Identification of T-kinin-Leu(T-kinin-containing peptide) released from T-kininogen by cathepsin D of granulomatous tissues in rats

Acid proteinases of granulomatous tissues in rats with carrageenin-induced inflammation released kinin from T-kininogen. By column chromatography on pepstatin-Sepharose 4B, two types of acid proteinase seems to be responsible for kinin release. One of the acid proteinase was identified as cathepsin D from SDS-polyacrylamide gel electrophoresis and Western-blot analysis, using anti-rat liver cathepsin D IgG. Cathepsin D alone could not release T-kinin, but T-kinin-containing peptides. The T-kinin-containing peptides were separated into two peptides by reverse-phase high-performance liquid chromatography. From determination of its amino acid composition and its immunoreactivity toward anti-bradykinin antiserum, one of the T-kinin-containing peptides was identified as T-kinin-Leu.     

Development of metal affinity-immobilized liposome chromatography and its basic characteristics

Metal affinity-immobilized liposome chromatography (MA-ILC) was newly developed as a chromatographic technique to separate and analyze peptides. The MA-ILC matrix gel was first prepared by immobilizing liposomes modified with functional ligands. The functional ligand used to adsorb metal ions was N-hexadecyl iminodiacetic acid (HIDA), which is obtained by attaching a long alkyl chain to an iminodiacetic acid (IDA). Cu(II) ion was first adsorbed on the gel matrix through its complex formation with the HIDA on the surface of the immobilized liposome. Synthetic peptides of various types ranging in size from 5 to 40 residues were then used, and their retention properties on the MA-ILC were evaluated. The retention property of peptides on the MA-ILC by using a usual imidazole elution was compared with the retention property in the case of the immobilized metal affinity chromatography (IMAC) and an immobilized liposome chromatography (ILC). It was found that the retention property of peptides on the MA-ILC has the features of both the IMAC and the ILC; the retention ability of peptides depends on both the number of histidine residues in peptides and the liposome membrane affinity of the peptides. Histidine and tryptophan residues among amino acid residues in peptides indicated a high contribution coefficient for the peptide retention on the MA-ILC, probably due to their metal ion and membrane interaction properties, respectively.     

Pleurostrin, an antifungal peptide from the oyster mushroom

A 7kDa peptide, with inhibitory activity on mycelial growth in the fungi Fusaerium oxysporum, Mycosphaerella arachidicola and Physalospora piricola, was isolated from fresh fruiting bodies of the oyster mushroom. The isolation procedure entailed extraction with an aqueous buffer, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 75. The protein was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel. It demonstrated an N-terminal sequence different from known antifungal proteins and peptides.     

Quantitation of type I, III, and V collagens in human tissue samples by high-performance liquid chromatography of selected cyanogen bromide peptides

A method to determine the proportions of the major fiber-forming collagens (types I, III, and V) in noncartilaginous human tissues is presented. The procedure relies on direct solubilization of tissue collagen as cyanogen bromide peptides. The peptides are subjected to cation exchange chromatography followed by gel permeation chromatography in a manner consistent with the rapid resolution and quantitation of relatively low-molecular-weight marker peptides for each collagen. The marker peptides utilized for type I, III, and V collagens are alpha 1 (I)-CB2, alpha 1 (III)-CB2, and alpha 1 (V)-CB1, respectively. Quantitation of the peptides is attained as a function of ultraviolet absorbance during gel permeation chromatography. The nature of the marker peptides, the use of high-performance liquid chromatography techniques, and quantitation of the peptides by ultraviolet absorbance renders the method suitably rapid, sensitive, and accurate for routine evaluations of collagen composition. The utility of the method is illustrated in the presentation of analyses on specimens of placental membranes and blood vessel walls.     

Applications of affinity chromatography in proteomics

Affinity chromatography is a powerful protein separation method that is based on the specific interaction between immobilized ligands and target proteins. Peptides can also be separated effectively by affinity chromatography through the use of peptide-specific ligands. Both two-dimensional electrophoresis (2-DE)- and non-2-DE-based proteomic approaches benefit from the application of affinity chromatography. Before protein separation by 2-DE, affinity separation is used primarily for preconcentration and pretreatment of samples. Those applications entail the removal of one protein or a class of proteins that might interfere with 2-DE resolution, the concentration of low-abundance proteins to enable them to be visualized in the gel, and the classification of total protein into two or more groups for further separation by gel electrophoresis. Non-2-DE-based approaches have extensively employed affinity chromatography to reduce the complexity of protein and peptide mixtures. Prior to mass spectrometry (MS), preconcentration and capture of specific proteins or peptides to enhance sensitivity can be accomplished by using affinity adsorption. Affinity purification of protein complexes followed by identification of proteins by MS serves as a powerful tool for generating a map of protein-protein interactions and cellular locations of complexes. Affinity chromatography of peptide mixtures, coupled with mass spectrometry, provides a tool for the study of protein posttranslational modification (PTM) sites and quantitative proteomics. Quantitation of proteomes is possible via the use of isotope-coded affinity tags and isolation of proteolytic peptides by affinity chromatography. An emerging area of proteomics technology development is miniaturization. Affinity chromatography is becoming more widely used for exploring PTM and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. More applications of affinity-based purification can be expected, including increasing the resolution in 2-DE, improving the sensitivity of MS quantification, and incorporating purification as part of multidimensional liquid chromatography experiments.     

Cicerarin,a novel antifungal peptide from the green chickpea

A peptide designated cicerarin, with an N-terminal amino acid sequence VKSTGRADDDLAVKTKYLPP dissimilar from known proteins and peptides and a molecular mass of 8kDa, was isolated from seeds of the green chickpea Cicer arietinum cv green chickpea. Cicerarin was isolated with a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and gel filtration by fast protein liquid chromatography on Superdex 75. Cicerarin was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel in 10mM Tris-HCl buffer (pH 7.3). Cicerarin exerted antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, and Physalospora piricola. The antifungal activity was preserved after exposure to 100 degrees C for 15min.     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from the edible mushroom Schizophyllum commune

An N-acetyl-D-galactosamine (GalNAc)-specific lectin was purified from the edible mushroom, Schizophyllum commune, using affinity chromatography on a porcine stomach mucin (PSM)-Sepharose 4B column. Under reducing and non-reducing conditions, SDS-polyacrylamide gel electrophoresis gave a major band of 31.5 kDa. The Schizophyllum commune lectin (SCL) showed high affinity toward rat erythrocytes and the sugar inhibition assay exhibited its sugar specificity highly toward lactose and N-acetyl-D-galactosamine. It was stable at 55 degrees C for 30 min and at pH 3-10 for 18-h test. The lectin was shown to be a glycoprotein with cytotoxic activity against human epidermoid carcinoma cells. The N-terminus of SCL was blocked but amino acid sequences of internal tryptic peptides showed moderately sequence similarities with some other fungal and plant lectins. Crystals of SCL were obtained by the sitting drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant, and gave an X-ray diffraction pattern to approximately 3.8 angstroms resolution.     

Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore bacterial cytoplasmic membranes

Three peptides with pore-forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed-phase high-performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic α-helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore-forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram-positive bacteria. The amoebapores represent a distinct family of membrane-active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.     

Mapping of Peptides in Soy Sauce by an Ion Exchange Chromatography

Pattern analysis of peptides in soy sauce was tried by gel filtration and ion exchange chromatography. In gel filtration through Sephadex G-15, the separation of smaller peptides and amino acids in soy sauce was found to be not so critical. Subsequently the mapping of peptides in soy sauce was done by ion exchange chromatography with Dowex 50-X2. Consequently it was estimated that there existed more than ten kinds of peptides in soy sauce.

Moreover, in the mapping of peptides, the automated measurement by the use of a discrete type analyzer, EEL Auto Chemist, was tried. The method developed here was not fully automated but available and effective for the mapping of peptides eluted from an ion exchange resin column.     

A ubiquitin-like peptide from the mushroom Pleurotus sajor-caju exhibits relatively potent translation-inhibitory and ribonuclease activities

The fruiting bodies of the edible mushroom Pleurotus sajor-caju were extracted with an aqueous buffer and then subjected to affinity chromatography on Affi-gel Blue gel, ion exchange chromatography on DEAE-cellulose and gel filtration on Superdex 75. From the fraction of the extract adsorbed on Affi-gel Blue gel and unadsorbed on DEAE-cellulose, a 9.5 kDa peptide with an N-terminal sequence similar to ubiquitin was isolated with a yield of 0.25 mg/kg mushroom. The peptide inhibited cell-free translation with an IC(50) of 30 nM. It exhibited a ribonuclease activity of 450 U/mg toward yeast transfer RNA. The activities were substantially more potent than those of previously isolated mushroom ubiquitin-like protein and peptide.     

Chromatography of peptides related to beta-endorphin

Chromatographic procedures are described here for the resolution of beta-endorphin and its related peptides at picomolar concentration. Initially gel filtration is carried out on Sephadex G75 in 50% acetic acid, providing peptides with the approximate molecular size of beta-endorphin. The group of beta-endorphin-related peptides is resolved by ion-exchange chromatography on the pyridinium form of sulfopropyl Sephadex C25 in the presence of 50% acetic acid. The addition of 125I-labeled marker peptides prior to chromatography allows the recovery of each peptide to be calculated and provides a guide for identifying the elution positions of the endogenous peptides. Additional resolution can be obtained by high-pressure liquid chromatography (HPLC) of the sulfoxide forms of the peptides on muBondapak C18 under acidic conditions. The advantages and disadvantages of ion-exchange chromatography and high-pressure liquid chromatography are discussed for the purification of small amounts of basic, hydrophobic peptides.     

The primary structure of the alpha subunit of protocatechuate 3,4-dioxygenase. I. Isolation and sequence of the tryptic peptides.

The carboxymethylated alpha subunit of protocatechuate 3,4-dioxygenase was digested with trypsin. The 14 tryptic peptides were isolated by ion exchange chromatography on DEAE-Sephadex and by gel filtration chromatography. Automated Edman degradation and carboxypeptidase Y and B digestion were used to establish the sequence of these peptides. Further fragmentation of two tryptic peptides, T3 and T5, by Staphylococcus aureus protease and cyanogen bromide, respectively, was necessary to complete the sequences. The tryptic peptides accounted for a minimum of 199 residues out of a total of 202 residues predicted by amino acid analysis.     

Tripeptide-specific aminopeptidase from Escherichia coli AJ005.

A tripeptidase, TP, from the ribosome-free fraction of Escherichia coli AJ005, a peptidase-deficient mutant of strain K-12, has been obtained using gel electrophoresis and chromatography on DEAE-Sephadex A-50, hydroxylapatite, and Sephadex G-200. Characterization studies on tripeptidase TP, freed of other detectable peptidases, indicate that this enzyme is capable of cleaving an amino-terminal leucine, lysine, methionine, or phenylalanine residue from certain tripeptides. Only one band of activity toward several tripeptides (and no activity toward dipeptides) was detected following gel electrophoresis of this preparation. Tripeptidase TP, the only strain AJ005 peptidase known to attack trilysine, was inactive toward all dipeptides, peptide amides, substituted peptides, esters, and tetrapeptides tested as substrates. Trilysine cleavage is optimal at about pH 8.5, as determined in Tris, borate, or phosphate buffers. Tripeptidase TP activity tested under a number of conditions was not inhibited by soybean trypsin inhibitor (3 mg/mL), phenylmethanesulfonyl fluoride (25 micrometer), or iodoacetate (9 mM). p-Mercuribenzoate (10 micrometer), divalent copper, cobalt, calcium (2.5 mM), zinc (25 micrometer), and mercury (10 micrometer) are inhibitory. Based on Sephadex G-200 chromatography tripeptidase TP has a particle weight of approximately 80 000 daltons. An apparent Km of 5.3 mM was determined for methionylglycylglycine cleavage.