Tyrosinase exhibits lag at pH 6.8 under steady state concentrations of tyrosine and 3,4-dihydroxy phenyl alanine in melanoma tissue.

Tyrosine to dopa ratio determines the extent of lag in cresolase activity of tyrosinase when assayed at pH 6.8. The levels of tyrosine and dopa in B-16 murine melanoma tissue were found to be 213 and 13 mmoles/g fresh wt of tissue respectively. Cresolase activity of tyrosinase, when assayed at the above steady state levels of tyrosine and dopa at pH 6.8, exhibited a lag of 5-15 min depending on the amount of enzyme used in the assay mixture and the initial enzyme activity was zero. Under in vivo conditions, the enzyme with zero initial activity can not be active and therefore a far reaching conclusion is that tyrosine to dopa ratio may not regulate the enzyme activity, unlike under in vitro conditions. Possible modes of the regulation of tyrosinase under in vivo conditions are discussed.     

Potent quinoxaline-based inhibitors of PDGF receptor tyrosine kinase activity. Part 1: SAR exploration and effective bioisosteric replacement of a phenyl substituent

Novel substituted 2-anilino- and 2-cycloalkylaminoquinoxalines have been found to be useful and selective inhibitors of PDGF-R autophosphorylation. Replacement of an anilino-substituent with substituted cyclohexylamino- or norbornylamino substituents led to significant improvements in the pharmacokinetic profile of these analogues.     

Identification of tyrosine O-sulfate in proteins by reverse-phase high-performance liquid chromatography: use of base hydrolysis combined with precolumn derivatization using phenyl isothiocyanate

A procedure has been developed for the analysis of tyrosine O-sulfate in proteins. Samples are subjected to base hydrolysis with Ba(OH)2, neutralized with sulfuric acid, and the majority of other amino acids removed by chromatography on Dowex AG 50 X 8. The average recovery of tyrosine O-sulfate from these procedures was 43%. Tyrosine O-sulfate was identified by reverse-phase HPLC as the phenylthiocarbamyl derivative following precolumn derivatization with phenyl isothiocyanate. The method has been applied to bovine fibrinogen giving a tyrosine O-sulfate content ranging from 0.59 to 1.23 mol/mol. These procedures were also shown to be suitable for the analysis of the incorporation of [35S]sulfate into tyrosine O-sulfate residues in proteins by intact cells.     

Maintenance of alpha-helical structures by phenyl rings in the active-site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from Pseudomonas putida biotype B.

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Delta5-3-ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14.Tyr55.Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of alpha-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimination of the phenyl ring of the tyrosine reduced significantly the content of alpha-helices. Urea-induced equilibrium unfolding experiments revealed that the DeltaG(U)H2O values of Y14S, Y30S, and Y55S were significantly decreased by 11.9, 13.7, and 9.5 kcal/mol, respectively, as compared with that of the wild type. A characterization of the unfolding kinetics based on PhiU-value analysis indicates that the interactions mediated by the tyrosine triad in the native state are very resistant to unfolding. Taken together, our results demonstrate that the internal packing by the phenyl rings in the active-site tyrosine triad contributes to the conformational stability and catalytic activity of KSI by maintaining the structural integrity of the alpha-helices.     

Action of the phosphonic analogue of tyrosine and of other tyrosine derivatives on rat liver tyrosine aminotransferase

Summary Tyrosine transamination has been investigatedin vitro with a preparation of rat liver tyrosine aminotransferase in the presence of several structural derivatives of the substrate, including the phosphonic analogue. The transamination by tyrosine aminotransferase (TAT) needs the presence in the substrate molecule of free amino and carboxylic groups, a three-carbon aliphatic chain, a para-phenolic hydroxylic function and al-configuration. Some tyrosine analogues can markedly disturb the Tyr-TAT association: the chief structural modifications are (i) the removal of the free amine function in a compound still possessing a para-hydroxylic and a carboxylic group, (ii) the change of the carboxylic function by another acidic group, especially a phosphonic one, (iii) a disubstitution in positions 3 and 5. In every situation, the presence of a parahydroxylic group is compulsory to observe an inhibitory effect.     

Analysis of tyrosine and deuterium labelled tyrosine in tissues and body fluids

A gas chromatographic mass spectrometric method has been developed to determine tyrosine and deuterium labelled tyrosine in biological samples using alpha-methyltyrosine (alpha-Me Ty) or m-hydroxyphenylalanine as internal standards. With the latter standard both labelled and unlabelled tyrosine as well as alpha-Me Ty can be determined simultaneously, in for example human blood, following administration of alpha-Me Ty to inhibit catecholamine synthesis. After isolation of the amino acids on an ion exchange column (Amberlite IR 120) the butyl ester pentafluoropropionyl derivatives were prepared. In human plasma the precision of the method was determined at a level of 80 nmol tyrosine ml-1 and found to be +/- 5% (coefficient of variation, n = 10).     

Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase

The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKbeta/FAK2, is a cytoskeleton-associated tyrosine kinase. We compared CADTK regulation with that of the highly homologous focal adhesion tyrosine kinase (FAK). First, we generated site-specific CADTK mutants. Mutation of Tyr402 eliminated autophosphorylation and significantly decreased kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation site predicted to bind Grb2, had little effect on CADTK regulation. Src family tyrosine kinases resulted in CADTK tyrosine phosphorylation even when co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK tyrosine-phosphorylated FAK when both were transiently expressed, but FAK did not phosphorylate CADTK. Biochemical experiments confirmed direct CADTK phosphorylation of FAK. This phosphorylation utilized tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Last, expression of the CADTK carboxyl terminus (CRNK) abolished CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibited both FAK and CADTK autophosphorylation, suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling.     

Induction of tyrosine aminotransferase by blastomogenic metabolites of tryptophan and tyrosine]

A comparitive study was made of the effect produced by endogenous blastomogenic agents (3-oxyanthranylic and paraoxphenyl-lactic acids) and their nonblastomogenic anaogues (anthranylic and phenyl-lactic acids) on the activity of tyrosine-aminotranspherase in the rat liver. Blastomogenic metabolites proved to be capable of inducing sharply the enzyme activity. This phenomenon and data on the role played by the increase in the activity of tyrosine-aminotranspherase and tryptophane-oxygenase in tyrosine and tryptophane catabolism on the way of a possible formation of blastomogenic metabolites permitted to put forward a suggestion on the "chain reaction" of accumulation of the endogenous blastomogenic agents in the organism.     

Photosensitized oxidation of phenyl and tert-butyl sulfides.

The photosensitized oxygenation of diphenyl (1), di-tert-butyl (2) and phenyl tert-butyl sulfide (3) was studied. Bimolecular rate constants of singlet oxygen quenching are low (1 to 5 x 10(4) M(-1)s(-1)) since the sulfides are poor nucleophiles due to sterical hindrance (2, 3) or the HOMO on the sulfur atom being a less accessible p(z) orbital (1). The quenching is mainly physical, but chemical reaction leading to sulfoxides also takes place in methanol and, to a lower degree, in acetonitrile. Catalysis by carboxylic acids considerably enhances the rate of sulfoxidation. Inefficiency in the chemical reaction is again due to the poor nucleophilicity of the sulfides, which limits oxygen transfer by electrophilic intermediates such as the protonated persulfoxide.     

Resonance Raman enhancement of phenyl ring vibrational modes in phenyl iron complex of myoglobin.

Resonance Raman spectra are reported for the organometallic phenyl-FeIII complexes of horse heart myoglobin. We observed the resonance enhancement of the ring vibrational modes of the bound phenyl group. They were identified at 642, 996, 1,009, and 1,048 cm-1, which shift to 619, 961, 972, and 1,030 cm-1, respectively, upon phenyl 13C substitution. The lines at 642 and 996 cm-1 are assigned, respectively, as in-plane phenyl ring deformation mode (derived from benzene vibration No. 6a at 606 cm-1) and out-of-plane CH deformation (derived from benzene vibration No. 5 at 995 cm-1). The frequencies of the ring "breathing" modes at 1,009 and 1,048 cm-1 are higher than the corresponding ones in phenylalanine (at 1,004 and 1,033 cm-1) and benzene (at 992 and 1,010 cm-1), indicating that the ring C--C bonds are strengthened (or shortened) when coordinated to the heme iron. The excitation profiles of these phenyl ring modes and a porphyrin ring vibrational mode at 674 cm-1 exhibit peaks near its Soret absorption maximum at 431 nm. This appears to indicate that these phenyl ring modes may be enhanced via resonance with the Soret pi-pi transition. The FeIII--C bond stretching vibration has not been detected with excitation wavelengths in the 406.7-457.9-nm region.     

Insecticidal activity of substituted phenyl N-methylcarbamates

The insecticidal and the acaricidal activities of a number of substituted phenyl N-methylcarbamates have been determined on the housefly (Musca domestica), the black bean aphid (Aphis fabae), the Colorado potato beetle (Leptinotarsa decemlineata), the cabbage worm (Pieris brassicae) and the carmine spider mite (Tetranychus cinnabarinus).It is demonstrated that the thesis of Kolbezen, Metcalf & Fukuto (1954), Metcalf, Fukuto & Winton (1962) and Kohn, Ospenson & Moore (1965), that the meta-isomers of alkylphenyl N-methylcarbamates are the most active, has to be restricted to some insect groups (e.g. flies and caterpillars). In the case of the Colorado potato beetle the o- and m-isomers were equally active; for aphids the o-isomer was the most toxic one.This fact as well as the different responses of the test insects if the compounds are further alkylated indicate that various carbamates exhibit more or less selective activities. Most striking was the high level of activity in the new group of p-dimethylaminomethylphenyl N-methylcarbamates on most of the insects, in combination with a complete lack of toxicity to houseflies.As previously pointed out by Kolbezen et al. (1954) and Metcalf et al. (1962), it was found that lengthening of the N-methyl group or N,N-dialkylation resulted in loss of insecticidal activity.The most active dimethylaminophenyl compounds were those with a p-dimethylamino group in combination with alkyl substituents in the 2,5- and 3,5-positions. Several p-dimethylaminomethylphenyl N-methylcarbamates with two or three alkyl substituents (except the 2,6-combination) proved to be highly active, except on flies, to which they were virtually nontoxic. Greatest broad-spectrum activity was shown by 2,3-dimethyl-4-dimethylaminomethylphenyl N-methylcarbamate. It is demonstrated that by introducing a p-dimethylamino- or a p-dimethylaminomethyl group in alkylphenyl N-methylcarbamates a considerable gain in anticholinesterase and insecticidal activity is obtained.

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Résumé p L'activité insecticide et acaricide de plusieurs N-méthylcarbamates de phényle substitués a été examinée sur la mouche domestique (Musca domestica), le puceron noir (Aphis fabae), le doryphore (Leptinotarsa decemlineata), la chenille de la piéride du chou (Pieris brassicae) et l'araignée rouge des serres (Tetranychus cinnabarinus). Les N-méthylcarbamates de phényle monosubstitués ne présentent qu'une faible activité acaricide. Les substances avec des substitutions en position méta ou ortho montrent une meilleure activité insecticide que celles avec la substitution en position para. Pour les mouches domestiques l'alcoylation en position méta se trouve donner les composés les plus actifs; pour les pucerons l'alcoylation en position ortho était la plus efficace et pour les doryphores les combinaisons o- et m-étaient d'une activité égale. Les isomères ortho des dérivés alcoxylés étaient plus efficaces. En cas de substitution par un groupe diméthylamino il n'y a pas de différence importante quant à l'activité insecticide entre les positions ortho ou méta. En général il en est de même pour les substitutions par diméthylaminométhyle, bien que le m-isomère soit le plus efficace sur les chenilles.L'introduction d'un deuxième groupe alcoyle dans un N-méthylcarbamate de o- ou m-isopropylphényle change le spectre d'action. Un groupe 5-méthyle diminue beaucoup l'activité de l'o-isomère. Un groupe 5-isopropyle diminue l'activité sur les pucerons, mais pas sur les mouches. Quand un groupe 5-méthyle ou 5-isopropyle est introduit dans un N-méthylcarbamate de m-isopropyl-phényle l'activité est généralement augmentée. L'introduction d'un groupe 6-méthyle diminue toutefois l'activité sur les mouches, les pucerons et les chenilles, mais pas sur les Coléoptères et les araignées rouges.Les N-méthylcarbamates de phényle s'avèrent posséder une meilleure activité que les combinaisons N,N-diméthylcarbamates correspondantes. Une prolongation du groupe N-alcoyle diminue également l'activité.Les dérivés de N-méthylcarbamates de 2-diméthylamino-phényle ont une plus grande activité que les isomères méta correspondants. On trouve toutefois la meilleure activité dans les N-méthylcarbamates de 4-diméthylamino-phényle alcoylés. Tout groupe alcoyle, à condition d'être introduit en position 2-, 3-, ou 5-augmente l'activité insecticide. Les composés métasubstitués semblent encore un peu plus actifs que les dérivés d'o-alcoyle. La grande activité sur les pucerons contraste nettement avec la faible activité sur les mouches et les araignées rouges. Le seul dérivé présentant une bonne activité sur les araignées rouges est le N-méthylcarbamate de 3-isopropyl-4-diméthylamino-5-méthylphényle.Les N-méthylcarbamates de p-diméthylaminométhyl-phényle alcoylés présentent également une forte action insecticide, sauf sur les mouches. Le N-méthylcarbamate de 2, 3-diméthyl-4-diméthylaminométhyl-phényle présente la plus grande activité et le plus large spectre d'action.A partir des N-méthylcarbamates d'alcoylphényle l'introduction d'un groupe p-diméthylamino ou p-diméthylamino-méthyle augmente considérablement l'activité anticholinestérasique aussi bien que l'activité insecticide.

     

Fungicidal action and structure correlation of monosubstituted phenyl isothiocyanates

The fungicidal activity of 10 monosubstituted phenyl isothiocyanates was observed with four plant pathogens. The ortho- and meta-substituted derivatives possessed a fair activity while the para-substituted ones proved to be highly fungicidal when compared to the control Agrosan GN.     

Effect of the phosphonic analogue of tyrosine on tyrosine iodination

Summary Depositing ofdl-1-amino-2-(p-hydroxyphenyl)-ethylphosphonic acid (Tyr-P) on the chicken embryo induced a dose dependent decrease of the iodine uptake by the embryonic thyroid. Tyr-P interfered on iodination of tyrosine when tested with hog thyroid peroxidase (TPO) and with bovine lactoperoxidase (LPO); the analogue was recognized by the two enzymes but its affinity for TPO and LPO was respectively 3 and 7 fold higher compared with that of the natural substrate, suggesting that Tyr-P may act as an iodine trap.     

Mutation of serine 395 of tyrosine hydroxylase decouples oxygen-oxygen bond cleavage and tyrosine hydroxylation

Ser395 and Ser396 in the active site of rat tyrosine hydroxylase are conserved in all three members of the family of pterin-dependent hydroxylases, phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Ser395 is appropriately positioned to form a hydrogen bond to the imidazole nitrogen of His331, an axial ligand to the active site iron, while Ser396 is located on the wall of the active site cleft. Site-directed mutagenesis has been used to analyze the roles of these two residues in catalysis. The specific activities for formation of dihydroxyphenylalanine by the S395A, S395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respectively. Both the S395A and S396A enzymes bind a stoichiometric amount of iron and exhibit wild-type spectra when complexed with dopamine. The K(M) values for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are unaffected by replacement of either residue with alanine. Although the V(max) value for tyrosine hydroxylation by the S395A enzyme is decreased by 2 orders of magnitude, the V(max) value for tetrahydropterin oxidation by either the S395A or the S396A enzyme is unchanged from the wild-type value. With both mutant enzymes, there is quantitative formation of 4a-hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required for amino acid hydroxylation but not for cleavage of the oxygen-oxygen bond, while Ser396 is not essential. These results also establish that cleavage of the oxygen-oxygen bond occurs in a separate step from amino acid hydroxylation.