Amino-acid sequence of a tetrameric, manganese superoxide dismutase from Thermus thermophilus HB8

The amino-acid sequence of a tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been determined. The protein was cleaved with cyanogen bromide (BrCN) into four peptides and their alignment was deduced through the fragment of partial cleavage with BrCN and the peptides were produced by cleavage of the protein with o-iodosobenzoic acid. Most of the peptides were sequenced by solid phase Edman degradation. Some of the peptides were sequenced by the Edman dansyl method after sub-fragmentation by proteinase digestion. The amino-acid sequence consists of 203 residues corresponding to a subunit molecular weight of 23,144.     

Studies on cytochrome-c oxidase, XIII. Amino-acid sequence of the small membrane polypeptide VIIIc from bovine heart respiratory complex IV

The isolation and complete amino-acid sequence analysis of the cytoplasmically synthesized polypeptide VIIIc from bovine heart cytochrome-c oxidase is described. The protein is a stoichiometric constituent of the mitochondrial respiratory complex IV. Its primary structure is deduced from N-terminal sequencing and peptides obtained by enzymatic cleavage with Staphylococcus aureus proteinase and chemical cleavage with cyanogen bromide. The small protein consists of 56 amino acids summing up to a total Mr of 6243. From position 34 to 51 the chain contains a hydrophobic sequence of 18 residues. This probably membrane-spanning segment also contains the 2 cysteine residues of the chain. The function of this subunit in the respiratory complex IV is still unknown.     

The amino-acid sequence of rat Cu-Zn superoxide dismutase

The primary structure of Cu-Zn superoxide dismutase isolated from rat liver was determined. The enzyme was reduced, carboxymethylated and fragmented by treatment with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by gel filtration or high performance liquid chromatography and sequenced by automated Edman degradation. The total sequence of 153 amino-acid residues per subunit was reconstructed from overlapping peptides. Rat Cu-Zn superoxide dismutase proved to be closely related to the corresponding sequences of other mammals in having more than 80% identical amino-acid residues in homologous position and an acetylated N-terminus. Comparison of the rat Cu-Zn superoxide dismutase structure with those of other species suggests a similar phylogenetic distance between rat, man, pig, cattle and horse and a rapid molecular divergence during vertebrate development compared to earlier evolutionary periods.     

Primary structure of streptococcal proteinase. II. Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragment 5.

The amino acid sequence of the cyanogen bromide fragment 5 of streptococcal proteinase has been determined. This fragment comprises residues 130 to 253 of the proteinase chain. Six tryptic peptides were isolated from maleylated cyanogen bromide fragment 5, and their alignment was obtained by the overlap of chymotryptic peptides. Sequence analysis of tryptic, chymotryptic, and thermolysin peptides was performed by the 5-deimethylaminoaphthalene-1-sulfonyl technique and carboxypeptidases digestion.     

Chymotryptic inhibitor I from potatoes. The amino acid sequence of subunit A

The amino acid sequence of subunit A of the potato chymotryptic inhibitor I was determined. The sequence was deduced from analysis of fragments and peptides derived from the protein by cleavage with cyanogen bromide, N-bromosuccinimide and dilute acid, and by digestion with trypsin, thermolysin, pepsin and papain. The molecule consists of a single polypeptide chain of 84 residues, which contains two homologous regions each of 13 amino acids. The protein does not appear to be homologous with any other known proteinase inhibitors.     

Protein inhibitors of cysteine proteinases. III. Amino-acid sequence of cystatin from chicken egg white

Cystatin, the protein inhibitor of cysteine proteinases from chicken egg white was purified by a new method. The two major forms with pI 6.5 (Peak I) and 5.6 (Peak II) were separated. Molecular masses of both forms are approx. 12700 Da as determined by gel chromatography; Form A from Peak I has a molecular mass of 12191 Da as calculated from its amino-acid sequence. The complete amino-acid sequence of Form A was determined by automated solid-phase Edman degradation of the whole inhibitor and its cyanogen bromide fragments. It contains 108 amino-acid residues. Form B from Peak II represents an elongation of Form A by 8 amino-acid residues at the N-terminus. Cystatin contains four cysteine residues, presumably forming two disulphide bridges. Comparison of the amino-acid sequences and near ultraviolet circular dichroism spectra of stefin, the cysteine proteinase inhibitor from human granulocytes, and cystatin shows that the two proteins are entirely different. According to the primary structures, probably neither proteinase inhibitor is involved in a thiol-disulphide exchange mechanism in the interaction with its target enzyme.     

The covalent structure of the elastase inhibitor from Anemonia sulcata--a "non-classical" Kazal-type protein

The amino-acid sequence of the proteinase inhibitor specific for elastases from the sea anemone Anemonia sulcata was determined from performic-acid oxidized inhibitor and from three cyanogen bromide fragments of reduced and carboxymethylated inhibitor. The molecule consists of a single polypeptide chain formed from 48 amino-acid residues and is stabilized by three intramolecular disulfide bridges. After cyanogen bromide cleavage of the native protein at methionines 10 and 28 followed by chymotryptic cleavage two fragments each containing a single disulfide bridge were isolated. These indicated the location of three intramolecular disulfide linkages between Cys4 and Cys34 (part of A-loop), Cys8 and Cys27 (B-loop) and Cys16 and Cys48 (C-loop). The sequential homology and the disulfide pattern identified the elastase inhibitor as a Kazal-type inhibitor in which, however, not only the CysI-CysII segment is rather short but interestingly the Cys4-Cys34 disulfide anchoring point (i.e. CysI-CysV) in the C-loop is shifted by one turn in the alpha-helical segment towards the C-terminus. Thus, the elastase inhibitor is a non-classical Kazal-type inhibitor with respect to the positioning of the half-cystines. The inhibitor molecule was modelled based on the known three-dimensional structure of the silver pheasant ovomucoid third domain. The shortened amino-terminal segment was arranged in such a manner to allow disulfide bridge formation between the first cysteine Cys4 and the replaced Cys34 under maintenance of a suitable binding loop conformation. The characteristic ovomucoid scaffold consisting of a central alpha-helix, an adjacent three-stranded beta-sheet and the proteinase-binding loop cross-connected through disulfide bridges CysI-CysV and CysIII-CysVI was conserved.     

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 rat kidney acylase I, characterization and molecular cloning. Differences with other acylases I.

The soluble acylase I from rat kidney was purified to homogeneity using a five-step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-L-methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224-bp open reading frame encoding a protein consisting of 408 amino-acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.     

Primary structure of streptococcal proteinase. I Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragments 1 to 4.

Tryptic and chymotroptic peptides were isolated and characterized from cyanogen bromide fragments 1 to 4 of streptococcal proteinase and subjected to sequence analysis by the Edman degradation, carboxy-peptidase digestion, and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. The results, together with the sequence data of the cyanogen bromide fragment 5 reported in the accompanying papers, provide the structural formula of streptococcal proteinase.     

A new inhibitor of elastase from the sea anemone (Anemonia sulcata)

A proteinase inhibitor for elastases was isolated from extracts of the sea anemone Anemonia sulcata and purified to apparent homogeneity. The procedure comprises ethanolic extraction of the deep-frozen animals followed by gel filtration on Sephadex G-50 and by ion exchange chromatography on DEAE-Sephadex A-25 and SP-Sephadex C-25 and by hydroxylapatite chromatography. The slightly acidic inhibitor (isoelectric point 5.9) is a small protein consisting of 48 amino-acid residues without tryptophan and phenylalanine. The single chain molecule contains two methionines and no free sulfhydryl group but six cysteines presumably forming disulfide bonds. Reaction with cyanogen bromide abolishes the inhibitory properties. The inhibitor exhibits a rather narrow specificity for elastases. It strongly inhibits porcine pancreatic elastase in a permanent fashion with an equilibrium dissociation constant Ki of about 10(-10)M and somewhat weaker the elastase from human leucocytes with a Ki of about 10(-7)M. No obvious inhibition is observed of other serine proteinase such as bovine trypsin, bovine chymotrypsin, subtilisin from Bacillus subtilis and cathepsin G from human leucocytes when tested with synthetic substrates.     

The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins

The amino-acid sequence of a lectin isolated from the coelomic fluid of the acorn barnacle Megabalanus rosa has been determined. The lectin (Mr 140,000) is a multimeric protein whose subunit consists of 173 amino acids and one carbohydrate chain attached to Asn-39. The amino-acid sequence was determined by the manual sequencing of peptides derived from the protein by digestion with Staphylococcus aureus V8 proteinase, lysine endopeptidase and chymotrypsin, as well as fragments produced by cleavage with cyanogen bromide. The amino-acid sequence of the lectin was compared with the sequence of one (Mr 64,000) of the multiple lectins of M. rosa. They are distinct molecules in spite of a significant homology in their amino-acid sequences. The amino-acid sequence includes some regions homologous to those in other invertebrate lectins, such as sea urchin and flesh fly lectins, and vertebrate lectins. This is the first report to show the amino-acid sequence of multiple lectins isolated from an invertebrate.     

Partial amino acid sequence of human plasma retinol-binding protein. Isolation and alignment of the five cyanogen bromide fragments and the amino acid sequences of four of the fragments

Studies are reported on the primary structure of human retinol-binding protein (RBP), the specific plasma transport protein for vitamin A. The protein consists of a single polypeptide chain of 186-187 amino acids. RBP was cleaved by cyanogen bromide into five fragments, CB-I (27 residues), CB-11 (25 residues), CB-III (20 residues), CB-IV (15 residues), and CB-V (99-100 residues). The cyanogen bromide fragments were isolated, their compositions were determined, and they were aligned after studies that included the tryptic digestion of maleylated, reduced, and carboxymethylated RBP and subsequent enzymatic digestion of some of the resulting tryptic peptides. The amino acid sequences of four of the five cyanogen bromide fragments were determined, and the sequence of almost two-thirds of the NH2-terminal portion of the RBP molecule was determined as: H2N-GLU-Arg-Asp-Cys-Arg-Val-Ser-ser-Phe-Arg-Val-Lys-Glu-Asn-Phe-Asp-Lys-Ala-Arg-Phe-Ser-Gly-Thr-Trp-Tyr-Ala-Met-Ala-Lys-Lys-Asp-Pro-Glu-Gly-Leu-Phe-Leu-Gln-Asp-Asx-Ile-Val-Ala-Glu-Phe-Ser-Val-Asx-Glx-Gly-Thr-Met-Ser-Ala-Thr-Ala-Gly-Lys-Arg-Val-Arg-Leu-Leu-Asn-Asn-Trp-Asp-Val-Cys-Ala-Asp-Met-Val-Gly-thr-Phe-Thr-Asp-Thr-Glu-Asp-Pro-Ala-Lys-Phe-Lys-Met-Lys-Tyr-Trp-Gly-Val-Ala-Ser-Phe-Leu-Gln-Lys-Gyl-Asn-Asp-Asx-His-Trp-Ile-Val-Asp-Thr-Asx-Thr-Tyr-Tyr-Ala-Val-Glu-Tyr-Cys-Ser-Arg---.     

Amino-acid sequence of the short subfragment-2 in adult chicken skeletal muscle myosin

The amino-acid sequence of a short subfragment-2 in the amino-terminal portion of subfragment-2 (S-2) derived from adult chicken skeletal muscle myosin was completely determined. Peptides cleaved by cyanogen bromide and by lysyl endopeptidase of S-carboxymethylated S-2, and hydrolytic peptides obtained with trypsin or dilute acetic acid of larger CNBr fragments were isolated and sequenced. This region was composed of 257 amino-acid residues, and hydrophobic and charged residue repeat units were found highly conserved and with a periodicity in 7 or 28 residues. This sequence of the short S-2 fragment of chicken skeletal muscle myosin was compared with the sequence of chicken and rat embryonic skeletal muscle myosins, rabbit skeletal and rabbit cardiac muscle myosin (alpha-myosin heavy chain), and 95.3%, 86.8%, 89.9% and 94.2% sequence identities were observed, respectively.     

Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. IV. Structure of peptides of thermolytic and limited tryptic hydrolysis of the fragment F1; peptides of cyanogen bromide hydrolysis of cytochrome P-450. Complete amino acid sequence

A thermolytic hydrolysis of maleinated fragment F1 has been performed, resulted in isolation of 44 peptides; their complete amino acid sequence has been determined. Non-overlapping thermolytic peptides of fragment F1 involve 178 amino acid residues, which comprises about 71% of its amino acid sequence. Also, the cleavage and structural investigation of some tryptophan-containing peptides obtained from the limited trypsinolysis of fragment F1 were carried out; reconstitution of the polypeptide chain of the fragment is completed. The cyanogen bromide cleavage of carboxymethylated cytochrome P-450 was achieved and 17 peptides, comprising almost the whole polypeptide chain of the protein molecule (91%), was isolated. To investigate structure of the cyanogen bromide peptides, we hydrolysed them at tryptophan residues with trypsin, chymotrypsin, proteinase from Staphylococcus aureus, and BNPS-skatole. The data obtained and those published earlier led to the complete primary structure of the cholesterol-hydroxylating cytochrome P-450. The proteins polypeptide chain consists of 481 amino acid residues and has the precise molecular mass 56 407.7.     

The primary structure of porcine Cu-Zn superoxide dismutase. Evidence for allotypes of superoxide dismutase in pigs

Cu-Zn superoxide dismutase was purified to homogeneity from mixed pig blood and from a single pig. The isolated product had an absorption ratio 280/260 nm of 0.91, a specific activity of 3 000 +/- 200 units (cytochrome c reduction test), and an isoelectric point of 7.5 (chromatofocusing) or 7.25 (isoelectric focusing), respectively. Sequence determination was performed by automated solid-phase Edman degradation of fragments of the reduced S-carboxymethylated proteins obtained by digestion with trypsin or Staphylococcus aureus proteinase V8 or treatment with cyanogen bromide. Acetylation of the N-terminus was confirmed by comparing high performance liquid chromatography retention times of N-terminal peptides with those of authentic samples. Sequencing of the superoxide dismutase of mixed porcine blood revealed heterogeneity (70% Leu; 30% Val at position 29), whereas the sample derived from a single French pig proved to be homogeneous (100% Leu at position 29). The complete sequence of pig superoxide dismutase comprised 152 amino-acid residues, which corresponds to a theoretical molecular mass of 15 800 Da per subunit, and exhibited the expected high homology with those of other mammals. The aspartate and all 7 histidine residues known to complex the metal ions in bovine superoxide dismutase are conserved in the porcine sequence at the homologous positions Asp82 and His45, His47, His62, His70, His79, His119, respectively.     

Identification and synthesis of a novel 15 kDa beta-galactoside-binding lectin in human leukocytes.

Knowledge of the identity, synthesis and secretion of beta-galactoside-binding lectins by leukocytes is of importance because lactosaminoglycans present at the leukocyte cell surface may be physiologically significant lectin receptors that could mediate autocrine or paracrine functions and/or cell adhesion. This paper presents data that show that a previously identified 15.5-16.5 kDa lactose-binding protein synthesized in vitro by human peripheral leukocytes is actually comprised of three different polypeptides. One of these is related to a novel 15 kDa lectin isolated from human spleen and which is synthesized by B lymphoblastoid cells. Spleen contains at least six lactose-binding polypeptides for which the carbohydrate-binding activity is independent of the presence of divalent cations and mercaptoethanol. The splenic 15 kDa polypeptide does not appear to be immunologically related to previously characterized beta-galactoside-binding lectins. It is separable from galaptin, another galactoside-binding lectin (subunit mol. wt 14.5 kDa) by chromatography on DEAE-Sephacel. Western blot analyses and immunoprecipitation/fluorography experiments with metabolically labelled cells showed the presence of the 15 kDa lectin in peripheral leukocytes and in Epstein-Barr virus-immortalized B lymphoblastoid cells. The 15 kDa lectin yielded polypeptide fragments of approximately 6.2 and approximately 8.6 kDa after cyanogen bromide (CNBr) degradation. These fragments were partially sequenced and 12 residues/fragment were identified. A similarity search of the SWISS PROT protein data base did not reveal a relationship of the 15 kDa polypeptide to known lectins, including galaptin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete amino-acid sequence of subfragment-2 in adult chicken skeletal muscle myosin

Although the complete amino-acid sequence of the short subfragment-2 (short S-2) and the partial sequence of the hinge region derived from adult chicken skeletal muscle myosin have been reported previously, the sequence of the N-terminal portion of subfragment-2 (S-2) and the connective portion between the above two regions could not be determined. In this study, the amino-acid sequence of these undetermined portions were completely sequenced. Furthermore, overlaps of cyanogen bromide (CNBr) peptides in the hinge region were also isolated and sequenced. Peptides obtained by hydrolysis with dilute formic acid and by digestion with lysyl endopeptidase of S-2 were purified and sequenced. These results established the complete amino-acid sequence of S-2 composed of 429 amino-acid residues. This sequence of adult chicken skeletal muscle myosin was compared with that of chicken embryonic skeletal muscle, chicken gizzard muscle and rabbit cardiac muscle myosin (alpha-myosin heavy chain) and shows degrees of 96%, 38% and 84% sequence identities, respectively. The frequency with which hydrophobic residues are present at position "a" in seven-residues repeats of the hinge region was markedly reduced when compared to the short S-2 sequence of the chicken skeletal muscle myosin.     

Amino acid sequence of the beta chain of human fibrinogen.

The beta chain of human fibrinogen contains 461 amino acid residues, 15 of which are methionines. The calculated molecular weight, independent of a single carbohydrate cluster, is 52 230. In this regard, we have isolated and characterized all 16 cyanogen bromide fragments. In one case (CNI), we have concentrated on a disputed portion of a previously reported fragment. The arrangement of the cyanogen bromide peptides was deduced by the use of overlap fragments obtained from the tryptic digestion of modified and unmodified beta-chains and from digestions with staphylococcal protease, as well as by considerations involving the plasmic digestion products of fibrinogen. In one case two adjacent fragments were aligned by homology with the corresponding segments of the gamma chain. The homology of the beta chain with the gamma chain is especially strong over the course of the carboxy-terminal two-thirds of the sequence. Neither of these chains appears to be homologous with the alpha chain in these regions. With a few minor exceptions, the sequence reported in this article is in agreement with data reported by other groups in Stockholm and Munich.