The complete amino acid sequence of the pediocin-like antimicrobial peptide leucocin C

The pediocin-like antimicrobial peptide leucocin C produced by a strain of Leuconostoc mesenteroides has been purified using a recently developed rapid two-step procedure. The complete and corrected amino acid sequence of the peptide has been determined by Edman degradation of the intact peptide and a C-terminal fragment generated by cleavage with Asp-N endoprotease. Leucocin C contained 43 residues with the following sequence: KNYGNGVHCTKKGCSVDWGYAWTNIANNSVMNGLTGGNAGWHN. The molecular weight of leucocin C as determined by mass spectrometry was 4595, which is consistent with the theoretical molecular weight of 4596 calculated from the sequence. Moreover, the molecular weights of the two fragments generated by cleavage with Asp-N were also consistent with the determined sequence.     

Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin

A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.     

Primary structure of Paim I, an alpha-amylase inhibitor from Streptomyces corchorushii, determined by the combination of Edman degradation and fast atom bombardment mass spectrometry

Paim I, a protein alpha-amylase inhibitor, inhibits animal alpha-amylases from pig, dog, cow, horse, etc. but has no activity against human salivary and pancreatic amylases. The primary structure of Paim I has been determined by Edman degradation and fast atom bombardment mass spectrometry (FABMS). This protein is a single-chain polypeptide of 73 amino acid residues with a calculated molecular weight from the sequence data of 7415.3 (monoisotopic molecular weight) and 7420.2 (average molecular weight). The sequencing strategy chosen for Paim I consists of four steps. First, the accurate molecular weights of the intact and tetra-S-carboxymethylated Paim I are determined by fast atom bombardment mass spectrometry. Second, the primary fragments generated by Staphylococcus aureus V8 protease are isolated by reversed-phase high-performance liquid chromatography. The molecular weights of these subpeptides are determined by FABMS. The peptides that must be sequenced are selected by the molecular weights of these subpeptides and the tetra-S-carboxymethylated Paim I. Third, these subpeptides and the whole protein are sequenced by automated Edman degradation. Finally, the primary structure of tetra-S-carboxymethylated Paim I is confirmed by the combination of tryptic, chymotryptic, and S. aureus V8 protease digestion and FABMS. The sequence of Paim I is compared with those of Haim II, Hoe-467A, Z-2685, and AI-3688 because they have different alpha-amylase inhibition spectra against mammalian alpha-amylases but belong to a family of related proteins.     

Amino acid sequence of bovine gamma E (IVa) lens crystallin.

When electrospray ionization mass spectrometry (ESMS) was used to analyze purified bovine gamma E (gamma IVa)-crystallin, it yielded a relative molecular mass (M(r)) of 20.955 +/- 5. This mass is significantly different from that calculated from the published sequence (M(r) 20.894) (White HE et al., 1989, J Mol Biol 207:217-235). Further, ES-MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203-208). The entire protein sequence of gamma E crystallin has therefore been studied via a combination of ES-MS, ES-MS/MS, and Edman amino acid sequencing. The corrected sequence gives an M(r) of 20.955.3, which matches that obtained by ES-MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine gamma-crystallin by R. Hay (pers. comm.).     

Characteristics and antifungal activity of a chitin binding protein from Ginkgo biloba

An antifungal peptide from leaves of Ginkgo biloba, designated GAFP, has been isolated. Its molecular mass of 4244.0 Da was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation. GAFP exhibited antifungal activity towards Pellicularia sasakii Ito, Alternaria alternata (Fries) Keissler, Fusarium graminearum Schw. and Fusarium moniliforme. Its activities differed among various fungi. GAFP could also cause increased hyphal membrane permeabilization and a rapid alkalization of the medium when applied at 100 microgram/ml to Pellicularia sasakii Ito hyphae. The amino acid sequence of GAFP shows characteristics of the cysteine/glycine-rich chitin binding domain of many chitin binding proteins. The cysteine residues are well conserved.     

Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry.

We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.     

A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning

An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.     

Sequence determination of lychnin, a type 1 ribosome-inactivating protein from Lychnis chalcedonica seeds

The complete amino acid sequence of lychnin, a type 1 ribosome-inactivating protein (RIP) isolated from Lychnis chalcedonica seeds, has been determined by automated Edman degradation and ESI-QTOF mass spectrometry. Lychnin consists of 234 amino acid residues with a molecular mass of 26 131.14 Da. All amino acid residues involved in the formation of the RIP active site (Tyr69, Tyr119, Glu170, Arg173 and Trp203) are fully conserved. Furthermore, a fast MALDI-TOF experiment showed that two out of three cysteinyl residues (Cys32 and Cys115) form a disulfide bridge, while Cys214 is in the thiol form, which makes it suitable for linking carrier molecules to generate immunotoxins and other conjugates.     

Amino acid sequence of rubber elongation factor protein associated with rubber particles in Hevea latex

The amino acid sequence of rubber elongation factor, a recently discovered protein tightly bound to rubber particles isolated from the commercial rubber tree Hevea brasiliensis, is presented. The role of this protein in rubber elongation and its interaction with prenyltransferase and rubber particles have been discussed in the preceding paper in this series (Dennis, M. S., and Light, D. R. (1989) J. Biol. Chem. 264, 18608-18617). Trypsin, Staphylococcus protease, chymotrypsin, acetic acid, and hydroxylamine cleavage were used to generate peptide fragments that were isolated by reverse phase high pressure liquid chromatography and analyzed by amino acid composition and automated Edman degradation. Each digest contained one blocked peptide identified as the amino terminus. The blocked amino-terminal peptide from the tryptic digest was analyzed by amino acid composition, fast atom bombardment mass spectrometry (molecular ion 1659.9), subdigested with Staphylococcus protease for partial sequence analysis, and finally deblocked with bovine liver acyl-peptide hydrolase removing an acetylalanine to allow analysis by Edman degradation. Rubber elongation factor is 137 amino acids long, has a molecular mass of 14,600 daltons, and lacks four amino acids: cysteine, methionine, histidine, and tryptophan. The NH2 terminus is highly charged and contains only acidic residues (5 of the first 12 amino acids). The first four amino acids are highly represented in other known NH2-terminally acetylated proteins. Comparison of the sequence of rubber elongation factor with other known sequences does not reveal significant sequence similarities that would suggest an evolutionary relationship.     

Isolation and characterization of a cell-associated protein of Bacillus pumilus PH-01

A cell-associated protein released from Bacillus pumilus PH-01 showed an affinity for some dioxins, like 1,2,3,4-tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4-tetrachlorodibenzofuran (TCDF), and the concentration of the protein increased when B. pumilus PH-01 was boiled in minimal salts medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-mass spectrometry revealed that the boiled culture supernatant contained a major protein with a molecular mass of 5,313.4 Da. The adsorption behavior of the protein for 1,2,3,4-TCDD and 1,2,3,4-TCDF was examined by digesting it with proteinase K and trypsin, showing that the proteolyzed protein lost the ability to adsorb the compounds. The amino acid sequence of the protein was determined by automated Edman degradation and tandem mass spectrometry. A search of the protein databases showed no existence of proteins with an homologous sequence.     

Sequence determination of three cuticular proteins and isoforms from the migratory locust,Locusta migratoria,using a combination of Edman degradation and mass spectrometric techniques

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.     

Hainantoxin-Ⅱ的分离纯化及其结构与功能分析(英文)

从分布于我国海南省的海南捕鸟蛛(Haplopelma hainanum)毒素中,利用阳离子交换色谱和反相高效液相色谱分离得到一种多肽神经毒素,命名为Hainantoxin-Ⅱ(HnTx-Ⅱ)。MALDI-TOF质谱分析表明该多肽毒素相对分子质量为4253.135,其氨基酸序列经Edman降解测序为LFECS VSCEI EKEGN KDCKK KKCKG GWKCK FNMCV KV,其中的6个Cys形成3对二硫键。同源性搜索表明,HnTx-Ⅱ与Huwentoxin-Ⅱ(HwTx-Ⅱ)的同源性高达91%,仅有3个氨基酸残基不同。HwTx-Ⅱ为从虎纹捕鸟蛛中提取的杀虫肽,该多肽具有独特的结构模体。活性分析表明HnTx-Ⅱ与HwTx-Ⅱ具有相似的生物学活性。经腹腔注射HnTx-Ⅱ,美洲蜚蠊可被麻痹,其ED50为16μg/g;而当剂量增加到60μg/g时,可立即杀死美洲蜚蠊,其杀死昆虫活性明显强于HwTx-Ⅱ。此外,HnTx-Ⅱ经脑室注射可杀死小鼠,其LD50为1.41μg/g,该活性却明显低于HwTx-Ⅱ。这两种多肽毒素的结构与功能的差异为进一步阐明多肽毒素的结构与功能之间的关系提供良好的研究模型。     

Apolipophorin-III-like protein expressed in the antenna of the red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae)

Antennal proteins of the male fire ant (Solenopsis invicta) were analyzed by two-dimensional gel electrophoresis, with the objective of identifying pheromone-binding proteins, which have not previously been found in ant antennae. The major low-molecular weight protein found in the male fire ant antenna was subjected to Edman degradation to determine the N-terminal amino acid sequence. Degenerate PCR primers based on this sequence were used to obtain a cDNA sequence corresponding to the full-length protein sequence. In-gel trypsin digestion followed by MALDI-TOF mass spectrometry and HPLC-ESI/MS/MS demonstrated that the protein gel spot contained only the protein corresponding to the cDNA sequence obtained by PCR. The sequence is similar to apolipophorin-III, an exchangeable lipid-binding protein. Fire ant apolipophorin-III is expressed in the antenna as well as the head, thorax and abdomen.     

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.     

Isolation and characterization of a new bacteriocin,termed enterocin M,produced by environmental isolate <Emphasis Type="Italic">Enterococcus faecium</Emphasis> AL41

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH₄)₂SO₄ precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4 628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4 701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.     

Isolation and partial characterization of a bacteriocin produced by Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product

Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product was found to produce a novel bacteriocin that is active against a wide range of gram‐positive and gram‐negative bacteria. Production of the bacteriocin BM‐1 started early in the exponential phase and its maximum activity (5120 AU/mL) was recorded early during the stationary phase (16 hr). Bacteriocin BM‐1 is sensitive to proteolytic enzymes but stable in the pH range of 2.0–10.0 and heat‐resistant (15 min at 121°C). This bacteriocin was purified through pH‐mediated cell adsorption–desorption and cation‐exchange chromatography on an SP Sepharose Fast Flow column. The molecular weight of the purified bacteriocin BM‐1 was determined to be 4638.142 Da by electrospray ionization Fourier transform mass spectrometry. Furthermore, the N‐terminal amino acid sequence was obtained through automated Edman degradation and found to comprise the following 15 amino acid residues: H₂N‐Lys‐Tyr‐Tyr‐Gly‐Asn‐Gly‐Val‐Tyr‐Val‐Gly‐Lys‐His‐Ser‐Cys‐Ser. Comparison of this sequence with that of other bacteriocins revealed that bacteriocin BM‐1 contains the consensus YGNGV amino acid motif near the N‐terminus. Based on its physicochemical characteristics, molecular weight, and N‐terminal amino acid sequence, plantaricin BM‐1 is a novel class IIa bacteriocin.     

Purification, characterization, and amino acid sequence of the mating pheromone Er-10 of the ciliate Euplotes raikovi

The mating pheromone Er-10 from mat-10 homozygous Euplotes raikovi was purified by a three-step purification procedure with an overall yield of 62%. It was identified as a protein of molecular weight 8000 having an isoelectic point of 3.9. Its complete primary structure was determined by automated Edman degradation of the whole protein after performic acid oxidation and of peptides generated by cyanogen bromide and Staphylococcus aureus V8 protease. The proposed sequence is Asp1-Leu-Cys-Glu-Gln-Ser-Ala-Leu-Gln-Cys10-Asn-Glu-Gln-Gly-Cys-His -Asn-Phe-Cys- Ser20-Pro-Glu-Asp-Lys-Pro-Gly-Cys-Leu-Gly-Met30-Val-Trp-Asn- Pro-Glu-Leu-Cys- Pro38. The calculated molecular weight of 4191.7, which is in good agreement with the value of m/z 4190.7 obtained by fission fragment ionization mass spectrometry, suggests that the native structure is a dimer with three intrachain disulfide bonds in each subunit. The amino acid sequence is 43% identical with that of the E. raikovi mating pheromone Er-1, with the identities concentrated in the amino-terminal half. The half-cystine locations are conserved, but Er-10 is two residues shorter than Er-1. Prediction of the secondary structure suggests that Er-10 may also contain a helical structure at the amino terminus. These results indicate that the mating pheromones of E. raikovi form a homologous family.     

Sequence determination of three cuticular proteins and isoforms from the migratory locust,Locusta migratoria,using a combination of Edman degradation and mass spectrometric techniques

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.     

Amino acid sequence of human plasma apolipoprotein C-III from normolipidemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-III (apoC-III) isolated from normal subjects is described. ApoC-III is a linear polypeptide chain of 79 amino acids. Tryptic digestion of intact apoC-III produced 5 major peptides, while tryptic digestion of the citraconylated protein yielded two peptides. The complete amino acid sequence of apoC-III was determined by the automated Edman degradation of the intact protein as well as the various tryptic peptides. Phenylthiohydantoin amino acids were identified by high-performance liquid chromatography and chemical ionization mass spectrometry. The amino acid sequence of apoC-III isolated from normolipidemic subjects is identical to the apoC-III sequence derived from the cDNA sequence and differs at 4 positions from the previously reported sequence of apoC-III derived from a patient with type V hyperlipoproteinemia.     

Amino acid sequence of VlF: identification in the C-terminal domain of residues common to non-hemorrhagic metalloproteinases from snake venoms

The complete amino acid sequence of a non-hemorrhagic fibrino(geno)lytic enzyme (VlF) isolated from Vipera lebetina venom has been determined. VlF was subjected to separate enzymatic and chemical digestions. Resulting fragments were purified by RP-HPLC and subjected for sequencing by automated Edman degradation. The amino terminus of VlF was determined by mass spectrometry. VlF was shown to be composed of 202 residues having a relative molecular mass of 22,826 Da and containing a zinc-binding site and a catalytically active residue. It displayed significant sequence similarities with many other mature metalloproteinases reported from snake venoms. Sequence comparison of hemorrhagic and non-hemorrhagic mature metalloproteinases revealed the presence at the C-terminal part of the enzymes of two residues common to only hemorrhagic metalloproteinases and two others shared by only non-hemorrhagic ones.