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.     

Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae.

The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the ATP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 80% formic acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 50% homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 gene in Aspergillus nidulans.     

Amino acid sequence of ovine 6-phosphogluconate dehydrogenase

The amino acid sequence of the NADP+-dependent enzyme ovine 6-phosphogluconate dehydrogenase has been determined by conventional direct protein sequence analysis of peptides resulting from digestion of the protein with trypsin and chemical cleavages with cyanogen bromide, hydroxylamine, and iodosobenzoic acid. The polypeptide contains 466 amino acids and its NH2 terminus is acetylated. The Candida utilis enzyme is inactivated by reaction of pyridoxal phosphate with two lysine residues (Minchiotti, L., Ronchi, S., and Rippa, M. (1981) Biochim. Biophys. Acta 657, 232-242). These residues are conserved in the ovine enzyme. In contrast to NAD+ dehydrogenases which have weakly related sequences and spatially related folds in their nucleotide-binding sites, no significant sequence homologies were detected between 6-phosphogluconate dehydrogenase and any of three other NADP+-requiring enzymes, glutamate dehydrogenase, p-hydroxybenzoate hydroxylase, and dihydrofolate reductase. This is in accord with structural data that show no spatial relationship between NADP+-binding sites in these enzymes.     

Amino acid sequence of the proteolipid subunit of the proton-translocating ATPase complex from the thermophilic bacterium PS-3

The proteolipid subunit of the ATPase complex was identified in whole membranes of the thermophilic bacterium PS-3 by means of a covalent modification with the 14C-labelled inhibitor dicyclohexylcarbodiimide. The proteolipid could be purified from the membrane in free and carbodiimide-modified form by extraction with chloroform/methanol and subsequent carboxymethylcellulose chromatography in mixtures of chloroform/methanol/water. The complete amino acid sequence of the 72-residue polypeptide could be determined by automated solid-phase Edman degradation of the whole protein, and of fragments obtained after cleavage with cyanogen bromide and N-bromosuccinimide. Chemical cleavages and separations of the resulting fragments by gel chromatography were performed in 80% formic acid. The amino acid sequence shows a concentration of hydrophobic amino acids in two segments of about 25 residues at the amino-terminal and carboxy-terminal ends. The polar residues are clustered in the middle of the polypeptide chain. The bound [14C]dicyclohexylcarbodiimide label is recovered exclusively at position 56, which is occupied by a glutamyl residue. The proteolipid from PS-3 exhibits homology to the corresponding ATPase subunit from mitochondria. The carbodiimide-reactive glutamyl residue occurs at the position as in the mitochondrial proteins.     

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---.     

The complete amino acid sequence of horse muscle acylphosphatase

The amino acid sequence of horse muscle acylphosphatase is given in the present paper. The carboxymethylated enzyme consists of a single polypeptide chain of 98 amino acid residues with an acetyl group blocking the NH2 terminus and a tyrosine at the COOH terminus. The calculated molecular weight of the native protein, a mixed disulfide with glutathione, is 11,365. The carboxymethylated protein was cleaved by cyanogen bromide. The three expected fragments were purified; moreover, an additional fragment, derived from a partial failure of cleavage at methionine-24, was purified and characterized. The structures of the cyanogen bromide fragments were established by subfragmentation with endopeptidases, and the sequences of the overlapping subfragments were determined. From the results, it was possible to order the peptides within the sequence and then to establish the complete primary structure of the enzyme.     

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.     

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen HLA-B7. 1. Isolation and amino acid composition of fragments and of tryptic and chymotryptic peptides

As a part of the overall strategy for determining the complete covalent structure of the papain-solubilized portion of the heavy chain of the human histocompatibility antigen HLA-B7, the protein was dissected into various fragments by a combination of partial acid hydrolysis and cyanogen bromide cleavage. After purification by chromatographic procedures, these fragments have been used as a source for tryptic and chymotryptic peptides. Thirty-three major tryptic and twenty-two major chymotryptic peptides were purified in nanomole amounts and their amino acid compositions determined. These peptides account for the whole extent of the polypeptide chain with the exception of the amino-terminal CNBr pentapeptide. They provide the basis for the formal alignment of the acid cleavage and cyanogen bromide fragments of the molecule as well as the source material for the elucidation of the primary structure of the HLA-B7 heavy chain.     

Isolation and characteristics of cyanogen bromide peptides of transducin alpha and beta subunits

The alpha- and beta-subunits of the GTP-binding protein (transducin) from cattle retina were cleaved with cyanogen bromide. 21 peptides covering 90-100% of the amino acid sequence of the alpha- and beta-subunits were isolated from the hydrolyzate. Cyanogen bromide peptides complete or partial amino acid sequence was determined, the results were compared with those by Numa and coworkers [1] and Lochrie et al. [2] at the primary structure of the transducin alpha-subunit deduced from the nucleotide sequence of the cDNA. The structure by Lochrie is shown to differ much from the true structure of the alpha-subunit; probably, the investigators isolated cDNA, corresponding to the gene for some GTP-binding protein homologous to transducin, but not to the gene for the transducin alpha-subunit. The Numa's structure also contains an error. The final primary structure of the transducin alpha-subunit is given. The protein polypeptide chain consists of 349 amino acid residues and has an acetylmethionine residue as the N-terminal residue.     

Complete amino acid sequence of a subunit from rapeseed high molecular weight protein

A subunit (Mr 15,600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.     

Purification, characterization, and primary structure of a novel cell wall hydrolytic amidase, CwhA, from Achromobacter lyticus

A novel bacteriolytic enzyme CwhA (cell wall hydrolytic amidase) was purified by ion exchange and gel-filtration chromatographies from a commercial bacteriolytic preparation from Achromobacter lyticus. CwhA exhibited optimal pH at 8.5 and lysed CHCl(3)-treated Escherichia coli more efficiently than Micrococcus luteus, Staphylococcus aureus, Enterococcus faecalis, and Pediococcus acidilactici. The enzyme was inhibited by 1,10-phenanthroline strongly and by EDTA to a lesser extent, suggesting that it is probably a metalloenzyme. Amino acid composition and mass spectrometric analyses for the CwhA-derived M. luteus muropeptides revealed that CwhA is N-acetylmuramoyl-L-alanine amidase [EC 3.5.1. 28]. The complete amino acid sequence of CwhA was established by a combination of Edman degradation and mass spectrometry for peptides obtained by Achromobacter protease I (API) digestion and cyanogen bromide (CNBr) cleavage. The enzyme consists of a single polypeptide chain of 177 amino acid residues with one disulfide bond, Cys114-Cys121. CwhA was found to be homologous to N-acetylmuramoyl-L-alanine amidase from bacteriophage T7 (BPT7). Its sequence identity with BPT7 is 35%, but the amino acid residues functioning as zinc ligands in BPT7 are absent in CwhA. These results suggest that CwhA is a new type of N-acetylmuramoyl-L-alanine amidase.     

Complete Amino Acid Sequence of a Subunit from Rapeseed Protein

A subunit of molecular weight 18300 has been separated and isolated from seeds of Brassica campestris L. This subunit was cleaved by using cyanogen bromide, trypsin, Staphylococcus aureus V8 protease and chymotrypsin; the fragments obtained from enzymatlc and chemical cleavages were separated and isolated by polyacrylamide gel electrophoresis and gel filtration. The amino acid analyses were carried out. The complete amino acid sequence of the subunit containing 172 amino acid residues has been established by manual Edman method.     

Studies on cytochrome c oxidase, IX. The primary structure of polypeptide VIa

The complete amino acid sequence of the cytoplasmic polypeptide VIa of cytochrome c oxidase from beef heart is described. The primary structure of this component of complex IV of the respiratory chain is elucidated by isolation and sequencing of overlapping glutamic acid, arginine, tryptophan and methionine fragments obtained by cleavage with Staphylococcus aureus protease, protease from submaxillaris glands of mice, 2-iodosylbenzoic acid and cyanogen bromide. The chain length of polypeptide VIa is 98 amino acids, the resulting molecular mass of 10670 Da. The hydrophilic protein does not contain a hydrophobic membrane penetrating sequence domain. Its function in the respiratory complex IV is unknown.     

Sequence homology analysis of proteins by chemical cleavages: using a mono and two dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The examination of possible sequence homology in proteins using SDS-PAGE systems after chemical cleavage is described. After SDS-PAGE, the establishment of amino acid compositions, the techniques of staining gel and five different methods of chemical cleavages (cyanogen bromide, BNPS-skatole, hydroxylamine, formic acid and nitrothiocyano benzoic acid) have been used for peptide mapping studies. Potential applications of this technique are discussed from both the biochemical and immunochemical point of view.     

Primary structure of the elongation factor G from Escherichia coli. IX. Structure of peptides generated by cyanogen bromide cleavage of the G-factor isolated on thiol-activated sepharose and of the products of the G-factor cleavage at Asp-Pro bonds. Complete primary structure

The amino acid sequence of cysteine- and cystine-containing peptides resulting from cleavage of the G-factor by cyanogen bromide has been determined. For structure analysis cyanogen bromide peptides were further degradated using trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease, or limited acid hydrolysis. The products of the G-factor cleavage at Asp-Pro bonds were also studied. The obtained data together with those published earlier permitted to establish the complete primary structure of the elongation factor G. The polypeptide chain consists of 701 amino acid residues and has molecular mass of 77321,46.     

Covalent structural analysis of yeast inorganic pyrophosphatase.

The present paper describes the amino acid sequence analysis of the internal and COOH-terminal cyanogen bromide fragments of yeast inorganic pyrophosphatase (Sterner, R., Noyes, C., and Heinrikson, R.L. (1974) Biochemistry 13, 91-99). This information coupled with that derived from earlier structural studies of the enzyme (Sterner, R., AND Heinrikson, R.L. (1975) Arch. Biochem. Biophys. 165, 693-703) provides the complete covalent structure of the pyrophosphatase subunit. The majority of the sequence data was derived from automated Edman degradation of the intact cyanogen bromide fragments and the large tryptic peptides obtained from citraconylated derivates in which cleavages were restricted to arginyl residues. The structural determination was completed by analysis of tryptic and chymotryptic peptides from the decitraconylated fragments. The monomer peptide chain contains 285 amino acid residues and the molecular weight calculated from the sequence analysis is 32,042.     

The primary structure of bovine cellular retinoic acid-binding protein

The complete amino acid sequence of bovine adrenal gland cellular retinoic acid-binding protein (CRABP) has been determined. The primary structure was established by analyses of cyanogen bromide fragments and peptides obtained by trypsin and Staphylococcus aureus protease digestions. The polypeptide chain of bovine CRABP comprises 136 amino acid residues. From partial sequence information, CRABP has been shown to be homologous to cellular retinol-binding protein, myelin protein P2, and the fatty acid-binding Z-protein. A comparison of the complete amino acid sequences of the members of this protein family, which also includes the rat intestinal fatty acid-binding protein, shows that CRABP is more similar to cellular retinol-binding protein and protein P2 than to the fatty acid-binding proteins. All five proteins are very similar in their NH2-terminal regions, suggesting that this part is important for a property common to the members of this protein family. This is the first report of a complete amino acid sequence of a CRABP.     

The primary structure of rabbit liver mitochondrial serine hydroxymethyltransferase

The complete amino acid sequence of mitochondrial serine hydroxymethyltransferase from rabbit liver was determined. The sequence was obtained from analysis of peptides isolated from chymotryptic, cyanogen bromide, and limited acid cleavages of the protein. The enzyme consists of four identical subunits, each of 475 residues, i.e. 8 residues shorter than the subunit of the corresponding cytosolic isoenzyme. The sequences of the two rabbit proteins are easily aligned, provided a gap of 5 residues near the amino terminus and a gap of 3 residues near the carboxyl terminus are included in the mitochondrial sequence. The overall degree of identity between the two isoenzymes is 61.9%, whereas the structural identity of each eukaryotic isoenzyme with the corresponding Escherichia coli enzyme is about 40%. The rabbit isoenzymes are about 70 residues longer than the E. coli enzyme, with one-half of these residues accounted for by insertions in both isoenzymes near their carboxyl terminus. Predictions of secondary structure and calculations of hydropathy profiles are also presented, suggesting an even more extensive degree of identity in the three-dimensional folding of the three proteins, in accord with the known similarity of their catalytic properties. Evidence was obtained for the existence of additional molecular forms of the mitochondrial protein, differing in the absence of some amino acid residues at the amino terminus of the polypeptide chain.     

Studies on cytochrome c oxidase, V. Polypeptide IV: alignment and amino acid sequences on cyanogen bromide fragments.

The isolation and chemical characterization of polypeptide IV from beef heart cytochrome oxidase is described. The protein is one of the main (stoichiometric) components of the oxidase. It is the largest polypeptide of the enzyme synthezised in the cytoplasm and has, as such, also been identified in enzyme preparations from yeast and Neurospora. A partial sequence, consisting of 105 amino acid residues which give a frame work of the covalent structure of the polypeptide is obtained from N- anc C-terminal sequencing and from the cyanogen bromide fragments of the chain. The isolation and sequencing of the fragments of this membrane protein are discussed.     

Use of thiol sorbents for obtaining human recombinant proinsulin

A convenient route of obtaining recombinant human proinsulin from the hybrid protein produced by the bacteria was developed. Chimeric protein was prepared by ultra- or gel-filtration, immobilized on thiol-support at cysteine residues and cleaved by cyanogen bromide to liberate purified proinsulin. Conditions of treatment hybrid protein with cyanogen bromide at methionine residues without affecting disulfide bonds between proinsulin and support are described. Proinsulin with correct disulfide bonds, directly obtained from polymer--attached polypeptide, followed was converted into insulin.