Bioactive Sequences Research Articles

Latent Bioactive Peptides in Milk Proteins: Proteolytic Activation and Significance in Dairy Processing

Referee: Mr. Dick Fitzgerald, Ph.D., Life Science Department, University of Limerick, Limerick, IrelandAfter a brief description of the properties of bioactive peptides, the proteolytic activation of the bioactive sequences from milk protein precursors is discussed. The ability of proteolytic enzymes from various sources, especially from lactic acid bacteria, to release bioactive peptides and the physiological and biotechnological significance of these peptides in dairy products are reviewed.

Tumor cell surface heparan sulfate as cryptic promoters or inhibitors of tumor growth and metastasis

Heparan sulfate glycosaminoglycans, present at the cell surface and in the extracellular matrix that surrounds cells, are important mediators of complex biological processes. Furthermore, it is now apparent that cells dynamically regulate the structure of their heparan sulfate "coat" to differentially regulate extracellular signals. In the present study, the importance of sequence information contained within tumor cell-surface heparan sulfate was investigated. Herein, we demonstrate that the heparan sulfate glycosaminoglycan coat present on tumor cells contains bioactive sequences that impinge on tumor-cell growth and metastasis. Importantly, we find that growth promoting as well as growth inhibiting sequences are contained within the polysaccharide coat. Furthermore, we find that the dynamic balance between these distinct polysaccharide populations regulates specific intracellular signal-transduction pathways. This study not only provides a framework for the development of polysaccharide-based anti-cancer molecules but also underscores the importance of understanding a cell's polysaccharide array in addition to its protein complement, to understand how genotype translates to phenotype in this post-genomic age.

Constitutive production of interleukin-1alpha mRNA and protein in the developing rat testis.

Interleukin-1 (IL-1), a multifunctional cytokine produced mainly by activated macrophages, is also produced in the intact testis. Rat testicular IL-1 was found to be identical to IL-1alpha, judged by immunoneutralization of the bioactive protein and sequence comparison of cloned rat testicular and macrophage pro-IL-1alpha cDNA. Testicular IL-1alpha mRNA was first demonstrated on postnatal day 15, and the corresponding bioactive protein from day 20. IL-1alpha mRNA was still low on day 20, but then increased rapidly in parallel with the bioactive protein to establish a plateau level from day 25. In adult testes, IL-1alpha mRNA and immunoreactive protein were low in stage VII of the seminiferous epithelial cycle, whereas other stages showed a clearly detectable expression. In the adult testis, the concentration of IL-1alpha was 75 pg/mg testicular protein (approximately 200 pM). In conclusion, production of testicular IL-1alpha is developmentally and stage-dependently regulated, probably at the transcriptional level, emphasizing an important paracrine role in testicular function.

Processing of chromogranin A in the parathyroid: generation of parastatin-related peptides

Chromogranin A (CgA) is a glycoprotein present in secretory granules of endocrine cells. In the parathyroid, it is costored and cosecreted with parathormone (PTH) in response to hypocalcemia. CgA is the precursor of several bioactive peptides including pancreastatin and betagranin. Parastatin (PARA, pCgA 347–419) is a novel peptide that we generated in vitro by enzymatic digestion of pCgA. In vitro, it inhibits low Ca 2+-stimulated parathyroid secretion. Full activity resides in its first 19 residues. In order to determine if PARA or PARA-derived peptides are natural products of the parathyroid, we generated an antiserum directed against pCgA 347–359 corresponding to the bioactive N-terminal sequence of pPARA (pPARA 1–13 antiserum), and developed a specific radioimmunoassay that we used in conjunction with various chromatographic separations. We identified small peptides carrying the pPARA 1–13 immunoactivity in extracts and secretion medium of porcine parathyroid glands. Continuous and pulse-chase radiolabeling studies, along with immunoprecipitation using PARA 1–13 antiserum demonstrate that a newly-synthesized PARA-related peptide fraction with a Mr of 11 kDa is secreted by the parathyroid cells and accumulates in the secretion medium. Edman degradation of the 11 kDa PARA-related peptide band by Edman degradation yielded three major N-terminal sequences: S-K-M-D-R-L-A-K-E-L—(residues 313–322), D-R-L-A-K-E-L-T-A-E—(residues 316–325), and A-K-E-L-T-A-E-K-R-L—(residues 319–329), in a molar ratio of approximately 1:2:1. The peptide bonds required to be cleaved to yield these peptides, Trp-Ser, Met-Asp and Leu-Ala, suggest that a chymotrypsin-like endopeptidase participated in their formation. The molecular size and the results of amino acid compositional analysis, indicate that the C-termini of these peptides extended variably to residues 384–401 of pCgA. These results demonstrate that processing of CgA by the parathyroid gland generates bioactive PARA-related peptides that could affect the gland’s secretory activity.

Template-constrained cyclic peptide analogues of somatostatin: subtype-selective binding to somatostatin receptors and antiangiogenic activity

β-Turns are a common secondary structure motif found in proteins that play a role in protein folding and stability and participate in molecular recognition interactions. Somatostatin, a peptide hormone possessing a variety of therapeutically-interesting biological acitivities, contains a β-turn in its bioactive conformation. The β-turn and biological activities of somatostatin have been succesfully mimicked in cyclic hexapeptide analogues. Two novel, structured, non-peptidic molecules were developed that are capable of holding the bioactive tetrapeptide sequence of somatostatin analogues in a β-turn conformation, as measured by somatostatin receptor (SSTR) binding. Template-constrained cyclic peptides in which the ends of the -Tyr- d-Trp-Lys-Val-tetrapeptide were linked by scaffolds based on either an N,N′-dimethyl- N,N′-diphenylurea or a substituted biphenyl system ( DJS631 and DJS811, respectively), bound selectively to mouse SSTR2B and rat and human SSTR5 with affinities as high as 1 nM. DJS811, at a dose of 3 mg/kg/day, was shown in a mouse Matrigel model to inhibit angiogenesis to a level of 79%. The development of structured turn scaffolds allows β-turn sequences to be contained in the context of a compact structure, with less peptidic nature and potentially greater bioavailability than cyclic hexapeptides. These systems can be used to study the determinants of β-turn formation, as well as to probe the importance of turn sequences occurring in molecular recognition interactions. The antiangiogenic activity of DJS811 suggests that it may have antitumor activity as well. In addition, because SSTR2 is overexpressed on many types of tumors, DJS631 and DJS811 may be useful in the development of agents for tumor imaging or the radiotherapy of cancer.

Chimeric Peptides of Statherin and Osteopontin That Bind Hydroxyapatite and Mediate Cell Adhesion

Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moalpha(v) melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moalpha(v) subpopulation demonstrated that the alpha(v)beta(3) integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.

In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope.

Celiac disease (CD) is an increasingly diagnosed enteropathy (prevalence, 1:200-1:300) that is induced by dietary exposure to wheat gliadins (as well as related proteins in rye and barley) and is strongly associated with HLA-DQ2 (alpha1*0501, beta1*0201), which is present in over 90% of CD patients. Because a variety of gliadin peptides have been identified as epitopes for gliadin-specific T-cell clones and as bioactive sequences in feeding studies and in ex vivo CD intestinal biopsy challenge, it has been unclear whether a 'dominant' T-cell epitope is associated with CD. Here, we used fresh peripheral blood lymphocytes from individual subjects undergoing short-term antigen challenge and tissue transglutaminase-treated, overlapping synthetic peptides spanning A-gliadin to demonstrate a transient, disease-specific, DQ2-restricted, CD4 T-cell response to a single dominant epitope. Optimal gamma interferon release in an ELISPOT assay was elicited by a 17-amino-acid peptide corresponding to the partially deamidated peptide of A-gliadin amino acids 57-73 (Q65E). Consistent with earlier reports indicating that host tissue transglutaminase modification of gliadin enhances gliadin-specific CD T-cell responses, tissue transglutaminase specifically deamidated Q65 in the peptide of A-gliadin amino acids 56-75. Discovery of this dominant epitope may allow development of antigen-specific immunotherapy for CD.

Structure and dynamics of peptide-amphiphiles incorporating triple-helical proteinlike molecular architecture.

Organized polymeric assemblies that incorporate bioactive sequences and structures are finding important applications for the study of protein structure-function relationships. We have recently described a heteropolymeric peptide-amphiphile system that forms organized structures in solution and on surfaces. While the overall three-dimensional features of peptide-amphiphiles have been studied previously, the precise environment of specific residues, particularly those within biologically active regions, have not been examined in detail. In the present study, we have used heteronuclear single quantum coherence (HSQC) and inverse-detected 1H-15N NMR spectroscopy to examine the structure and dynamics of a peptide and peptide-amphiphile that incorporate the alpha1(IV)1263-1277 ([IV-H1]) amino acid sequence from type IV collagen. Three variants of the sequence (Gly-Pro-Hyp)4-[IV-H1]-(Gly-Pro-Hyp)4 were constructed with a single 15N-labeled Gly placed in the middle of the N-terminal (Gly-Pro-Hyp)4 region (residue Gly7), in the middle of the [IV-H1] sequence (residue Gly19), or in the middle of the C-terminal (Gly-Pro-Hyp)4 region (residue Gly34). These peptides were also N-terminally acylated with hexanoic acid to create an analogous series of 15N-labeled peptide-amphiphiles. HSQC spectra indicated that both the peptide and the peptide-amphiphile were in triple-helical conformation at low temperature, supporting prior circular dichroism (CD) spectroscopic results. The intensities of the triple-helical cross-peaks were stronger for the peptide-amphiphile, consistent with an enhanced triple-helical thermal stability within the peptide-amphiphile construct compared to that of the peptide alone. Relative relaxation values for the peptide-amphiphile monomeric and trimeric species were consistent with those reported previously for other triple-helical peptides. Relaxation measurements indicated that the triple-helical [IV-H1] region did not appear to be dramatically more flexible than the Gly-Pro-Hyp regions. The angle between Gly N-H bonds and the helix dyad axis, determined from the relaxation data, was within the range expected for triple helices. Overall, the peptide headgroup of the C6-(Gly-Pro-Hyp)4-[IV-H1]-(Gly-Pro-Hyp)4 peptide-amphiphile appears to form a continuous triple helix that behaves similarly, in a dynamic sense, to a triple-helical peptide. The enhanced thermal stability of the peptide-amphiphile compared to the analogous triple-helical peptide, along with the multitude of organized structures formed by lipidlike compounds, suggest that peptide-amphiphiles could be utilized as targeted liposomes, sensors, receptors, or enzymes.

Cross-linking exogenous bifunctional peptides into fibrin gels with factor XIIIa.

Bi-domain peptides with a factor XIIIa substrate in one domain and a bioactive peptide in another domain were covalently incorporated into fibrin gels during coagulation through the action of the transglutaminase factor XIIIa. The cross-linking characteristics were determined for two bi-domain peptides with factor XIIIa substrates based on fibrinogen, dYRGDTIGEGQQHHLGG-NH2, and dLRGDGAKDV-NH2, as well as one bi-domain peptide with a substrate sequence based on alpha2-plasmin inhibitor, dLNQEQVSPLRGD-NH2, and another with a nonbiological, oligolysine substrate, dLRGDKKKKG-NH2 (substrate domains in italic). Each of these peptides was able to cross-link into the fibrin gels during coagulation, with the peptide containing the factor XIIIa substrate based on alpha2-plasmin inhibitor being incorporated at levels in excess of 8 mol/mol fibrinogen. The structural characteristics of these peptide-modified gels proved to be the same as those for a native fibrin gel. The bioactivity of the incorporated active factors was tested in a neuronal culture model with day 8 chicken dorsal root ganglia using two bioactive sequences, RGD and DGEA, and one inactive control sequence, RDG. Each of these peptides influenced the extension of neurites from the ganglia as expected, indicating that the incorporated factors retained their activity. With the use of soluble competitive inhibitors, it was shown that this effect was due to the covalently incorporated peptides. Through exploiting the role of factor XIIIa in coagulation, we have developed a method by which to impart the character of nonfibrin proteins, such as extracellular matrix proteins, to fibrin, a biological material with many potential therapeutic and academic applications.

Structural Modification of Fibroblast Growth Factor-binding Heparan Sulfate at a Determinative Stage of Neural Development

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity and appear to act by coupling particular forms of FGF to appropriate FGF receptors. During neural development, one particular HS proteoglycan is able to rapidly switch its potentiating activity from FGF-2, as neural precursor cell proliferation occurs, to FGF-1, as neuronal differentiation occurs. Using various analytical techniques, including chemical and enzymatic cleavage, low pressure chromatography, and strong anion-exchange high performance liquid chromatography, we have analyzed the different HSs expressed during these crucial developmental stages. There are distinct alterations in patterns of 6-O-sulfation, total chain length, and the number of sulfated domains of the HS from the more mature embryonic brain. These changes correlate with a switch in the ability of the HS to potentiate the actions of FGF-1 in triggering cell differentiation. It thus appears that each HS pool is designed to function in the modulation of an intricate interaction with a specific growth factor and its cognate receptor, and suggests tightly regulated expression of specific, bioactive disaccharide sequences. The data can be used to construct a simple model of controlled variations in HS chain structure which have functional consequences at a crucial stage of neuronal maturation.

Potential metabolic bioactivation pathways involving cyclic tertiary amines and azaarenes.

A major theme explored in this review is the MAO-and cytochrome P450-catalyzed alpha-carbon oxidations of selected cyclic tertiary amines to give iminium metabolites that undergo further chemical modifications to form known or potentially toxic products. The most dramatic illustration of this type of bioactivation process is the conversion of the parkinsonian-inducing neurotoxin MPTP (23) by brain MAO-B to the iminium (dihydropyridinium) metabolite 24 which is oxidized further to the pyridinium species MPP+ (25). The selective destruction of nigrostriatal neurons by MPP+ is dependent on a unique sequence of events (transport into the nerve terminals by the dopamine transporter, localization in the inner mitochondrial membrane by electromotive forces, and inhibition of complex I of the mitochondrial electron transport chain) that, fortunately, are unlikely to be encountered with many substances. A second example of a well-documented metabolic bioactivation sequence involves the highly toxic pyrrolizidine alkaloids (102). These compounds undergo cytochrome P450-catalyzed alpha-carbon oxidation which converts the 3-pyrrolinyl moiety present in the parent alkaloids into a pyrrolyl-containing metabolite (105). The presence of labile functional groups results in the spontaneous conversion of 105 to reactive electrophilic products (106 and 108) that undergo Michael addition reactions with nucleophiles on biomacromolecules leading to a variety of toxic outcomes. Less clearly defined are the potential contributions to neurodegenerative processes that may be mediated by low-level, long term exposure to less potent toxins. Examples of potential proneurotoxins are the endogenously formed tetrahydroisoquinolines (such as 40-50) and tetrahydro-beta-carbolines (such as 54) that may be biotransformed to neurotoxic isoquinolinium (such as 51) and beta-carbolinium (such as 52) species in the brain. A similar argument can be made for 4-piperidinols (compounds that are at the same oxidation state as the tetrahydropyridines) which may be metabolized via iminium intermediates to amino enols that spontaneously convert to dihydropyridinium species and hence to pyridinium metabolites (67-->68-->69-->70-->71, Scheme 10). This type of reaction sequence has been well documented with the parkinsonian-inducing neuroleptic agent haloperidol (72) which is metabolized in humans, baboons, and rodents to the pyridinium species HPP+ (75), a potent inhibitor of mitochondrial respiration. Finally, an appreciation of the alpha-carbon oxidations of fully reduced azacycles such as (S)-nicotine (61) and phencyclidine (82) to chemically reactive metabolites that form covalent adducts with proteins, including the enzymes that are responsible for their formation, may prove of toxicological importance when attempting to account for the effects of chronic abuse of these potent drugs.1

Synthesis of multiple peptides on plastic pins.

Scanning protein sequences by bioassay for smaller bioactive peptide sequences requires a source of many peptides homologous with the parent protein sequence. This unit deals with one of the synthetic methods for making such sets of peptides. The strategy of the multiple peptide approach to biological scanning is discussed, along with the synthetic protocols, and the handling of peptides after synthesis, i.e., cleavage, preliminary purification, storage, and analysis. It is specific for the multipin technique using equipment obtained from Chiron Technologies, although some of the approaches are applicable to other multiple synthesis techniques. Procedures for multipin equipment obtained from other suppliers may differ from the procedures described here, and the manufacturer's literature should be consulted. This unit also includes protocols for preparing Fmoc-amino acid solutions and for acetylating or biotinylating synthesized peptides.

ChemInform Abstract: Conformation of Cyclic Peptides. Principle Concepts and the Design of Selectivity and Superactivity in Bioactive Sequences by Spatial Screening

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Conformation of cyclic peptides. Principle concepts and the design of selectivity and superactivity in bioactive sequences by 'spatial screening'

Abstract

Bioactive peptides derived from milk proteins. Structural, physiological and analytical aspects.

The primary function of dietary proteins is to supply the body adequately with indispensable amino acids and organic nitrogen. Little attention has been paid up to date to milk proteins, in particular caseins, that are currently the main source of biologically active peptides, although other animal as well as vegetable proteins are known to contain potentially bioactive sequences. Such regulatory peptides can be released by enzymatic proteolysis of caseins in vitro and in vivo and may act as potential physiological modulators of metabolism during the intestinal digestion of the diet. It has been proved that bioactive peptides derived from caseins, such as beta-casomorphins and phosphopeptides, can be released during gastrointestinal passage. It is also evident that peptides originating from food proteins should be taken into account as potential modulators of various regulatory processes in the body. The possible regulatory effects concern nutrient uptake (phosphopeptides, casomorphins), postprandial hormone secretion (casomorphins), immune defense (immunopeptides, casokinins, casomorphins) and neuroendocrine information transfer (casokinins). The advances in the research field of bioactive peptides are driven by a molecular understanding of biological processes, and analytical techniques are a critical component of this understanding. Different up-to-date methods, including peptide synthesis and immunochemistry, have been applied to the chemical characterization of bioactive peptides. Especially casein derived peptides have already found interesting applications, both as dietary supplements (phosphopeptides) and as pharmaceutical preparations (phosphopeptides, beta-casomorphins). The question of 'what kinds of bioactive peptides are beneficial and desirable as food constituents or as drugs' should be always carefully examined. However, the possibilities for the design of dietary products and 'natural' drugs look promising.

Conformational Properties of the Unnatural Amino Acid .beta.-Methylphenylalanine in a Linear Octapeptide System; Correlations of 13C-NMR Chemical Shifts with the Side-Chain Stereochemistry of These Amino Acid Residues

Conformational properties of the four stereoisomers ([2S,3S], [2S,3R], [2R,3S], and [2R,3R]) of a synthetic amino acid, β-methylphenylanaline (β-MePhe), in a bioactive octapeptide sequence of CCK, H-Asp 1 -Tyr 2 -β-MePhe 3 -Gly 4 -Trp 5 -Nle 6 -Asp 7 -Phe 8 -NH 2 , have been studied by using 1 H and 13 C-2D NMR spectroscopy. β-MePhe 3 residues introduce significant perturbations to the side-chain conformations. On the basis of the rotamer populations determined by a combination of homonuclear and heteronuclear vicinal coupling constants, each of the four different stereoisomers of β-MePhe residues virtually eliminates one of the three staggered side-chain conformations, trans for (2S,3S)- and (2R,3R)-β-MePhe, gauche(+) for (2S,3R)-β-MePhe, and gauche(-) for (2R,3S)-β-MePhe, respectively

βVI Turns in peptides and proteins: A model peptide mimicry

To investigate the role of proline in defining beta turn conformations within cyclic hexa- and pentapeptides we synthesized and determined the conformations of a series of L- and D-proline-containing peptides by means of 2D NMR spectroscopy and restrained molecular dynamics simulations. Due to cis/trans isomerism the L-proline peptides adopt at least two different conformations that are analyzed and compared to the structures of the corresponding D-proline peptides. The cis conformations of the compounds cyclo(-Pro-Ala-Ala-Pro-Ala-Ala), cyclo(-Arg-Gly-Asp-Phe-Pro-Gly-), cyclo(-Arg-Gly-Asp-Phe-Pro-Ala-), cyclo (-Pro-Ala-Ala-Ala-Ala-), and cyclo(-Pro-Ala-Pro-Ala-Ala-) form uncommon beta VI turns that mimic the turn geometries found in crystallographically refined protein structures at such a detailed level that even preferred side chain orientations are reproduced. The ratios of the cis/trans isomers are analyzed in terms of the steric demand of the proline-following residue. The conformational details derived from this study illustrate the importance of the examination of small model compounds derived from protein loop regions, especially if bioactive recognition sequences, such as RGD (Arg-Gly-Asp), are incorporated.

Synthetic immunogens.

Chimeras of the double chain bis-cystinyl hinge fragment 225-232/225'-232' of the human IgG1 and of peptides related to human little-gastrin were synthesized, whereby the fully bioactive gastrin sequences 2-17 and 5-17 were amide-bond-linked N- and N- or C-terminally, respectively, to the hinge peptide. All the dimeric constructs proved to be efficient immunogens; however, both the configuration of the constructs and the length of the haptenic gastrin molecule were found to drastically affect the specificity of the antibody response and, thus, the type of dominant immune epitope expressed. The different degree of accessibility of the gastrin chains in the dimers is similarly reflected by their binding affinities to gastrin receptors and their bioactivities in vivo. Molecular dynamics simulations of the chimeric compounds clearly revealed that the conformational space of the gastrin peptide chains 2-17 and 5-17 is strongly restricted upon linkage to the hinge peptide. Only in the gastrin-(2-17) construct does sufficient free conformational space seem to be retained, at least for one of the two gastrin chains, in order to allow folding into the bioactive structure. This also agrees with the observation that the dimeric gastrin-(2-17) behaves like a gastrin monomer in terms of receptor binding affinity and biopotency in vivo; but it could additionally explain why an antibody response of gastrin receptor-like specificity could only be induced with this construct. The experimental data may therefore suggest a high degree of parallelism between the mechanism of recognition of the gastrin peptides in the dimeric constructs as hormonal ligands by the gastrin receptors and as haptens by the immune competent cells.

Thymosin-like peptides as potential immunostimulants. Synthesis via the polymeric-reagent method

This paper reports our attempt at designing new immunostimulating peptides which are chemically related to the bioactive peptides thymosin alpha 1 and thymopentin. Three peptides were synthesized, Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr (3), Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (2), and Asp-Leu-Lys-Glu-Arg-Lys-Asp-Val-Tyr-Glu-Glu-Ala-Glu-Asn (1), each of which contains the thymopentin sequence and portions of the bioactive sequence of thymosin alpha 1. Peptides 1-3 were assembled from selected blocked fragments that were synthesized by the polymeric-reagent method, using PHBT (polystyrene-bound 1-hydroxybenzotriazole) as the activating polymer. The ability of peptides 1-3 to enhance the activation (RNA synthesis) and proliferation (DNA synthesis) of human T lymphocytes was determined. In comparison to thymosin alpha 1, thymosin alpha 1 (15-28), and thymopentin, peptides 1-3 did not show significant enhancement of these processes.

Biologically active peptides in milk proteins.

Bioactive peptides have been identified as digestion products of several food proteins. All the bioactive sequences are hidden in an inactive state inside the polypeptide chain of the larger protein. Milk proteins are a rich source of biologically active peptides such as exorphins (casomorphins), phosphopeptides and immunopeptides. Such peptides are released during intestinal digestion of caseins and whey proteins. They may be involved in regulation of nutrient entry and influence the postprandial metabolism via stimulation of the secretion of hormones. Furthermore, they may exert a stimulating effect on the immune system. These findings offer new aspects for evaluating the nutritive value of food proteins. Moreover, bioactive peptides have already found interesting applications as dietary supplements and as pharmaceutical preparations.