4-Methyleneglutamic acid and 4-methyleneglutamine: isolation from extracts of peanut seedlings and determination by high-performance liquid chromatography

The non-protein amino acids, 4-methyleneglutamic acid and 4-methyleneglutamine, are isolated from aqueous extracts of peanut seedlings in good yield and high purity using a simple HCl-gradient elution from a column of cation-exchange resin followed, in some instances, by a gradient elution with acetic acid from a column of an anion-exchange resin. All of the 4-substituted glutamic acids commonly found in legume species are resolved by a combination of these two system. For analytical purposes, resolution of the acidic amino acids as their phenylthiocarbamoyl derivatives is achieved by HPLC but not by conventional ion-exchange amino acid analysis. Although 4-methyleneglutamine undergoes cyclic deamidation in acidic medium at a slower rate than glutamine, this reaction occurs to a significant extent at 22 degrees C but not a 4 degrees C during the cation-exchange chromatographic fractionation.     

Isolation and sequence of rat peptide YY and neuropeptide Y

Rat peptide YY and rat neuropeptide Y have been isolated in parallel from colon and brain extracts respectively, using salt fractionation, gel filtration chromatography, cation-exchange HPLC, and reverse phase phenyl-silica HPLC. Immunoreactivity was identified using a combination of 3 NPY immunoassays which exhibit differing cross-reactivities for PYY (90%, less than 0.01% and 30% respectively). The yield at the final purification step was 1.2 nmol rPYY and 0.5 nmol rNPY. Half of each purified peptide was subjected to complete microsequence analysis. This showed that while rat NPY was structurally identical to human NPY, the sequence of PYY from rat colon was the same as porcine PYY isolated from extracts of duodenum.     

Insulin-stimulating peptide from tryptic digest of bovine serum albumin

Insulin-stimulating peptide was isolated from a tryptic digest of bovine serum albumin by gel permeation, SP Sephadex column chromatography, reversed phase HPLC and cation-exchange HPLC. This peptide, with a molecular weight of about 8,400, had no insulin-like activity by itself, but enhanced fatty acid synthesis from glucose in rat adipose tissue explants in the presence of suboptimal concentrations of insulin. It also stimulated the effect of insulin on CO2 production from glucose in rat adipocytes, without affecting insulin binding. These stimulations were dose-dependent and were observed at concentrations of more than 2 X 10(-7) M peptide only in the presence of a suboptimal concentration of insulin.     

The molecular defect of albumin Castel di Sangro: 536 Lys----Glu

Albumin Castel di Sangro is a rare fast-moving variant of human serum albumin which has been discovered in heterozygous form in the serum of an 85-year-old woman living in Castel di Sangro (Abruzzo, Italy). Isoelectric focusing analysis of CNBr fragments from the purified variant allowed us to localize the mutation in fragment CNBr VI (residues 447-548). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by reverse-phase and cation-exchange HPLC, revealed that the variant arises from the substitution of lysine 536 by glutamic acid. This amino acid replacement, probably due to a single-base substitution in the structural gene, causes a change in the net charge of -2 units, which is in keeping with both the increased electrophoretic mobility of the native protein and the isoelectric point of the modified CNBr fragment.     

N-Acetylhistidine, Glutamate, and β-Alanine Are Concentrated in a Receptor Cell Layer of the Trout Inner Ear

An epithelial sheet isolated from the trout saccular macula, highly enriched in acousticolateralis receptor cells (hair cells), has been analyzed for primary amine-containing compounds. The hair cell preparation, compared to the saccular nerve, was found to contain elevated levels of the presumptive receptoneural transmitter, glutamate, as well as beta-alanine, and components eluting in the positions of the standards phosphoserine and phosphoethanolamine on cation-exchange HPLC. Saccular nerve contained a different spectrum of primary amines and was elevated specifically in carnosine/homocarnosine. Acid hydrolysis of perchlorate extracts of both hair cell and nerve fractions yielded large amounts of histidine. For the saccular nerve fraction, production of histidine by acid hydrolysis was matched by production of beta-alanine and gamma-aminobutyric acid (GABA) and disappearance of carnosine/homocarnosine. The dipeptides carnosine and homocarnosine have been chromatographically resolved by expanded HPLC and found to be present in saccular nerve in a ratio of approximately 10:1, respectively. Production of histidine in the hair cell extract was not coupled with production of beta-alanine and GABA. The hair cell histidine-containing unknown, present in millimolar concentration, has been identified as N-acetylhistidine by the hydrolysis and rechromatography of fractions from cation-exchange HPLC. The large and specific presence of N-acetylhistidine in the hair cell preparation, together with electrophysiological evidence for its facilitatory action on afferent fibers in the frog semicircular canal, is suggestive of a role for this molecule as well as glutamate in acousticolateralis receptoneural transmission.     

Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae.

Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa. Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction. Production levels are relatively low (ca. 1 mg/liter). Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting. The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS. From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca. 1% of the purified r-ATS), were characterized. Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form. RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing. Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system. Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay. Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide. The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.     

A Highly Sensitive Assay for Histamine Using Ion-Pair HPLC Coupled with Postcolumn Fluorescent Derivatization: Its Application to Biological Specimens

A simple and highly sensitive method for the determination of histamine (HA) was developed using ion-pair, reversed-phase HPLC coupled with postcolumn o-phthalaldehyde derivatization fluorometry, and it was applied to the unpurified extracts of human and rat plasma, and brains of rats and mice. The HA concentrations both in the plasma and brains determined by the present method were well consistent with the values obtained by cation-exchange HPLC with postcolumn fluorescent derivatization currently in use. The present method was more advantageous than the assay using cation-exchange HPLC: (1) it was three to four times more sensitive (the detection limit was 0.5 pg of HA), and (2) it enabled the measurement of HA in samples containing (R)alpha-methylhistamine, a potent and specific H3-receptor agonist, which could not be separated from HA by cation-exchange chromatography. Using the present method coupled with intracerebral microdialysis, we found in the rat hypothalamus that (R)alpha-methylhistamine (5 mg/kg i.p.) markedly decreased the extracellular concentration of HA with a maximal effect (83% reduction) during 30-60 min after injection, suggesting that most of HA in the microdialysate fraction is neuronal in origin.     

Automated multi-dimensional liquid chromatography: sample preparation and identification of peptides from human blood filtrate

A comprehensive on-line sample clean-up with an integrated two-dimensional HPLC system was developed for the analysis of natural peptides. Samples comprised of endogenous peptides with molecular weights up to 20 kDa were generated from human hemofiltrate (HF) obtained from patients with chronic renal failure. The (poly-)peptides were separated using novel silica-based restricted access materials with strong cation-exchange functionalities (SCX-RAM). The size-selective sample fractionation step is followed by cation-exchange chromatography as the first dimension. The subsequent second dimension of separation is based on hydrophobic interaction using four parallel short reversed-phase (RP) columns implemented via a fully automated column switching technique. More than 1000 peaks were resolved within the total analysis time of 96 min. Substances of selected peaks were sampled to analyse their molecular weights by off-line MALDI-TOF mass spectrometry and to determine their amino acid sequence by Edman degradation. The potential for comprehensive peptide mapping and identification is demonstrated.     

Characterisation of a type-I collagen trimeric cross-linked peptide from calf aorta and its cross-linked structure. Detection of pyridinoline by time-of-flight secondary ion-mass spectroscopy and evidence for a new cross-link

A collagenous trimeric cross-linked peptide has been isolated from the insoluble matrix of calf aorta, using trypsin solubilisation, and purified by gel filtration, cation-exchange chromatography and reversed-phase HPLC. Molecular mass and amino acid composition indicated that the C-terminal, non-helical region of type I collagen in its dimer form, designated as [ColC(I)]2, is cross-linked to a tryptic peptide TN(I) from the N-terminal helical cross-link region of an adjacent type I molecule, forming the cross-linked peptide [ColC(I)]2 X TN(I). Amino acid sequence analysis of the peptide yielded a series of sequences corresponding to the cross-linking domains ColC(I) and TN(I) and furnished the first direct chemical evidence for the 4D staggered arrangement of type I molecules within native fibers. The trifunctional cross-linking amino acid pyridinoline was shown to occur in the peptide, confirming the peptides three-chain structure. Pyridinoline was isolated from the cross-linked peptide by preparative amino acid analysis and reversed-phase HPLC and identified by its ultraviolet absorption spectra, its fluorescence excitation and emission spectra and, for the first time, its time-of-flight secondary ion-mass spectrum. The high sensitivity of the latter method, exceeding that of fast-atom-bombardment mass spectroscopy by three orders of magnitude, allowed detection of pyridinoline in the picomole range. The occurrence of pyridinoline in non-stoichiometric amounts, the presence of hydroxylysine in hydrolysates of all cross-linked peptides and the finding that hydrolysates also contained an unidentified component indicated that there is at least one cross-link form that is different from pyridinoline and is hydrolysable.     

Isolation of Lysozyme from the Hemolymph of Sweet Potato Hornworm, Agrius convolvuli Larvae

ABSTRACT Lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. A new family member of insect lysozyme, an antibacterial peptide, has been isolated from the hemolymph of the fifth instar larvae of Agrius convolvuli . Vaccination was proceeded with E. coli K12 D21 (4x10⁶ cells of log phase) injection into the abdomen of the larvae. Agrius lysozyme was isolated by cation-exchange chromatography and RP-FPLC, and sequenced by HPLC system. It was observed that the purified Agrius lysozyme was heat-stable and had a molecular weight of approximately 15 kDa by SDS-PAGE. The N-terminal amino acid sequence of Agrius lysozyme is K-H-F-S-R-C-G-L-V-Q-E-L-F-W-Q-G-F-P with the highest similarity to that of Heliothis virescence , and it has been identified that the Agrius lysozyme belongs to c type lysozyme.     

Expression of Met-(-1) angiogenin in Escherichia coli: conversion to the authentic less than Glu-1 protein

A method for obtaining authentic human angiogenin utilizing an Escherichia coli recombinant expression system is described. A synthetic gene encoding angiogenin was placed into a vector for direct expression under the control of a modified E. coli trp promoter. The protein was produced by the bacteria in an insoluble form and purified to homogeneity by cation-exchange and reversed-phase HPLC following reduction/solubilization and reoxidation. The protein isolated was identified as Met-(-1) angiogenin by amino acid analysis and tryptic peptide mapping; the latter demonstrated that all three disulfide bonds had formed correctly. Both the enzymatic and angiogenic activities of the Met-(-1) protein were equivalent to those of native angiogenin. A Met-(-1) Leu-30 derivative of angiogenin was also isolated and found to be fully active. Conversion of Met-(-1) angiogenin to the authentic less than Glu-1 protein was achieved by treatment with Aeromonas aminopeptidase under conditions in which the new N-terminal glutamine readily cyclizes nonenzymatically. This aminopeptidase treatment may have more general applicability for removal of undesirable N-terminal methionine residues from foreign proteins expressed in bacteria.     

Determination of carbohydrates in fermentation processes by high-performance liquid chromatography

Summary HPLC is a universal, fast, accurate and selective method for the quantification of carbohydrates during fermentation processes. HPLC is not affected by complex constituents of fermentation media, such as meat extract, soybean meal or distillers solubles. The detection limit of the different investigated carbohydrates by refractive index monitoring ranges between 20 and 40 mg/l using a cation-exchange resin and between 50 and 100 mg/l using amino- or diol-bonded phases.Offprint requests to: H.-P. Fiedler     

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.     

Comparison of amino acid levels in rat blood obtained by catheterization and decapitation

A novel, sensitive and highly resolving amino acid analysis procedure was developed and used to compare two methods of obtaining blood from experimental animals. The procedure, utilizing a meter long microbore HPLC column containing spherical cation-exchange resin and fluorescence detection following postcolumn reaction with o-phthaldialdehyde, was shown to reliably measure forty-one primary amine components in rat plasma.Comparison of values from blood obtained by decapitation and by catheterization documented the significant artifactual influence of the decapitation procedure on approximately half of the measured constituents.     

Endothelial cell growth factors in embryonic and adult chick brain are related to human acidic fibroblast growth factor.

We have investigated the nature of endothelial cell growth factors in 14-day embryonic and adult chick brain extracts. Mitogenic activity was isolated by a combination of cation-exchange, heparin-Sepharose affinity, and reverse-phase HPLC. Two major mitogenic fractions eluted from heparin-Sepharose at 0.8-1.3 M and 1.5-2 M. Biologically active proteins eluting at 0.8-1.3 M NaCl, after purification to homogeneity from embryonic and adult brain, were found to possess the same amino-terminal sequence as human acidic fibroblast growth factor (aFGF). The notion that the isolated mitogens represent chick aFGF is further supported by the findings that their affinity for heparin and their retention behavior in highly resolutive HPLC are indistinguishable from those of genuine aFGF. Mitogenic activities eluting at 1.5-2 M NaCl were also present in embryonic and adult brain, but in quantities insufficient for preliminary characterization. The high specific mitogenic activity for endothelial cells, high affinity for heparin and cross-reactivity with antibodies against bovine basic FGF (bFGF) suggest a relationship of those materials with basic FGF. Our data also suggest that the sequence of aFGF is highly conserved among vertebrates. While angiogenesis occurs predominantly in the embryonic brain, the absence of notable differences in the contents of the potent angiogenic factors aFGF and bFGF in embryonic versus adult chick brain is interesting.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Isolation and purification of mature bovine adrenocortical ferredoxin with an elongated carboxyl end

Mature bovine adrenocortical ferredoxin (adreno-ferredoxin) was extracted from fresh adrenal glands at pH 9.0. Extraction and purification at this alkaline pH protected the mature adreno-ferredoxin molecule from proteolytic degradation. The mature adreno-ferredoxin was extensively purified by a rapid procedure including two kinds of column chromatography, hydrophobic and ion exchange. The purified adreno-ferredoxin was homogeneous on the basis of two HPLC analyses, hydrophobic and ion exchange, and had the highest purity so far reported. Then it was digested by trypsin and the carboxyl-terminal peptide was isolated from the tryptic digest by a novel column chromatographic method using a cation-exchange HPLC column, TSK-gel SP-5PW. The carboxyl-terminal amino acid was isoleucine, so the adreno-ferredoxin had 127 amino acid residues, the longest polypeptide so far determined chemically for bovine adreno-ferredoxin. Only Glu-128 was lacking within the carboxyl-terminal elongated peptide that was found by nucleotide sequencing of the adreno-ferredoxin gene. There was no evidence obtained on whether the deletion of Glu-128 was due to so-called carboxyl-terminal processing or to proteolytic degradation during storage and purification.     

Antibacterial and hemolytic activity of the skin of the terrestrial salamander, Plethodon cinereus

As resistance increases against fungal antibiotics, antimicrobial peptides are receiving attention as possible replacements. The dermal glands of frogs secrete, among other things, antimicrobial peptides. As part of the innate immune system, stressors may affect the production of antimicrobial peptides by dermal glands. The dermal secretions of some salamanders have been examined for their toxic secretions, but little attention has been given to salamander antimicrobial peptides. This study examines the skin from the tail region for the production of antimicrobial peptides in the terrestrial salamander, Plethodon cinereus. Fractions of tail extracts were isolated using cation-exchange chromatography and reverse-phase HPLC. An HPLC fraction eluting at 15.75 min (HPLC run: 30 min, 30-80% acetonitrile/water gradient, Aquapore RP-300 C18 column) showed activity against Staphylococcus aureus but not against Escherichia coli. The antibacterial activity gradually increased over a 4-hr incubation time up to about 85% inhibition of bacterial growth. Lysis of guinea pig red blood cells also increased gradually over a 1-hr time period. J. Exp. Zool. 287:340-345, 2000.     

Characterization of a new electrophoretically silent hemoglobin variant. Hb saale OR alpha 2beta 2 84(EF8)Thr --> Ala

A new abnormal hemoglobin was detected in a young German anemic patient by cation-exchange high performance liquid chromatography (HPLC). Using a combination of electrospray mass spectrometry, HPLC, direct sequencing, and family screening with polymerase chain reaction/restriction digestion approach, we have characterized this hemoglobin variant as resulting from a Thr --> Ala replacement at beta84(EF8). It could be separated neither by electrophoresis nor by isoelectric focusing. Hb Saale is slightly unstable, exhibiting a moderate tendency to auto-oxidize. Functional properties and the heterotropic interactions are similar to those of Hb A.     

Dependence of lysozyme-catalysed solubilization of Proteus mirabilis peptidoglycan on the extent of O-acetylation

The degree of peptidoglycan O-acetylation in 14 strains of Proteus mirabilis has been accurately determined by a procedure which employs the quantitation of mild-base-released acetic acid by HPLC, and the estimation of peptidoglycan concentration by cation-exchange amino acid analysis. The beta-D-N,6-O-diacetylmuramyl content of all isolated and purified peptidoglycans was ranged 20-52.8%, relative to the total muramic acid concentration. Each of the O-acetylated peptidoglycans was found to be resistant to solubilization by both human and hen egg-white lysozymes and for hen egg-white lysozyme, the extent of this resistance was dependent upon the degree of O-acetylation. The steady-state parameters, Km and V, for the hen-egg-white-lysozyme-catalysed solubilization of various peptidoglycan preparations were determined at pH 6.61 and 25 degrees C. Values of Km for the different peptidoglycan samples were found to increase with increasing O-acetylation, whereas with V no such relationship appeared to exist. An increase in the overall change in the standard Gibbs free energy of activation [delta(delta G#)], a consequence of increasing O-acetylation, was observed, and is shown to result from the weaker affinity of the enzyme for the modified substrates.