Isolation and amino acid sequence of the 'Rieske' iron sulfur protein of beef heart ubiquinol:cytochrome c reductase

The sequence of the 'Rieske' iron sulfur protein from the bc1 complex of beef heart mitochondria has been determined by solid phase Edman degradation of the whole protein and of various proteolytic fragments. The protein consists of 196 amino acid residues. The molecular mass of the apoprotein was calculated to be 21,536 Da, that of the holo-protein including the Fe2S2 cluster as 21,708 Da. The protein is mainly hydrophilic with a polarity index of 42.9% and 25% of charged residues. It contains a hydrophobic membrane anchor which is predicted to form a 'hairpin' structure. The iron sulfur cluster is bound near the C-terminus of the protein between a hydrophobic and a more amphipathic domain. This reflects the fact that the cluster is located near the outer surface of the inner mitochondrial membrane. A folding pattern describing all known features of the protein is proposed.     

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.     

Primary structure of the CFA1 fimbrial protein from human enterotoxigenic Escherichia coli strains

The complete amino acid sequence of the structural protein that constitutes the subunit of the CFA1 fimbria has been elucidated. The protein was fragmented by cyanogen bromide cleavage, and by enzymatic cleavage with trypsin. Secondary cleavage of the resulting peptides was performed with chymotrypsin, Staphylococcus aureus protease, and thermolysin. Sequential Edman degradation was performed manually. The CFA1 protein comprises 147 amino acid residues, with a molecular weight of 15058.     

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.     

Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi

Transferrin was isolated from plasma of the ascidian Halocynthia roretzi by ion-exchange chromatography. The molecular weight of the plasma transferrin was determined to be 52K by SDS-polyacrylamide gel electrophoresis and gel filtration. Ascidian plasma transferrin was found to bind one mole of iron ion per mole of protein. The reductive S-pyridylethylated transferrin was subjected to Edman degradation analysis for determination of the N-terminal amino acid sequence, and it was also subjected to proteolytic fragmentation to yield peptide fragments, whose amino acid sequences were determined by Edman degradation analysis. Using the above amino acid sequences, a cDNA clone (1880 base pairs) encoding a protein of 372 amino acids containing a signal peptide of 21 amino acids was isolated from an H. roretzi hepatopancreas cDNA library. The reduced amino acid sequence contains the same sequences of the peptide fragments. A comparison of the amino acid sequence of ascidian transferrin with those of other members of the transferrin family revealed that the ascidian transferrin is composed of only the N-terminal lobe of two-lobed vertebrate transferrins. Thus, a one-lobed transferrin is present in the ascidian H. roretzi.     

Ribonuclease Fl1 from Fusarium lateriticum. Isolation, substrate specificity and amino acid sequence

Extracellular RNase Fl1 has been purified from the culture filtrate of Fusarium lateritium. The enzyme has been obtained in the electrophoretically homogeneous state with the yield about 90% and 300 fdd degree of purification. RNase Fl1 is a guanyl specific enzyme (EC 3.1.27.3) with the specific activity on RNA 1420 units/mg of protein. The total primary structure of the RNase has been determined by the automated Edman degradation of two non-fractionated peptide hydrolysates produced by trypsin and Staphylococcus aureus protease and of the hydroxylamine cleavage products of the protein. It was shown that hydroxylamine converts the RNase Fl1 N-terminal residue, pyroglutamic acid, into the hydroxyamic acid derivative sensitive to Edman degradation. RNase Fl1 consists of 105 amino acid residues (Mr 10,852) and is a structural homologue of the Fus. moniliforme RNase F1, differing from the latter by 15 amino acid substitutions outside the enzyme active site.     

The amino acid sequence of human beta-microseminoprotein

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.     

The amino acid sequences of two 13 kDa polypeptides and partial amino acid sequence of 30 kDa polypeptide of complex I from bovine heart mitochondria: possible location of iron-sulfur clusters

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated system in the respiratory chain. It consists of many subunits, some of which hold iron-sulfur clusters, but structural information is still limited. The amino acid sequences of two 13 kDa polypeptides, 13 kDa-A and 13 kDa-B polypeptides, of iron-sulfur protein fraction (IP) of bovine heart mitochondrial complex I were determined by a combination of protease digestion, Edman degradation, and carboxypeptidase digestion. The 13 kDa-A polypeptide was composed of 96 amino acids with a molecular weight of 10,536. The 13 kDa-B polypeptide consisted of 114 amino acids and had an acetylated amino terminus. The molecular weight of this protein was calculated to be 13,130 including the acetyl group. These proteins had no obvious sequence similarity to other known proteins. The partial amino acid sequence of 30 kDa-B polypeptide of IP was also determined to reveal a characteristic arrangement of cysteine residues that could be involved in iron-sulfur cluster formation.     

Isolation and amino acid sequence of the 8 kDa DCCD-binding protein of beef heart ubiquinol:cytochrome c reductase

The 8 kDa protein of beef heart ubiquinol:cytochrome c reductase was detected by means of a new SDS-PAGE [(1985) FEBS Lett. 190, 89-94] system and was isolated by a series of chromatographic steps involving dissociation of the complex by salt treatment. The amino acid sequence was determined by solid-phase Edman degradation of both the N-terminal part of the whole protein and proteolytic cleavage fragments of the protein. The protein consists of 78 amino acid residues: its Mr was calculated to be 7998. Structure predictions have been made from average and sided hydropathy profiles. The suggested structure encompasses an alpha-helix and a beta-strand, the latter comprising a glutamic acid residue situated in a relatively hydrophobic neighbourhood. This residue may be responsible for the fact that the 8 kDa protein is the first subunit of the whole reductase (consisting of 11 subunits) to be labelled by DCCD when the reductase is in free form or inlaid in phospholipid vesicles.     

Amino acid sequence of p15 from avian myeloblastosis virus complex

The complete amino acid sequence of the p15 gag protein from avian myeloblastosis virus (AMV) complex has been determined by sequential Edman degradation of the intact molecule and of peptide fragments generated by limited tryptic cleavage, cleavage with staphylococcal protease, and cyanogen bromide cleavage. AMV p15 is a single-chain protein containing 124 amino acids. The charged amino acids tend to be clustered in the primary structure. p15 contains a single cysteine at position 113 which may be essential for the p15 associated proteolytic activity. However, p15 shows no appreciable sequence homology with papain or other classical thiol proteases.     

Primary structure of human lymphotoxin derived from 1788 lymphoblastoid cell line

The amino acid sequence of human lymphotoxin derived from a 1788 lymphoblastoid cell line was determined. Peptide fragments obtained by trypsin, lysine-C peptidase, cyanogen bromide, and acetic acid cleavage of the intact protein were purified by reverse-phase high performance liquid chromatography and analyzed by amino acid composition and by automated Edman degradation. The protein is 171 amino acids long with a molecular weight of 18,664. It contains one asparagine-linked glycosylation site and lacks cysteine. The salient features of the amino acid sequence of lymphotoxin are described.     

Complete amino acid sequence of amelogenin in developing bovine enamel

Pure amelogenin protein in developing bovine incisor enamel was isolated and its primary structure was investigated by sequencing the peptides obtained after clostripain and chymotrypsin digestions and CNBr degradation with an automated Edman sequencer. The enamel protein was found to be composed of 170 amino acid residues with one phosphate having a molecular weight of 19,350 and its complete amino acid sequence was elucidated. This protein has no sequence homology with any other tissue or secretory protein of known structure.     

Single amino acid substitution between SHV-1 beta-lactamase and cefotaxime-hydrolyzing SHV-2 enzyme

SHV-2 beta-lactamase was purified from an overproducing variant of a clinical isolate of Escherichia coli resistant to cefotaxime. Pure protein was digested by trypsin and Lys-C endoproteinase. Proteolytic peptides, isolated by reverse-phase HPLC, were submitted to manual Edman degradation and aligned by homology with the sequence of SHV-1 beta-lactamase. A putative amino acid sequence was deduced. Structural comparison revealed that SHV-2 differed from SHV-1 by only one amino acid, Gly----Ser, at position 213 of the mature protein.     

Cross-linking of nitrogenase components. Structure and activity of the covalent complex

The nitrogenase complex from Azotobacter vinelandii is composed of the MoFe protein (Av1), an alpha 2 beta 2 tetramer, and the Fe protein (Av2), a gamma 2 dimer. During turnover of the enzyme, electrons are transferred from Av2 to Av1 in parallel with the hydrolysis of MgATP. Using the cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, we have identified some of the properties of the complex between the two components. The cross-linking reaction was highly specific yielding a single apparent Mr = 97,000 protein. The amount of cross-linked product was essentially independent of whether MgATP or MgADP were in the reaction. Also, the amount was maximum at high ratios of Av2 to Av1. The Mr = 97,000 protein was characterized by amino acid analysis and Edman degradation and was found to be consistent with a 1:1 complex of an Av2 gamma subunit and an Av1 beta subunit (the amino terminal serine subunit). The complex was no longer active in the nitrogenase reaction which supports, but does not prove, the requirement for dissociation of the complex after each electron transferred. Nitrogenase activity and cross-linking were inhibited in an identical way by NaCl, which suggests that electrostatic forces are critical to the formation of the electron transfer complex.     

Isolation and characterization of okadaic acid binding proteins from the marine sponge Halichondria okadai

Okadaic acid, first isolated from the marine sponge Halichondria okadai, is a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A, respectively). Photoaffinity labeling experiments previously performed with biotinylated photoreactive okadaic acid revealed the presence of okadaic acid binding protein (OABP) in the crude extract of H. okadai. In this article, OABP1 and OABP2 were purified from H. okadai as guided by the binding affinity of [27-3H]okadaic acid. OABP1 has an approximate molecular mass of 37 kDa in SDS-PAGE analysis. Edman degradation followed by molecular cloning and sequencing identified OABP1 as being 88% identical to the rabbit PP2Abeta catalytic subunit. On the other hand, HPLC analysis revealed that OABP2 consists of three 22 kDa proteins (OABP2.1, OABP2.2, and OABP2.3). Electrospray ionization mass spectrometry indicated that OABP2.1 and OABP2.2 form a complex with okadaic acid. The complete amino acid sequence of OABP2, determined by Edman degradation and molecular cloning, showed that OABP2.1 is 96% identical to OABP2.2 and 66% identical to OABP2.3, while being very slightly homologous to any protein phosphatases known to date. OABP2 did not exhibit phosphatase activity, though it bound to okadaic acid with a Kd of 0.97 nM. Furthermore, OABP2 was not detected in the sponge Halichondria japonica or the dinoflagellate Prorocentrum lima. We thus speculated that OABP2 might be involved in detoxifying okadaic acid.     

Detection of DBD-carbamoyl amino acids in amino acid sequence and D/L configuration determination of peptides with fluorogenic Edman reagent 7-[(N,N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate.

A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals.     

Complete amino acid sequence of yeast thioltransferase (glutaredoxin)

The amino acid sequence of a thioltransferase isolated from Saccharomyces cerevisiae was determined. The protein was cleaved by trypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. The peptides generated were purified by reverse phase HPLC. Sequencing of intact protein and its fragments were achieved by automated Edman degradation. The protein contains 106 amino acid residues with two cysteines. Yeast thioltransferase showed 51% structural similarity to pig liver thioltransferase and 34% to E. coli glutaredoxin.     

Partial amino acid sequence of human thyroxine-binding globulin. Further evidence for a single polypeptide chain.

The NH₂-terminal amino acid of highly purified thyroxine-binding globulin has been identified by dansyl chloride, cyanate and Edman degradation methods. All three gave alanine as the only amino terminal residue. Carbamylation and Edman degradation of the denatured protein yielded 0.86 and 0.98 – 1.05 mole of alanine per mole of protein, respectively. These data further indicate that thyroxine-binding globulin is composed of a single polypeptide chain. Automated Edman degradation gave the partial sequence as: Ala-Ser-Pro-Glu-Gly-Lys-Val-Thr-Ala-Asp-Ser-Ser-Ser-Gln-(Pro)-X-Ala-(Ser)-Leu-Tyr- A computer search revealed no homology of the NH₂-terminal segment of thyroxine-binding globulin with human prealbumin. The NH₂-terminal portion of prealbumin contains part of the thyroxine binding site.     

Complete Amino Acid Sequence of the Sweet Protein Monellin

The sweet protein monellin consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of these chains. The complete amino acid sequence of monellin was determined by a combination of FAB- and ESI-mass spectrometry, and by automatic Edman degradation.     

Hepatocyte plasma membrane ECTO-ATPase (pp120/HA4) is a substrate for tyrosine kinase activity of the insulin receptor

pp120/HA4, a membrane protein found in hepatocyte plasma membranes and a substrate for the insulin receptor tyrosine kinase, was purified to homogeneity, subjected to partial proteolysis, and peptides were sequenced by Edman degradation. Six amino acid sequences were obtained, and they matched the deduced amino acid sequences of six regions of a hepatocyte membrane protein called ecto-ATPase.