Purification and primary structure of cytochrome c-552 from the cyanobacterium, Synechococcus PCC 6312.

Cytochrome c-552 (soluble 'cytochrome f') from the unicellular cyanobacterium Synechococcus PCC 6312 (ATCC 27167) was purified and the primary structure determined. The proposed sequence consists of one polypeptide chain of 87 residues. The sequence was determined by a combination of chemical and enzymatic cleavage, manual and automatic sequencing and mass spectroscopy. This is the first amino acid sequence of this cytochrome from a unicellular cyanobacterium to be determined in a study of the variation in primary structure between phylogenetically distant cyanobacteria. The sequence is compared to the primary structures of the cytochrome from filamentous cyanobacteria and from eukaryotic algae. The significance of these sequence comparisons to the current hypotheses concerning the origin of eukaryotic cells and their chloroplasts is discussed.     

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.     

Isolation, characterization, and amino acid sequence of a ubiquitin-like protein from insect eggs

A protein with a primary structure identical to that of human and bovine ubiquitin has been purified from insect eggs. The isolation, secondary structure, and amino acid sequence of this ubiquitin-like protein are reported. The sequence was determined by automatic Edman degradation of the intact molecule as well as by the manual sequence analysis of the enzymatic cleavage products. The polypeptide has 74 amino acid residues and internal homology regions. Interactions of the protein with peptides results in protective effects against proteolysis. This paper reports for the first time the presence of the ubiquitin molecule in invertebrates.     

Studies on cytochrome c oxidase, XII. Isolation and primary structure of polypeptide VIb from bovine heart

The isolation and complete sequence analysis of the cytoplasmically synthesized polypeptide VIb from bovine heart cytochrome c oxidase is described. The protein is a stoichiometric constituent of the respiratory complex IV. Its primary structure is deduced from N-terminal sequencing and overlapping peptides obtained from tryptic cleavage and specific cleavage at arginyl and tryptophyl peptide bonds. The polypeptide chain consists of 84 amino acids from which a Mr of 9419 is derived. It has a relatively high content of histidine and proline and contains a single cysteine. A hydrophobic sequence of 20 amino acids points to a membrane-penetrating structure similar to that found in polypeptides I, II, III, IV and VIIIa, VIIIb, VIIIc of the bovine oxidase. The sequence of VIb is tissue-specific, it contributes to the formation of nuclear coded isoenzymes of cytochrome c oxidase. The protein thus may be involved in a tissue-specific regulation of cellular respiration.     

The complete amino-acid sequence of the low-spin class II cytochrome c-556 from Agrobacterium tumefaciens strain B2a

The amino acid sequence of the soluble monohaem cytochrome c-556 from Agrobacterium tumefaciens, strain B2a, has been determined. The sequence was derived from peptides obtained by digestion of the apoprotein with trypsin and chymotrypsin, and by subdigestion of some of the peptides with Staphylococcus aureus protease and thermolysin. Sequencing of the various peptides was achieved by a combination of manual dansyl-Edman degradation and automatic liquid-phase sequence analysis. The main characteristic of this cytochrome is that the haem-binding sequence Cys-Xaa-Yaa-Cys-His occurs in the C-terminal region of the polypeptide chain, the first cysteine being located 11 residues ahead of the C-terminal lysine-122. As such, the protein belongs to cytochrome c sequence class II (sensu Ambler). The cytochrome c-556 is the first example known of a class II cytochrome of the low-spin type isolated from an obligate aerobic organism.     

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.     

Carboxy terminal fragment of human alpha-1-antitrypsin from hydroxylamine cleavage: homology with antithrombin III.

Human α-1-antitrypsin (AT) was reacted with hydroxylamine at pH 9.0 giving cleavage at an Asn-Gly bond. A fragment of molecular weight 8,500 was released and this was isolated and sequenced. The fragment had the same carboxy terminal amino acid sequence as intact AT. The 80 residue polypeptide contained the Z variant mutation site and a portion of sequence identical to that found by others for the reactive site, inferring the presence in AT of two active sites. This sequence combined with prviously published work gives a continuous sequence of 152 amino acid residues from the carboxy terminal end of the AT molecule, including the mutation site of the S variant. The sequence shows strong homology with human antithrombin III.     

Amino-acid sequence of the smallest protein of the cytochrome c1 subcomplex from beef heart mitochondria

A cytochrome c1 subcomplex was obtained from purified complex III. The subcomplex consists of three protein subunits, present at an equimolar stoichiometry. The primary structures of two of these proteins, the heme-carrying cytochrome c1 and a protein with a molecular mass of 9175 Da, have been published by Wakabayashi et al. The covalent structure of the smallest subunit is presented in this paper. This protein consists of 62 amino acid residues; its molecular mass was calculated to be 7189 Da. The sequence was determined by complete solid-phase Edman degradation of the uncleaved polypeptide and was confirmed by sequencing the C-terminal fragment resulting from cleavage of a single tryptophyl bond.     

Primary structure of hemoglobin from monitor lizard (Varanus exanthematicus albigularis--Squamata)

The primary structure of the major hemoglobin component from the Monitor Lizard Varanus exanthematicus albigularis is presented. The polypeptide subunits were separated by reversed-phase high-performance liquid chromatography on Nucleosil C-4 column. The amino-acid sequence was established by automatic Edman degradation of the native polypeptide and its tryptic and hydrolytic cleavage products in a spinning cup sequencer. The structural data are discussed with reference to other reptiles.     

Action of neocarzinostatin on cell nuclei: release of specific chromatin

We report the first complete sequence of a P450 monoxygenase cytochrome. The P450_CAM from $\text{Pseudomonas}\text{putida}$ is a single polypeptide of 412 residues as determined from the isolated tryptic, clostripain, CNBr, and mild acid cleavage fragments. Significant molecular features, including secondary structure, are discussed.     

Chromatographic and protein chemical analysis of the ubiquinol-cytochrome c2 oxidoreductase isolated from Rhodobacter sphaeroides

The ubiquinol-cytochrome c2 oxidoreductase (cytochrome bc1 complex) purified from chromatophores of Rhodobacter sphaeroides consists of four polypeptide subunits corresponding to cytochrome b, c1, and the Rieske iron-sulfur protein, as well as a 14-kDa polypeptide of unknown function, respectively. In contrast, the complex isolated from Rhodospirillum rubrum by the same procedure lacked a polypeptide corresponding to the 14-kDa subunit. Gel-permeation chromatography of the R. sphaeroides cytochrome bc1 complex in the presence of 200 mM NaCl removed the iron-sulfur protein, while the 14-kDa polypeptide remained tightly bound to the cytochromes; this is consistent with the possibility that the latter protein is an authentic component of the complex rather than an artifact of the isolation procedure. The individual polypeptides of the R. sphaeroides complex were purified to homogeneity by gel-permeation chromatography in the presence of 50% aqueous formic acid and their amino acid compositions determined. The 14-kDa polypeptide was found to be rich in charged and polar residues. Edman degradation analysis indicated that its N terminus is blocked and not rendered accessible by de-blocking procedures. Cyanogen bromide cleavage gave rise to a blocked N-terminal fragment as well as a C-terminal peptide comprising more than one-third of the protein. Gas-phase sequence analysis of this peptide established a sequence of 48 residues and identified a putative trans-membrane segment near the C terminus. The blocked N-terminal fragment was cleaved at tryptophan with BNPS-skatole. The resulting peptides, together with tryptic fragments derived from the intact protein, yielded additional sequence information; however, none of the sequences exhibited significant homologies to any known proteins. Tryptic fragments were also used to generate sequence information for cytochrome c1.     

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.     

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site.     

Primary structure of the membrane-binding segment of rabbit cytochrome b5.

The primary structure of the membrane-binding segment of rabbit cytochrome b5 has been determined. This segment, prepared by trypsin digestion of the intact protein, consists of 43 amino acid residues and corresponds to the COOH-terminal end (residues 91-133) of the parent molecule. Deduction of the primary structure was based on automated sequence analysis of the whole segment as well as manual and dansyl-Edman degradations of peptide fragments produced by CNBr cleavage and partial acid hydrolysis. The sequence obtained is: Leu-Ser-Lys-Pro-Met-Glu-Thr-Leu-Ile-Thr-Thr-Val-Asn-Ser-Asn-Ser-Ser-Trp-Trp-Thr-Asn-Trp-Val-Ile-Pro-Ala-Ile-Ser-Ala-Leu-Ile-Val-Ala-Leu-Met-Tyr-Arg-Leu-Tyr-Met-Ala-Asp-Asp. This sequence is 63 to 81% homologous with respect to those determined for the membrane-binding segments of equine, porcine and bovine cytochrome b5. The interaction of this segment with phospholipid bilayer membranes is discussed, and a prediction of its secondary structure is also presented.     

The primary structure of the cytotoxin restrictocin

The complete amino acid sequence of the single polypeptide chain of cytotoxin restrictocin has been determined. Its structure was established by automated Edman degradation of the intact molecule reduced and [14C]carboxymethylated and of fragments obtained by chemical cleavage of the protein with cyanogen bromide and BNPS-skatole and by enzymatic cleavage of the polypeptide chain with trypsin. The molecule consists of 149 amino acid residues with a calculated relative molecular mass of 16836. The protein presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and the other one formed by cysteine residues at positions 75 and 131. The amino acid sequence of restrictocin shows a high degree of homology (86%) with that of the cytotoxin named alpha-sarcin.     

Cytochrome c from Schizosaccharomyces pombe. 2. Amino-acid sequence.

The amino acid sequence of Schizosaccharomyces pombe cytochrome c has been established by automatic degradation of the protein and by manual degradation of fragments obtained by cyanogen bromide cleavage and chymotryptic digestion. The chymotryptic peptides were aligned by homology with other known cytochrome c sequences. The protein is 108 residues long, with a four-residue amino-terminal tail. It has only one methionine residue and differs from other fungal cytochromes c in lacking the one-residue deletion at the C-terminal end. After a cyanogen bromide step, an unexpected cleavage of the peptide chain before a cysteine residue was observed. This is ascribed to formation of a dehydroalanyl residue during an incomplete S-carboxymethylation of the apoprotein, and subsequent cleavage under acidic conditions. Experimental evidence is presented in favour of the proposed mechanisms.     

Complete amino acid sequence of an allosteric enzyme, T2 bacteriophage deoxycytidylate deaminase

The amino acid sequence of deoxycytidylate deaminase isolated from T2 phage-infected Escherichia coli has been determined. The enzyme is a hexamer, consisting of identical polypeptide subunits, each composed of 188 amino acids with a calculated Mr = 20,560. The primary structure was established by automatic Edman degradation of the intact carboxymethylated protein and of peptides derived from the protein by cleavage with cyanogen bromide, trypsin, chymotrypsin, the Staphylococcus aureus V8 protease, and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Knowledge of the primary structure of deoxycytidylate deaminase should aid in determining the allosteric binding site of the negative effector, dTTP, recently reported (Maley, F., and Maley, G.F. (1982) J. Biol. Chem. 257, 11876-11878), and eventually that of the enzyme's positive regulator, dCTP, as well as its substrate. The deaminase has been crystallized through the use of polyethylene glycol; a scanning electron micrograph is presented.     

Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus.

The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.     

Amino acid sequence of the sex steroid binding protein of human blood plasma

The amino acid sequence of the sex steroid binding protein (SBP) from human plasma has been determined. The SBP subunit consists of a 373-residue polypeptide chain containing two disulfide bonds and three oligosaccharide chains. The sequence was solved primarily by analysis of peptides derived by cleavage at either lysyl or methionyl residues. In our preparations, approximately half of the protein molecules have the amino-terminal sequence Arg-Pro-Val-Leu-Pro; the other half lack Arg-Pro and begin with the valine. Preparations of Hammond et al. [Hammond, G. L., Robinson, P. A., Sugino, H., Ward, D. N., & Finne, J. (1986) J. Steroid Biochem. 24, 815] have an additional leucine at the amino terminus, making a total of 373 residues in the chain. Oligosaccharide chains are placed at Thr-7 and at Asn residues 351 and 367. The two disulfide bonds connect Cys-164 to Cys-188 and Cys-333 to Cys-361. The reported heterogeneity of preparations of the molecule may result in part from the amino-terminal microheterogeneity, in part from variations in the oligosaccharide moieties, and possibly in part from rearrangements involving cyclic imide formation in two Asn-Gly sequences. Certain hydrophobic segments are suggested as possible components of the steroid-binding sites. The protein shows no homology either with the cDNA-derived sequences of the estrogen and glucocorticoid receptors found by others to be homologous with each other or with any other protein sequence in the 1986 data base.