Thyroid hormonogenesis. Identification of a sequence containing iodophenyl donor site(s) in calf thyroglobulin

The formation of dehydroalanine in thyroglobulin is the result of the side chain elimination of an iodophenyl group during the thyroid hormone formation from two iodotyrosyl residues. This amino acid is easily converted to labeled alanine (upon reduction with [3H] borohydride) or changed to labeled aspartic acid (upon addition of Na14CN and subsequent acid hydrolysis). The cleavage of the protein by CNBr produced many stainable electrophoretic bands, but the autoradiography indicated the presence of a much smaller number of radioactive species. Although three major species raised attention, because they could be all jointly labeled and were present in all preparations, only a species of 15,900 Da was fully studied. It was isolated and its sequence partially determined by Edman degradation. It was established that this species corresponded to the thyroglobulin fragment between methionines 2,432 and 2,578. This peptide contains two hormonogenic sites (positions 2,555 and 2,569) which are either tyrosyl residues or hormone residues arising from them, and five additional tyrosines all potentially involved as donor sites in the hormonogenesis. Upon treatment with N-chlorosuccinimide, the fragment was split into three smaller peptides of about 2,900, 8,500, and 4,600 Da containing 1, 2, and 2 tyrosyl residues, respectively. Only the 8,500-Da subfragment contained [3H]Ala. This finding strongly suggests that at least some of the tyrosines involved as donor sites in thyroid hormonogenesis are within this peptide and possibly map at positions 2,469 and/or 2,522. Moreover, at minimum levels of iodination, when thyroglobulin contains the lowest number of hormone molecules, dehydroalanine is mostly found in the 15,900-Da peptide.     

The sequence of 967 amino acids at the carboxyl-end of rat thyroglobulin. Location and surroundings of two thyroxine-forming sites

The entire rat thyroglobulin mRNA sequence (about 8500 nucleotides) has been cloned in five recombinant plasmids containing overlapping cDNA inserts. The 3' end of the mRNA is precisely defined by the poly (A) tail found in the furthest 3' end clone. Evidence that most of the 5' end is cloned come from size considerations and from a primer extension experiment. At the 3' end of the mRNA only one long open reading frame is present in the sequence of 3018 nucleotides that has been established. In the deduced protein sequence we have localized two thyroxine-forming sites in a region containing a high concentration of tyrosine residues.     

The amino acid substrate of bovine tyrosine hydroxylase.

Tyrosine hydroxylase catalyzes the tetrahydropterin-dependent hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine. Several nonphysiological aromatic amino acids have been examined as inhibitors and substrates for bovine adrenal tyrosine hydroxylase. The Ki values for para-substituted phenylalanines increase as the size of the substituent increases. For each A2 increase in surface area of the substituent, the free energy of binding becomes 50 cal more positive. Replacement of the phenyl ring with a pyridyl ring decreases the affinity about one order of magnitude. A number of these aromatic amino acids are also substrates for the enzyme. The KM values again increase in size with increasing size of the substituent, but the Vmax value is independent of the reactivity of the amino acid. The effect of size on binding is consistent with a tight interaction between the para position region of the substrate and the enzyme. The lack of a change in the Vmax value is consistent with the rate-limiting step in catalysis by bovine tyrosine hydroxylase being formation of the hydroxylating intermediate rather than hydroxylation of the amino acid. These results will be useful in designing mechanism-based inhibitors of catecholamine biosynthesis and establish that the mechanisms of rat and bovine tyrosine hydroxylase do not differ significantly.     

Structure-function relationship in thyroglobulin: amino acid sequence of two different thyroxine-containing peptides from porcine thyroglobulin.

From the cyanogen bromide (CNBr) treatment of porcine thyroglobulin a peptide of mol. wt. 15 000, CNBr-b1, was purified by gel filtration and ion-exchange chromatography. CNBr-b1 contained 50% of the thyroxine (T4) content of the protein. After digestion with trypsin and protease from Staphylococcus aureus V-8, thyroxine-containing peptides were purified and analyzed by microsequence analysis using the colored Edman's reagent dimethylaminoazobenzeneisothiocyanate . Two different sequences harboring T4 were identified: sequence 1, His-Asp-Asp-Asp-T4-Ala-Thr-(Glx,Gly)-Leu-Tyr-Phe-Ser-Ser-Arg, which contains 1 mol T4/mol peptide and sequence 2, Asp-(Tyr/MIT/DIT/T4)-Phe-Ile-Leu-X-Pro-Val-, which is a mixture of the same peptide at different levels of iodination and coupling. These sequences are likely to be representative of distinct hormonogenic sites, the former giving evidence of early iodinated tyrosine residues where preferential coupling into hormonal residues occurs especially at low iodine levels and the latter representing less reactive site(s) operative at higher iodine levels.     

Search for folding initiation sites from amino acid sequence

A crucial event in protein folding is the formation of a folding nucleus, which is a structured part of the protein chain in the transition state. We demonstrate a correlation between locations of residues involved in the folding nuclei and locations of predicted amyloidogenic regions. The average Phi-values are significantly greater inside amyloidogenic regions than outside them. We have found that fibril formation and normal folding involve many of the same key residues, giving an opportunity to outline the folding initiation site in protein chains. The search for folding initiation sites for apomyoglobin and ribonuclease. A coincides with the predictions made by other approaches.     

Identification of the amino acid residues in trichosanthin crucial for IgE response

Trichosanthin (TCS) is the major effective component from Chinese herb Trichosanthes kirilowii. TCS has been approved to be effective in clinical treatment of HIV infection and leukemia, but its allergenicity has limited its clinical usage. To identify amino acid residues in TCS with an important role in IgE induction, TCS-specific IgE mAb (TE1) was used to serve as a probe and TE1 epitope was determined by a random phage-peptide library. Based on phage peptide sequences, TE1 epitope was predicted at amino acid residues 169-174 (QQIGKR) of TCS protein. Based on modeling data, two amino acids (Lys173 and Arg174) on TCS were considered to have a crucial role in binding to TE1. After lysine 173 and arginine 174 were mutated to glycine, the mutant TCS protein specifically lost the binding activity to TE1 mAb and exhibited reduced IgE induction in the immunized mice. The data showed that the IgE epitope of TCS was determined and shown to play a critical role in induction of IgE, and the modification of IgE-epitope may be a useful strategy to reduce the allergenicity of an allergen.     

Primary amino acid sequence of bovine dopamine beta-hydroxylase

Fifty-eight tryptic and Staphylococcus aureus V8 protease generated peptides from bovine dopamine beta-hydroxylase were isolated by reverse-phase high pressure liquid chromatography and sequenced. These peptide sequences were compared with the deduced amino acid sequences of bovine and human dopamine beta-hydroxylase obtained from the cloned cDNAs. Bovine peptide sequences had five differences with the sequence derived from the bovine cDNA, and four of the changes could be accounted for by a single base change in the DNA. N-terminal sequence analysis of the bovine enzyme indicated that it contained two N termini, one of which is 3 amino acids longer than the other and begins with the sequence Ser-Ala-Pro. The amino acid sequences deduced from the bovine and human cDNAs are 19 and 25 amino acids longer, respectively, and these additional amino acids represent leader peptide sequences. Two bovine peptide sequences contained glycosylation sites and gave positive tests for carbohydrate residues, and two others contained the consensus sequence for a glycosylation site but were negative in the carbohydrate test. The bovine enzyme contains 6 Trp, as compared with 7 in the bovine cDNA and 8 in the human cDNA. The protein and bovine cDNA contain 24 Tyr each, as compared with 26 in the human cDNA. These numbers indicate that the true epsilon 1% 280 = 8.95, and, therefore, that it is 28% lower than the previously determined value. The data also identify 5 His-containing regions that may be involved in Cu2+ coordination at the active site.     

Calcium-binding protein of bovine intestine. The complete amino acid sequence.

The amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.     

Identification of amino acid residues required for Ras p21 target activation.

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.