Chemical properties of the histidine residue of secretin: evidence for a specific intramolecular interaction

Secretin has a single histidine residue located at the amino terminus which plays a crucial role in its biological activity. The chemical properties, viz. pK and reactivity, of the alpha-amino and imidazole groups of this residue were determined at a secretin concentration of 10(-6) M in 0.1 M KCl at 37 degrees C. Competitive labelling using tritiated 1-fluoro-2,4-dinitrobenzene (DNP-F) as the labelling reagent was the experimental approach employed. The alpha-amino group was found to have a pK value of 8.83 and a reactivity 5-times that of the alpha-amino group in the model compound, histidylglycine. For the imidazole function a pK value of 8.24 and a reactivity 26-times that of the imidazole function in histidylglycine was found. Both these groups in secretin had pK values which were shifted one pK unit higher than in histidylglycine, but like the model compound the reactivity of the imidazole function was still linked to the state of ionization of the alpha-amino group. These observations are interpreted as evidence for the existence of a major conformational state in dilute aqueous solution in which the amino-terminal histidine of secretion is interacting with a negatively charged carboxyl group.     

o-Quinones formed in plant extracts. Their reactions with amino acids and peptides

1. The reactions of amino acids and peptides with the o-quinones produced by the enzymic oxidation of chlorogenic acid and caffeic acid have been studied manometrically and spectrophotometrically. 2. Amino acids, except lysine and cysteine, react primarily through their alpha-amino groups to give red or brown products. These reactions, which compete with the polymerization of the quinones, are followed by secondary reactions that may absorb oxygen and give products with other colours. 3. The in-amino group of lysine reacts with the o-quinones in a similar way. The thiol group of cysteine reacts with the quinones, without absorbing oxygen, giving colourless products. 4. Peptides containing cysteine react with the o-quinones through their thiol group. 5. Other peptides, such as glycyl-leucine and leucylglycine, react primarily through their alpha-amino group and the overall reaction resembles that of the N-terminal amino acid except that it is quicker. 6. With some peptides, the secondary reactions differ from those that occur between the o-quinones and the N-terminal amino acids. The colours produced from carnosine resemble those produced from histidine rather than those from beta-alanine, and the reactions of prolylalanine with o-quinones are more complex than those of proline.     

Selective isolation of free and blocked amino-terminal peptides from enzymatic digestion of proteins

A general method for the selective isolation of free and blocked amino-terminal peptides from proteins is described. The rationale behind the methodology is based on the reasoning that if a protein, which has all its free amino groups blocked by citraconylation, is digested with a protease, all peptides, except those derived from the amino terminus, will have a free amino group. Reaction of such a digest with 1-fluoro-2,4-dinitrobenzene (Dnp-F) followed by removal of citraconyl groups by acid treatment and removal of dinitrophenyl (Dnp) groups from histidine and tyrosine side chains by thiolysis will result in dinitrophenylation of all alpha-amino groups of peptides generated from internal cleavages, leaving only peptides derived from the amino terminus without a Dnp group. The strong adsorption of Dnp groups to polystyrene is used to selectively elute the underivatized amino-terminal peptides from such a column. It is also demonstrated how selective isolation of amino-terminal peptides can be used to determine whether a protein has a free or blocked amino terminus.     

Amino acid specificity of glycation and protein-AGE crosslinking reactivities determined with a dipeptide SPOT library.

Advanced glycation end products (AGEs) contribute to changes in protein conformation, loss of function, and irreversible crosslinking. Using a library of dipeptides on cellulose membranes (SPOT library), we have developed an approach to systematically assay the relative reactivities of amino acid side chains and the N-terminal amino group to sugars and protein-AGEs. The sugars react preferentially with cysteine or tryptophan when both the alpha-amino group and the side chains are free. In peptides with blocked N-terminus and free side chains, cysteine, lysine, and histidine were preferred. Crosslinking of protein-AGEs to dipeptides with free side chains and blocked N termini occurred preferentially to arginine and tryptophan. Dipeptide SPOT libraries are excellent tools for comparing individual reactivities of amino acids for nonenzymatic modifications, and could be extended to other chemically reactive molecules.     

Quantitative identification of N-terminal amino acids in proteins by radiolabeled reductive methylation and amino acid analysis: application to human erythrocyte acetylcholinesterase

A novel method of determining N-terminal amino acids in proteins is introduced. Reductive methylation of a protein with radiolabeled formaldehyde methylates both the alpha-amino group of the N-terminal amino acid and the epsilon-amino groups of Lys residues. The radiomethylated amino acids are stable to acid hydrolysis, and each of 16 possible hydrolysis-stable N-terminal amino acids can be identified by the unique elution positions of its N alpha-methyl and N alpha,N alpha-dimethyl derivatives with an appropriate amino acid analyzer elution schedule. The technique is at least as sensitive as other N-terminal amino acid determinations and, in addition, permits a quantitative evaluation of the number of N-terminal groups in a sample. Reductive methylation of bovine serum albumin revealed N-terminal Asp at a stoichiometry of 0.97 amino acid residue per polypeptide, while methylation of prolactin resulted in 0.86 residue of N-terminal Thr per polypeptide. Human erythrocyte acetylcholinesterase contained two N-terminal amino acids with stoichiometries of 0.66 Glu and 0.34 Arg per 70-kDa subunit. Identification of Glu as the principal N-terminus of acetylcholinesterase was confirmed by Edman sequencing.     

In vitro demonstration of histamine biosynthesis from carnosine by kidneys of pregnant mice

Kidneys of pregnant mice synthesize histamine when incubated in the presence of carnosine, manganese, and pyridoxal phosphate. Intensity of biosynthesis increases linearly with the amount of enzyme and the incubation time. The reaction can only be catalysed by two enzymes that are located in kidneys and act in succession: carnosinase, which hydrolyzes carnosine into its two moieties, and histidine decarboxylase, which transforms histidine, a product of carnosine degradation, into histamine. The biosynthesis of histamine from carnosine seems to increase with the progress of pregnancy. In nonpregnant mice, kidneys do not effect this biosynthesis. The above results directly demonstrate that carnosine may be used for histamine synthesis when the activity of histidine decarboxylase is high, as in pregnant mouse kidney. Vertebrate carnosine, its role still enigmatic, might thus be mainly a potential histidine reservoir that would be mobilized any time there is a significant requirement for histidine, such as for histamine biosynthesis.     

Reactions of N-hydroxysulfosuccinimide active esters

N-Hydroxysulfosuccinimide esters are reactive functional groups employed in a variety of protein modification reagents, especially cross-linking reagents. For these compounds, hydrolysis is the most important reaction competing for reaction of the esters with nucleophilic groups in proteins. We have employed model compounds to investigate the rates of hydrolysis of N-hydroxysulfosuccinimide esters and their reactions with the alpha-amino group and the side chains of naturally occurring amino acids, under conditions comparable to those used for protein modification studies. The rats of hydrolysis observed were found to be very low, as compared with their rates of reaction with nitrogen nucleophiles found in proteins. Further, within the ranges investigated, the rate of aminolysis was observed to increase more rapidly than the rate of hydrolysis with increasing pH or with increasing temperature. Four amino acid side chains and the alpha-amino group were found to react measurably with N-hydroxysulfosuccinimide esters. At pH 7.4 and room temperature, the order of reactivity was found to be N alpha-Cbz-histidine greater than N alpha-Cbz-lysine approximately phenylalanine (alpha-amino group) much greater than N-acetylcysteine approximately N-acetyltyrosine; however, the acylimidazole adduct formed with the side chain of histidine was found to be a transient product, subject to hydrolysis or reaction with another nucleophile.     

Inactivation of the intestinal uptake system for beta-lactam antibiotics by diethylpyrocarbonate

The uptake system for beta-lactam antibiotics in the rabbit small intestine was investigated using brush-border membrane vesicles. After treatment of membrane vesicles with the reagent diethylpyrocarbonate (DEP), the uptake of orally active beta-lactam antibiotics with an alpha-amino group in the substituent at position 6 or 7 of the penam or cephem nucleus was significantly inhibited, whereas DEP-treatment had no inhibitory effect on the uptake of beta-lactam antibiotics without an alpha-amino group. The kinetic analysis revealed an apparent competitive inhibition indicating a decreased affinity of the transport system for alpha-amino-beta-lactam antibiotics. Substrates of the intestinal dipeptide transport system - dipeptides and alpha-amino-beta-lactam antibiotics - could protect the transport system from irreversible inhibition by DEP, whereas beta-lactam antibiotics without an alpha-amino group as well as amino acids or bile acids had no effect. Incubation of DEP-treated vesicles with hydroxylamine led to a partial restoration of the transport activity indicating that DEP may have led to a modification of a histidine residue of the transport protein. From the data presented we conclude that a specific interaction of the alpha-amino group in the substituent at position 6 or 7 of the penam or cephem nucleus presumably with a histidine residue of the transport protein is involved in the translocation process of orally active alpha-amino-beta-lactam antibiotics across the intestinal brush-border membrane.     

Proton-magnetic-resonance studies of the lysine residues of ribonuclease A.

The amino groups of ribonuclease A (RNase-A) have been methylated with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance (PMR) studies. Although enzymatic activity is lost, PMR difference spectroscopy and PMR studies of thermal denaturation show native conformation is largely preserved in methylated RNase-A. Resonances corresponding to the NH2-terminal alpha-amino and 10 xi-amino N-methyl groups are titrated at 220 MHz to obtain pK values. After correction for the effects of methylation, using values previously derived from model compound studies, a pK of 6.6 is found for the alpha-amino group, a pK of 8.6 for the xi-amino group of lysine-41 and pK values ranging from 10.6 to 11.2 for the other lysine xi-amino groups. Interactions between lysine-7 and lysine-41 or between the alpha-amino and xi-amino groups of lysine-1 have been proposed to account for deviations from simple titration behaviour. The correct continuities for the titration curves of the histidine H-2 proton resonances have been confirmed by selective deuteration of the H-2 protons. Titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A show deviations from the titration curves for the native enzyme, indicating some alteration of the active-site conformation. In the presence of phosphate, titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A indicate binding of phosphate at the active site, but these curves continue to show deviations from the titration behaviour of native RNase-A. The titration curve for the N-methyl resonance of lysine-41 is perturbed considerably by the presence of phosphate, which indicates a possible catalytic role for lysine-41.     

Rubisco small and large subunit N-methyltransferases. Bi- and mono-functional methyltransferases that methylate the small and large subunits of Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)is methylated at the alpha-amino group of the N-terminal methionine of the processed form of the small subunit (SS), and at the epsilon-amino group of lysine-14 of the large subunit (LS) in some species. The Rubisco LS methyltransferase (LSMT) gene has been cloned and expressed from pea and specifically methylates lysine-14 of the LS of Rubisco. We determine here that both pea and tobacco Rubisco LSMT also exhibit (alpha)N-methyltransferase activity toward the SS of Rubisco, suggesting that a single gene product can produce a bifunctional protein methyltransferase capable of catalyzing both (alpha)N-methylation of the SS and (epsilon)N-methylation of the LS. A homologue of the Rubisco LSMT gene (rbcMT-S) has also been identified in spinach that is closely related to Rubisco LSMT sequences from pea and tobacco. Two mRNAs are produced from rbcMT-S, and both long and short forms of the spinach cDNAs were expressed in Escherichia coli cells and shown to catalyze methylation of the alpha-amino group of the N-terminal methionine of the SS of Rubisco. Thus, the absence of lysine-14 methylation in species like spinach is apparently a consequence of a monofunctional protein methyltransferase incapable of methylating Lys-14, with activity limited to methylation of the SS.     

The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and alpha-amino groups

As previous studies have indicated a multiple electrophilic reactivity of patulin (PAT) towards simple thiol nucleophiles, we have methodically investigated the ability of PAT to covalently crosslink proteins in vitro. By means of sodium dodecylsulphate polyacrylamide gel electrophoresis, the formation of PAT-induced intermolecular protein-protein crosslinks was clearly demonstrated for bovine serum albumin containing one thiol group per molecule, but also for the thiol-free hen egg lysozyme. Characterization of the crosslink sites was carried out by (1) modulation of the thiol groups with N-ethylimaleimide and 2-iminothiolane; (2) comparison with various known crosslinking agents, i.e. phenylenedimaleimide, glutardialdehyde, and dimethylsuberimidate, and (3) fluorescence incorporation studies using dansyl-labeled amino acids and a fluorescent glutathione derivative. The thiol group of cysteine was preferred for PAT-mediated crosslink reactions, but the side chains of lysine and histidine, and alpha-amino groups also exhibited reactivity. PAT can act both as a homobifunctional as well as a heterobifunctional crosslinking agent. The initial formation of a monoadduct with a thiol group appears to activate PAT for the subsequent reaction with an amino group, but also leads to rapid loss of further electrophilic properties when no second nucleophile for crosslink completion is available. Studies using microtubule proteins as a protein with experimentally controllable quarternary structure and a proposed cellular target for PAT toxicity emphasized the influence of specific sterical conditions on crosslink formation at low protein concentrations. Non-polymerized microtubule proteins, i.e. tubulin alpha,beta-dimers, formed a defined product with PAT consisting of an intramolecularly crosslinked beta-tubulin, whereas guanosine triphosphate- or paclitaxel-induced polymerization to microtubule-like quarternary structures prior to treatment with PAT gave rise to intermolecular crosslink formation between alpha- and beta-tubulin. In contrast, denaturated tubulin yielded none of those two new protein species, but only unspecific intramolecular crosslinks and highly crosslinked aggregates. Thus, in addition to the amino acid composition, the tertiary and quarternary superstructures of proteins appear to markedly influence their reactivity towards PAT. Under appropriate conditions, the generation of protein crosslinks could easily be observed at concentrations of PAT equal to or even below the concentration of the protein. The relevance of these novel reaction pathways of PAT demonstrated in vitro for its in vivo mechanisms of toxicity remains to be investigated.     

Differential Proteome and Interactome Analysis Reveal the Basis of Pleiotropy Associated With the Histidine Methyltransferase Hpm1p

The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates H243 in the ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extraribosomal phenotypes including improved growth rates in alternative carbon sources. Here, we investigate how the loss of histidine methyltransferase Hpm1p results in diverse phenotypes, through use of targeted mass spectrometry (MS), growth assays, quantitative proteomics, and differential crosslinking MS. We confirmed the localization and stoichiometry of the H243 methylation site, found unreported sensitivities of Δhpm1 yeast to nonribosomal stressors, and identified differentially abundant proteins upon hpm1 knockout with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale stable isotope labeling of amino acids in cell culture crosslinking MS for yeast, which resulted in the identification of 1267 unique in vivo lysine–lysine crosslinks. By reproducibly monitoring over 350 of these in WT and Δhpm1, we detected changes to protein structure or protein–protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria. Importantly, these occurred independently of changes in protein abundance and could explain a number of phenotypes of Δhpm1, not addressed by expression analysis. Further to this, some phenotypes were predicted solely from changes in protein structure or interactions and could be validated by orthogonal techniques. Taken together, these studies reveal a broad role for Hpm1p in yeast and illustrate how crosslinking MS will be an essential tool for understanding complex phenotypes.     

Reductive methylation and 13C NMR studies of the lysyl residues of fd gene 5 protein. Lysines 24, 46, and 69 may be involved in nucleic acid binding

We have examined the role of lysyl residues in the binding of fd gene 5 protein to a nucleic acid polymer. The lysyl residues of the protein were chemically modified to form N epsilon, N epsilon-dimethyllysyl derivatives containing 13C-enriched methyl groups. The 13C NMR spectrum of the modified protein was studied as a function of pH and salt concentration. Differences in the local magnetic environment of the six dimethyllysyl amino groups allowed all six 13C resonances to be resolved for samples in the pH range 8.5-9.0 at less than 50 mM ionic strength. One of the dimethylamino resonances was split at low pH, indicating that the two methyl groups were nonequivalent and that the corresponding lysyl residue (either Lys-3 or Lys-7) might be involved in an ion-pairing interaction. Specific lysyl residues were protected from methylation when the protein was bound to poly(rU). The level of protection of individual lysyl residues was quantitated using peptide mapping and sequencing of gene 5 protein labeled with 3H and 14C radioactive labels. Lysines 24, 46, and 69 showed significant protection (33-52%) from methylation in the protein-polynucleotide complex, suggesting that these 3 residues form part of the nucleic acid-binding site. The alpha-amino group of Met-1 was relatively unreactive in both the free and bound protein, which indicated that the amino terminus is not as exposed in solution as in the crystal structure (Brayer, G.D., and McPherson, A. (1983) J. Mol. Biol. 169, 565-596).     

Amino acid substitution and modification resulting from Escherichia coli expression of recombinant Plasmodium falciparum histidine-rich protein II

The histidine-rich protein II (HRP II) from Plasmodium falciparum is an unusual protein composed of 40% alanine, 36% histidine, and 11% aspartate residues. Expression of HRP II in Escherichia coli results in the isolation of a heterogeneous protein. Mass spectrometry reveals a reduction in mass by multiples of 9 Da from the expected molecular mass that can be attributed to the substitution of glutamine for some histidine residues in the sequence. The extent of the glutamine for histidine substitution can be reduced by slowing the expression rate. Mass spectral analysis of HRP II also revealed alpha-amino methylation of the N-terminal alanine residue of HRP II.     

肌肽、丙氨酸和组氨酸抗氧化性的比较研究

肌肽是一种发现于脊椎动物骨骼肌和大脑中的二肽(β-丙氨酰-L-组氨酸).为了探讨肌肤的抗氧化性与其结构之间的关系,试验研究了肌肽、丙氨酸和组氨酸对DPPH自由基的清除作用和对牛血清白蛋白(BSA)氧化修饰的抑制作用.结果表明肌肽对DPPH自由基有显著的清除效果(P＜0.01),组氨酸清除率低于肌肤,而丙氨酸基本无清除自...     

Peptide utilization in Escherichia coli: Studies with peptides containing β-alanyl residues

A glycine auxotroph of Escherichia coli can utilize glycine oligopeptides as a source of its required amino acid. Glycylglycyl-β-alanine and β-alanylglycylglycine are both readily hydrolysed by intracellular peptidases, but only the former supports growth of the glycine auxotroph. Glycylglycyl-β-alanine is not nutritionally active towards a glycine mutant that is unable to transport oligopeptides. The nutritional responses to these β-alanine peptides are interpreted in terms of the structural requirements of the oligopeptide transport system, for which an α-peptide bond is required but the C-terminal α-carboxyl group is not essential. Dipeptides of β-alanine are generally poor sources of amino acids for auxotrophs of E. coli, although β-alanylhistidine (carnosine) is as effective as the free amino acid in supporting growth of a histidine auxotroph; this observation does not accord with the structural requirements established for dipeptide transport in general, and may indicate a separate uptake process. The results are related to the occurrence of β-alanyl peptides in the normal environment of enteric bacteria, and to the known ability of the intestine to transport carnosine.     

Metabolism of thyrotropin releasing hormone in brain extracts. Isolation and characterization of an imidopeptidase for histidylprolineamide.

An extract of porcine brain acetone powder incubated with thyrotropin-releasing hormone (TRH; pGlu-His-ProNH2) produces acid TRH (pGlu-His-Pro), histidine, and prolineamide. Fractionation of the brain extract by DEAE-cellulose chromatography produces three protein fractions which metabolize TRH. The activity of these fractions was characterized using TRH with a 3H-label on the histidine or proline as well as [His-3H]His-ProNH2. Fraction I contains pyroglutamate aminopeptidase and Fraction II contains TRH deamidase. Fraction III was found to contain a previously unrecognized enzyme which cleaves His-ProNH2 to histidine and proline. The histidylprolineamide imidopeptidase has been characterized. A competition study using a variety of compounds containing histidine or proline suggests that the best substrates for the imidopeptidase contain a free alpha-amino group on histidine and a blocked carboxyl group on proline, as is found in His-ProNH2. A survey of a variety of polypeptide hormones indicates that many of them inhibit the imidopeptidase activity. A kinetic study of the inhibition of the enzyme by adrenocorticotropic hormone (1-24) shows that the inhibition by polypeptide hormones is noncompetitive. We hypothesize that pituitary hormones may stimulate the production of (cyclo)-His-Pro by inhibiting alternate routes of TRH metabolism.     

In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine.

Carnosine, a beta-alanyl-L-histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)-generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and beta-alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe-nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Anti-Aggregating Effect of the Naturally Occurring Dipeptide Carnosine on Aβ1-42 Fibril Formation

Carnosine is an endogenous dipeptide abundant in the central nervous system, where by acting as intracellular pH buffering molecule, Zn/Cu ion chelator, antioxidant and anti-crosslinking agent, it exerts a well-recognized multi-protective homeostatic function for neuronal and non-neuronal cells. Carnosine seems to counteract proteotoxicity and protein accumulation in neurodegenerative conditions, such as Alzheimer’s Disease (AD). However, its direct impact on the dynamics of AD-related fibril formation remains uninvestigated. We considered the effects of carnosine on the formation of fibrils/aggregates of the amyloidogenic peptide fragment Aβ1-42, a major hallmark of AD injury. Atomic force microscopy and thioflavin T assays showed inhibition of Aβ1-42 fibrillogenesis in vitro and differences in the aggregation state of Aβ1-42 small pre-fibrillar structures (monomers and small oligomers) in the presence of carnosine. in silico molecular docking supported the experimental data, calculating possible conformational carnosine/Aβ1-42 interactions. Overall, our results suggest an effective role of carnosine against Aβ1-42 aggregation.     

Neuroprotective actions of a histidine analogue in models of ischemic stroke

Histidine is a naturally occurring amino acid with antioxidant properties, which is present in low amounts in tissues throughout the body. We recently synthesized and characterized histidine analogues related to the natural dipeptide carnosine, which selectively scavenge the toxic lipid peroxidation product 4-hydroxynonenal (HNE). We now report that the histidine analogue histidyl hydrazide is effective in reducing brain damage and improving functional outcome in a mouse model of focal ischemic stroke when administered intravenously at a dose of 20 mg/kg, either 30 min before or 60 min and 3 h after the onset of middle cerebral artery occlusion. The histidine analogue also protected cultured rat primary neurons against death induced by HNE, chemical hypoxia, glucose deprivation, and combined oxygen and glucose deprivation. The histidine analogue prevented neuronal apoptosis as indicated by decreased production of cleaved caspase-3 protein. These findings suggest a therapeutic potential for HNE-scavenging histidine analogues in the treatment of stroke and related neurodegenerative conditions.