Tyrosyl Residues Research Articles

High-performance liquid chromatography of N-terminal tyrosine-containing oligopeptides by pre-column fluorescence derivatization with hydroxylamine, cobalt(II) and borate reagents

A pre-column fluorescence derivatization method is described for the high performance liquid chromatographic determination of N-terminal tyrosine-containing oligopeptides involving methionine-enkephalin and leucine-enkephalin. The peptides are converted into fluorescent derivatives by heating in a weakly alkaline medium (pH 8.5) containing hydroxylamine, cobalt(II) ion and borate. The derivatives are separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution of acetonitrile in a mobile phase containing borate buffer (pH 8.5) and tetra- n-butylammonium chloride, and then determined by fluorimetry. The derivatization provides a single fluorescent product for each N-terminal tyrosine-containing oligopeptide, but does not allow the production of fluorescent derivatives for peptides having no tyrosyl residue at the N-terminal. The method is selective and sensitive; the lower limits of detection for the N-terminal tyrosine-containing oligopeptides tested were 140–310 fmol per 100 μl injected.

Tyrosine-specific dephosphorylation-phosphorylation with alkaline phosphatases and epidermal growth factor receptor kinase as evidenced by 31P NMR spectroscopy.

The dephosphorylation of phospho-amino acids with alkaline phosphatase (AlPase) from calf intestine or Escherichia coli and the phosphorylation of bovine serum albumin (BSA) with epidermal growth factor (EGF) receptor kinase from human A431 epidermoid carcinoma cells were investigated by 31P NMR spectroscopy. The initial rates of the dephosphorylation of phospho-tyrosine (P-Tyr) and phosphoserine (P-Ser) with AlPase were essentially the same in the one-substrate system. In the two-substrate system (P-Tyr plus P-Ser), however, the ratio of the initial rate for P-Tyr vs. P-Ser was 2.4 to 4.5 depending on the buffer and pH conditions employed. This substantiates for the first time the specificity of AlPases to P-Tyr over P-Ser at the free amino acid level. In the stationary phase of the overall process, the dephosphorylation of P-Ser became slow compared to that of P-Tyr in the one-substrate system. The decrease in the rate for P-Ser was further pronounced in the two-substrate system. For this remarkable effect, the rephosphorylation of serine was responsible, as demonstrated in the reaction mixture containing serine, Pi, and AlPase. BSA phosphorylated by EGF receptor kinase exhibited sharp 31P resonances around 0 ppm at neutral pH, far distant from the peak positions (4.9 ppm) of histone H1 phosphorylated by cAMP-dependent protein kinase. These NMR data are directed evidence that BSA was phosphorylated exclusively at the tyrosyl residues, whereas the phosphorylation of histone H1 was at the seryl residues.

COOH-terminal propeptides of the major human procollagens: Structural, functional and genetic comparisons

The sequences of the carboxy-terminal extensions (COOH-propeptides) of at least one chain of all of the major human procollagens have only recently been deduced, and include those of the interstitial (α1(I), α2(I), α1(II), α1(III)). basement membrane (αl(IV)) and pericellular (α2(V)) procollagens. Comparisons of DNA and protein sequences, corresponding to these COOH-propeptides domains, established the early divergence of the basement membrane α1(IV) COOH-propeptide from the corresponding sequences of the interstitial and pericellular procollagens. The latter are relatively highly conserved and share 58% primary peptide sequence similarities, whereas sequence similarities relative to α1(IV) are limited. Hydropathy profiles and secondary structure potentials further emphasize the clustering of conserved and variable regions among the interstitial and pericellular COOH-propeptides, and provided further evidence for significant structural differences between these sequences and the α1(IV) COOH-propeptide. The most highly conserved sequences of the α1(I), α2(I), α1(II), α1(III) and α2(V) COOH-propeptides include regions surrounding the carbohydrate attachment site, cysteine-containing regions and the COOH-terminal sequences. Cysteinyl, tyrosyl and tryptophanyl residues were found to be highly conserved as were most charged residues. Localization of variable regions, in general, occurs within hydrophilic sequences with high β-turn potentials that are proximal to intron/exon splice junctions. The most variable sequences are associated with the telopeptides and adjoining NH 2-terminal portions of the COOH-propeptides as demonstrated by predictive secondary structure analyses. These results, combined with similar analyses of abnormal α2(I) COOH-propeptide ( osteogenesis imperfecta) permitted the identification of subsequences that are likely to be a prerequisite for COOH-propeptide functions, namely procollagen chain recognition and nucleation sites for triple helix formation. These functions are also common to the αl(IV) COOH-propeptide; however, the lack of cleavage of this region and its additional postulated structural role in extracellular matrix interactions likely account for its divergent primary and secondary structure.

Total synthesis of porcine cholecystokinin-33 (CCK-33)

Porcine cholecystokinin-pancreozymin (CCK), which is a 33 amino acid peptide having a sulphated tyrosyl residue at position 27, was synthesized by the classical solution procedure applying our maximum protection strategy, for which the phenoxyacetyl group was used for the first time for protection of Ser residues.

Polyclonal antibodies to the encephalitogenic neighborhoods of myelin basic protein: Singular affinity populations neutralized by specific synthetic peptide probes

Specific ligand neutralization was used to probe the extent to which singular antibody affinity populations signified specific determinants in the neighborhood of myelin basic protein (MBP) encephalitogens. The probes were individual members of a panel of synthetic peptide analogs subsuming encephalitogenic regions. Comparative Scatchard analyses of neutralized and unneutralized antisera helped to identify the particular peptide determinants involved in the original polyclonal antibody responses to the multiple antigenic determinants of encephalitogenic peptides. The range of affinities for an antibody population against a singular MBP peptide determinant was found to be relatively restricted while the range of affinities overall for all populations within a given antipeptide antiserum was found to be relatively wide and invariably discontinuous. Consequently, the individual discontinuous affinity populations could readily be dissected by application of the Rosenthal method of Scatchard curve analysis. It was found that the singular high affinity antibody population (5.6 × 10 17 M −1) of a Lewis rat antiserum to rat encephalitogenic GSLPQKAQRPQDENG (S49) was against a determinant near the N-terminal non-encephalitogenic end of the peptide. Only the low affinity antibody populations were found that had reactivity for determinants within the encephalitogenic region itself. The singular high affinity antibody population (5.97 × 10 7 M −) of a rabbit antiserum to rabbit encephalitogenic TTHYGSLPQKAQGHRPQDEG (S82) was against a determinant centered about the tyrosyl residue, within the encephalitogenic region for the rabbit, but was completely cross-reactive with a specific circulating endogenous inhibitor. The results obtained with the rat and rabbit EAE sera were consistent with a previously advanced hypothesis that antibodies to determinants within encephalitogenic neighborhoods would effectively block the onset of EAE if high enough in affinity and not neutralized by an endogenous inhibitor.

Investigation of 4-(3-hydroxyphenyl)-4-methylpipecolic acid as a conformationally restricted mimic of the tyrosyl residue of leucine-enkephalinamide.

Analogues of leucine-enkephalinamide containing N-terminal cis- or trans-4-(3-hydroxyphenyl)-4-methylpipecolic acid were prepared to examine the conformational requirements of the N-terminal tyrosyl residue in opioid activity. The diastereomeric amino acids were prepared and purified by semipreparative HPLC before incorporation into the peptide. Spectroscopic analysis based on proton nuclear Overhauser enhanced differential spectroscopy (NOEDS) allowed assignment of the cis and trans stereochemistry. Despite spatial analogy between the trans isomer 5 and leucine-enkephalinamide, it possessed neither opioid agonist nor antagonist activity in the guinea pig ileal longitudinal muscle (GPI) or mouse vas deferens (MVD) preparations. Possible explanations for this inactivity are discussed.

Phosphorylation of protein tyrosine by human erythrocyte casein kinase A

Human erythrocyte casein kinase A, previously identified as a seryl, threonyl kinase, was found also to catalyze the phosphorylation of protein tyrosine. Phosphorylation of tyrosyl residues was detected in angiotensin-II and in several tyrosine containing synthetic peptides. In addition, phosphorylation on tyrosyl residues was also observed in alkylated bovine serum albumin and in band 3 and ankyrin purified from human erythrocyte membranes. The identification of phosphotyrosine was conducted using two-dimensional thin layer electrophoresis at pH 1.9 and 3.5 after acid hydrolysis of the phosphoproteins. It should be noted, however, that the major phosphorylation sites in band 3 and ankyrin catalyzed by casein kinase A were seryl and threonyl residues.

Linked-function analysis of fluorescence decay kinetics: resolution of side-chain rotamer populations of a single aromatic amino acid in small polypeptides.

AbstractA linked‐function approach to fluorescence decay data analysis is presented that permits complex systems to be resolved from a single decay curve. The method involves linking fluorescence decay parameters based on a relationship established by independent physical measurements. As an example, by correlating the fluorescence data with 1H‐NMR results, the complex fluorescence decay kinetics of tyrosine analogs and single tyrosyl residues in simple polypeptides can be explained by ground‐state rotameric populations of the phenol ring about the Cα‐Cβ bond.

Properties of two chymotrypsins from the digestive gland of prawn Penaeus monodon

For the first time chymotrypsins have been isolated from the hepatopancreas of prawn. Two purified chymotrypsins from Penaeus monodon were found to be single-chained with molecular masses of 27 and 26 kDa, respectively. They have chymotrypsin-like specificities, and cleave the insulin chain B preferentially on the carboxyl side of tyrosyl, phenylalanyl and leucyi residues. They were effectively inhibited by soybean trypsin inhibitor, turkey and chicken ovomucoid, chymostatin, PMSF, Z-AlaGlyPhe chloromethyl ketone but not by Z-Phe chloromethyl ketone and many other small protease inhibitors. The shrimp enzymes hydrolyze protein substrates as rapidly as but small substrates much slower than bovine chymotrypsin, suggesting the importance of an extended substrate interacting site.

La gestion des acides aminés aromatiques chez les Lépidoptères

Lepidoptera, like other insects (sensu stricto, i.e., without their possible symbionts), require an exogenous input of aromatic nuclei. Phenylalanine must thus be considered as an essential amino acid; however, the most important aromatic metabolite is tyrosine. In addition to being incorporated into proteins, tyrosine goes through specific stages that are either transitory (storage or mobilization forms) or terminal in some of the major functional pathways in insects (generally through previous hydroxylation into catecholic substances). Some polypeptids incorporate tyrosyl residues either for structural or for metabolic storage purposes. Studies on insect neurochemistry have revealed the existence of neuroactive phenolamines and catecholamines. Although the chemistry of melanin formation is important in understanding certain physiological phenomena that affect both larval and adult Lepidoptera, it has never been studied in detail because of its complexity. Biochemical studies of sclerotization, which have been extensive during the past 20 years, have recently revealed several specific lepidopteran characteristics. Lepidoptera use a specific transitory storage form of tyrosine, β-D-glycopyranosyl-O-tyrosine, and the process of N-acylation, which affects dopamine at the time of nymphal ecdysis, leads to the formation of N-β-alanyldopamine instead of N-acetyldopamine as was shown by classic studies on Diptera. Moreover, differential activation of these metabolic pathways in the course of ontogenesis brings about a marked temporal compartmentalization. Many phases of tyrosine biochemistry are thus recognized in the development of Lepidoptera.[Journal translation]

Catalytic function of a tyrosyl residue in tryptophanase

Tryptophanase has an essential tyrosyl residue/active site which can be modified by tetranitromethane. Pyridoxal 5′-phosphate can prevent this modification efficiently, whereas pyridoxal 5′-phosphate N-oxide cannot, indicating that the free pyridinium N is required for the interaction of the coenzyme with the tyrosyl residue, probably via a hydrogen bond. The weakened binding of the coenzyme to the modified enzyme was confirmed on gel filtration, the modified enzyme being dissociated from the coenzyme seven-fold faster than the native enzyme. Furthermore, absorption spectral analyses demonstrated that the modified enzyme can catalyze the transaldimination step, but fails to abstract the α-H of substrates. The tyrosyl residue, therefore, not only participates in coenzyme binding, but also contributes to α-H labilization.

1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis.

Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.

Different DNA-binding modes and cooperativities for bacteriophage M13 gene-5 protein revealed by means of fluorescence depolarisation studies.

The binding of the bacteriophage-M13-encoded gene-5 protein to oligo(deoxythymidylic acid)s and M13 DNA was studied by means of tyrosyl fluorescence decay and fluorescence anisotropy measurements. The observed fluorescence decays could be described with two exponentials, characterised by the lifetimes tau 1 = 2.2 ns and tau 2 = 0.8 ns respectively. Only the amplitude of the longer-lifetime component is influenced by binding of the protein to DNA. This indicates that a part of the tyrosyl residues is involved in the binding. By means of fluorescence depolarisation measurements the rotational correlation time of the protein dimer is found to be 12.9 ns. In contrast to earlier measurements, carried out on the DNA-binding protein of phage Pf1 [Kneale, G. G. and Wijnaendts van Resandt, R. W. (1985) Eur. J. Biochem. 149, 85-93], the observed rotational correlation times of the gene-5 protein pass through a maximum when the protein is titrated with oligo(deoxythymidylic acid)s. This is not observed upon titration with M13 DNA. Our measurements showed that for the oligo(deoxythymidylic acid)s there clearly is a decrease in the number of clustered proteins on the lattice in the case of excess nucleotide. This is a direct consequence of the much lower cooperativity of the binding to the oligonucleotides compared to the cooperativity characteristic of binding to polynucleotides. The number of nucleotides covered by a protein monomer is found to be less than or equal to 3 for the oligonucleotides and approximately equal to 4 for M13 DNA. Model calculations show that the 'time-window' through which the fluorescence depolarisation can be observed (i.e. the fluorescence lifetime) in this case significantly affects the 'measured' effective rotational correlation times.

Histochemical modification of the active site of succinate dehydrogenase with N-acetylimidazole.

The kinetics of acetylation of mitochondrial succinate dehydrogenase [EC 1.3.99.1] in the two fibre types (A and C) of rat gastrocnemius with N-acetylimidazole was studied by a newly modified histochemical technique. Acetylimidazole partially inactivated the enzyme, but subsequent deacetylation with hydroxylamine restored the enzyme activity completely. Inactivation of the enzyme by acetylimidazole was prevented by malonate, which is a competitive inhibitor of the enzyme. The value of the inhibition constant (Ki = 34 microM) for malonate, obtained from the dependence of the pseudo-first order rate constant of acetylation of the enzyme with acetylimidazole on the malonate concentration, was in good agreement with the Ki value (33 microM) obtained by a different method, the dependence of the initial velocity of succinate oxidation by the dehydrogenase on the substrate concentration in the presence of malonate. These findings suggest that a tyrosyl residue is located in the malonate binding site (the active site) of succinate dehydrogenase in the gastrocnemius and plays a role in substrate binding, but is not a catalytic group.

Interactions of antibiotics of the iturin group with human erythrocytes

The peptidolipid antibiotics, iturin A and bacillomycin L have a disrupting effect on erythrocyte membrane leading to a simultaneous release of K + ions and hemoglobin. The formation of ghosts is accompanied by a partial solubilisation of lipid components. Binding experiments with radioactive antibiotics show that about 7·10 7 molecules of iturin A and 4·10 7 molecules of bacillomycin L are bound to one erythrocyte at the concentration giving 100% hemolysis. This concentration is reduced by about 20% after treatment of erythrocytes by phospholipase A 2. Scatchard plots show that the affinity for erythrocyte membrane is higher with bacillomycin L than with iturin A. This difference is probably correlated to the differences in their peptide moieties. The substitution of tyrosyl residue leads to a ten-fold increase of the concentrations giving 100% hemolysis, probably due to a low distribution coefficient of derivatives between the membrane and the solvent. This result and the humped Scatchard curves obtained with both antibiotics may be related to the self-association of the compounds in aqueous solutions.

Complete amino acid sequence of a cytochrome P-450 isolated from beta-naphthoflavone-induced rabbit liver microsomes. Comparison with phenobarbital-induced and constitutive isozymes and identification of invariant residues.

The complete covalent structure of a cytochrome P-450, form 4, isolated from liver microsomes of beta-naphthoflavone-induced rabbits was determined. The S-carboxyamidomethylated protein was cleaved with cyanogen bromide, endoproteinase Lys-C, and trypsin before and after succinylation. Selected peptides from CNBr digests of alkylated rabbit cytochrome P-450 forms 3a and 3c were also isolated and sequenced. Form 4 exhibited microheterogeneity due to the presence of several truncated forms. The existence of multiple NH2-terminal residues for form 4 was confirmed by the isolation and sequence analysis of the corresponding tryptic peptides. The predominant form contained 514 residues, corresponding to Mr 58,030. A peptide having Gly-232 and Gln-246 replaced by Ser and Asn residues, respectively, was also found in the isozyme preparation investigated here. The amino acid sequences of form 4 and selected peptide sequences from forms 3a and 3c were compared with the primary structures of forms 2 and 3b (previously determined in this laboratory). This comparison identified some 90 invariant residues. A cysteinyl residue at position 456, earlier reported as the heme-binding cysteine 436 (Heineman, F. S., and Ozols, J. (1982) J. Biol. Chem. 257, 14988-14999), was also present in forms 4, 3a, and 3c. Other single invariant residues identified were form 4/forms 2,3b, Trp-132/121, and His 270/252. The tyrosyl residues at positions 71/62 and 365/348 were also invariant. The latter is present in the "conserved segment" of the protein, residues 363/346 to 375/359, and may be involved in the substrate binding of cytochrome P-450. Also a lysyl residue, formerly identified by other laboratories to be involved in the electron transfer between the reductase and cytochrome P-450 form 2, was invariant in all five species. This lysyl residue corresponds to Lys-402 in form 4 or Lys-384 in the other forms.

Localization of the structural difference between bovine blood coagulation factors X1 and X2 to tyrosine 18 in the activation peptide.

Bovine Factor X is isolated in two chromatographically separable forms, Factor X1 and Factor X2. Whereas only a single form of Factor Xa, the active protease, exists, the activation peptides also exist as two chromatographically distinct species. These peptides have been shown to differ at a tyrosyl residue by ultraviolet spectrophotometry, and in their composition after alkaline hydrolysis. On the basis of the spectral properties, and elution position of the modified tyrosine on Dowex 1 columns and on an amino acid analyzer, it has been concluded that Factor X2 contains a tyrosyl-O-SO4 residue at position 18 in the activation peptide whereas Factor X1 contains only tyrosine. Alternative explanations such as differences in carbohydrate composition, differences in phosphate content, or differences in the number of gamma-carboxyglutamic acid residues were demonstrated to be unrelated to the difference in chromatographic behavior between bovine Factors X1 and X2.

Comparative study of the cytotoxins of the venom of the central asian cobraNaja naja oxiana by the intrinsic-fluorescence method

The intrinsic fluorescence of cytotoxin VC1 and VC5 from the venom of the Central Asian cobra due to the tyrosine residues and differing with respect to quantum yield for these two polypeptides because of the nonidentity of the microenvironments of the tyrosyl residues has been measured and characterized. The dependence of the quantum yield on the pH and the temperature also indicate this nonidentity.

Time-resolved fluorescence and 1H NMR studies of tyrosyl residues in oxytocin and small peptides: correlation of NMR-determined conformations of tyrosyl residues and fluorescence decay kinetics.

Steady-state and time-resolved fluorescence properties of the single tyrosyl residue in oxytocin and two oxytocin derivatives at pH 3 are presented. The decay kinetics of the tyrosyl residue are complex for each compound. By use of a linked-function analysis, the fluorescence kinetics can be explained by a ground-state rotamer model. The linked function assumes that the preexponential weighting factors (amplitudes) of the fluorescence decay constants have the same relative relationship as the 1H NMR determined phenol side-chain rotamer populations. According to this model, the static quenching of the oxytocin fluorescence can be attributed to an interaction between one specific rotamer population of the tyrosine ring and the internal disulfide bridge.

Production and characterization of a rat monoclonal antibody against leu 5 enkephalin

A rat × mouse hybridoma line producing a monoclonal antibody against leu 5 enkephalin has been obtained. The monoclonal antibody belongs to the IgG2b class and has an affinity constant of 8.0 × 10 8 M −1 at 4°C. This antibody exhibits ~40% cross-reactivity with 1–6 dynorphin but very weak cross-reactivities with met 5 enkephalin (1.4%), 1–13 dynorphin (1.3%) and β-endorphin (0.0045%). The detailed study, by competitive assay, of the interaction between this antibody and various enkephalin derivatives shows that the carboxy-terminal part of the molecule and, particularly, the leu side chain constitutes the immunodominant group. Nevertheless, the tyrosyl residue also contribute considerably to the binding, probably via a peculiar conformation effect, although we can not exclude the tyrosyl residue acting as a secondary contact residue. This monoclonal antibody has been used in a radioimmunoassay to determine leu 5 enkephalin like immunoreactive material present in rat brain and hypothalamus.