Phosphofructokinase: complete amino-acid sequence of the enzyme from Bacillus stearothermophilus

The entire amino acid sequence of the protein subunit of phosphofructokinase from Bacillus stearothermophilus has been established mainly by sequence analysis of cyanogen bromide fragments and of peptides derived from these fragments by further digestion with proteolytic enzymes. Overlaps of the cyanogen bromide fragments as well as peptide sequences necessary to complement and to confirm tentative assignments within the larger peptide fragments were obtained from the sequences of selected peptides isolated from tryptic and chymotryptic digests of the intact S-[14C]-carboxymethylated protein. Sequence information was also provided by automated sequence analysis of the intact protein subunit and of some of the larger peptide fragments. The sequence is as follows: (See Text).     

The complete amino-acid sequence of the sweet protein thaumatin I.

The primary structure of the sweet-tasting protein thaumatin has been elucidated. The protein consists of a single polypeptide chain of 207 residues. The sequence of the N-terminal part of the chain was determined by sequenator analysis. As the protein contains only one methionine residue, it was possible to deduce the N-terminal sequence of the C-terminal cyanogen bromide fragment by automatic sequencing of the cyanogen-bromide-cleaved, succinylated protein. To arrive at the sequence of the whole protein tryptic and Staphylococcus protease peptides, together with chymotryptic peptides and a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole) fragment were also sequenced. Comparing the amino acid sequence of thaumatin with that of the other sweet-tasting protein, monellin, we have located five sets of identical tripeptides. Since immunological cross-reactivity of thaumatin antibodies with monellin has recently been described, one or more of these tripeptides might be part of a common antibody recombination site and possibly be involved in the interaction with the sweet-taste receptor.     

The complete amino-acid sequence of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon

The amino-acid sequences of both subunits of C-phycoerythrin from the cyanobacterium Fremyella diplosiphon have been determined. The alpha-subunit contains 164 amino acid residues, two phycoerythrobilin (PEB) chromophores and has a molecular mass of 18,368 Da (protein: 17,192 Da + 2 PEB, one PEB accounting for 588 Da). The beta-subunit consists of 184 residues, three PEB chromophores and has a molecular mass of 20,931 Da (protein: 19,168 Da and 3 PEB: 1,764 Da). The five PEB chromophores (open chain tetrapyrroles) are covalently bound to six cysteine residues (one of them doubly bound to two cysteine residues). On the alpha-subunit, the first chromophore was found at position 84, homologous to the chromophore binding site of the other biliproteins APC, PC and PEC. The second chromophore, unique for the alpha-subunit of PE, is inserted together with a pentapeptide at position 143 a. On the beta-subunit, a doubly bound chromophore is attached to cysteine residues 50 and 61, similar to the rhodophytan phycoerythrins (B-PE and R-PE). The second and third chromophores were found at positions 84 and 155, homologous to the other biliproteins. A unique peptide insertion of 14 amino acid residues (without chromophore) was found at position 141 a-o in the beta-subunit and probably is located in the three-dimensional model near the additional chromophores of the C-PE alpha- and beta-subunits. Both additional chromophores of the C-PE alpha- and beta-subunit may be located at the periphery of the C-PE-trimer. The amino-acid sequence homology between C-PE alpha- and beta-subunit is 26% and to the alpha- and beta-subunits of C-PC from Mastigocladus laminosus 49% and 48%, respectively.     

The complete amino-acid sequence of histone H2B from the mollusc Patella granatina.

1. From the marine mollusc, Patella granatina, a histone has been isolated. Its primary structure has been established and it has been designated histone H2Bpatella. It consists of a polypeptide chain of 121 amino acids. 2. In the carboxy-terminal two thirds of the molecule there is a highly degree of sequence homology to the corresponding region in calf histone H2B with identical residues in 95% of the positions. 3. In the N-terminal 22 amino acids histone H2Bpatella differs considerably from the mammalian histone H2B and it is shorter by four residues.     

The complete amino-acid sequence of a basic phospholipase A2 in the venom of Bungarus fasciatus

A basic phospholipase A2 (PLA2) was isolated and purified from the venom of Bungarus fasciatus. Four kinds of enzymes, lysyl endopeptidase, endoproteinase Asp-N, endoproteinase Glu-C and trypsin, were employed to elucidate the complete primary structure by means of gas-phase sequencing. The amino-acid sequence reveals 118 amino-acid residues containing seven pairs of half-cystine. It has 78% and 61% structural identities with PLA2 from Bungarus multicinctus and Naja melanoleuca DE-II, respectively.     

Determination of the complete amino-acid sequence of subtilisin DY and its comparison with the primary structures of the subtilisins BPN', Carlsberg and amylosacchariticus

The complete amino-acid sequence of subtilisin DY, an extracellular alkaline proteinase produced by Bacillus subtilis strain DY was determined. This included automated sequence analysis of the whole molecule and its large fragments such as tryptic peptides obtained from the inactivated enzyme, peptides generated by cyanogen bromide, by o-iodosobenzoic acid and by hydroxylamine. The peptides were isolated by gel filtration and by reversed-phase high performance liquid chromatography. The amino-acid sequence of subtilisin DY was determined by overlapping the isolated peptides. It consists of 274 amino-acid residues, like that of subtilisin Carlsberg. By comparison with the structures of the subtilisins Carlsberg, amylosacchariticus and BPN' 32, 80 and 82 amino-acid substitutions were found, which are caused by 37, 102 and 106 nucleotide mutations, respectively. It was found also that 62.5% of the amino-acid residues in the molecules of these four subtilisins are identical with respect to kind and position of the residue, which suggests that these molecules have had a common ancestral precursor. The amino-acid replacement analysis of the four subtilisins leads to the conclusion that they have evolved almost independently.     

The complete amino-acid sequence of the rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis

Summary SummarySeveral cDNA clones encoding the entire Rieske FeS-precursor protein of the chloroplast cytochrome b ₆ f-complex have been isolated by high density plaque immunoscreening of a phage lambda gt11 cDNA expression library, made from poly A⁺-RNA of spinach seedlings. The identity of the cDNAs has been confirmed by N-terminal amino acid sequencing of the purified protein. The nucleotide sequence indicates a protein of 247 amino acid residues including a putative transit sequence of 68 amino acids corresponding to molecular masses of 26.3 kDa (precursor) and 18.8 kDa (mature protein; 179 amino acid residues). Alignteins of the sequence with sequences from Rieske FeS-proteins of respiratory electron transport chains, two of bacterial and three of mitochondrial origin, shows little sequence homology, but remarkable similarity in secondary structure including a putative N-terminal transmembrane segment of about 25 residues and the peptides CTHLGCV and CPCHGS in the C-terminal region of the protein that are involved in the binding of the Fe₂S₂-cluster.     

The complete amino-acid sequence and the phylogenetic origin of phycocyanin-645 from the cryptophytan alga Chroomonas sp

The first complete amino-acid sequence of the cryptomonad phycobiliprotein phycocyanin-645 from Chroomonas sp. is presented. The alpha 1-subunit contains 70 amino-acid residues and the alpha 2-subunit 80 residues. In each of the alpha-subunits a green, 697-nm absorbing chromophore is covalently bound to Cys18. Both alpha-subunits contain a high number of charged residues. The phycocyanin-645 beta-subunit consists of 177 amino-acid residues. Two phycocyanobilin chromophores are singly bound to Cys beta 82 and Cys beta 158. A purple cryptoviolin-like chromophore is doubly bound to Cys beta 50 and Cys beta 61. Sequence comparisons revealed that the phycocyanin-645 beta-subunit is closely related to red algal phycoerythrin (73% identical amino-acid residues) and not so close to C-phycocyanin (55% identical amino-acid residues). The phycocyanin-645 alpha-subunits represent a special type of phycobiliprotein and a direct relationship to other phycobiliproteins or any light-harvesting polypeptide-pigment complexes could not be derived by sequence comparisons.     

The complete amino-acid sequence of dimeric beta-lactoglobulin from mouflon (Ovis ammon musimon) milk

beta-Lactoglobulin from Mouflon (Ovis ammon musimon) milk has been isolated and its complete primary structure determined. This protein has been isolated in dimeric form and has a molecular mass of 37 kDa. The amino-acid sequence has been determined by microsequencing of the native protein and the peptides were obtained after tryptic cleavage. The tryptic peptides were isolated by reversed phase high-performance liquid chromatography. The primary structure of mouflon beta-lactoglobulin shows close similarity to ruminant beta-lactoglobulins. The presence of His at position 20 indicates that this protein belongs to the B-type of dimeric ovine beta-lactoglobulins. Mouflon beta-lactoglobulin is a 162 amino acid long polypeptide chain with two disulphide bridges and one free thiol group. Structural similarities to the bilin-binding protein, BG protein from olfactory epithelium and retinol-binding protein are discussed.     

The complete amino-acid sequence of the alpha and beta subunits of B-phycoerythrin from the rhodophytan alga Porphyridium cruentum

Determination of the complete amino-acid sequence of the subunits of B-phycoerythrin from Porphyridium cruentum has shown that the alpha subunit contains 164 amino-acid residues and the beta subunit contains 177 residues. When the sequences of B- and C-phycoerythrins are aligned with those of other phycobiliproteins, it is obvious that B-phycoerythrin lacks a deletion at beta-21-22 present in C-phycoerythrin. However, relative to C-phycoerythrin from Fremyella diplosiphon (Calothrix) (Sidler, W., Kumpf, B., Rüdiger, W. and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, 627-642), B-phycoerythrin has deletions at beta-141k-o, beta-142, beta-143, beta-147 and beta-148. The four singly-linked phycoerythrobilins at positions alpha-84, alpha-143a, beta-84 and beta-155, and the doubly-linked phycoerythrobilin at position beta-50/61 are at sites homologous to the attachment sites in C-phycoerythrin. The aspartyl residues (alpha-87, beta-87, and beta-39), that interact with the bilins at alpha-84, beta-84, and beta-155 in C-phycocyanin, are found in the homologous positions in B-phycoerythrin. B-Phycoerythrin, in common with other phycobiliproteins, contains a N gamma-methylasparagine residue at position beta-72.     

The complete amino-acid sequence of the heavy chain of the human myeloma protein WIE, an immunoglobulin D.

The human myeloma protein WIE is a lambda-type immunoglobulin D; the amino-acid sequence of its Fc part and aminoethylated heavy chain was completely determined. The VH-part (subgroup III) begins N-terminally with 5-oxoproline, and it contains a long, unique CDR3 region. Since the constant part differs from known delta chains by one amino-acid substitution in the hinge region, IgD WIE probably represents an allotypic variant. As in other protein delta chains, O-glycosylations are confined to the hinge region. Furthermore, the ratios of N-glycosylations at the three positions are identical in IgD WAH [Takahashi, N. et al. (1984) J. Chromatogr. 317, 11-26.] and IgD WIE (100%, 50%, 100%). From the most conserved constant domain, C delta 3, a three-dimensional model was constructed to clarify the role of its delta-specific substitutions and glycosylation.     

Partial amino-acid sequence of the epidermal growth-factor-binding protein

The partial amino acid sequence of the epidermal growth-factor-binding protein was determined. Residues in 108 unique positions, corresponding to 45% of the molecule, were identified. The protein is a serine protease, closely related to the nerve growth factor gamma subunit. It is suggested that the epidermal growth-factor-binding protein, like other serine protease, is synthesized as a single polypeptide chain which undergoes limited endoproteolysis. The isolated material also contained minor amounts of a second serine protease. This protease is closely related to the epidermal growth-factor-binding protein, differing from it in 7 out of the 45 amino acid positions available for comparison. The latter protease may be identical to the previously described protease A.     

The phycobiliprotein beta 16.2 of the allophycocyanin core from the cyanobacterium Mastigocladus laminosus. Characterization and complete amino-acid sequence

The minor phycobiliprotein beta 16.2 was isolated from the APC-core of phycobilisomes from the cyanobacterium Mastigocladus laminosus. Its complete amino-acid sequence and some spectral characteristics are presented. beta 16.2 consists of 169 amino acids and its molecular mass is 19,390 Da. A phycocyanobilin chromophore is covalently bound to a cysteine at position 84 as in all other phycobiliproteins. The first 138 amino acids are highly homologous to the N-terminal part of beta AP (62.5%), whereas the C-terminal part has no homology. The absorption maximum is situated at 624 nm, the fluorescence emission maximum at 644 nm in phosphate buffer, pH 7.0. Both values are slightly redshifted compared with alpha AP and beta AP.     

The complete amino-acid sequence of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.)

After reduction and alkylation of the disulfide bonds of the proteinase inhibitor B from the root of the arrowhead (Sagittaria sagittifolia L.) followed by CNBr cleavage three peptide fragments with 68, 62 and 11 amino-acid residues could be separated on DEAE-Sepharose CL-6B. The peptides or the inhibitor itself were further specifically cleaved either by trypsin or by the mixture of (CH3)2SO/HCl/HBr at the arginyl- and the tryptophyl-peptide bond, respectively. The complete amino-acid sequences of the peptides were determined by manual solid phase DABITC/PITC double coupling micro-method and the primary structure of the arrowhead inhibitor B consisting of 141 amino-acid residues was then elucidated. Twenty pairs of amino-acid residues are repeated in the molecule of this inhibitor, three of these pairs even occur three times. The possible locations of the reactive sites are discussed. On the basis of sequence comparisons between this inhibitor and all other serine proteinase inhibitors the arrowhead inhibitor may belong to a new family.