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1.
The reduction of methionine sulfoxide to methionine in peptides and proteins has been systematically investigated in terms of specific reducing agent, concentration of reducing agent, temperature, pH of the solution, and the presence of denaturing agents. While several of the reagents examined had a greater rate of reduction, N-methylmercaptoacetamide was found to be the reducing agent of choice as it was the reagent with the highest rate of reduction having no adverse interaction with other residues in peptides and proteins. Its rate of reduction increased until its concentration reached approximately 50% (vv). Its reducing ability was relatively independent of pH changes but decreased with increases in acetic acid concentration. Using this reagent under acid, neutral, or basic conditions at a concentration of 0.7–2.8 m, methionine sulfoxide can be completely reduced to methionine in peptides and proteins at 37°C in 12 to 24 h. The sulfoxide form of S-carbamoylmethylcysteine in peptide and proteins takes approximately five times longer to reduce than methionine sulfoxide.  相似文献   

2.
  • 1.1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone.
  • 2.2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved.
  • 3.3. Methionine 4 is the most reactive group, followed by methionines 72 and 178—methionine 123 being the less reactive residue.
  • 4.4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues.
  • 5.5. Results agree with data previously obtained with bovine growth hormone.
  相似文献   

3.
The enzymatic conversion of 5-methylthioribose to methionine and its deaminated derivatives, 2-keto-4-methylmercaptobutyric acid and 2-hydroxy-4-methylmercaptobutyric acid by cell-free extracts of Enterobacteraerogenes has been demonstrated. 14C-Labeled methionine was isolated from incubation mixtures with 5-methylthio[U-14C]ribose. The carbohydrate part of this compound furnishes at least part, if not all, of the four carbon chain of methionine.  相似文献   

4.
Human alpha-1-proteinase inhibitor (α-1-PI) from synovial fluid has been isolated to near 90% purity. The preparation has a molecular weight near 52,000, contains 3.5 residues of methionine sulfoxide, and an amino terminal glutamine residue. Sequence studies indicate that the first 17 residues, normally present in plasma α-1-PI, are missing from this protein. The inhibitor did not form a complex with porcine pancreatic elastase but, instead, was converted to a lower molecular weight form. Sequence studies on the latter indicated that two methionyl residues, one at the P1 reactive site and the other at P8, had been oxidized. These data confirm the fact that oxidized α-1-PI may be formed in vivo, presumably by the action of myeloperoxidase. This latter effect may alter the proteinase-proteinase inhibitor balance in tissues so that excess proteolysis and abnormal tissue degradation may occur.  相似文献   

5.
The amino acid sequence of the 11.6 K dalton heme a subunit of bovine heart cytochrome oxidase has been completed and is presented here. The sequence investigation has established the positions in the protein of all the possible heme ligands, namely cysteine, methionine, histidine and lysine residues. However, the isolation conditions may have caused the heme a to migrate from its original site or the heme is caged by peptides as pointed out in Reference 6. The sequence of the heme a subunit and the β-chain of hemoglobin shows homology. It is possible that these two proteins have arisen from a common ancestor in the distant past.  相似文献   

6.
A new c-type cytochrome containing a single heme group, cytochrome c553(550) has been purified from Desulfovibrio desulfuricans (Norway strain) and some of its properties have been investigated. It has an isoelectric point of 6.6 and a higher redox potential than cytochrome c3 isolated from the same bacteria. Its molecular weight was estimated to be 9,200 by gel filtration. The main absorption peaks are at 553, 522.5 and 417 nm in the reduced form and at 690, 529, 411, 357 and 280 nm in the oxidized form. The asymmetric α band of the reduced state is similar to the one reported for socalled “split α” cytochromes c. The cytochrome contains 86 amino acid residues with 5 methionine, two cysteine and two histidine residues. The N terminal sequence of D. desulfuricans Norway cytochrome c553(550) presents no evident homology with that of Desulfovibrio vulgaris Hildenborough cytochrome c553.  相似文献   

7.
Two biologically active aziridine ring-containing compounds, N,N-ethylene urethane (I) and N,N-ethylene urea (II), have been shown to react with methionine in dilute phosphate buffer (pH 7.4) at 37°C. Degradative procedures indicate that the aziridine ring effectively alkylates the thio ether group of methionine and other thio ether-containing amino acids to produce sulphonium salts (V). By using [35S]methionine, the sulphonium salts have been shown to be quite stable under physiological conditions (tbuilt12 7–9 days) hydrolysing to convert the methionine residue to homoserine.It is proposed that similar alkylations of methionyl residues in vivo by aziridine-alkylating agents may explain the complex, and as yet unknown, metabolic fate of the aziridine ring and could also be a factor contributing to the diverse effects that these agents have on living cells.  相似文献   

8.
A single DNA polymerase has been purified 965 fold from isolated chloroplasts of Euglenagracilis with a yield of 53%. The isolation methods include solubilization of the enzyme with 1M NaCl, ammonium sulfate precipitation, DNA affinity and DEAE-cellulose chromatography. The enzyme requires all four deoxynucleotide triphosphates, magnesium and denatured DNA for maximal activity. The chloroplast DNA polymerase is free of contaminating nucleases and phosphatases, has a sharp pH optimum at pH 7.2 and magnesium optimum of 6mM.  相似文献   

9.
The immobilization of glucose oxidase, a glycoenzyme from Aspergillusniger consisting of 16% carbohydrate, has been achieved by oxidizing its carbohydrate residues with periodic acid followed by coupling the activated enzyme to water-insoluble p-aminostyrene. At pH 5.6 and 25°, approximately 60% of the carbohydrate residues are oxidized, but the enzyme retains full activity. No oxidation of any amino acid residue is evident. The enzyme-polymer conjugate derived from this activated enzyme retains full activity and even shows a slightly enhanced thermal stability at 60° compared with the soluble native and oxidized glucose oxidases.  相似文献   

10.
Translation in a cell-free extract of Krebs II ascites cells of a mRNA fraction prepared from bovine parathyroid glands results in the synthesis of two radioactive products that appear identical to pre-proparathyroid hormone (Pre-ProPTH) (M.W. ~ 14,000), the suspected earliest biosynthetic precursor of parathyroid hormone (PTH) (M.W. 9,500), and to proparathyroid hormone (ProPTH) (M.W. 10,200), the immediate biosynthetic precursor of PTH. The two products of synthesis in the ascites extract co-electrophoresed on both urea-acetate and urea-SDS acrylamide gels with Pre-ProPTH obtained from cell-free translation of parathyroid RNA in extracts of wheat-germ and with ProPTH isolated from parathyroid slices. Both products were precipitated with an antiserum to PTH. Partial analysis of the amino acid sequence of [35S]methionine-labeled Pre-ProPTH synthesized by the ascites extract indicates that a substantial fraction of the product is lacking the two N-terminal methionines present in the Pre-ProPTH synthesized by the wheat-germ system. The results indicate that, (i), unlike the wheat-germ, ascites extracts contain enzymes that remove the initiator methionine from Pre-ProPTH and convert Pre-ProPTH into ProPTH (no ProPTH was observed in the wheat-germ system) and (ii) the cleavage processes appear to occur in association with synthesis, inasmuch as neither removal of NH2-terminal methionine nor formation of ProPTH was observed upon incubation of Pre-ProPTH isolated from either the wheat-germ system or from the ascites system when put back into the ascites system.  相似文献   

11.
The proximal colon of the new-born pig maintains a stable short-circuit current which is partly dependent upon the presence of methionine. This interaction between methionine and short circuit current shows Michaelis- Menten knetics with a Km of 0.24 mM and a V of 27 μA·cm?2. The net flux of methionine to the serosal surface of proximal colons also shows a hyperbolic relation to the external concentration of methionine (Km 0.38 mM; V 10.4 nmol·cm?2·min?1). The proximal colon concentrates methionine within its epithelium giving a mucosal to medium ratio of 11.2 ± 1.9 (90 min incubation in 1 mM methionine).The ability of the colon to transport methionine across and concentrate methionine within its mucosa is maintained for at least 24 h after birth. Colonic transport of amino acids could be physiologically important in the pig, where the immediate post-natal transfer of immune globulins has been shown to cause a temporary inhibition of normal intestinal function.  相似文献   

12.
Alkaline isomerization of ferricytochrome C from Euglena gracilis   总被引:1,自引:0,他引:1  
Euglena gracilis ferricytochrome c has a small absorption maximum at about 700 nm having an extinction of 850 ± 10 M?1cm?1. This absorption band is analogous to the more commonly found maximum at 695 nm which is observed in ferricytochromes from other sources and which is characteristic of ligation of methionine 80 with the heme ion. The 700 nm band disappears upon raising the pH to 11 giving a transition involving a single proton having an apparent pK of about 10. These results demonstrate that the phenolic ionization of tyrosine 67 is not required to trigger the alkaline isomerization of ferricytochromes c since Euglena cytochrome has a phenylalanine residue at position 67.  相似文献   

13.
The amino acid sequence of the Spirulina maxima ferredoxin has been determined. Spirulina maxima is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The S. maxima ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.  相似文献   

14.
The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a given protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the Escherichia coli proteome using several proteomic methodologies and globally measured oxidation rates of methionine residues in the presence and absence of tertiary structure, as well as the folding stabilities of methionine-containing domains. These data indicated that buried methionines have a wide range of protection factors against oxidation that correlate strongly with folding stabilities. Consistent with this, we show that in comparison to E. coli, the proteome of the thermophile Thermus thermophilus is significantly more stable and thus more resistant to methionine oxidation. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and propose a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures. Overall, these results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability.  相似文献   

15.
Chicken liver microsomes contain an albumin having an isoelectric point approximately 0.2 pH unit in excess of that of chicken serum albumin. Although the serum protein is also present in microsomes, only the basic albumin there becomes labelled and undergoes turnover in vivo. Sequence analysis of the purified basic microsomal albumin indicates that the first twelve residues are: Arg-Asn-Leu-Gln-Arg-Met-Ala-Arg-Asp-Ala-Glu-His. The data suggest that the octapeptide (underlined) is attached to the amino terminus of chicken serum albumin (the last four residues). The amino terminal sequence of the serum albumin precursor in chicken liver is thus markedly different from that of the rat and bovine proalbumins.  相似文献   

16.
The packing of α-helices and β-sheets in six αβ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an αβ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (106 to 1014) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 αβ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced Cα atoms averaged 5.6 Å for 100 residues. For the αβ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.  相似文献   

17.
Photoactivated cross-linking of prolactin to hepatic membrane binding sites   总被引:3,自引:0,他引:3  
The oxidized chemotactic peptide N-formyl-L-methionyl-sulfoxide-L-leucyl-L-phenylalanine can be reduced using either a partially purified methionine sulfoxide peptide reductase from Escherichiacoli or a neutrophil extract. The product of the enzymatic reduction shows chemotactic activity.  相似文献   

18.
Potato virus X (PVX) is modified by incubation with chlorogenic acid and polyphenoloxidase. The product made at pH 7 (PVX-Q1) is grey in colour, retains about 2/3 of its initial infectivity, and contains, on average, 1 molecule of bound chlorogenic acid per protein subunit. The product made at pH 7.8 (PVX-Q2) is blue, retains at least 13 of its infectivity, and contains approximately 2 molecules of chlorogenic acid per subunit. Both preparations contain a proportion (18–42%) of cross linked subunits. Brief exposure to trypsin converts subunits of both types of PVX-Q to a form with a slightly lower MW; the reaction goes more extensively with PVX-Q1 (80% converted) than with PVX-Q2 (45% converted). Prolonged exposure to trypsin degrades both forms of PVX-Q to free quinic acid and peptides, apparently only one of which contains chlorogenic acid. It is argued that, in PVX-Q1, predominantly one specific lysine ε-NH2 has been modified with chlorogenoquinone. The structure of PVX-Q2 is less clear.  相似文献   

19.
In proteins, all amino acid residues are susceptible to oxidation by various reactive oxygen species (ROS), with methionine and cysteine residues being particularly sensitive to oxidation. Methionine oxidation is known to lead to destabilization and inactivation of proteins, and oxidatively modified proteins can accumulate during aging, oxidative stress, and in various age-related diseases. Although the efficiency of a given methionine oxidation can depend on its solvent accessibility (evaluated from a protein structure as the accessible surface area of the corresponding methionine residue), many experimental results on oxidation rate and oxidation sites cannot be unequivocally explained by the methionine solvent accessible surface area alone. In order to explore other possible mechanisms, we analyzed a set of seventy-one oxidized methionines contained in thirty-one proteins by various bioinformatics tools. In which, 41% of the methionines are exposed, 15% are buried but with various degree of flexibility, and the rest 44% are buried and structured. Buried but highly flexible methionines can be oxidized. Buried and less flexible methionines can acquire additional local structural flexibility from flanking regions to facilitate the oxidation. Oxidation of buried and structured methionine can also be promoted by the oxidation of neighboring methionine that is more exposed and/or flexible. Our data are consistent with the hypothesis that protein structural flexibility represents another important factor favoring the oxidation process.  相似文献   

20.
Bovine liver microsomes contain an albumin having an apparent isoelectric point approximately 0.3 pH unit in excess of bovine serum albumin. Sequence analysis of the purified protein shows that the first ten residues at the amino terminus are: Arg-Gly-Val-Phe-Arg-Arg-Asp-Thr-His-Lys. The data suggest that the hexapeptide (underlined), identical to that found in proalbumin from rat liver, is attached to the amino terminus of bovine serum albumin (the last four residues). By analogy with the rat liver system, this protein therefore is bovine proalbumin, a precursor of bovine serum albumin.  相似文献   

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