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1.
Methylation at specific altered aspartyl and asparaginyl residues in glucagon by the erythrocyte protein carboxyl methyltransferase 总被引:1,自引:0,他引:1
Protein carboxyl methyltransferases from erythrocytes and brain appear to catalyze the esterification of L-isoaspartyl and/or D-aspartyl residues but not of normal L-aspartyl residues. In order to identify the origin of these unusual residues which occur in subpopulations of a variety of cellular proteins, we studied the in vitro methylation by the erythrocyte enzyme of glucagon, a peptide hormone of 29 amino acids containing 3 aspartyl residues and a single asparagine residue. Methylated glucagon was digested with either trypsin, chymotrypsin, pepsin, or endoproteinase Arg C, and the labeled fragments were separated by high-performance liquid chromatography and identified. In separate experiments, methyl acceptor sites were determined by digesting glucagon first with proteases and then assaying purified glucagon fragments for methyl acceptor activity. Using both approaches, we found that the major site of methylation, accounting for about 62% of the total, was at the position of Asp-9. Chemical analysis of fragments containing this residue indicated that this site represents an L-isoaspartyl residue. A second site of methylation, representing about 23% of the total, was detected at the position of Asn-28 and was also shown to represent an L-isoaspartyl residue. Methyl acceptor sites were not detected at the positions of Asp-15 or Asp-21. Preincubation of glucagon under basic conditions (0.1 M NH4OH, 3 h, 37 degrees C) increased methylation at the Asn-28 site by 4-8-fold while methylation at the Asp-9 site remained unchanged. These results suggest that methylation sites can originate from both aspartyl and asparaginyl residues and that these sites may be distinguished by the effect of base treatment. 相似文献
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Kornelia M. Mikula Luisa Krumwiede Andreas Plückthun Hideo Iwaï 《Journal of biomolecular NMR》2018,71(4):225-235
Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling. 相似文献
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Inka Brockhausen Arthur A Grey Henrianna Pang Harry Schachter Jeremy P Carver 《Glycoconjugate journal》1988,5(4):419-448
Sixteen asparagine-linked oligosaccharides ranging in size from (Man)2(GlcNAc)2 (Fuc)1 to (GlcNAc)6(Man)3(GlcNAc)2 were obtained from human 1-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis
bisecting GlcNAc
- DMSO
dimethylsulfoxide
- FAB
fast atom bombardment
- Fuc
l-fucose
- Gal
d-galactose
- GLC
gas-liquid chromatography
- GlcNAc or Gn
N-acetyl-d-glucosamine
- HPLC
high performance liquid chromatography
- Man or M
d-mannose
- MES
2-(N-morpholino)ethanesulfonate
- MS
mass spectrometry
- NMR
nuclear magnetic resonance
- PIPES
piperazine-N,N-bis(2-ethane sulfonic acid)
the nomenclature of the oligosaccharides is shown in Table 1. 相似文献
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Saska I Gillon AD Hatsugai N Dietzgen RG Hara-Nishimura I Anderson MA Craik DJ 《The Journal of biological chemistry》2007,282(40):29721-29728
Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event. 相似文献
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Reactions that are relevant to the cleavage, by hydrazinolysis, of O‐linked oligosaccharides from glycoproteins were studied using dehydroalanine derivatives as models of the intermediates formed from O‐glycosylated serine residues. Conjugate addition of hydrazine followed by cyclisation to form pyrazolidinones, if occurring during glycoprotein hydrazinolysis, could reduce the yield of released oligosaccharide. However, N‐acetyldehydroalanine amide derivatives, which modelled the dehydroalanine derivatives believed to be intermediates in the hydrazinolysis of glycoproteins containing O‐linked oligosaccharides, underwent conjugate addition but no cyclisation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
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Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation 总被引:27,自引:0,他引:27
Aspartyl and asparaginyl deamidation, isomerization, and racemization reactions have been studied in synthetic peptides to model these spontaneous processes that alter protein structure and function. We show here that the peptide L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala undergoes a rapid deamidation reaction with a half-life of only 1.4 days at 37 degrees C, pH 7.4, to give an aspartyl succinimide product. Under these conditions, the succinimide product can further react by hydrolysis (half-time, 2.3h) and by racemization (half-time, 19.5 h). The net product of the deamidation reaction is a mixture of L- and D-normal aspartyl and beta-transpeptidation (isoaspartyl) hexapeptides. Replacement of the asparagine residue by an aspartic acid residue results in a 34-fold decrease in the rate of succinimide formation. Significant racemization was found to accompany the deamidation and isomerization reactions, and most of this could be accounted for by the rapid racemization of the succinimide intermediate. Replacement of the glycyl residue in the asparagine-containing peptide with a bulky leucyl or prolyl residue results in a 33-50-fold decrease in the rate of degradation. Peptide cleavage products are observed when these Asn-Leu and Asn-Pro-containing peptides are incubated. Our studies indicate that both aspartic acid and asparagine residues may be hot spots for the nonenzymatic degradation of proteins, especially in cells such as erythrocytes and eye lens, where these macromolecules must function for periods of about 120 days and 80 years, respectively. 相似文献
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Spontaneous degradation of polypeptides at aspartyl and asparaginyl residues: effects of the solvent dielectric. 总被引:4,自引:1,他引:3
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We have investigated the spontaneous degradation of aspartate and asparagine residues via succinimide intermediates in model peptides in organic co-solvents. We find that the rate of deamidation at asparagine residues is markedly reduced in solvents of low dielectric strength. Theoretical considerations suggest that this decrease in rate is due to the destabilization of the deprotonated peptide bond nitrogen anion that is the postulated attacking species in succinimide formation. This result suggests that asparagine residues in regions with low dielectric constants, such as the interior of a protein or in a membrane bilayer, are protected from this type of degradation reaction. On the other hand, we found little or no effect on the rate of succinimide-mediated isomerization of aspartate residues when subjected to the same changes in dielectric constant. In this case, the destabilization of the attacking peptide bond nitrogen anion may be balanced by increased protonation of the aspartyl side chain carboxyl group, a reaction that results in a superior leaving group. Consequently, any protein structure or conformation that would increase the protonation of an aspartate side chain carboxyl group can be expected to render that residue more labile. These results may help explain why particular aspartate residues have been found to degrade in proteins at rates comparable to those of asparagine residues, even though aspartyl-containing peptides degrade more slowly than corresponding asparaginyl-containing peptides in aqueous solutions. 相似文献
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The linkage of heparin to protein 总被引:4,自引:0,他引:4
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The analysis of O-glycans is essential for better understanding their functions in biological processes. Although many techniques for O-glycan release have been developed, the hydrazinolysis release method is the best for producing O-glycans with free reducing termini in high yield. This release technique allows the glycans to be labeled with a fluorophore and analyzed by fluorescence detection. Under the hydrazinolysis release conditions, a side reaction is observed and causes the loss of monosaccharides from the reducing terminus of the glycans (known as peeling). Using bovine fetuin (because it contains the sialylated O-glycans most commonly found on biopharmaceuticals) and bovine submaxillary gland mucin (BSM), here we demonstrate that peeling can be greatly reduced when the sample is buffer exchanged prior to hydrazinolysis with solutions of either 0.1% trifluoroacetic acid (TFA) or low-molarity (100, 50, 20, and 5 mM) ethylenediaminetetraacetic acid (EDTA). The addition of calcium chloride to fetuin resulted in an increase in peeling, whereas subsequent washing with EDTA abolished this effect, suggesting a role of calcium and possibly other cations in causing peeling. The presented technique for sample preparation prior to hydrazinolysis greatly reduces the level of undesirable cleavage products in O-glycan analysis and increases the robustness of the method. 相似文献
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Pinin is a cell adhesion-associated and nuclear protein that has been shown to localize in the vicinity of intermediate filament (IF) convergence upon the cytoplasmic face of the desmosomal plaque as well as in the nucleus. The localization of pinin to the desmosomes has been correlated with the reinforcement of intercellular adhesion and increased IF organization. In this study, keratins 18, 8, and 19 were identified to interact with the amino end domain of pinin in a two-hybrid screening. Further truncation analyses indicated that the 2B domain of keratin contains the sequence responsible for interacting with pinin. The amino end of pinin (residues 1-98) is sufficient to bind to keratin. Point mutation analyses revealed two essential residues within the pinin fragment 1-98, leucine 8 and leucine 19, for the interaction with keratin. Finally, in vitro protein overlay binding assays confirmed the direct interaction of the amino end domain of pinin with keratins, while pinin mutant L8P GST fusion protein failed to bind to keratins in the overlay assay. Coupled with our previous morphological observations and transfection studies, these data suggest that pinin may play a role in epithelial cell adhesion and the IF complex through a direct interaction with the keratin filaments. 相似文献
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《Process Biochemistry》2010,45(4):586-592
Immobilized lipase-catalyzed synthesis of benzoic acid hydrazide from hydrazine and phenyl benzoate is reported in this work. A series of immobilized lipases such as Candida antarctica lipase B, Mucor miehei lipase and Thermomyces lanuginosus lipase were screened to establish that C. antarctica lipase B was the best lipase for hydrazinolysis. When phenyl benzoate (0.01 mol) and hydrazine (0.02 mol) in toluene (15 ml) were reacted with C. antarctica lipase B (Novozym 435) at 50 °C, 95% of phenyl benzoate was converted to benzoic acid hydrazide after 2 h. The effects of various parameters such as speed of agitation, concentration of the substrates, temperature, enzyme concentration, and reusability of the enzyme were studied to deduce kinetics and mechanism of the reaction. A mechanism based on an ordered bi–bi dead end complex with hydrazine was found to fit the data. Systematic deactivation studies indicated that the enzyme was deactivated due to the hydrazine and phenol, enzyme deactivation obeys first-order series model. The kinetic parameters deduced from these models were used to simulate the lipase activity. There was a very good agreement between the simulated and experimental values. 相似文献
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The partial amino acid sequence of the epidermal growth-factor-binding protein was determined. Residues in 108 unique positions, corresponding to 45% of the molecule, were identified. The protein is a serine protease, closely related to the nerve growth factor gamma subunit. It is suggested that the epidermal growth-factor-binding protein, like other serine protease, is synthesized as a single polypeptide chain which undergoes limited endoproteolysis. The isolated material also contained minor amounts of a second serine protease. This protease is closely related to the epidermal growth-factor-binding protein, differing from it in 7 out of the 45 amino acid positions available for comparison. The latter protease may be identical to the previously described protease A. 相似文献
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