Chemical modification of erythropoietin: an increase in in vitro activity by guanidination
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Succinylation
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N-lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously.
Acetyltransferases
Acetyltransferases
Rhodopseudomonas palustris
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Abstract The arginine and lysine residues of calf thymus histone H1 were modified with large molar excesses of 2,3‐butanedione and O ‐methylisourea, respectively. Kinetic study of the modification reaction of the arginine residue revealed that the reaction is divided into the two pseudo‐first‐order processes. About a third (1 Arg) of the total arginine residues of the H1 molecule was rapidly modified without causing any detectable structural change of the molecule, and the slow modification of the remaining arginine residues (2 Arg) led to a loss of the folded structure of H1. In the case of lysine residue modification, 93% (56 Lys) of the total lysine residues of the H1 was modified with the same rate constant, while 7% (4 Lys) of lysine residue remained unmodified. When the reaction was performed in the presence of 6 M guanidine‐HCl, all of lysine residues were modified. It is concluded that the 2 arginine and 4 lysine residues resistant to modification are buried in interior regions of the H1 molecule and play an important role in the formation of the H1 globular structure, while the other 1 arginine and 56 lysine residues are exposed to solvent.
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Guanidine
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Chemical modification
Cryoglobulins
Tetranitromethane
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Abstract Lysine acetylation refers to addition of an acetyl moiety to the epsilon‐amino group of a lysine residue and is important for regulating protein functions in various organisms from bacteria to humans. This is a reversible and precisely controlled covalent modification that either serves as an on/off switch or participates in a codified manner with other post‐translational modifications to regulate different cellular and developmental processes in normal and pathological states. This unit describes methods for in vitro and in vivo determination of lysine acetylation. Such methods can be easily extended for analysis of other acylations (such as propionylation, butyrylation, crotonylation, and succinylation) that are also present in histones and many other proteins. © 2017 by John Wiley & Sons, Inc.
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By specific chemical modification of amino acid residues of egg white lysozyme it is demonstrated that the carbamylation of the α amino group and carboxymethylation of the unique histidine residue do not affect the reaction of the enzyme with the anti‐lysozyme antibodies. In contrast, it appears from this work that carbamylation or acetylation of the ɛ amino groups, carboxymethylation of the methionine residues as well as CNBr cleavage of the polypeptide chain do alterate the antigenic structure of lysozyme.
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Muramidase
Chemical modification
Cleavage (geology)
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Formylation
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Reaction of ovine prolactin (oPRL) with a 150‐fold molar excess of N ‐acetylimidazole over protein content resulted in the modification of 2.5 tyrosine residues and 1.2 lysine residues. Acetylation greatly decreased the in vitro binding capacity to lactogenic sites. This binding capacity was partially restored by ammonium bicarbonate treatment, which removes O ‐acetyl groups from tyrosine residues but not N ‐acetyl groups from lysine residues. The modification extent of the tyrosine residues was determined. The results suggest that acetylation of tyrosine 44 or of tyrosine 96 is likely to be responsible for the decrease in binding activity of acetylated oPRL, and that one of these residues may play a role in the interaction of oPRL with lactogenic receptors.
Ammonium bicarbonate
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Bicarbonate
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Tyrosine residues in ricin D were modified with N-acetylimidazole and the saccharide binding properties of the resulting derivatives were examined. The cytoagglutinating activity of ricin D was not altered by acetylation of one tyrosine residue/mol, but decreased greatly upon modification of two tyrosine residues/mol. In the cytoagglutination test, only 6% residual activity was found in the derivative, 2-Ac-ricin D, in which 2.4 tyrosine residues/mol were acetylated. In the presence of lactose, however, one tyrosine residue/mol was protected from acetylation by N-acetylimidazole with a retention of the saccharide binding ability, suggesting the involvement of one tyrosine residue in the saccharide binding. Fluorescence and UV-difference spectroscopic data indicate that the binding ability of the low affinity saccharide-binding site (LA-site) of ricin D remained unchanged after modification of 2 tyrosine residues/mol. The affinity chromatography of the acetylated derivatives of ricin D on the lactamyl- and galactosamine-cellulofine columns suggests that 2-Ac-ricin D binds galactopyranosides only at the LA-site, but lacks the binding ability at the high affinity saccharide-binding site (HA-site). It is postulated that introduction of the bulky acetyl group into the hydroxyl group of the tyrosine residue at the HA-site of ricin D may sterically hinder the binding of saccharides.
Ricin
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