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1.
1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein. 相似文献
2.
J Y Chang 《Biochimica et biophysica acta》1979,578(1):175-187
1. A mechanism for the destruction of serine and threonine thiohydantoins during protein sequence analysis by the Edman-type degradation is proposed. The mechanism begins with the dehydration of serine and threonine side chains (at the cyclization stage) which occurs mainly in anhydrous acid solution. The dehydrated derivatives finally polymerize by way of the reactive methylene group (enamine) to form polymers with various molecular weights. In aqueous acid solution, the dehydrated thiohydantoins of serine and threonine undergo hydration (according to the Markovnikov rule) and ring fission, which leads to the irreversible breakdown of thiohydantoin ring. The serine derivative shows a much greater tendency to undergo these side-reactions than the threonine derivative. 2. In the presence of oxygen, the alkaline hydrolysis of amino acid thiohydantoins goes through an oxidation-deamination reaction at the C-N bond of the thiohydantoin ring and leads to the formation of thiourea derivative and keto acids. This reaction mechanism accounts for the low recoveries of amino acid obtained from the alkaline hydrolysis of amino acid thiohydantoins. 相似文献
3.
Pseudomonas 7A glutaminase-asparaginase (PGA) catalyzes the hydrolysis of D and L isomers of glutamine and asparagine. Crystals of PGA were reacted with diazo analogues of glutamine (6-diazo-5-oxo-L-norleucine, DON) and asparagine (5-diazo-4-oxo-L-norvaline, DONV), which are known inhibitors of the enzyme. The derivatized crystals remained isomorphous to native PGA crystals. Their structures were refined to crystallographic R = 0.20 and R(free) = 0.24 for PGA-DON and R = 0.19 and R = 0.23 for PGA-DONV. Difference Fourier electron density maps clearly showed that both DON and DONV inactivate PGA through covalent inhibition. Continuous electron density connecting the inhibitor to both Thr20 and Tyr34 of the flexible loop was observed providing strong evidence that Thr20 is the primary catalytic nucleophile and that Tyr34 plays an important role in catalysis as well. The unexpected covalent binding observed in the PGA-DON and PGA-DONV complexes shows that a secondary reaction involving the formation of a Tyr34-inhibitor bond takes place with concomitant inactivation of PGA. The predicted covalent linkage is not seen, however, suggesting an alternative method of inhibition not yet seen for these diazo analogues. These surprising results give insight as to the role of the flexible loop Thr and Tyr in the catalytic mechanism. 相似文献
4.
It is known that plasma serine and threonine concentrations are elevated in rats chronically fed an essential amino acid deficient
diet, but the underlying mechanisms including related gene expressions or serine and threonine concentrations in liver remained
to be elucidated. We fed rats lysine or valine deficient diet for 4 weeks and examined the mRNA expressions of serine synthesising
(3-phosphoglycerate dehydrogenase, PHGDH) and serine/threonine degrading enzymes (serine dehydratase, SDS) in the liver. Dietary
deficiency induced marked elevation of hepatic serine and threonine levels associated with enhancement of PHGDH mRNA expression
and repression of SDS mRNA expression. Increases in plasma serine and threonine levels due to essential amino acid deficiency
in diet were caused by marked increases in hepatic serine and threonine levels. Proteolytic responses to the amino acid deficiency
may be lessened by storing amino radicals as serine and inducing anorexia through elevation of threonine. 相似文献
5.
A model study directed towards the preparation of nucleopeptides via H-phosphonate intermediates. 总被引:5,自引:5,他引:0
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E Kuyl-Yeheskiely C M Tromp A H Schaeffer G A van der Marel J H van Boom 《Nucleic acids research》1987,15(4):1807-1818
The monofunctional phosphitylating reagents bis-(N,N-diethylamino)chlorophosphine and salicylchlorophosphine have been applied for the preparation of H-phosphonates of the amino acids serine, threonine and tyrosine. Experimental evidence showed that the latter reagent was less effective for the synthesis of a tyrosine H-phosphonate. The amino acids (peptide) H-phosphonates of serine or threonine proved to be suitable starting compounds for the formation of a phosphate diester bond with the 5'-OH of a d-nucleoside derivative using pivaloyl chloride as the activating reagent. 相似文献
6.
K Watanabe 《Journal of biochemistry》1976,80(2):379-387
Mild alkaline treatment of glycopeptide (GP-II) resulted in the loss of 1 mole of serine and 5 moles of threonine per mole of GP-II, suggesting the presence of O-glycosyl bonds between 1 serine and 5 threonine residues and carbohydrate chains. Treatment of GP-II with alkaline borohydride released only disaccharide. Methylation studies of the carbohydrate moiety gave 2,3,4,6-tetra-O-methyl and 2,4,6-tri-O-methyl derivatives of mannose in a ratio of approximately 1:1. In addition, one step of Smith degradation resulted in the loss of about 6 residues of mannose per mole of GP-II. Moreover, alpha-mannosidase [EC 3.2.1.24] liberated about 6 residles of mannose per mole of GP-II. On the basis of these data, the structure of the carbohydrate moiety of GP-II was confirmed to be 3-O-alpha-mannosylmannose. The amino- and carboxyl-terminal amino acids of GP-II were determined to be threonine and serine, respectively. On reductive cleavage of N-proline bonds with metallic sodium in liquid ammonia, 2 moles of alanine per mole of GP-II were lost. From the compositions of three fragments isolated from the reductive cleavage products, the amino acid sequence of the peptide portion of GP-II was determined. Based on these data, a probable structure was proposed for GP-II. 相似文献
7.
To investigate the influence of flanking amino acid sequence on the O-glycosylation of a single threonine residue in vitro, we have examined a series of 52 related peptides. The substrates were based upon a sequence from human von Willebrand factor which is known to be glycosylated in vivo (-6PHMAQVTVGPGL+5). Each residue of the parent peptide was substituted, in turn, with isoleucine, alanine, proline, glutamic acid, or arginine. Peptides were glycosylated using a UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase purified 15,000-fold from bovine colostrum by chromatography on DEAE-Sephacel, SP-Sephadex, Sephacryl S-300, Affi-Gel Blue, and 5-mercuri-UDP-GalNAc thiopropyl-Sepharose. Single amino acid changes in the sequences flanking the threonine could profoundly alter the glycosylation of the substrate peptides. Substitution of any amino acid tested at positions +3, -3, and -2 markedly decreased O-glycosylation, as did the presence of a charged residue at position -1. The substitution of amino acids at the other positions of the peptide substrate had little effect on the incorporation of GalNAc. Statistical analysis of sequences flanking known glycosylated threonine and serine residues suggests that they should be glycosylated with equal efficiency in the same sequence context (O'Connell et al., 1991). However, the bovine colostrum transferase failed to glycosylate a peptide derived from human erythropoietin which contains a serine that is glycosylated in vivo (-5PPDAASAAPLR+5). When a threonine was substituted for the serine in this peptide (-5PPDAATAAPLR+5), the substrate proved to be an excellent acceptor of GalNAc. These observations indicate that although flanking amino acid sequence is important for the O-glycosylation of specific hydroxyamino acids, discrete threonine- and serine-specific transferases may exist. 相似文献
8.
Fish surface mucin from Pampus argenteus was extracted with different organic solvents and the residue passed through Sephadex G-200. The major peak was purified by DEAE-Cellulose chromatography and five fractions were obtained. Carbohydrate and protein contents showed that major peak is a glycoprotein. Rechromatography of this component on the Sephadex G-200 column gave a single peak, with an estimated minimal molecular weight of 6.9 X 10(5). Analysis of individual sugar components revealed the presence of galactose, glucose, mannose, arabinose, N-acetyl glucosamine, N-acetyl galactosamine and sialic acid. The most represented amino acids are threonine, serine, proline, glutamic acid and glycine. The N-terminal amino acid end was blocked. Nearly 47% of sulphate was acid labile. Sialic acid and fucose were released rapidly by mild acid hydrolysis. The presence of blood group-A activity suggests that some kind of terminal alpha-Gal-NAC may be present. 相似文献
9.
Horse B-active and non-B-active glycoproteins from gastric mucosae are indistinguishable in their precipitating abilities with concanavalin A, anti-BP1, type XIV horse antipneumococcal serum, the lectin from Lotus tetragonolobus and a group 1 anti-I serum, Ma; no Lea or Leb activity was found. Each was subjected to catalyzed release of its oligosaccharide chains by 0.05 n NaOH in 1 m NaBH4. Destruction of serine, threonine and 2-acetamido-2-deoxygalactopyranose (dGalNAc) was associated with production of alanine, α-aminobutyric acid and N-acetyl-d-galactosaminitol, as expected for a carbohydrate to peptide linkage via dGalNAc to serine or threonine. No evidence of basecatalyzed peeling was seen. Bio-Gel P-2 elution patterns of the salt-free oligosaccharides from the two preparations were compared. Unlike results obtained with human ovarian cyst substances, very little material was excluded. The largest-size chains are in the range of deca- or dodecasaccharides, and a reduced octasaccharide was isolated. The four most abundant amino acids in both B-active and non-B-active materials are threonine, serine, proline and glutamic acid, which together account for 60% of the weight of amino acids. 相似文献
10.
The nature of amino acid-sugar linkages in cell walls was investigatedin a monocotyledonous tissue, rice coleoptiles. The molar ratiosof aspartic acid, threonine, and serine in cell walls were decreasedby hydrazinolysis in coleoptiles grown both on and under water.The molar ratios of threonine and serine were decreased alsoby a NaOHNaBH4 treatment, while the alanine content was increased,and -aminobutyric acid was not formed. The cell walls were treated with NaOH in the presence of NaB3H4,hydrolyzed, then divided into amino acid and sugar fractions.Two distinct radioactive peaks were detected in the thin-layerchromatography of the amino acid fractions. One was identifiedas alanine derived from glycosylated serine; the other was confirmedto be an oxidation product of glucosaminitol. There was justone 3H-labeled product in the sugar fractions, galactitol. Theseresults suggest the presence of serine-O-galactose and asparagine-N-N-acetylglucosamine linkages in rice coleoptile cell walls. The existence of glucosamine linked to amino acids was furthersupported by the incorporation of 14C-glucosamine into cellwalls. These linkages were also detected in the cell walls ofa dicotyledonous tissue, Vicia epicotyls. (Received April 2, 1981; Accepted June 24, 1981) 相似文献
11.
Luo X Crawley SW Steimle PA Egelhoff TT Cote GP 《The Journal of biological chemistry》2001,276(21):17836-17843
Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine. 相似文献
12.
Carole E. Schanté Guy ZuberCorinne Herlin Thierry F. Vandamme 《Carbohydrate polymers》2012,87(3):2211-2216
Injection of hyaluronic acid (HA)-based hydrogels has proven to provide many therapeutic benefits. To increase the stability of HA-based products against enzymatic digestion, we modified hyaluronic acid by grafting various amino acids on its carboxylic group and then evaluated the enzymatic stability of the various conjugates in presence of a hyaluronidase. Our results showed that all amino acid-modified HA polymers were more resistant to degradation compared to the native HA albeit with variation according to the amino acids. Amino acids with carboxylate groups such as aspartic acid or with hydroxyl functions (threonine, serine or tyrosine) conferred a particularly strong resistance to HA towards enzymatic digestion. The HA-amino acid products were then cross-linked with butanediol diglycidyl ether (BDDE). The swelling properties of the formed hydrogels appeared close to native HA whereas the increased resistance towards hyaluronidase digestion remained. These results suggest that amino acid-modified HA derivatives can become promising material for viscosupplementation or drug delivery. 相似文献
13.
Golan G Zharkov DO Grollman AP Dodson ML McCullough AK Lloyd RS Shoham G 《Journal of molecular biology》2006,362(2):241-258
The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane pyrimidine dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 pyrimidine dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the alpha-amino group of the N-terminal threonine residue and the C1' of the deoxyribose sugar of the 5' pyrimidine at the dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA-protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing pyrimidine dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (approximately 66 degrees ) in the DNA at the dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction. 相似文献
14.
Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis. 相似文献
15.
1. An analysis of bovine bone sialoprotein, a homogeneous glycoprotein isolated from cortical bone, is presented. 2. Analytical results agree with earlier physical measurements indicating a molecular weight of about 23000. 3. Mild acid hydrolysis and treatment with neuraminidase showed that fucose and sialic acid occupy terminal positions on oligosaccharide chains. 4. Treatment of the sialic acid-free glycoprotein with beta-galactosidase showed that much of the galactose occupies a sub-terminal location in the intact glycoprotein. 5. The polypeptide chain is rich in aspartic acid, glutamic acid, serine, threonine and glycine, and has no detectable free terminal amino group. 6. Glycopeptides were studied after proteolytic digestion. 7. It is considered that the carbohydrate moiety is highly branched and is probably linked by an acid- and alkali-stable glycosylamine bond involving aspartic acid. 相似文献
16.
Two polyclonal antibodies were raised to synthetic peptides corresponding to amino acids Ser21-Tyr35 and Lys247-Phe261 of cytochrome b561. These antibodies were used to test the native orientation of the amino and carboxyl termini of this transmembrane electron transport protein. Carboxyl-terminal epitopes were lost when intact chromaffin granules were treated with Pronase. This result indicates that the carboxyl terminus is cytoplasmically exposed and confirms a theoretical prediction obtained from hydropathy plots. Epitopes that were recognized by an amino-terminal antipeptide antibody were not removed under the same conditions. This finding implied that the amino terminus was not proteolytically accessible on the exterior of the granule. The abundance of threonine and serine residues in the amino-terminal region suggested that the amino terminus could be held in the membrane by covalent fatty acylation. Treatment of purified delipidated cytochrome b561 with hydroxylamine resulted in the release of a fatty acid hydroxamate. Sulfhydryl analysis of purified cytochrome b561 showed that all 3 cysteine residues were in the free sulfhydryl form. These observations indicate that cytochrome b561 is covalently fatty acylated and that the lipid is bound through ester linkages of serine or threonine residues. 相似文献
17.
Hydroxy amino acid metabolism in Pseudomonas cepacia: role of L-serine deaminase in dissimilation of serine, glycine, and threonine.
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Growth of Pseudomonas cepacia (P. multivorans) on serine depended upon induction of a previously undescribed L-serine deaminase distinct from threonine deaminase. Formation of the enzyme was induced during growth on serine, glycine, or threonine. The induction pattern reflected a role of the enzyme in catabolism of these three amino acids. Both threonine and glycine supported growth of serine auxotrophs and were presumably converted to serine and pyruvate in the course of their degradation. Mutant strains deficient in serine deaminase, or unable to use pyruvate as a carbon source, failed to utilize serine or glycine and grew poorly with threonine, whereas strains deficient in threonine dehydrogenase or alpha-amino beta-ketobutyrate:coenzyme A ligase (which together convert threonine to glycine and acetyl coenzyme A) failed to utilize threonine or derepress serine deaminase in the presence of this amino acid. The results confirm for the first time the role of alpha-amin beta-ketobutyrate:coenzyme A ligase in threonine degradation and indicate that threonine does not mimic serine as an inducer of serine deaminase. 相似文献
18.
J S Culp H J Blytt M Hermodson L G Butler 《The Journal of biological chemistry》1985,260(14):8320-8324
We report here the identification of the amino acid residue which forms the covalent intermediate in the catalytic mechanism of bovine intestinal 5'-nucleotide phosphodiesterase and the sequence of the neighboring amino acids. The active site of 5'-nucleotide phosphodiesterase was labeled using thymidine 5'-[alpha-32P]triphosphate as substrate. A single labeled cyanogen bromide peptide was isolated using reversed-phase high performance liquid chromatography. After subdigestion with endoproteinase Lys-C and chymotrypsin, the entire amino acid sequence of the 60-residue active site peptide was obtained using automated Edman degradation. All of the radioactivity of the active site peptide was localized to a hexapeptide with sequence Thr-Phe-Pro-Asn-His-Tyr. Phosphoamino acid analysis of this peptide indicated that the labeled residue was threonine. We are not aware of any other enzymes in which threonine is phosphorylated as a covalent intermediate in the catalytic mechanism. 相似文献
19.
Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies. 总被引:21,自引:15,他引:6
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J Peters M Peters F Lottspeich W Schfer W Baumeister 《Journal of bacteriology》1987,169(11):5216-5223
The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate [HPI])-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids. Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids. The N terminus was blocked to Edman degradation. The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence. The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%). The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids. 相似文献
20.
R. N. Trigiano R. A. May B. V. Conger 《In vitro cellular & developmental biology. Plant》1992,28(4):187-191
Summary Production of somatic embryos in suspension cultures ofDactylis glomerata L. (orchardgrass) was stimulated by the addition of various compounds containing reduced nitrogen to Schenk and Hildebrandt
(SH) basal medium. Equimolar concentrations of combinations of proline and either serine or threonine supported embryogenesis,
whereas, these amino acids individually did not promote embryogenesis. SH medium supplemented with 6 to 25 mM ammonium ion or Murashige-Skoog basal medium also supported embryo production. Ammonium ion did not act synergistically with
either proline or serine to enhance embryogenesis. Embryos produced in media containing amino acid combinations were formed
singly, did not exhibit secondary embryogenesis, and had significantly higher conversion rates compared to those formed in
either SH medium supplemented with ammonium ion only or in combination with amino acids. 相似文献