Partial nucleotide sequence of the parvalbumin from chicken thymus designated "avian thymic hormone"

The incomplete amino acid sequence of the protein identified as avian thymic hormone was recently reported [Brewer et al. (1989), Biochem. Biophys. Res. Commun. 160, 11555-1161], and a very high degree of homology to the parvalbumins was apparent. Using mixed oligonucleotide primers based on the reported protein sequence, we have succeeded in amplifying and cloning a 188 bp fragment of the coding region for this protein, beginning with double-stranded cDNA prepared from chicken thymus mRNA. The translated nucleotide sequence of this fragment and the reported amino acid sequence display substantial disagreement. Most notably, the nucleotide sequence indicates that the CD site of avian thymic hormone is a typical parvalbumin CD site.     

Identification of a novel parvalbumin in avian thymic tissue

A novel calcium-binding protein has been isolated from chicken thymus tissue. Its molecular weight (approximately 11,500) and characteristic interactions with Tb3+ and Eu3+ identify the protein as a member of the parvalbumin family. Electrophoretically distinct from both chicken (muscle) parvalbumin and avian thymic hormone, it represents the third parvalbumin to be identified in avian tissues and the second to be identified in the avian thymus gland.     

Avian thymic hormone (ATH) is a parvalbumin

Amino acid sequence analysis of a protein from chicken thymus tissue which promotes immunological maturity in chicken bone marrow cells in culture has established sequences of a 45-residue fragment, a 24-residue fragment and a 9-residue and an 8-residue peptide. Independent comparison of the 45- and 24-residue fragments with known amino acid sequences by computer search has unequivocally identified avian thymic hormone as a parvalbumin. This is the first demonstration that a protein previously identified by a biological function is a parvalbumin.     

The amino acid sequence of the major parvalbumin of the whiting (Gadus merlangus).

1. The amino acid sequence of the major parvalbumin of the Whiting has been determined; the polypeptide chain is made of 108 residues, the terminal amino acid group is acetylated, there is no disulfide bridges, the structure of the two calcium binding sites is preserved and the distribution along the polypeptide chain of the hydrophobic residues implicated in the compact hydrophobic core of the protein is also maintained. 2. The comparison of this amino acid sequence with other parvalbumins indicates that it belongs to the beta type and that within the Gadidae family two types of parvalbumins also occur.     

Metal ion-binding properties of avian thymic hormone

Lanthanide ion luminescence studies and 45Ca2(+)-binding measurements were used to study the metal ion-binding properties of avian thymic hormone. The procedure used to isolate the protein--involving heat-treatment at 80 degrees C, trichloroacetic acid precipitation, DEAE-agarose chromatography, and gel filtration--affords material that is deemed homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as well as the absence of a detectable tryptophan signal in the fluorescence emission spectrum. Avian thymic hormone exhibits a pI = 4.35 when subjected to isoelectric focusing through polyacrylamide gels. The two ion-binding sites are indistinguishable in their interactions with Ca2+ and Mg2+, displaying KCa = 8 nM and KMg = 68 microM. The Eu3+ 7Fo----5Do excitation spectrum at pH 6 displays a peak at 5795.4 A, with a shoulder at 5792.8 A and is replaced at higher pH values by a broader spectrum with a maximum at 5784.8 A and a shoulder at 5777.1 A. The pKa governing this spectral interconversion is 8.21. All of these properties are very similar to those observed with other parvalbumins. However, polyclonal antibodies to avian thymic hormone do not cross-react with the parvalbumin from chicken leg muscle, as judged by Western blot analysis-further evidence that avian thymic hormone and the muscle-associated chicken parvalbumin are indeed distinct proteins.     

The amino acid sequence of the rabbit glycoprotein hormone alpha subunit

The amino acid sequence of the alpha subunit of rabbit (lagomorph) lutropin (lLH) has been determined. Overlapping peptides from trypsin and chymotrypsin digestions were isolated by reverse-phase high-pressure liquid chromatography (HPLC). Sequencing was by the dansyl-Edman procedure. Amide placements were established by HPLC analysis of the PTH amino acid derivatives. The proposed sequence of lLH alpha subunit is (asterisks denote carbohydrate attachment sites): This proposed sequence is highly homologous with the porcine, murine, ovine, and bovine glycoprotein hormone alpha subunit sequences. Two unusual proteolytic cleavages were observed: (1) a cleavage by trypsin between Asn-77 and Ala-78, and (2) a cleavage by chymotrypsin between Ala-45 and Arg-46. Similar enzymatic cleavages were previously reported for equine chorionic gonadotropin alpha subunit by Wardet al. and for these sites in the ovine LH alpha subunit by Liuet al. Chymotrypsin cleaved on the carboxyl side of methionine sulfone residues at positions 51 and 75.     

The amino acid sequence of a hypothalamic peptide, neurotensin.

The amino acid sequence of neurotensin, a hypotensive peptide isolated from acid-acetone extracts of bovine hypothalami, has been established as less than Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Oh. (The nomenclature and symbols follow the suggestions of the IUPAC-IUB Commission on Biochemical Nomenclature (1972) J. Biol. Chem. 247, 977). This was accomplished by sequence analyses performed on the intact peptide as well as its isolated tryptic, chymotryptic, and papain-generated fragments. The results of enzymic hydrolyses were consistent with the specificities of the enzymes used and indicated that all of the amino acids are unsubstituted and in the L configuration. The absence of non-amino acid constituents was further supported by analyses of electrophoretic mobility-molecular weight relationships of neurotensin and its fragments.     

Porcine proparathyroid hormone. Identification, biosynthesis, and partial amino acid sequence.

Porcine parathyroid gland slices were incubated with 3H-labeled amino acids in order to label tissue proteins. After incubation a crude hormonal extract was prepared and analyzed by chromatography on carboxymethylcellulose. Among the three radioactive peaks which were detected in the eluate, two were identified as parathyroid hormone and proparathyroid hormone. Based on thin layer gel filtration in the presence of 6 M guanidine-HCl, the proparathyroid hormone had a molecular weight of 11,500 compared to about 9600 for parathyroid hormone. Radioisotope sequence analysis of the proparathyroid hormone revealed a partial sequence of: Lys1-Pro2-Ile3-Lys4-Lys5-Arg6-Ser7-Val8-Ser9--Ile11--Met14--Gly18--Ser22--Ser23---. Thus, from position 7 onward the relative position of each amino acid tested in this molecule corresponded exactly to that in the porcine parathyroid hormone sequence. The conservation of a similar, though not identical, basic hexapeptide grouping Lys-X-Y-Lys-Lys-Arg- at the amino terminal region of the prohormone in all species examined thus far (porcine, human, and bovine) suggests that this segment of the molecule may play an important role in the conversion of the prohormone to the hormone.     

Rapid determination of parvalbumin amino acid sequence from Rana catesbeiana (pI 4.78) by combination of ESI mass spectrometry, protein sequencing, and amino acid analysis

The complete amino acid sequence of beta-type parvalbumin (PA) from bullfrog Rana catesbeiana (pI 4.78) was determined by tandem mass spectrometry in combination with amino acid analysis and peptide sequencing following Arg-C and V(8) protease digestion. The primary structure of the protein was compared with that of beta-type PA from R. esculenta (pI 4.50), with which it is highly homologous. Compared with R. esculenta beta-type PA4.50, R. catesbeiana beta-type parvalbumin (PA 4.78) differed in 15 out of 108 amino acid residues (14% displacement), PA4.78 had Cys at residue 64 and was acetylated at the amino terminus, but 25 residues of the carboxyl terminus were completely conserved. Several amino acid displacements were found between residues 51 and 80 (30% displacement), although the functionally important sequence of PA was completely conserved. The amino acids residues of putative calcium-binding sites were Asp-51, Asp-53, Ser-55, Phe-57, Glu-59, Glu-62, Asp-90, Asp-92, Asp-94, Lys-96, and Glu-101, which were conserved in all a and b-types of R. catesbeiana as well as other parvalbumins. In addition, Arg-75 and Glu-81, which are thought to form a salt bridge located in the interior of the molecule [Coffee, C.J. et al. (1976) Biochim. Biophys. Acta 453, 67-80], were also conserved in PA4.78.