Pepsin Treatment Research Articles

Analysis of Matrix Protein Components of the Dermis-Like Structure Formed in a Long-Term Culture of Human Fibroblasts: Type VI Collagen Is a Major Component

Formation of a dermis-like structure by a long-term culture of fibroblasts in the presence of ascorbic acid is a potential model for tissue organization or wound healing, and has its practical use as a skin graft. In the present study, solubilization of the dermis-like structure without pepsin treatment was attempted for analysis of pepsin-labile matrix components that might be involved in the formation of the dermis-like structure, as well as quantification of mutated type I collagen that could be susceptible to pepsin. The whole dermis-like structure was dissolved in a Tris buffer containing SDS and urea at 80 degreesC. Analysis of the extract by SDS-PAGE revealed several protein bands that were not found in the pepsin-treated extract. Among them, the polypeptide band migrating at 140k under reducing condition showed a similar intensity of protein staining to the alpha2(I) chain band. The N-terminal amino acid sequences of cyanogen bromide peptides derived from the 140k polypeptide band as well as the amino acid composition of the band suggested that the band essentially consisted of alpha1(VI) and alpha2(VI) chains. The results demonstrated that the type VI collagen was a major component, being a comparable in amount to type I collagen, in the dermis-like structure.

Thermal Stability of Bone Collagen as an Indicator of Bone Turnover in Gonadectomized and Multiparous Rats

Previous Findings indicate that the thermal stability of bone collagen is related to age. In this study, collagen from rat bone with reported different turnover rates was investigated. Cortical and trabecular bone from femur were obtained from intact, ovariectomized, orchidectomized and multiparous breeder rats. Thermal stabilities of fibrillar collagen in decalcified bone matrix and molecular collagen obtained by pepsin treatment were measured as shrinkage (Ts) and 'melting' temperature (Tm), respectively. Both Ts and Tm of cortical collagen from intact female rats decreased in parallel with age as previously found in male rats indicating that Ts and Tm measurements are interchangeable techniques in characterizing the thermal stability of bone collagen. Tm of trabecular collagen from intact rats decreased with age, however, with a decay only one-third of that for cortical collagen. The different rates possibly reflect different ages of collagen due to remodeling activity present in trabecular and minimal in cortical bone. Compared with control rats the Tm of trabecular collagen from gonadectomized and multiparous rats with a reported increased trabecular turnover rate was elevated, whereas only minor variations in Tm of cortical collagen were found. In conclusion, the thermal stability of bone collagen decreases with the age of the collagen. Increased bone turnover implies elevated thermal stability of bone collagen. Thus, thermal stability of bone collagen appears to be an indicator of bone turnover.

Gelation of Porcine Globin by Pepsin Treatment.

The effects of pepsin treatment on the gelation of porcine globin were studied by measurements of surface hydrophobicity, extent of hydrolysis, gel strength and polyacrylamide gel electrophoresis. Gel formation occurred below pH 4.0 at 30-50°C above 3% globin concentration. After 48-h incubation at pH 3.0 and 50°C in the pepsin concentration used (0.005-1.0% (E/S)), 0.01% (enzyme-substrate ratio: E/S) pepsin gave the highest gel strength. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed several peptides with molecular weight of 4000-6000 to be present in the resultant gel after 48 h of pepsin treatment. Gel strength after 12-h incubation was highly correlated with surface hydrophobicity and markedly elevated at 2-3% hydrolysis. As longer incubation time (over 12 h) is required for maximum gel strength, the gelation of globin would thus appear to occur as follows: pepsin yields peptides from globin, which aggregate to produce a three-dimensional gel network during incubation.

Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that catalyzes the first step leading to the C-terminal amidation of glycine-extended peptides. The catalytic core of PHM (PHMcc), refined to residues 42-356 of the PHM protein, was expressed at high levels in CHO (DG44) (dhfr-) cells. PHMcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified PHMcc under nonreducing conditions cleaved the protein at Lys219, indicating that the protein consists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-linked peptides generated by sequential CNBr and pepsin treatment of radiolabeled PHMcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal domain (Cys47-Cys186, Cys81-Cys126, and Cys114-Cys131), along with three of the His residues critical to catalytic activity (His107, His108, and His172). Two disulfide linkages (Cys227-Cys334 and Cys293-Cys315) occur in the C-terminal domain, along with the remaining two essential His residues (His242, His244) and Met314, thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr79 or Tyr318 with Phe increased the Km of PHM for its peptidylglycine substrate without affecting the Vmax. Replacement of Glu313 with Asp increased the Km 8-fold and decreased the kcat 7-fold, again identifying this region of the C-terminal domain as critical to catalytic activity. Taking into account information on the copper ligands in PHM, we propose a two-domain model with a copper site in each domain that allows spatial proximity between previously described copper ligands and residues identified as catalytically important.

Effect of a Wheat Protein on pH‐Dependent Water‐Binding Capacity and Viscosity of Wheat Tailings Fractions

ABSTRACTWheat flour was fractionated with acetic acid using a mortar and pestle method or a blender method. Higher pH‐dependent water‐binding capacity (WBC) and viscosity were obtained only in the tailings fraction. The higher pH‐dependent WBC was rather stable at 5–37°C, however it decreased with salt addition. Pepsin or bromelain treatment stopped the pH‐dependent changes in WBC and viscosity, which suggests that this characteristic of the tailings fraction is due to the presence of proteins. HPLC indicated that the Mr of the proteins associated with high WBC at low pH was >200 kDa.

Cytoprotective effect of epidermal growth factor on acid- and pepsin-induced damage to rat gastric epithelial cells: roles of Na+/H+ exchangers.

We have previously established a cell damage model, with damage induced by either acid or pepsin treatment for 30 min, involving a rat gastric epithelial cell line (RGM1). In the present study, pretreatment of cells with epidermal growth factor (EGF; 0.1-10 ng/mL) or sucralfate (0.1-3 mg/mL) for 4 h prevented such cell damage in a concentration-dependent manner. Protection of cells by these drugs was not affected by pretreatment with indomethacin (10(-5) mol/L) for 4 h. Removal of Na+, but not Ca2+, from the acidified medium totally abolished the inhibitory effect of EGF, but not that of sucralfate. Genistein (a tyrosine kinase inhibitor) apparently reduced the inhibitory effect of EGF. DNA synthesis by RGM1 cells did not increase when cells were incubated with EGF for 4 h. We conclude that both EGF and sucralfate protect RGM1 cells from acid- and pepsin-induced damage and that the mechanism of protection by EGF against acid-induced damage seems to be via activation of Na+/H+ exchangers.

Virus inactivation by pepsin treatment at pH 4 of IgG solutions: factors affecting the rate of virus inactivation.

IgG preparations have rarely transmitted infectious diseases; however, because such transmission has occurred a few times, manufacturers are required to present experimental proof that their specific production process removes and/or inactivates viruses that may be present in the starting material. The kinetics of virus inactivation mediated by pepsin treatment at pH 4 during the production of intravenous immunoglobulin was assessed with spiking experiments using human immunodeficiency virus, bovine viral diarrhea virus, Semliki Forest virus, and pseudorabies virus. The influence of various factors on the rate of virus inactivation also was studied by modifying the composition of the IgG solutions with respect to IgG, sucrose, and NaCl content. Virus inactivation at 37 degrees C was extremely rapid and resulted in a complete loss of infectivity within 5 minutes to 1 hour. Inactivation was much slower at lower temperatures. Furthermore, inactivation was dependent on the solute composition. Increasing the sucrose content from 0 to 15 percent reduced the rate of inactivation of pseudorabies virus but did not affect the rate of inactivation of Semliki Forest virus. In contrast, increasing the NaCl content from 0 to 150 mM resulted in a reduction in the rate of inactivation of Semliki Forest virus, whereas the rate of inactivation of pseudorabies virus remained unaffected. Moreover, increasing the IgG concentration from 0 to 10 percent resulted in an increased rate of inactivation of pseudorabies virus but a decreased rate of inactivation of Semliki Forest virus. Inactivation of viruses by pepsin treatment at pH 4 essentially is temperature-dependent, and the reaction rate is selectively influenced by the solute composition of the IgG solution. This has to be taken into account when safety data for different products are compared.

Collagen expression in chicken tibial dyschondroplasia.

Collagen expression in growth plate cartilage derived from broiler chickens with tibial dyschondroplasia was studied and compared with samples from unaffected birds. Normal growth plate contains 12% collagen (dry weight) and dyschondroplastic growth plate 19% collagen compared with articular cartilage, which contains 55%. Dyschondroplastic growth plate collagens were more resistant to extraction by pepsin treatment than were those from unaffected growth plate. Normal and dyschondroplastic growth plate cartilages contain similar amounts of type I collagen (5% of the total collagen) but dyschondroplastic growth plate cartilage contains slightly less type II and type XI collagens, and significantly more type X collagen (25% as compared to 11%) than in normal growth plate. The levels of the mature collagen cross-link, hydroxylysyl-pyridinoline, are very low in normal growth plate but are six times higher in dyschondroplastic lesions. Immunolocalisation studies show that there is little change to the normal patterns of collagen organisation in dyschondroplastic growth plate. Investigation of metalloproteinase activity showed there to be a reduction in MMP-2 levels in dyschondroplastic growth plate compared to normal growth plate. In vitro studies on articular, normal growth plate and dyschondroplastic growth plate chondrocytes cultured in alginate or on plastic revealed differences between the cell types. When plated on plastic, articular chondrocytes rapidly assume a fibroblastic morphology. In contrast, normal growth plate chondrocytes retain their polygonal morphology whereas chondrocytes derived from dyschondroplastic cartilage initially exhibit both fibroblastic and polygonal phenotypes but gradually change to totally fibroblastic. These morphological changes are reflected by the collagen synthesis in vitro. Chondrocytes derived from normal articular cartilage synthesised collagen types I, II and X when cultured in alginate but type X synthesis was lost when cultured on plastic. Chondrocytes derived from normal growth plate cartilage synthesised predominantly type X collagen when cultured in either system. Chondrocytes derived from dyschondroplastic growth plate exhibited a similar phenotype to normal growth plate chondrocytes when cultured in alginate beads, but showed signs of dedifferentiation with reduced type X collagen and increased type I collagen when plated on plastic. These results suggest that the chondrocytes in dyschondroplastic growth plate cartilage are at a different stage of maturity than normal resulting in a cartilage that is failing to turn over at a normal rate.

Purification of β-Lactoglobulin from Whey Protein Concentrate by Pepsin Treatment

β-Lactoglobulin was purified from whey protein concentrate by a combination of pepsin treatment and membrane filtration. Porcine pepsin was added to whey protein (1:200, wt/wt), and the mixture was then incubated at pH 2.0 and 37°C for 60min. The protein fraction was collected by ammonium sulfate precipitation, and the precipitate was either dialyzed against water using a dialysis membrane (20-kDa pore size) of filtered using an UF membrane (30-kDa pore size). The β-LG did not differ from standard β-LG as measured by chromatography, SDS-PAGE, native PAGE, differential scanning calorimetry, or UV spectrum. Based on the results, a simplified procedure was developed, consisting of pepsin treatment and UF, to purify β-LG directly from whey.

Evaluation of pepsin treatment for electron microscopic RNA in situ hybridization on ultra-thin cryosections of cultured cells

The in situ hybridization (ISH) technique, as applied to electron microscopic detection of RNAs, was evaluated for ultra-thin cryosections of cultured rat fibroblasts (rat 9G). Experimental variables to balance penetration of detection reagents and preservation of ultrastructural morphology included various strengths of aldehyde fixation and pepsin treatment. We performed ISH for 28S ribosomal RNA (rRNA) followed by ultra-small colloidal gold immunocytochemistry and silver enhancement. An acceptable balance for 28S rRNA ISH detection was obtained using mild cross-linking fixation followed by treatment with a relative high concentration of pepsin for a short time. The ISH method presented in this study was compatible with immunocytochemical detection of protein as demonstrated by double-labeling experiments.

Influence of Streptococcus dysgalactiae surface hydrophobicity on adherence to mammary epithelial cells and phagocytosis by mammary macrophages.

Bacterial surface hydrophobicity (SH) plays a role in adhesion of bacteria to host surfaces and ingestion by phagocytic cells. Streptococcus dysgalactiae (n = 60) isolated from bovine intramammary infections were examined for expression of SH after growth in Todd-Hewitt broth (THB) and THB supplemented with skim milk, whey, lactose, and casein. Strains were significantly more hydrophobic after growth in THB and THB plus whey and more hydrophilic after growth in THB plus skim milk. Both trypsin and proteinase K abolished SH in three strains tested. Mild pepsin treatment had little effect on SH, while heat treatment at 70 degrees or 80 degrees C abolished SH in two strains tested. A hydrophilic strain of S. dysgalactiae did not adhere as well to bovine mammary epithelial cells as a hydrophobic strain. Trypsin treatment significantly reduced adherence of a hydrophobic strain of S. dysgalactiae to epithelial cells while adherence of a hydrophilic strain remained unaltered. A hydrophilic strain of S. dysgalactiae was significantly more resistant to phagocytosis by bovine mammary gland macrophages than a hydrophobic strain. Differences in expression of SH may play an important role in determining the ability of S. dysgalactiae to establish successfully within the mammary gland.

The avian eggshell extracellular matrix as a model for biomineralization.

The avian eggshell is a complex, extracellularly assembled structure which contains both mineralized and non-mineralized regions. The composition of the hen eggshell organic matrix was examined by immunohistochemistry with antibodies to different extracellular matrix molecules. Type I collagen is found in the shell membranes, but only after treatment of the tissue sections with pepsin. When incomplete eggshells are removed from the oviduct and immunostained, type I collagen can be detected in the shell membranes without pepsin treatment. The shell membranes, which are non-mineralized, also contain type X collagen, and this immunostaining does not require pepsin treatment. The occurrence of type X collagen in the shell membranes is surprising, since this collagen has not been found in any tissue other than hypertrophic cartilage. Immunostaining for various glycosaminoglycans shows the presence of keratan sulfate and dermatan sulfate. Several different antibodies to keratan sulfate stain different regions of the eggshell; one keratan sulfate epitope is prominent in the calcium reserve assemblies. Dermatan sulfate staining is very intense in the palisade region. Demineralized matrix from the palisade region was extracted with guanidine and fractionated by ion exchange chromatography. A approximately 200-kDa dermatan sulfate proteoglycan is found in these extracts, along with a number of protein components. This preparation was tested for its ability to affect calcium carbonate crystal formation in vitro. Pieces of demineralized shell membranes were used as a substrate for crystal formation and various amounts of the palisade matrix dermatan sulfate proteoglycan preparation were added to the solution from which the crystals were formed. This material causes a concentration-dependent change in crystal morphology to one in which the crystals are smaller and more rounded, which more closely approximates the crystals normally observed in eggshells. These results suggest that the dermatan sulfate proteoglycans may be important in modulating crystal morphology in the hen eggshell and correlate with mineralization-modulating biomolecules from other calcified tissue, which are generally anionic.

Monitoring morphology and signal during non-radioactive in situ hybridization procedures by reflection-contrast microscopy and transmission electron microscopy.

We analyzed the effects of steps in RNA in situ hybridization (ISH) procedures on morphology and hybridization signal with reflection-contrast microscopy (RCM) and transmission electron microscopy (TEM). In chessboard experiments, a range of fixatives containing formaldehyde, glutaraldehyde, or both, and various permeabilization protocols, including ethanol and pepsin treatment, were investigated. A transfected rat fibroblast cell line that harbors an inducible human cytomegalovirus immediate early (IE) transcription unit, and specific probes for 28S ribosomal RNA and IE messenger RNA were used for this purpose. Probes were labeled with digoxigenin and hybrids were detected with anti-digoxigenin F(ab)2 fragments conjugated to horseradish peroxidase, followed by diaminobenzidine/H2O2 reaction. Effects of fixation and pre-treatments on RNA detection efficiency and morphology were monitored by RCM on whole cells. After Epon embedding and ultra-thin cross-sectioning, the corresponding TEM images were obtained. With the pre-treatments analyzed, it appeared impossible to find an acceptable balance between ISH signals and preservation of ultrastructural morphology: when good signal-to-noise ratios are obtained, the ultrastructural morphology is already deteriorated. We discuss the parameters that influence the fragile balance between high RNA detection efficiency and good preservation of ultrastructure and the benefit of RCM monitoring in the development and procedures for pre-embedding electron microscopic ISH.

VV-hemorphin-7 and LVV-hemorphin-7 released during in vitro peptic hemoglobin hydrolysis are morphinomimetic peptides

Two opioid peptides were generated by in vitro pepsin treatment of bovine hemoglobin. These peptides were identified using a GPI test and purified using HPLC chromatographic techniques. They correspond to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin-7) of the beta-chain of bovine hemoglobin. Binding experiments strongly confirm that VV-hemorphin-7 and LVV-hemorphin-7 are opioid peptides since they inhibited [3H]naloxone binding to rat brain membranes. Our results indicate that VV-hemorphin-7 and LVV-hemorphin-7 exhibit a lesser potency both in GPI and binding tests. Selectivity and affinity of these purified peptides and synthetic hemorphin-7 for opioid receptors is discussed.

The preparation of catalytically active human cathepsin B from its precursor expressed in Escherichia coli in the form of inclusion bodies.

A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/l E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three-amino-acid extension at its N-terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.

Recognition of cryptic sites in human and mouse laminins by rat osteoclasts is mediated by β3 and β1 integrins

Laminins may be encountered by osteoclasts and their precursors in basement membranes when they migrate from periosteal vasculature during skeletal development and in pathological situations. We have examined the recognition by osteoclasts of intact laminins and their proteolytic derivatives, and analysed the mechanism of adhesion. Rat osteoclasts fail to bind intact mouse Engelbreth-Holm-Swarm (EHS) laminin (3% adhesion relative to adhesion to foetal calf serum proteins) and bind only weakly to native human placental laminin (13%) or human merosin (9%). Pepsin treatment of native mouse EHS and human laminins increased osteoclast adhesion. Rat osteoclasts adhered to mouse EHS laminin-derived P1 fragment (70%), but failed to bind the E8 fragment, which contains adhesion sites recognised by some integrins. Binding to human and mouse P1 laminins was abolished by treatment with RGD-containing peptides and required divalent cations, but not by YIGSR peptide. Combinations of monoclonal antibodies to rat β3 and ∝v integrins reduced binding to P1 fragment by 91% and to human laminin by 72%, demonstrating that the major integrin involved in rat osteoclast adhesion to proteolysed laminin is ∝vβ3. Antiserum to β1 integrin inhibited adhesion to human laminin by 40%, but to P1 fragment by only 8%; this suggests that β1 integrin(s) contribute to osteoclast adhesion to human laminin but probably not to P1 fragment. The involvement of ∝vβ3 integrin was confirmed using a recombinant human ∝vβ3 solid phase binding assay. ∝vβ3 bound to mouse P1 fragment and proteolytically digested human laminin, but not intact lamming. Binding of the ∝vβ3 receptor to both laminins was significantly inhibited by RGD peptides and by monoclonal antibody 9C9 to human β3 integrin. In conclusion, our findings demonstrate that rat osteoclasts bind to laminin only when cryptic peptide sites become functional after exposure by proteolytic cleavage, and that both β3 and, to a lesser degree, β1 integrins are involved in this process.

A Novel Proteoglycan Synthesized and Secreted by Chondrocytes of the Superficial Zone of Articular Cartilage

A novel proteoglycan (PG) has been identified in culture medium from thin slices of the superficial zone of bovine articular cartilage. This PG is synthesized and secreted selectively by chondrocytes of this zone but has not been demonstrated in culture medium from slices deeper in the same tissue. There is little, if any, incorporation of this PG into the extracellular matrix. The PG has been partially purified by isopycnic CsCl density gradient ultracentrifugation, ion-exchange chromatography on DEAE Sephacel, and gel filtration chromatography on Sepharose CL-2B. It migrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of approximately 345 kDa. The molecule is degraded by papain, trypsin, or pronase; however, limited pepsin treatment performed at 4°C only decreases its molecular weight to approximately 315 kDa. The molecule is substituted with keratan sulfate and chondroitin sulfate, which are largely removed by limited pepsin treatment. In addition, this PG, or a very similar molecule, has been demonstrated in synovial fluid. This novel PG may serve as a functional metabolic marker for chondrocytes of the superficial zone of articular cartilage.

Stimulation of calcification of growth plate cartilage matrix vesicles by binding to type II and X collagens

Matrix vesicles (MV), microstructures which rapidly accumulate Ca2+ and induce mineral formation in vitro, are linked to type II and X collagens and proteoglycans in the hypertrophic cartilage. However, the roles of these matrix proteins on MV function are not known. This led us to investigate the influence of type II and X collagen binding on Ca2+ uptake by MV. MV isolated from chicken growth plate cartilage were treated with pure bacterial collagenase and 1 M NaCl in synthetic cartilage lymph to selectively and completely remove associated type II and X collagens. Uptake of 45Ca2+ by these collagen-depleted vesicles was markedly reduced. Further treatment with detergent, which disrupted the membrane, restored Ca2+ uptake, indicating that the vesicle membrane structure and the nucleational core inside the vesicle lumen were still intact after the collagenase and 1 M NaCl treatments. Readdition of either native type II or X collagen to the collagenase, 1 M NaCl-treated MV stimulated their Ca2+ uptake to levels similar to those of untreated vesicles. Pepsin-treated type II and X collagens were less effective in stimulating Ca2+ uptake, indicating that non-triple helical domains of these collagens were involved. The pepsin treatment of these collagens also decreased their binding to annexin V (anchorin CII), one of three annexins found in MV, suggesting that annexin V is involved in mediating the binding of type II and X collagens to the MV surface. Furthermore, treatment of collagenase, 1 M NaCl-treated MV with chymotrypsin, which damaged annexin V as well as many other MV proteins, prevented the stimulation of Ca2+ uptake by these collagens. Thus, the interaction between type II and X collagens with MV activates the influx of Ca2+ into MV and may play an important role in calcification of the vesicles.

Proliferative responses towards native, heat-denatured and pepsin-treated ovalbumin by peripheral blood mononuclear cells from patients with hen's egg-sensitive atopic dermatitis.

In order to clarify the mechanism of food-antigen recognition, the proliferative responses of peripheral blood mononuclear cells (PBMCs) to native, heat-denatured or pepsin-treated ovalbumin (OA) were investigated in 16 hen's egg-sensitive patients with atopic dermatitis (AD). Seven of them had hypersensitivity to boiled hen's egg and others had not. The responses of PBMCs to heat-denatured OA were lower than those to native OA in the patients without hypersensitivity to boiled hen's egg. However, there were no differences of the responses of PBMCs between heat-denatured OA and native OA in the patients with hypersensitivity to boiled hen's egg. Moreover, the reduction of the responses of PBMCs to pepsin-treated OA was recognized in six out of seven patients. The primary structure of OA did not change by heating or pepsin treatment according to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results suggested that the secondary structure of OA changed in connection with the reduction of the responses of PBMCs to denatured OA. In addition, we demonstrated the suppressive effect of anti-HLA-DR and anti-HLA-DQ monoclonal antibodies on the proliferative response of PBMCs to OA. The results suggested that the proliferative responses of PBMCs to OA were restricted by HLA-DR or HLA-DQ in hen's egg-sensitive patients with AD.

Purification and characterization of enkephalin-related peptides released by in vitro peptic digestion of bovine plasma proteins

In vitro pepsin treatment of plasma proteins generates biologically active peptides such as enkephalin-related peptides. These peptides were characterized using chromatographic techniques along with a radioimmunoassay procedure involving the use of Leu-enkephalin and Met-enkephalin antisera. Serum albumin is the only existing source of Met-enkephalin-immunoreactive peptides. One of these peptides consists of nine residues with the sequence NH 2-Glu-Lys-Leu-Gly-Glu-Tyr-Gly-Phe-Gln; a second immunoreactive peptide might be the hexapeptide NH 2-Gly-Glu-Tyr-Gly-Phe-Gln, which has been already identified in a rat serum albumin hydrolysate. Our results indicate that immunoglobulins constitute the main source of Leu-enkephalin-immunoreactive peptides. Immunoreative NH 2-Tyr-Phe-Leu was isolated from pepsin-treated bovine immunoglobulins. Binding experiments and cyclic nucleotide measurements suggested that this peptide was an enkephalin-related peptide. Similar experiments could be carried out to identify the proteins that contain enkephalin-like peptide sequences with the view to investigating the various biological processes occurring in enzymatically treated proteins.