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.     

Contribution of the B16 and B26 tyrosine residues to the biological activity of insulin

We report the synthesis and biological evaluation of five insulin analogues in which one or both of the B-chain tyrosine residues have been substituted. [B16 Phe]insulin and [B16 Trp]insulin display a very modest reduction in potency (c. 65%) relative to porcine insulin; [B26 Phe]insulin is less active (30–50%), and the doubly substituted [B16 Phe, B26 Phe]insulin displays still lower potency (c. 35%). The further substitution of Asp for B10 His in [B16 Phe, B26 Phe]insulin raises its activity to approximately twofold greater than natural insulin, an increase of approximately fivefold over the parent compound. We conclude that the bulk and/or aromaticity of the amino acid residue at position B16, but not its hydrogen-bonding capacity, contributes to the biological activity of the hormone. We further conclude that hydrogen-bonding capacity or special side-chain packing characteristics are required at the B26 position for insulin to display high biological activity.     

On the mechanism of iodination of tyrosine

Existing data contain proof that the iodinating species of tyrosine and its derivatives contained in mixtures of iodine and iodide is hypoiodous acid, HOI. It appears likely that the peroxidase-catalyzed iodination reaction with hydrogen peroxide, tyrosine or a tyrosine derivative and either iodide or iodine as substrates involves enzyme-activated HOI.     

Identification of insulin receptor tyrosine residues autophosphorylated in vitro

To identify the autophosphorylation sites on the human insulin receptor (IR), partially purified human IR was incubated in vitro in the presence of insulin and manganese [gamma-32P]ATP so as to achieve near-maximal activation of the histone 2b kinase activity. Approximately 70% of all beta subunit [32P]phosphotyrosine resides on two tryptic peptide segments identified by microsequencing as IR precursor (Ullrich, A., Bell, J. R., Chen, E.-Y., Herrera, R., Petruzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) 1144-1152 (tyrosine at 1146, 1150, 1151, designated peptide 5) and 1315-1329 (tyrosine at 1316, 1322, designated peptide 8), which were recovered in approximately equal amounts. Half of the remaining unidentified [32P]phosphotyrosine residues reside on another tryptic peptide of Mr 4000-5000. Assignment of [32P]phosphotyrosine to specific residues required subdigestion and Edman degradation of 32P peptides covalently coupled to solid supports. Peptide 5 was recovered in triple and double phosphorylated forms in a molar ratio of about 2:1. Tyr-1146 contained 32P in both forms of peptide 5; in the double phosphorylated form, phenylthiohydantoin-[32P]phosphotyrosine was recovered at both Tyr-1150 and Tyr-1151, in a ratio of about 1:2. Thus, the double phosphorylated peptide 5 is presumably a mixture of Tyr-P-1146/1150 and Tyr-P-1146/1151, predominantly the latter. Peptide 8 was recovered only as the double phosphorylated form. We conclude that autophosphorylation of human IR in vitro leads to the phosphorylation of at least 6 of the 13 tyrosine residues on the beta subunit intracellular extension. Five of these tyrosines are clustered in two domains; one domain is in the structurally unique C-terminal tail and contains Tyr-1316 and -1322 which are both phosphorylated. The second domain is located in the segment of the tyrosine kinase region homologous to the major in vitro autophosphorylation site of pp60 v-src and contains Tyr-1146, which is fully phosphorylated, and Tyr-1150 and -1151; although the majority of IR beta subunits exhibit phosphorylation of both tyrosine 1150 and 1151, up to 20-25% of Tyr-1150 remains unphosphorylated at complete kinase activation.     

Studies on the mechanism of the iodination of tyrosine by lactoperoxidase

Studies with lactoperoxidase showed that a highly reactive intermediate is produced (on the enzyme) from I- and H2O2 which then diffuses from the enzyme and very rapidly and indiscriminately iodinates any Tyr or peptides containing Tyr which are in the same solution. The evidence supporting these conclusions follows. 1) The rate followed the Michaelis-Menten pattern with I- and H2O2 while the concentration of Tyr peptides had no measurable effect on the rate; 2) the rates of reaction were independent of the type of peptide in which Tyr was located; 3) the amount of iodination which had occurred after the reaction had gone to completion and the amounts of monoiodination and diiodination after completion of the reaction were independent of the peptide type, the pH, the solvent polarity, or the ionic strength; 4) competition for reaction by two very different Tyr peptides depended only on their initial concentrations; and 5) iodination of a large protein occurred through a dialysis membrane. Free Tyr was iodinated at the same rate as Tyr peptides by lactoperoxidase, but monoiodotyrosine and m-fluorotyrosine were iodinated at one-half that rate. The results also showed that one can choose ratios of [peptide] to [H2O2] such that monoiodination is maximized relative to diiodination. It was also found that the iodination capacity of a mixture of I- and H2O2 with lactoperoxidase (when Tyr was absent) was only slowly dissipated. Finally, the results showed that lactoperoxidase can be used to brominate and chlorinate Tyr peptides at a slow rate.     

Identification of tyrosine residues that are susceptible to lactoperoxidase-catalyzed iodination on the surface of Escherichia coli 30S ribosomal subunit

The detailed surface topography of the Escherichia coli 30S ribosomal subunit has been investigated, with iodination catalyzed by immobilized lactoperoxidase as the surface probe. Under mild conditions, only proteins S3, S7, S9, S18, and S21 were iodinated to a significant and reproducible extent. These proteins were isolated from the iodinated subunits, and in each case, the individual tyrosine residues that had reacted were identified by standard protein sequencing techniques. The targets of iodination that could be positively established were as follows: in protein S3 (232 amino acids), the tyrosines at positions 167 and 192; in S7 (153 amino acids), tyrosines 84 and 152; in S9 (128 amino acids), tyrosine 89; in S18 (74 amino acids), tyrosine 3 (tentative); in S21 (70 amino acids), tyrosines 37 and 70. The results represent part of a broader program to investigate ribosomal topography at the amino acid-nucleotide level.     

The role of tyrosine residues in the RNA N-glycosidase activity of cinnamomin A-chain

Cinnamomin is a type II ribosome-inactivating protein (RIP) and its A-chain (CTA) is a RNA N-glycosidase. It is observed that modification of tyrosine residues by N-acetylimidazole (N-AI) causes almost complete loss of CTA activity. Adenine partially protects tyrosine residues from modification by N-AI. It is proposed that tyrosine residues are involved in the active site of CTA and they are crucial in recognition and binding of ribosomal RNA. Tryptophan residues of CTA are also studied by NBS modification.     

Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose

Insulin stimulates the autophosphorylation of tyrosine residues of the beta subunit of the insulin receptor (IR); this modified insulin-independent kinase has increased activity toward exogenous substrates in vitro. We show here that replacement of one or both of the twin tyrosines (residues 1162 and 1163) with phenylalanine results in a dramatic reduction in or loss of insulin-activated autophosphorylation and kinase activity in vitro. In vivo, these mutations not only result in a substantial decrease in insulin-stimulated IR autophosphorylation but also in a parallel decrease in the insulin-activated uptake of 2-deoxyglucose. Furthermore, a truncated IR protein (lacking the last 112 amino acids) has an unstable beta subunit; this mutant has no kinase activity in vitro or in vivo and does not mediate insulin-stimulated uptake of 2-deoxyglucose. IR autophosphorylation is thus implicated in the regulation of IR activities, with tyrosines 1162 and 1163 as major sites of this regulation.     

The tyrosine kinase activity of the chicken insulin receptor is similar to that of the human insulin receptor

The tyrosine kinase activity of a chimeric insulin receptor composed of the extracellular domain of the human insulin receptor (IR) and the intracellular domain of the chicken IR was compared with wild-type human IR. The degrees of autophosphorylation, phosphorylation of IRS-1, and in vitro phosphorylation of an exogenous substrate after stimulation by human insulin were similar to that seen with the human IR. We conclude that the insulin resistance of chickens is not attributable to a lower level of intrinsic tyrosine kinase activity of IR.