A study of elastase peptides from bovine white matter proteolipid

Bovine white matter proteolipid has been digested with elastase in the presence of deoxycholate. After acidification, the digest was separated into an acid-soluble and an acid-insoluble fraction. The acid-insoluble fraction was enriched in nonpolar amino acids and, by a combination of solvent fractionation and chromatography, a fraction was obtained which consisted of a mixture of two peptides with a molecular weight of approximately 4000 daltons. The acid-soluble peptides were separated by molecular sieve, ion exchange and high performance liquid chromatography (HPLC) in the reverse phase mode. The purified peptides were smaller than expected on the basis of their elution position from a molecular sieve column, suggesting they were in an aggregated state during the initial chromatography. Reverse phase HPLC was shown to be useful for fingerprinting these peptide mixtures. The data demonstrate the difficulties associated with the study of this proteolipid and emphasize the tendency of both the protein and the peptides derived from it to aggregate.     

Utilization of aspartate,glutamate, and their corresponding peptides byFusobacterium nucleatum subspecies andPorphyromonas gingivalis

Glutamate and aspartate are key amino acids for catabolism byFusobacterium nucleatum subspecies andPorphyromonas gingivalis respectively. However, peptides such as yeast extract are their preferred sources of energy. To determine more precisely the possible nature of these peptides, we examined the utilization of these amino acids and their corresponding peptides by cell suspension experiments with a fluorescamine labeling technique. High molecular weight (M.W.) polyglutamate (>40,000) was poorly utilized by all taxa, whereas 95% of its low-M.W. peptide (2,000–5,000) was used byF. nucleatum subspeciesnucleatum, but the remaining two subspecies utilized <30%.P. gingivalis used ca. 50% polyglutamate but >90% polyaspartate within the same period. ForF. nucleatum subspeciesnucleatum as the test organism, T_0.5 (the time taken to use 50% of the test substrate) was 1.7 h longer for glutamate than for the homopolymer. Furthermore, in the presence of both substrates, polyglutamate suppressed the uptake of glutamate until about 50% (ca. 1.5 mmol/L) of the peptide was used, after which the incorporation of the free amino acid started. A similar pattern of utilization was observed inP. gingivalis with its preferred peptide polyaspartate, for which the T_0.5 was three times shorter than its monomer, aspartate. Both species had the capacity to utilize the heteropolymer, poly aspartate/glutamate, but at a significantly slower rate than the corresponding homopolymer.     

Extracellular polypeptides ofAnabaena L-31: Evidence for their role in regulation of heterocyst formation

Extracellular polypeptides released by both N₂-grown [peptide I] and NO₃-grown [peptide II]Anabaena L-31 have molecular weight of approximately 3,500 but have distinctly different amino acid composition. Acid hydrolysis of the peptide I fraction (obtained by separation on Sephadex G-25) yielded ten amino acids whereas that from peptide II fraction yielded only 3 amino acids. On addition to a freshly inoculated N₂-grown culture, the peptide I fraction stimulated pro-heterocyst and to a lesser extent heterocyst differentiation, whereas the peptide II fraction strongly inhibited differentiation. The inhibitory effect of polypeptide II fraction could not be relieved by methionine sulphoximine, which by itself enhances differentiation, but was greatly relieved by addition of the peptide I fraction. The data suggest but does not prove, thatAnabaena L-31 synthesises “inducer” or “inhibitor” peptides which could possibly control pattern formation.     

CHANGES OF AMINO ACID COMPOSITION OF CHLORELLA CELLS DURING THEIR LIFE CYCLE

The cells of Chlorella ellipsoidea were grown synchronously,and at different stages of their life cycle, the cells wereanalysed for their contents in amino acids existing in freeforms as well as in the fractions of bulk protein and peptides.Throughout the algal life cycle, the content of bulk protein(per unit dry weight of cells) remained relatively constant,being about 20 to 40 times those of peptides and free aminoacids. The amino acid composition of the protein fraction alsoremained fairly constant, the predominant amino acids beingalanine, glutamic acid, glycine and leucine. The contents inthe bulk peptides increased appreciably during the periods ofgrowth and "ripening" (light period), and decreased markedlyduring the periods of "post-ripening" and cellular division(dark period). Similar modes of change in content were alsoobserved in most of the individual amino acids contained inthe peptide fraction. The most abundant component in the peptidefraction was arginine followed by glutamic acid, glycine andcyst(e)ine. Rather irregular was the mode of change of the levelsof individual free amino acids, although, as a whole, theirbehavior was similar to that of bulk peptides, increasing duringthe light period and decreasing during the dark period. Themost predominant free amino acids were glutamic acid and alaninefollowed by proline. Experimental evidence showed that the processes of formationof free amino acids and peptides are for the most part lightdependent, while the synthesis of protein, which is thoughtto be effected using as building blocks mostly free amino acids—formeddirectly or indirectly from early photosynthates or derivedfrom pre-formed peptides—is essentially a light-independentprocess. Peptides, as a whole, seem to have significance asreservoirs of building blocks for the syntheses in the darkof protein and other nitrogenous cellular substances. The synthesisof protein in the dark takes place not only by consuming thefree amino acids and peptides that have been accumulated duringthe light period, but also by assimilating the exogenous nitrogensource (nitrate). The distribution of individual amino acidsin the three main fractions mentioned above as it changed duringthe course of algal cell cycle was followed in detail, and theresults obtained were discussed in relation to various relevantdata reported by other workers. (Received June 29, 1964; )     

Structural Modification and Expression of Attacin A from Glossina morsitans morsitans in E. coli and its Antibacterial Activities

Attacin A from Glossina morsitans morsitans belongs to the type of Gly-rich antimicrobial peptides, with mature peptide of 188 amino acids and molecular weight of 19.4 kDa. In this work, a truncated form of Attacin A was studied to evaluate its N-terminal and G1 domain for antibacterial activities with the help of genetic engineering technology. Genes coding for mature full length Attacin A together with its truncated form Attacin AP1 containing 1–99 amino acids of Attacin A were cloned, expressed and purified. The expression level for Attacin A and Attacin AP1 were about 10 and 20% of total cell protein, respectively. The purity of bioactive recombinant proteins, which showed virtually homogenous as determined by SDS–PAGE, was over 90%. Antibacterial testing shows that Attacin A could inhibit the growth of Gram-negative bacteria such as E. coli, Enterobacter cloacae and Klebsiella pneumoniae at relatively high concentration comparing with Ampicillin, but had little activity against Gram-positive Staphylococcus aureus. Attacin AP1 maintained the inhibitory activities against E. coli, E. cloacae and K. pneumoniae. This result makes it possible for better understanding of the structure–activity relationship of antimicrobial peptides.     

The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides.

The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide. All peptides corresponding to each of the MH+ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from beta-casein by proteinase only.     

Large-scale production of peptides using the solid phase continuous flow method. Part 2: preparative synthesis of a 26-mer peptide thrombin inhibitor

A preparative method for the preparation of large peptides is described. An advantageous theoretical weight of peptide/weight of starting resin ratio (tP_w/R_w) of about 0.3 was successfully experimented. The esterification of the first amino acid was realized with a racemization of less than 1%. The study of the coupling conditions led to the use of a diluted acylating mixture that allowed a 56% consumption of the amino acid derivatives (percentage use of amino acids) introduced in the synthesis. The cost analysis of the synthesis showed that the recovery of the amino acid derivatives was not worthwhile. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

C-terminal pro-ghrelin peptides are present in the human circulation

We provide the first evidence for the existence in human plasma of peptides derived from the 66 carboxyl-terminal amino acids of pro-ghrelin (C-ghrelin). C-ghrelin immunoreactivity in plasma was higher than ghrelin, and did not significantly correlate with body mass index in normal health. In patients with myocardial infarction, plasma levels of both ghrelin and C-ghrelin were significantly decreased (approximately 30%, P<0.05), whereas in patients with heart failure, C-ghrelin levels were significantly elevated (approximately 32%, P<0.05) compared with controls. HPLC coupled with RIA showed circulating C-ghrelin to be primarily of low molecular weight (M(r) approximately 3500), but in chronic heart failure, a higher molecular weight form (M(r) approximately 7500) is also present. This is the first evidence for potential circulating hormones derived from the carboxyl terminus of pro-ghrelin and for their modulation in cardiovascular diseases.     

COMPARATIVE PEPTIDE MAPPING AND ISOELECTRIC FOCUSING OF ISOLATED SUBUNITS FROM CHICK EMBRYO BRAIN TUBULIN

The α- and β-subunits of chick embryo brain tubulin have been isolated under denaturing conditions and compared with respect to their molecular weight, amino acid composition, tryptic peptide maps, amide content and isoelectric focusing properties. An 8 M-Urea-containing polyacrylamide gel system with varying acrylamide concentrations was used for calculation of the retardation coefficients (K_R) of the tubulin subunits. A molecular weight of 53,000 was estimated for each subunit by comparison to K_R values for standard proteins. Amide contents of approx 41% of the carboxyl groups of α-tubulin and 48% of the carboxyl groups of β-tubulin were calculated using the average PI value, the pK_intrinsic for the ionizable side chains of the amino acids and the amino acid composition of each subunit. Comparative peptide maps of trypsin digested α- and β-tubulin demonstrated 16 peptides unique to each subunit and 23 peptides which comigrate. Both subunits give rise to multiple species on electrofocusing gels. The average isoelectric points for the α- and β-subunits are 5.4 and 5.2, respectively.     

Further studies on the peptido-galactomannan from the yeast form of Cladosporium werneckii. Identification of O-acetyl substituents and isolation of the peptide components following beta-elimination.

The pathogenic yeast Cladosporium werneckii produces a surface peptido-phosphogalactomannan (PPGM) with a peptide backbone rich in serine and threonine to which three types of carbohydrate chains are linked. These chains are: Type a, glactomannan units linked through phosphodiester bonds to produce long chains of molecular weight about 50,000; type b more numerous short mannosyl oligosaccharide units, and type c, more infrequent, long galactomannan chains. The first two are linked to the peptide through alkali-labile bonds to serine and threonine of the peptide whereas type c chains are linked through alkali-stable bonds (Lloyd, K. O. (1972) Biochemistry, 11, 3884–3890). The PPGM sample can be separated into three or four major components by diethylaminoethyl (DEAE-) Sephadex chromatography. By means of sequential degradations with alkali and acid, the structural basis for this heterogeneity has been demonstrated. It is due to the presence of different proportions of the three types of chains in the various fractions. The presence of O-acetyl groups, mainly on the a chains, was demonstrated in PPGM by chemical analysis and by proton and ¹³C nuclear magnetic resonance spectroscopy. Purified a chains were isolated from PPGM following Pronase digestion. By taking advantage of the alkali lability of the carbohydrate-protein linkages it has been possible to cleave the peptide moiety away from the carbohydrate. By sequential chromatography on Bio-Gel P-100, Dowex 1 and Bio-Gel P-100, five modified peptide fractions were isolated. The molecular weights of these fractions varied from 9,500 to 18,500 as judged by polyacrylamide-gel electrophoresis. Unlike the original peptide, the modified peptides contained low amounts of serine and threonine. The predominant amino acids were alanine, glycine, aspartic acid and glutamic acid which together make up between 51 and 55% of the peptides. The high content of the last two amino acids accounts for the acidic nature of the peptides. It appears that each fraction consists of a slightly heterogeneous population of peptides very similar in amino acid composition. It is not clear whether the compositional and size heterogeneity exists in the original peptide or whether it arose during the isolation procedure.     

Similar peptides from two beta cell autoantigens, proinsulin and glutamic acid decarboxylase, stimulate T cells of individuals at risk for insulin-dependent diabetes.

BACKGROUND: Insulin (1) and glutamic acid decarboxylase (GAD) (2) are both autoantigens in insulin-dependent diabetes mellitus (IDDM), but no molecular mechanism has been proposed for their association. We have identified a 13 amino acid peptide of proinsulin (amino acids 24-36) that bears marked similarity to a peptide of GAD65 (amino acids 506-518) (G. Rudy, unpublished). In order to test the hypothesis that this region of similarity is implicated in the pathogenesis of IDDM, we assayed T cell reactivity to these two peptides in subjects at risk for IDDM. MATERIALS AND METHODS: Subjects at risk for IDDM were islet cell antibody (ICA)-positive, first degree relatives of people with insulin-dependent diabetes. Peripheral blood mononuclear cells from 10 pairs of at-risk and HLA-DR matched control subjects were tested in an in vitro proliferation assay. RESULTS: Reactivity to both proinsulin and GAD peptides was significantly greater among at-risk subjects than controls (proinsulin; p < 0.008; GAD; p < 0.018). In contrast to reactivity to the GAD peptide, reactivity to the proinsulin peptide was almost entirely confined to the at-risk subjects. CONCLUSIONS: This is the first demonstration of T cell reactivity to a proinsulin-specific peptide. In addition, it is the first example of reactivity to a minimal peptide region shared between two human autoimmune disease-associated self antigens. Mimicry between these similar peptides may provide a molecular basis for the conjoint autoantigenicity of proinsulin and GAD in IDDM.     

Primary structure of internal peptide VII of T-even bacteriophages

As first shown by Hershey (1955,1957), osmotic rupture of the head of bacteriophage T2 releases, together with the DNA, an acid-insoluble protein fraction and an acid-soluble peptide fraction. Subsequent chromatographic analysis of the acid-soludle fraction of bacteriophage T4-infected Escherichia coli resolved two peptides, designated II and VII, corresponding to the peptide fraction of T2 described by Hershey (Eddleman &; Champe, 1966). Both peptides were found to be composed largely of acidic residues with only a limited number of other amino acids. The amino acid compositions predicted minimum molecular weights of 3900 (33 residues) and 2700 (23 residues) for peptides II and VII, respectively, consistent with their retardation on Sephadex G25 (Champe &; Eddleman, 1967). The formation of these peptides in T4-infected cells parallels the synthesis of late proteins. Pulse-labelling experiments showed, however, that the internal peptides are derived from acid-insoluble precursors, suggesting the probable involvement of proteolytic cleavage in their formation. The role of these peptides, if any, in phage replication is unclear. Eddleman &; Champe (1966) suggested that they may be inactive remnants of a core protein whose elimination is required for assembly. Alternatively, Laemmli &; Favre (1973) have proposed that the internal peptides may function to facilitate packaging of the DNA in the phage head.     

Identification of peptide epitopes of MAGE-1, -2, -3 that demonstrate HLA-A3-specific binding

 The MAGE gene family of tumour antigens are expressed in a wide variety of human cancers. We have identified 43 nonamer peptide sequences, from MAGE-1, -2 and -3 proteins that contain binding motifs for HLA-A3 MHC class I molecules. The T2 cell line, transfected with the cDNA for the HLA-A3 gene, was used in a MHC class I stabilisation assay performed at 37°C and 26°C. At 37°C, 2 peptides were identified that stabilised HLA-A3 with high affinity (fluorescence ratio, FR >1.5), 4 peptides with low affinity (FR 1.11 – 1.49) and 31 peptides that did not stabilise this HLA haplotype (FR <1.1). At 26°C, 12 peptides were identified that stabilised HLA-A3 with high affinity, 8 peptides with low affinity and 17 peptides that did not stabilise this HLA haplotype. Two peptides stabilised HLA-A3 at both temperatures. Small changes in one to three amino acids at positions distinct from the anchor residues altered peptide affinity. Data were compared to a similar study in which a peptide competition assay was used to investigate MAGE-1 peptide binding to several HLA haplotypes. This study demonstrates that anchor residues do not accurately predict peptide binding to specific HLA haplotypes, changes in one to three amino acids at positions distinct from anchor residues influence peptide binding and alternative methods of determining peptide binding yield different results. We are currently investigating the ability of these peptides to induce antitumour cytotoxic T lymphocyte activity as they may be of potential therapeutic value. Received: 4 January 1996 / Accepted: 20 March 1996     

Antifungal peptides homologous to the Penicillium chrysogenum antifungal protein (PAF) are widespread among Fusaria

Putative antifungal peptide encoding genes containing Penicillium chrysogenum antifungal protein (PAF) characteristic amino acid motifs were identified in 15 Fusarium isolates, representing 10 species. Based on the predicted sequences of mature peptides, discrepancy in one, two or three amino acids was observed between them. Phylogenetic investigations revealed that they show high amino acid sequence similarity to PAF and they belong to the group of fungal derived antifungal peptides with PAF-cluster. Ten from the 15 partially purified <10 kDa peptide fraction of Fusarium ferment broths showed antifungal activity. The presence of approximately 6.3 kDa molecular weight peptides was detected in all of the antifungally active ferment broths, and this peptide was isolated and purified from Fusarium polyphilaidicum. The minimal inhibitiory concentrations of F. polyphilaidicum antifungal protein (FPAP) were determined against different filamentous fungi, yeasts and bacteria. Filamentous fungal species were the most susceptible to FPAF, but some yeasts were also slightly sensitive.     

The processing of interleukin-1 beta studied with antibodies raised against synthetic peptides from the precursor N-terminal region

The exact sequence of events during processing of human interleukin-1 beta (IL-1 beta) and the fate of the N-terminal region are unknown. We have used anti-peptide sera specific for the precursor and mature regions of IL-1 beta to study biosynthesis. These were raised against peptides corresponding to amino acids 1-15, 17-32, and 43-54 of the precursor and a peptide corresponding to the C-terminal 33 amino acids of mature human IL-1 beta. Antiserum to the mature region peptide immunoprecipitated the 35-kD precursor from cell lysates and 17-kD mature IL-1 beta and a 31-kD protein from the culture supernatants from radiolabeled human peripheral blood monocytes stimulated with lipopolysaccharide (LPS). Antisera to peptides from the precursor region also immunoprecipitated the 35-kD IL-1 beta precursor but not the 31-kD or 17-kD forms. Of the precursor-specific sera, only antiserum to amino acids 1-15 specifically recognized any other proteins; a peptide of 18 kD and a low molecular weight peptide, both of which accumulated in the medium. The 18-kD protein was not recognized by any of the other antisera and is unlikely to be the N-terminal region of the precursor removed during processing. Pulse-chase experiments indicated that the 31-kD protein could be a processing intermediate and also that it was itself an end product along with full-length precursor. Only 17-kD mature IL-1 beta had biological activity.     

Peptide and amino acid oxidation in the presence of thiosulfate by members of the genus Thermoanaerobacter

Thermoanaerobacter brockii, T. ethanolicus, T. thermohydrosulfuricus, T. finnii, and Thermoanaerobacter strain SEBR 5268 (an isolate from an oil-producing well) were studied for their ability to oxidize proteinaceous compounds that included gelatin, peptides, and casamino acids. All bacteria tested used peptides and amino acids, but only slightly. However, in the presence of thiosulfate all the Thermoanaerobacter species showed a substantial improvement in growth and/or the production of acetate, isovalerate, isobutyrate, and sulfide. Propionate was a minor product of peptide or amino acid oxidation. The reduction of thiosulfate during growth on peptides by members of the Thermoanaerobacter species is a trait that closely resembles that of archaeal hyperthermophiles during growth on peptides and amino acids with elemental sulfur as electron acceptor.     

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25°C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l⁻¹, respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties. Journal of Industrial Microbiology & Biotechnology (2001) 26, 235–240. Received 02 August 2000/ Accepted in revised form 15 December 2000