment of rh-prorenin with endo-beta-N-acetylglucosaminidase converted the rather broad protein band to a sharp band on SDS-PAGE and reduced the number of multiple pI peaks on IEF. Amino-terminal sequence analysis of both the purified rh-prorenin and rh-renin revealed Leu-Pro-Thr-Asp- and Leu-Thr-Leu-Gly-, respectively, which agreed with those predicted from the base sequences of their cDNA. These data suggested that microheterogeneity of rh-prorenin is due to the carbohydrate moiety, but not to the protein moiety. Purified rh-prorenin was almost inactive, but was cleaved at the carboxyl end of a dibasic pair Lys-2-Arg-1 by trypsin and converted to active renin. However, at the early stage during trypsin activation, new intermediate forms between rh-prorenin and rh-renin were formed, suggesting multiple activation steps of rh-prorenin in addition to the one step activation.
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PMID:Isolation and characterization of recombinant human prorenin in Chinese hamster ovary cells. 201 71

Trimming glucosidase I has been purified about 400-fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N-glycoprotein as shown by its binding to concanavalin A-Sepharose and its susceptibility to endo-beta-N-acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non-specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N-linked high-mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal alpha 1-2-linked glucose residue from natural Glc3-Man9-GlcNAc2, but is inactive towards Glc2-Man9-GlcNAc2 or nitrophenyl-/methyl-umbelliferyl-alpha-glucosides. The enzyme displays a pH optimum close to 6.4, does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (Ki approximately 2.1 microM), N,N-dimethyl-1-deoxynojirimycin (Ki approximately 0.5 microM) and N-(5-carboxypentyl)-1-deoxynojirimycin (Ki approximately 0.45 microM), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross-react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross-reactivity.
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PMID:Purification and characterization of trimming glucosidase I from pig liver. 267 80

Thyroglobulin from colloid as well as from membrane fractions became radiolabeled upon incubation of calf thyroid slices with [35S]sulfate. The identity of the sulfate-labeled molecule was established by immunoprecipitation, polyacrylamide gel electrophoresis, Bio-Gel A-5m filtration, and DEAE-cellulose chromatography. Size analysis by gel filtration of [35S]glycopeptides and hydrazine-released oligosaccharides indicated that the sulfate was primarily located in the complex (unit B) carbohydrate units of thyroglobulin. Moreover, although [35S]sulfate-labeled oligosaccharides were cleaved by N-glycanase to the same extent as those labeled with [3H]mannose, they were not released by endo-beta-N-acetylglucosaminidase under conditions that led to the complete removal of polymannose carbohydrate (unit A). The failure of 35S-labeled glycopeptides and oligosaccharides to bind to immobilized Concanavalin-A indicated that the sulfate residues in calf thyroglobulin are located in carbohydrate units with three or more branches. No evidence for the occurrence of tyrosine sulfate was found upon examination of Pronase digests of radiolabeled thyroglobulin, and chemical analyses excluded the presence of this amino acid down to a level of 0.5 residues/polypeptide subunit. Studies with density gradient-separated membrane fractions as well as with puromycin indicated that sulfate addition is a late event in thyroglobulin biosynthesis which occurs in the Golgi compartment. Furthermore, it was observed that the nondimerized thyroglobulin subunit was much less sulfate labeled than the mature molecule. The location of the sulfated carbohydrate in a terminal portion of the calf thyroglobulin peptide chain was suggested by the observation that the subunit [mol wt (Mr) = 330,000] can undergo a transformation, presumably mediated by an endogenous protease, to a sulfate-free component (Mr = approximately 270,000) with the appearance of a 35S-labeled 60,000 Mr fragment; the release of a single sulfate-labeled peptide (Mr = 60,000) by mild trypsin treatment was consistent with a sequestration of sulfate groups in the thyroglobulin molecule.
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PMID:Biosynthesis of sulfated asparagine-linked complex carbohydrate units of calf thyroglobulin. 338 87

Three different carbohydrate-depleted enzymes were prepared from an endo-beta-1,4-glucanase of Aspergillus niger IFO31125 by treatment with endo-beta-N-acetylglucosaminidase or alpha-mannosidase. They were purified by Concanavalin A-Sepharose affinity and DEAE ion-exchange column chromatographies. The molecular sizes of these enzymes had been decreased from 40 kDa containing 9.0% carbohydrate to 39, 38, and 37 kDa with carbohydrate at 4.5, 1.3, and 0.8% (wt/wt), respectively. The native and these carbohydrate-depleted enzymes were compared in their enzymatic properties, and it was found that they were identical in their catalytic activities and both thermal and pH stabilities. However, the 37-kDa enzyme was more susceptible to proteolysis by Savinase, proteinase K, and Pronase E. On the other hand, the specific protease trypsin showed no such effect on activity of all enzymes. These results suggested that the core structure of the asparagine-linked sugar chain, which consisted of three monosaccharide residues, contributed to the high stability of the endo-beta-1,4-glucanase against protease digestion.
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PMID:Effects of size of carbohydrate chain on protease digestion of Aspergillus niger endo-beta-1,4-glucanase. 761 90

The Staphylococcus aureus autolysin gene, atl, encodes a unique 138-kDa protein (ATL) with amidase and glucosaminidase domains. ATL has been suggested to undergo proteolytic processing to generate two extracellular peptidoglycan hydrolases, 51-kDa endo-beta-N-acetylglucosaminidase (51-kDa GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (62-kDa AM). To investigate cell-associated bacteriolytic enzymes for atl gene products, proteins were extracted from the cells as follows. The cells were exposed to 3 M LiCl followed by 4% SDS. Thereafter, the cells were disrupted and again extracted with 4% SDS. Whole SDS-stable cell-associated bacteriolytic proteins were extracted without disrupting the cells. Exposure to 3 M LiCl released major 138-, 115-, 85-, 62- and 51-kDa bacteriolytic proteins, and subsequent 4% SDS extraction released major 138- and 115-kDa bacteriolytic proteins. These bacteriolytic proteins were missing in extracts of atl mutant RUSAL2 (S. aureus RN450 atl::Tn551). Immunoblotting studies suggest that these are all atl gene products: the 138-kDa protein is an ATL with a cleaved signal sequence; the 115- and 85-kDa proteins are intermediates; and the 51- and 62-kDa proteins are cell-associated 51-kDa GL and 62-kDa AM, respectively. The trypsin susceptibility of these proteins suggests that they are located outside the cell membrane. Differences in extractability and immunoelectron microscopic studies suggest that atl gene products are associated with cells in two different ways, LiCl extractable and non extractable. We suggest that the 138-kDa ATL undergoes processing through intermediate proteins (115- and 85-kDa proteins) to mature as the active cell cluster-dispersing enzymes 51-kDa GL and 62-kDa AM on the cell surface.
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PMID:Subcellular localization of the major autolysin, ATL and its processed proteins in Staphylococcus aureus. 925 Oct 58

A bacterial strain isolated from soil and identified as a Bacillus species produced two endo-beta-N-acetylglucosaminidases in the culture broth when it was cultivated on medium containing only hen ovomucoid. Almost no production of the enzymes occurred when the bacterium was grown on glucose medium. The two endo-beta-N-acetylglucosaminidases, named Endo-BI and Endo-BII, were separated and purified to homogeneity by preparative gel electrophoresis after partial purification by column chromatography on DEAE-resins. Endo-BI hydrolysed oligosaccharides of both hen ovalbumin and ovomucoid. In contrast, Endo-BII could act only on oligosaccharides of hen ovalbumin and showed almost no activity towards those of hen ovomucoid. Deglycosylation of hen ovomucoid was performed with the partly purified endo-beta-N-acetylglucosaminidase preparation, with the aid of contaminating beta-N-acetylhexosaminidase. The deglycosylated ovomucoid exhibited no changes in trypsin inhibitory activity but was very unstable to heat treatment in comparison with native ovomucoid. These results suggest that oligosaccharides of ovomucoid have an important role in the stabilization of the protein against heat.
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PMID:Characterization of Bacillus sp. endo-beta-N-acetylglucosaminidase and its application to deglycosylation of hen ovomucoid. 979 22

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).
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PMID:Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 1022 60