The complete amino acid sequence of human complement factor H.

The complete amino acid sequence of the human complement system regulatory protein, factor H, has been derived from sequencing three overlapping cDNA clones. The sequence consists of 1213 amino acids arranged in 20 homologous units, each about 60 amino acids long, and an 18-residue leader sequence. The 60-amino-acid-long repetitive units are homologous with those found in a large number of other complement and non-complement proteins. Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma. A tyrosine/histidine polymorphism was observed within the seventh homologous repeat unit of factor H. This is likely to represent a difference between the two major allelic variants of factor H. The nature of the cDNA clones indicates that there is likely to be an alternative splicing mechanism, resulting in the formation of at least two species of factor H mRNA.     

The complete amino acid sequence of human brain-derived acidic fibroblast growth factor

Acidic fibroblast growth factor is a potent mitogen for a variety of cells in culture, including vascular endothelial cells, and is angiogenic in vivo. The complete amino acid sequence of human brain-derived acidic fibroblast growth factor has been determined from amino terminal sequence analysis and carboxypeptidase A digestion of the whole protein and sequence analyses of peptides generated by tryptic, Staphylococcus aureus V8 protease and cyanogen bromide cleavages. A potential Asn-Gly-Ser glycosylation sequence is present in the human protein. The complete amino acid sequence is compared to that of the equivalent protein purified from bovine brain.     

Human adenosine deaminase. cDNA and complete primary amino acid sequence

A previously cloned partial adenosine deaminase cDNA insert (0.8 kilobase) was used to clone additional nucleotide sequences from human HPB ALL cDNA libraries. cDNA encompassing the entire coding, and 3'-untranslated regions as well as nearly all of the 5'-untranslated region was obtained. The complete amino acid sequence of the enzyme deduced from the cDNA sequence and protein sequencing consists of 362 amino acids, excluding the initiator Met, and accounts for Mr = 40,638. Secondary structure predictions assign adenosine deaminase to the alpha/beta class of proteins. Northern blot analysis with a cDNA probe showed adenosine deaminase mRNA to be present in normal to above normal amounts in B-lymphoblasts derived from adenosine deaminase-deficient patients with severe combined immunodeficiency disease. Knowledge of the cDNA and primary amino acid sequence of adenosine deaminase will be pivotal in further defining the genetic abnormality and its functional consequences in adenosine deaminase expression defects.     

The complete amino acid sequence of chicken skeletal-muscle enolase.

The complete amino acid sequence of chicken skeletal-muscle enolase, comprising 433 residues, was determined. The sequence was deduced by automated sequencing of hydroxylamine-cleavage, CNBr-cleavage, o-iodosobenzoic acid-cleavage, clostripain-digest and staphylococcal-proteinase-digest fragments. The presence of several acid-labile peptide bonds and the tenacious aggregation of most CNBr-cleavage fragments meant that a commonly used sequencing strategy involving initial CNBr cleavage was unproductive. Cleavage at the single Asn-Gly peptide bond with hydroxylamine proved to be particularly useful. Comparison of the sequence of chicken enolase with the two yeast enolase isoenzyme sequences shows that the enzyme is strongly conserved, with 60% of the residues identical. The histidine and arginine residues implicated as being important for the activity of yeast enolase are conserved in the chicken enzyme. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Russell (1986) Biochem. J. 236, 127-130].     

Spirulina ferredoxin-NADP+ reductase. The complete amino acid sequence

The amino acid sequence of ferredoxin-NADP+ oxidoreductase [EC 1.18.1.2, FNR] from Spirulina sp., a blue-green alga, was determined. Spirulina ferredoxin-NADP+ oxidoreductase was composed of 294 amino acid residues and the molecular weight of the holoenzyme was 34,135. An apparent homology of the amino(N)-terminal region was found between ferredoxin-NADP+ reductases from Spirulina and spinach. We also found some sequence similarities in human erythrocyte glutathione reductase and p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, both of which are NADPH-dependent FAD enzymes.     

Bovine testis acylphosphatase: Purification and amino acid sequence

Two acylphosphatase molecular forms have been isolated from bovine testis. Their amino acid sequence was determined. One (ACY1) consists of 98 amino acid residues, while the other one (ACY2) consists of 100 amino acid residues. Both molecular forms are N-acetylated and differ only in the amino terminus. ACY2 has an additional Ser-Met tail with respect to ACY1. Both ACY1 and ACY2 are organ-common type isoenzymes and thus differ for about half of the amino acid positions from the previously sequenced bovine muscle isoenzyme.     

The complete amino acid sequence of yeast phosphoglycerate kinase.

The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.     

Mouse UDP glucuronosyltransferase. cDNA and complete amino acid sequence and regulation

A cDNA clone (UDPGTm-1) encoding a mouse UDP glucuronosyltransferase (transferase) was isolated from pBR322 and lambda gt11 libraries by hybridization to a rat transferase clone. This cDNA is 1860 bp long and 65-87% similar at both the nucleotide and the deduced amino acid sequence levels to three different rat transferase clones [Mackenzie, P.I. et al. (1984) J. Biol. Chem. 259, 12153-12160; Mackenzie, P.I. (1986) J. Biol. Chem. 261, 6119-6125]. UDPGTm-1, like the rat transferase clones already described, contains an open reading frame of 1590 bp encoding 530 amino acids (unmodified Mr = 60,856), an N-terminus membrane-insertion signal sequence, a carboxy-terminus hydrophobic putative membrane-spanning region, and potential asparagine-linked glycosylation sites (residues 316 and 483). The cDNA contains two poly(A) addition consensus sequences at positions 1695-1837. UDPGTm-1 is complementary to a 2200-base mRNA and also cross-hybridizes to a 2000-base mRNA species due to sequence homology in the 5' region of the clone. Both the 2200-base and the 2000-base mRNA are induced approximately 2.5-fold by the hypolipidemic agent clofibrate, and also by phenobarbital and benzo[a]pyrene. A new and more potent hypolipidemic agent, perfluorooctanoic acid, is also shown to induce both mRNA species. Each of these compounds induces bilirubin transferase activity in a manner parallel to the effects on mRNA, i.e. perfluorooctanoic acid being the most effective, followed by phenobarbital, benzo[a]pyrene, and clofibrate. Southern blot hybridization of UDPGTm-1 to mouse genomic DNA showed sequence homology to a total DNA size of 40-50 kb. These data indicate that UDPGTm-1 is a member of a new subfamily of transferases in mouse with patterns of regulation of their mRNAs similar to that seen for bilirubin transferase activity.     

The complete amino acid sequence of monkey progastricsin

The complete amino acid sequence of progastricsin from the Japanese monkey (Macaca fuscata) was determined. Progastricsin is composed of 374 residues, including the gastricsin moiety of 331 residues and the activation segment of 43 residues. Upon activation under acidic conditions, progastricsin was converted to gastricsin via the intermediate protein species. NH2-terminal sequence determination of these protein species enabled us to deduce the NH2-terminal 78-residue sequence of progastricsin, including the 43-residue activation segment. The complete sequence of the gastricsin moiety was determined using peptide fragments obtained by several chemical and enzymatic cleavages. The molecular weight of progastricsin was determined to be 40,785. As compared with pepsinogen A of the same monkey species, deletion of 4 residues and insertion of 5 residues were observed. Although monkey progastricsin and pepsinogen A have highly homologous sequences around the two active site aspartyl residues, the homology between these proteins is rather small (49% identity). This indicates that progastricsin diverged from pepsinogen A in the early phase of the evolution of gastric aspartyl proteinases.     

The complete amino acid sequence of α-neo-endorphin

After re-purification by reverse phase high performance liquid chromatography, α-neo-endorphin was submitted to structural analyses, performed by dansyl-Edman degradation, as well as by C-terminal analysis by ³H-labeling. The full sequence of α-neo-endorphin has been determined to be : Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys, in which the 8th residue previously reported as Arg is found to be Tyr. A synthetic decapeptide with the above sequence was verified to be identical with natural α-neo-endorphin. For further structural confirmation, tryptic and chymotryptic peptides were also identified. Thus, the complete sequence of α-neo-endorphin has been definitely established. Its potent opioid activity in the guinea-pig ileum assay is also discussed.     

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.     

The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase.

The complete amino acid sequence of human skeletal-muscle fructose-bisphosphate aldolase, comprising 363 residues, was determined. The sequence was deduced by automated sequencing of CNBr-cleavage, o-iodosobenzoic acid-cleavage, trypsin-digest and staphylococcal-proteinase-digest fragments. Comparison of the sequence with other class I aldolase sequences shows that the mammalian muscle isoenzyme is one of the most highly conserved enzymes known, with only about 2% of the residues changing per 100 million years. Non-mammalian aldolases appear to be evolving at the same rate as other glycolytic enzymes, with about 4% of the residues changing per 100 million years. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Freemont (1988) Biochem. J. 249, 789-793].     

The complete amino acid sequence of barley trypsin inhibitor

The amino acid sequence of barley trypsin inhibitor has been determined. The protein is a single polypeptide consisting of 121 amino acid residues and has Mr = 13,305. No free sulfhydryl groups were detected by Ellman's reagent, which indicates the presence of five disulfide bridges in the molecule. The primary site of interaction with trypsin was tentatively assigned to the arginyl-leucyl residues at positions 33 and 34. On comparison of the sequence of this inhibitor with those of other proteinase inhibitors, we found that the barley trypsin inhibitor could not be classified into any of the established families of proteinase inhibitors (Laskowski, M., Jr., and Kato, I. (1980) Annu. Rev. Biochem. 49, 593-626) and that this inhibitor should represent a new inhibitor family. On the other hand, this trypsin inhibitor showed a considerable similarity to wheat alpha-amylase inhibitor (Kashlan, N., and Richardson, M. (1981) Phytochemistry (Oxf.) 20, 1781-1784) throughout the whole sequence, suggesting a common ancestry for both proteins. This is the first case of a possible evolutionary relationship between two inhibitors directed to totally different enzymes, a proteinase and a glycosidase.