Conserved domains in molybdenum hydroxylases. The amino acid sequence of chicken hepatic sulfite oxidase

The amino acid sequence of the molybdenum-containing domain of chicken hepatic sulfite oxidase has been determined by Edman degradation of the purified protein. Combining these data with those previously published for the heme-containing domain (Guiard, B., and Lederer, F. (1979) Eur. J. Biochem. 100, 441-453) indicates that each subunit of the homodimer comprises a single polypeptide chain containing 460 amino acid residues (Mr = 50,545). Comparison of the sequence with the cDNA-deduced sequence of assimilatory nitrate reductase from Arabidopsis thaliana shows a substantial degree of sequence conservation in the regions of the proteins that have been identified as comprising the Mo-pterin- and cytochrome b557-binding domains. These results suggest that the sequences forming the molybdenum-binding domains of the molybdenum hydroxylases may have evolved from a common ancestral gene.     

Amino acid sequence of chicken fibrinopeptide A

Chicken fibrinopeptide A was isolated and the complete amino acid sequence was established. It is a pentadecapeptide with the sequence of Gln(Pyr)-Asp-Gly-Lys-Thr-Thr-Phe-Glu-Lys-Glu-Gly-Gly-Gly-Gly-Arg and is homologous with mammalian fibrinopeptide A. Two peptides which appear to be derivatives of fibrinopeptide A were also isolated. One of these appeared to be fibrinopeptide A with NH2-terminal pyroglutamic acid; the other was fibrinopeptide A which lacked the COOH-terminal arginine residue.     

Amino acid sequence of chicken gizzard gamma-tropomyosin

Chicken gizzard muscle tropomyosin has been fractionated into its two major components, beta and gamma and the amino acid sequence of the gamma component established by the isolation and sequence analysis of fragments derived from cyanogen bromide cleavage and tryptic digestions. Despite its much slower mobility on sodium dodecyl sulfate-polyacrylamide electrophoretic gels, it has the same polypeptide chain length (284 residues) as the alpha and beta components of rabbit skeletal muscle. Evidence for microheterogeneity of the chicken gizzard component was detected both on electrophoretic gels and in the sequence analysis. The gamma component is more closely related to rabbit skeletal alpha-tropomyosin than to the beta component. While the protein is highly homologous to the rabbit skeletal tropomyosins, significant sequence differences are observed in two regions; between residues 42-83 and 258-284. In the latter region (COOH-terminal) the alterations in sequence are very similar to those seen in platelet tropomyosin when compared with the skeletal proteins.     

Amino-acid sequence of the cytochrome-b5-like heme-binding domain from Hansenula anomala flavocytochrome b2

Flavocytochrome b2 (L-lactate dehydrogenase) from baker's yeast is composed of two structural and functional domains. Its first 100 residues constitute the heme-binding core, which is homologous to cytochrome b5 [B. Guiard, O. Groudinsky & F. Lederer (1974) Proc. Natl Acad. Sci. USA 71, 2539-2543]. We report here the amino acid sequence of the heme-binding domain isolated by tryptic proteolysis of Hansenula anomala flavocytochrome b2. The sequence was established by automated degradation of the whole fragment and of peptides obtained by CNBr cleavage at the unique tryptophan and by proteolysis with thermolysin and endoproteinase Lys C. As isolated, the domain consists of 84 residues without any sulfur amino acids. It shows 49 identities with the heme-binding domain from Saccharomyces cerevisiae and 28 with beef microsomal cytochrome b5. Using the recently published three-dimensional structure of S. cerevisiae flavocytochrome b2 [Z-x. Xia, N. Shamala, P. H. Bethge, L. W. Lim, H. D. Bellamy, N. H. Xuong, F. Lederer and F. S. Mathews (1987) Proc. Natl Acad. Sci. USA 84, 2629-2633], it can be seen that there are only positively charged side chains close to the accessible heme edge, the only negative charges in that area being those of the heme propionates. The implications of this result are discussed in the light of Salemme's model for the cytochrome b5/cytochrome c complex [F. R. Salemme (1976) J. Mol. Biol. 102, 563-568].     

Amino acid sequence of chicken liver cathepsin L

The complete amino acid sequences of the heavy and light chains of chicken liver cathepsin L have been determined by automated gas-phase Edman degradation. The heavy and light chains contained 176 and 42 amino acid residues respectively. A glucosamine-based oligosaccharide group was attached to Asn-109 of the heavy chain. Chicken liver cathepsin L had high sequence homology with rat cathepsin H, but exhibited less similarity with rat cathepsin B. Comparisons of cathepsin L with plant cysteine proteinases, such as papain, actinidin and aleurain, reveal high degree of homology.     

Amino acid sequence of phosvitin derived from the nucleotide sequence of part of the chicken vitellogenin gene

The amino acid sequence of the egg yolk storage protein phosvitin has been deduced from the nucleotide sequence of part of the chicken vitellogenin gene. Of the phosvitin sequence, 210 amino acids including the N-terminal residue are contained on one large exon, whereas the remaining six amino acids are encoded on the next exon. Phosvitin contains a core region of 99 amino acids, consisting of 80 serines, grouped in runs of maximally 14 residues interspersed by arginines, lysines, and asparagines. The serines of the core region are encoded by AGC and AGT codons exclusively and the arginines by AGA and AGG, which results in a continuous stretch of 99 codons with adenine in the first position. The N-terminal quarter of the phosvitin sequence contains 16 serines grouped in a cluster with alanines and threonines and coded mainly by TCX triplets. The C-terminal part includes 27 serines, preferentially coded by AGC and AGT, 13 histidine residues, and the sequence ...Asn-Gly-Ser... at which the carbohydrate moiety of phosvitin is attached. Heteroduplex formation between cloned DNAs from chicken and Xenopus vitellogenin genes shows that the phosvitin sequence contains a stretch of highly conserved sequence.     

The kinetic behavior of chicken liver sulfite oxidase.

A comprehensive kinetic study of sulfite oxidase has been undertaken over the pH range 6.0-10.0, including conventional steady-state work as well as rapid kinetic studies of both the reaction of oxidized enzyme with sulfite and reduced enzyme with cytochrome c (III). A comparison of the pH dependence of kcat, kred, and kox indicates that kred is principally rate limiting above pH 7, but that below this pH the pH dependence of kcat is influenced by that of kox. The pH independence of kred is consistent with our previous proposal concerning the reaction mechanism, in which attack of the substrate lone pair of electrons on a Mo(VI)O2 unit initiates the catalytic sequence. The pH dependence of kred/Kdsulfite indicates that a group on the enzyme having a pKa of approximately 9.3 must be deprotonated for effective reaction of oxidized enzyme with sulfite, possibly Tyr 322, which from the crystal structure of the enzyme constitutes part of the substrate binding site. There is no evidence for the HSO3-/SO32- pKa of approximately 7 in the pH profile for kred/Kdsulfite, suggesting that enzyme is able to oxidize the two equally well. By contrast, kcat/Kmsulfite and kred/Kdsulfite exhibit distinct pH dependence (the former is bell-shaped, the latter sigmoidal), again consistent with the oxidative half-reaction contributing to the kinetic barrier to catalysis at low pH. The pH dependence of kcat/Km(cyt c) (reflecting the second-order rate of reaction of free enzyme with free cytochrome) is bell-shaped and closely resembles that of kox/Kd(cyt c), reflecting the importance of the oxidative half-reaction in the low substrate concentration regime. The pH profile for kox/Kd(cyt c) indicates that two groups with a pKa of approximately 8 are involved in the reaction of free reduced enzyme with cytochrome c, one of which must be deprotonated and the other protonated. These results are consistent with the known electrostatic nature of the interaction of cytochrome c with its physiological partners.     

Amino acid sequence of the alpha chain of chicken AI hemoglobin.

Adult chicken hemoglobin is heterogeneous and contains two major components, AI and AII (1). The amino acid sequence of the alpha chain of the AI component from white leghorns (small A type) was determined and compared with that of the alpha chain of the AII component, previously determined by the authors (2). An unexpectedly large difference of 65 amino acids was found between these two chains.     

Amino acid sequence of the phosphopyridoxyl peptide from P-protein of the chicken liver glycine cleavage system

A pyridoxal 5'-phosphate-containing peptide which contained 54 amino acid residues was isolated from chicken liver P-protein of the glycine cleavage system following reduction with NaB3H4, carboxymethylation, and proteolysis with lysylendopeptidase. Two peptides which comprise the two halves of the phosphopyridoxyl peptide were isolated from apo-P-protein. Sequence analysis of these three peptides provided the primary structure of the phosphopyridoxyl peptide and revealed that the cofactor is linked to Lys-35. The pyridoxal 5'-phosphate-binding site has the His-Lys(PLP)-X structure characteristic of known pyridoxal 5'-phosphate-dependent amino acid decarboxylases, tryptophan synthase, and serine hydroxymethyltransferase.     

Purification of prosthetically intact sulfite oxidase from chicken liver using a modified procedure

A modified procedure was used to purify sulfite oxidase (sulfite:O2 oxidoreductase; EC 1.8.3.1) from chicken liver in high yield. The modifications included dialysis of the enzyme against a buffered solution containing sodium molybdate (prior to ion-exchange chromatography), which apparently reconstituted any demolybdo enzyme present in the extract, and phenyl-Sepharose column chromatography. Analysis showed that the purified enzyme contained Mo and heme in a 1.03:1.00 ratio, indicating that the enzyme was prosthetically intact; exogenous heme and other colored proteins were absent from the final pool. Treatment of the sulfite-reduced enzyme with 50 mM cyanide at pH 8.5 resulted in a gradual loss of catalytic activity with a half-life of 19.7 min. Analysis of the cyanide-inactivated enzyme gave a Mo:heme ratio of 1.02:1.00, providing the first direct evidence that the enzyme does not lose molybdenum when inactivated with cyanide. This modified purification procedure provides enzyme in high yield which is well-suited for experiments requiring prosthetically intact enzyme and which is not contaminated with extraneous heme or with other redox active proteins.     

Amino acid sequence of rhizopuspepsin isozyme pI 5

The complete amino acid sequence of an aspartic protease from Rhizopus chinensis, rhizopuspepsin isozyme pI 5, has been determined. Partial sequences were first obtained from the isolated isozyme by a combination of chemical and proteolytic enzyme cleavages, peptide purifications, and Edman degradations. About one-half of the sequence was revealed by this approach. To complete the amino acid sequence, a cDNA library of R. chinensis in pBR322 was constructed. An oligonucleotide probe was synthesized based on the sequence Trp-Trp-Gly-Ile-Thr, and about 40 positive clones were identified by colony hybridization. A clone, 33E2, which had an insert size of about 1.1 kilobase pairs, was found to contain the entire coding region of rhizopuspepsin isozyme pI 5. The sequence of rhizopuspepsin contains 325 amino acid residues. The alignment of the rhizopuspepsin sequence against other aspartic proteases revealed expected homology, with the closest similarity to penicillopepsin which shares 39% identical residues. Porcine pepsin shares about 36% identical residues with rhizopuspepsin.     

Amino acid sequence of chicken gizzard smooth muscle SM22 alpha

The complete amino acid sequence of SM22 alpha, a novel and abundant 22-kDa protein from chicken gizzard smooth muscle, was determined by a combination of automated and manual Edman degradation methods on fragments produced by suitable chemical and proteolytic cleavages. The protein consists of a single polypeptide chain of 197 residues, has a Mr of 21, 978, and a net charge of +4.5 at neutral pH. The pattern of alternating hydrophilic and hydrophobic regions throughout the length of SM23 alpha is typical of a globular protein. The overall secondary structural analysis, using several algorithms based on the sequence, predicts approximately 31% alpha-helix, 24% beta-sheet, 18% beta-turn, and 27% random coil. A search against the National Biomedical Research Foundation Protein Sequence Databank (Washington) and GenBank (Los Alamos) failed to demonstrate significant similarity with any other protein of known sequence.     

Complete amino acid sequence of the thioesterase domain of chicken liver fatty acid synthase

The complete amino acid sequence of thioesterase domain of chicken liver fatty acid synthase has been determined by sequencing peptides produced by trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide cleavage. The thioesterase domain consists of 300 amino acid residues. All of the tryptic peptides of the thioesterase domain were isolated and sequenced, except the segment covered from position 109 to position 124. Peptides resulting from digestion by Staphylococcus aureus V8 protease and cyanogen bromide cleavage filled the missing part and overlapped the complete sequence of the entire thioesterase domain. The NH2 terminus of the thioesterase domain was determined to be lysine by sequencing the whole domain up to 20 residues while the COOH terminus was identified as serine through carboxyl peptidase Y cleavage. The active site of the thioesterase domain of chicken fatty acid synthase was suggested to be the serine on position 101 according to its homology with other serine-type esterases and proteases which have a common structure of -Gly-X-Ser-Y-Gly- with the variable amino acids X and Y disrupting the homology.     

Amino acid sequence of chicken calsequestrin deduced from cDNA: comparison of calsequestrin and aspartactin

We have previously reported the amino terminal sequence of adult chicken calsequestrin, an intraluminal Ca2(+)-binding protein isolated from fast-twitch skeletal muscle. The partial sequence showed homology with mammalian calsequestrins contained in the PIR data bank and complete identity with the amino terminus of a putative laminin-binding protein of the extracellular matrix, aspartactin. Based on these data, oligonucleotide primers were synthesized for PCR amplification and direct DNA sequencing. We report herein the primary sequence of chicken calsequestrin, deduced from cDNA. The sequence has been verified by amino acid sequencing of internal tryptic peptides. Importantly, the data show the primary structure of calsequestrin to be identical to the amino acid sequence reported for aspartactin, with the exception of a single amino acid difference (ileu vs. val) which may be animal strain-related. Based on these data, calsequestrin and aspartactin are the same protein.     

Amino acid sequence of the triple-helical domain of human collagen type VI

The complete amino acid sequence of the triple-helical domain of human collagen VI was deduced from sequences of appropriate cDNA clones and confirmed to about 50% by Edman degradation of tryptic peptides. This domain consists of three different peptide segments containing some 335-336 amino acid residues originating from central portions of the alpha 1 (VI), alpha 2(VI), and alpha 3(VI) chains, respectively. Sequence identity in the X/Y positions of the Gly-X-Y repeats is rather low (10-15%) between the chains. Peculiar features of these sequences include 3 cysteine residues about 50 (alpha 3(VI)) and 89 (alpha 1(VI), alpha 2(VI)) residues away from the N-terminus and several Gly-X-Y interruptions clustered in the C-terminal two-thirds of the triple helix. These structures are presumably required for cross-linking collagen VI oligomers and for super-coiling of triple helices in the dimers. Other features include 11 Arg-Gly-Asp sequences, some of which are likely to be used as cell-binding sites, and four Asn-X-Thr sequences, allowing N-linked glycosylation along the triple helix. Junctional areas close to the helix contain short, cysteine-rich segments which may seal the triple-helical domain through disulfide bond formation, endowing it with high stability. These features, together with a low sequence homology to fiber-forming and basement-membrane collagens, document the unique character of collagen VI, whose triple helix is specifically adjusted for forming microfibrils in tissues.     

Amino acid sequence of myoglobin from the molluscBursatella leachii

The complete amino acid sequence of myoglobin from the triturative stomach of gastropodic molluscBursatella leachii has been determined. It is composed of 146 amino acid residues, is acetylated at the N-terminus, and contains a single histidine residue at position 95 which corresponds to the heme-binding proximal histidine. The E7 distal histidine, which is conserved widely in myoglobins and hemoglobins, is replaced by valine inBursatella myoglobin. The amino acid sequence ofBursatella myoglobin shows strong homology (73–84%) with those ofAplysia andDolabella myoglobins.     

Amino acid sequence of a novel calmodulin from Paramecium tetraurelia that contains dimethyllysine in the first domain

A class of Paramecium behavioral mutants called pantophobiacs have a deficiency in calcium-dependent potassium efflux, and this deficiency can be corrected by the microinjection of wild-type Paramecium calmodulin (Hinrichsen, R. D., Burgess-Cassler, A., Soltvelt, B. C., Hennessey, T., and Kung, C. (1986) Science 232, 503-506). As a starting point in investigations of which features allow wild-type Paramecium calmodulin to fully restore this behavior while other calmodulins are inactive or poorly effective, we elucidated the amino acid sequence of the wild-type calmodulin. We utilized an approach that combined Edman chemistry with mass spectrometry. This approach resulted in the identification of a new post-translational modification in calmodulin: N epsilon,N epsilon-dimethyllysine at residue 13. This particular modification has not been described for calmodulins studied previously. The only other first-domain modification that has been described for any calmodulin is acetylation of the amino terminus (Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255, 962-975). These results along with analyses of pantophobiac calmodulin and calmodulin binding proteins will provide insight into calmodulin's role in a well-defined behavioral mutant.     

Amino acid sequence of lysozyme from baboon milk

Reduced alkylated baboon milk lysozyme was subjected to digestion with trypsin. The resulting peptides were purified by a combination of Dowex 1 (X2) and chromatography and electrophoresis on paper. The amino acid sequence of these peptides was determined in detail chiefly by the Edman procedure. Alignment of the tryptic peptides into a single chain containing 130 amino acids was established chiefly by homology with human milk lysozyme; 14 replacements were noted between the two enzymes. Baboon milk lysozyme was devoid of methionine and contained six basic amino acids (arginine residues) less than human milk lysozyme.