The elastin receptor shows structural and functional similarities to the 67-kDa tumor cell laminin receptor

Laminin- and elastin-binding proteins were isolated by ligand affinity chromatography from plasma membranes of fetal bovine auricular chondroblasts and human A2058 melanoma cells. From both cell types, a 67-kDa protein was identified which bound to either elastin or laminin affinity resins. Structural and functional similarities between the elastin and laminin-binding proteins were suggested by 1) cross-reactivity between antibodies directed against the two proteins; 2) elution of the laminin receptor from laminin columns with soluble elastin peptides; and 3) modulation of substrate binding by galactoside sugars. In addition, extraction properties indicate that both receptors are peripheral membrane proteins whose association with the cell surface is mediated by their lectin properties. Mapping of the binding site on laminin suggests that the 67-kDa chondroblast receptor interacts with a hydrophobic elastin-like sequence in domain V of the B1 chain, and chemotaxis studies indicate that cell migration to elastin peptides and laminin involves the same receptor.     

Development of an oxyntomodulin/glicentin C-terminal radioimmunoassay using a "thiol-maleoyl" coupling method for preparing the immunogen

Oxyntomodulin (OXM) and glicentin, two peptides processed from proglucagon, both contain the glucagon sequence and a C-terminal basic octapeptide, KRNRNNIA extension. A method to produce antibodies, directed specifically toward the C-terminal extension of these two peptides, was developed; it consisted of the use of thioled bovine serum albumin conjugated with the synthetic N-maleoyl C-terminal octapeptide as the immunogen. Three rabbits (FAN, LEG, and PIP) generated antisera with affinity constants close to 5 X 10(10) M-1. In the radioimmunoassay system, these antisera showed a 100% cross-reactivity with OXM, partially purified rat and human glicentin, and the C-terminal 19-37 OXM fragment. They displayed no cross-reactivity toward the glucagon molecule. The cross-reactivity of C-terminal fragments of OXM demonstrated that the epitope involves the C-terminal hexapeptide and that the two last amino acid residues are essential for the binding. The high-performance liquid chromatography elution profiles of human jejunum or rat intestinal extracts obtained by radioimmunoassay with LEG antiserum showed two major peaks which had the same retention times as OXM and glicentin markers. Thus, the major end products in the human and rat small intestine are OXM and glicentin. In human or rat pancreas, the two main peaks detected were glucagon and the C-terminal hexapeptide of OXM/glicentin. Small amounts of OXM were also found in pancreas, whereas no significant quantities of glicentin could be detected. The "thiol-maleoyl" coupling method described here, and applied to produce C-terminal OXM/glicentin specific antisera, might be of general use to obtain antibodies against a well-defined epitope.     

Alz 50, a monoclonal antibody to Alzheimer's disease antigen, cross-reacts with tau proteins from bovine and normal human brain

Microtubule-associated protein tau from bovine brain reacted on immunoblots and on enzyme-linked immunosorbent assay with a monoclonal antibody, Alz 50, which has previously been found to bind to an Alzheimer disease-specific antigen. The apparent affinity of binding of Alz 50 to tau was 2.1 X 10(-9) M on competitive enzyme-linked immunosorbent assay, and it was in the same range as for Tau-1 (0.5 X 10(-9) M), an antibody raised against purified bovine tau proteins. Immunoblotting of trypsin-digested tau revealed differences between Alz 50 and Tau-1 binding sites. The binding of both antibodies to tau was not affected by prior treatment with phosphatase, indicating that the cross-reactivity of Alz 50 with tau is due to the presence of phosphate-independent epitope. This epitope then differs from phosphate-dependent tau epitopes often shared with other cytoskeletal proteins. Alz 50 and Tau-1 binding sites were present in all isoelectric (pI 6-8) and molecular weight variants of tau. In contrast, phosphate-dependent epitopes recognized by another tau-reactive antibody (NP14) were found mostly in acidic tau variants. Similarly to tau proteins from bovine brain, tau-enriched preparations from normal human brain contained Alz 50 and Tau-1 reactive sites in all isoelectric (pI 6.5-8.5) and molecular weight variants. Our observation of Alz 50 cross-reactivity with tau suggests a relationship between tau and the novel protein identified recently in Alzheimer brains.     

Rabbit antibodies against the folate binding protein from cow's milk. Production,characterization and use for development of an enzyme-linked immunosorbent assay (ELISA)

Rabbit antibodies to purified folate binding protein from cow's milk whey were used for development of a two-site enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine folate binding proteins, The folate binding proteins in human milk and serum showed no cross-reactivity. A partial saturation of purified bovine folate binder with folate gave rise to an increased antigenicity probably due to a ligand (folate)-induced exposure of antigenic sites on the protein.     

Expression and characterization of human proinsulin fused to thioredoxin in Escherichia coli

Native proinsulin (PI) belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its high proteolytic decay and troubles to reproduce the native disulphide pattern. In the present study, human PI was produced in E. coli as a fusion thioredoxin protein (Trx-PI). Such chimeric protein was obtained from the intracellular soluble fraction, and it was purified in one step by affinity chromatography on immobilized phenylarsine oxide. Trx-PI was also recovered from inclusion bodies and purified by anion exchange chromatography. The product identity and integrity were verified by mass analysis (22,173.5 Da) and mapping with Staphylococcus aureus V8 protease. Native PI folding was evaluated by biochemical and also by immunochemical analysis using specific sera from PI antibody-positive diabetic patients that recognise conformational discontinue epitopes. Dose-response curves showed identity between standard PI and Trx-PI. Moreover, surface plasmon resonance technique verified the correct conformation of the recombinant protein. The biochemical and immunochemical assays demonstrated the integrity of the chimera and the epitopes involved in the interaction with antibodies. In conclusion, it was possible to obtain with high-yield purified human PI as a fusion protein in E. coli and useful for analytical purposes.     

The epitope space of the human proteome

In the post-genome era, there is a great need for protein-specific affinity reagents to explore the human proteome. Antibodies are suitable as reagents, but generation of antibodies with low cross-reactivity to other human proteins requires careful selection of antigens. Here we show the results from a proteome-wide effort to map linear epitopes based on uniqueness relative to the entire human proteome. The analysis was based on a sliding window sequence similarity search using short windows (8, 10, and 12 amino acid residues). A comparison of exact string matching (Hamming distance) and a heuristic method (BLAST) was performed, showing that the heuristic method combined with a grid strategy allows for whole proteome analysis with high accuracy and feasible run times. The analysis shows that it is possible to find unique antigens for a majority of the human proteins, with relatively strict rules involving low sequence identity of the possible linear epitopes. The implications for human antibody-based proteomics efforts are discussed.     

Purification and characterization of pregnant sheep myometrium myosin light chain kinase

Myosin light chain kinase (MLCK) has been purified from the myometrium of pregnant sheep. The Mr of the enzyme was determined from SDS-polyacrylamide gels to be 160,000. It requires Ca2+ and calmodulin for activation, and phosphorylates the 20,000-Da light chains of myosin at a rapid rate. The specific activity for the myosin light chains from turkey gizzards and rabbit uterine muscle are 7.7 and 5.4 mumol/min/mg, respectively. The Km for the former substrate is 40 microM and the Vmax of the reaction is 19 mumol/min/mg. Polyclonal antibodies raised against the enzyme cross-reacted with pregnant sheep myometrium (psm), turkey gizzard (tg), and chicken gizzard MLCK. Affinity purification of the antibodies on tg-MLCK Sepharose resulted in the preparation of two fractions of antibodies with different reactivity toward these proteins. Fraction A antibodies which did not bind to the affinity column cross-reacted only with psm-MLCK while Fraction B antibodies which bound to the column cross-reacted with all three proteins. Western blots of extracts of turkey gizzards, human myometrium, and various tissues from sheep showed cross-reactivity of both fractions of antibodies with a 160,000-Da protein in the extracts of sheep smooth muscles. Only Fraction B antibodies cross-reacted with a protein (130,000 Da) in turkey gizzards and human myometrium extracts. Prolonged tryptic digestion of psm-MLCK produced large fragments Mr approximately 60,000 which appears to be similar to that formed from tg-MLCK, and some smaller peptides. Fraction A antibodies cross-reacted with the small peptides while Fraction B antibodies cross-reacted with the large fragments but not vice versa. Further analysis of the tryptic peptides suggests that the epitopes of Fraction A antibodies are localized in a peptide which appears to be in the NH2-terminal region of the molecule.     

Reduced immunogenicity of beta-lactoglobulin by conjugating with chitosan

Bovine beta-lactoglobulin (beta-LG) was conjugated with chitosan (CHS) by means of a water-soluble carbodiimide to reduce the immunogenicity of beta-LG. Each beta-LG-CHS conjugate was purified by ion-exchange chromatography and hydrophobic chromatography. The conjugation between beta-LG and CHS was confirmed by SDS-PAGE, the isoelectric point of the conjugate being higher than that of beta-LG. Two types of the beta-LG-CHS conjugate were obtained with molar ratios of beta-LG to CHS of 1:1 (F1) and 1:2 (F2). Structural analyses by fluorescence measurement, ELISA with monoclonal antibodies and retinol-binding activity indicated that the conjugates had almost maintained the native structure of beta-LG. The antigenicity of the beta-LG-CHS conjugates was similar to that of beta-LG in C3H/He mice. Reduction of the immunogenicity of beta-LG was achieved by conjugation with CHS. In particular, F2 showed very low immunogenicity. B cell epitopes of beta-LG and the conjugates recognized in C3H/He mice were determined with 15-mer multi-pin peptide; the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, while the antibody response to each epitope was dramatically reduced. Conjugation of beta-LG with chitosan was effective for reducing the immunogenicity of beta-LG.     

Monoclonal antibodies to bovine UDP-galactosyltransferase. Characterization, cross-reactivity, and utilization as structural probes

A series of mouse monoclonal antibodies has been developed against a soluble form of bovine UDP-galactose:N-acetylglucosamine galactosyltransferase purified to apparent chemical homogeneity by a combination of affinity and immunoadsorption chromatography. The purified enzyme consists of two molecular mass variants of 42 and 48 kDa. Individual monoclonal antibodies were selected for by their ability to recognize immobilized affinity-purified galactosyltransferase and were not reactive against bovine alpha-lactalbumin and bovine immunoglobulins. Based on competitive binding assays and Western blot analysis with either galactosyltransferase or lactose synthetase (covalently cross-linked alpha-lactalbumin galactosyltransferase), these monoclonal antibodies can be subdivided into four groups. Group A (3 clones) recognize an epitope at or near the alpha-lactalbumin binding site. In addition, this group is cross-reactive with soluble galactosyltransferase from human milk and pleural effusion. Group B (6 clones) and D (1 clone) appear to recognize two different epitopes on the 6-kDa fragment which is released when the 48-kDa galactosyltransferase polypeptide is converted to the 42-kDa form, apparently by proteolysis. Groups A and C (1 clone) recognize epitopes found on both the 48- and 42-kDa polypeptide. Interestingly, immunofluorescence studies indicate that only two monoclonal antibody groups (C and D) are able to decorate membrane-bound galactosyltransferase (Golgi-associated) in formalin-fixed, methanol-, or detergent-permeabilized cells. Thus, these groups of monoclonal antibodies appear to identify four separate structural/functional domains on soluble galactosyltransferase, two of which are not readily accessible for binding in situ.     

Studies with antibodies against the conserved cysteine region of progesterone receptor

Polyclonal antibodies were generated against two synthetic peptides corresponding to sequences from the DNA-binding domain of steroid receptors. The sequence for peptide 1 (13 amino acids) lies between the two putative metal-binding loops of the conserved cysteine region while the sequence for peptide 2 (12 amino acids) lies within one loop. Peptide antibodies were generated by injecting rabbits with peptide conjugated to bovine serum albumin. By Western blot analysis, antibodies to peptide 2 recognized chick and human progesterone receptor and human glucocorticoid receptor, but peptide 1 antibodies did not. No cross-reactivity with native chick progesterone receptor was detected with either anti-peptide. These findings suggest that the epitopes for peptide 2 antibodies, and possibly for peptide 1 antibodies, are inaccessible to antibody in the native receptor.     

Targeting a Cross-Reactive Gly m 5 Soy Peptide as Responsible for Hypersensitivity Reactions in a Milk Allergy Mouse Model

Background

Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity.

Methods

Cow''s milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy.

Results

Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice.

Conclusions

Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.     

Modulation of the T cell response to beta-lactoglobulin by conjugation with carboxymethyl dextran

We have previously prepared beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates with water-soluble carbodiimide and achieved reduced immunogenicity of beta-LG. In the present study, to elucidate the mechanism for the reduced immunogenicity of beta-LG, we investigated changes in the T cell response to beta-LG after conjugation with CMDs differing in molecular weight (about 40 and 162 kDa). Lymph node cells from BALB/c, C3H/He, and C57BL/6 mice that had been immunized with beta-LG or the conjugates were stimulated with beta-LG, and the in vivo T cell response was then evaluated by BrdU (5-bromo-2'-deoxyuridine) ELISA as the ex vivo proliferative response. T cells from the conjugate-immunized mice showed a lower proliferative response than those from the beta-LG-immunized mice. T cell epitope scanning, using synthesized peptides, showed that the T cell epitope profiles of the conjugates were similar to those of beta-LG, whereas the proliferative response to each epitope was reduced. These results indicate that the lower in vivo T cell response with the conjugates was not due to induction of conjugate-specific T cells, but due to a decrease in the number of beta-LG-specific T cells. After the lymph node cells from beta-LG-immunized mice had been stimulated with beta-LG or the conjugates, the efficiency of the antigen presentation of the conjugate to beta-LG-specific T cells was evaluated by BrdU ELISA as the in vitro proliferative response. The antigen presentation of beta-LG to the T cells was reduced by conjugation with CMD. In addition, conjugation with CMD enhanced the resistance of beta-LG to cathepsin B and cathepsin D, which suggest that conjugation with CMD inhibited the degradation of beta-LG by proteases in APC and led to suppression of the generation of antigenic peptides including T cell epitopes from beta-LG. It is therefore considered that the suppressive effect on the generation of T cell epitopes reduced the antigen presentation of the conjugates and that this reduction led to a decrease in the number of beta-LG-specific T cells in vivo. As a result, the decreased help to B cells by T cells would have reduced the antibody response to beta-LG. We conclude that suppression of the generation of T cell epitopes by conjugation with CMD is important to the mechanism for the reduced immunogenicity of beta-LG.     

Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.     

Use of Wisteria floribunda agglutinin affinity chromatography in the structural analysis of the bovine lactoferrin N-linked glycosylation

Background

Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(α1-3)Gal(β1-4)GlcNAc (αGal), GalNAc(β1-4)GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with αGal, LacdiNAc, LacNAc [Gal(β1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes, and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.

Methods

For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-MS and HPLC-FD profiling, including sequential exoglycosidase treatments.

Results

This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) containing LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, αGal and sialic acid.

Conclusions

Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, αGal, and Neu5Gc epitopes, than was shown in previous studies.

General significance

Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics.     

Beneficial health effects of milk and fermented dairy products — Review

Milk is a complex physiological liquid that simultaneously provides nutrients and bioactive components that facilitate the successful postnatal adaptation of the newborn infant by stimulating cellular growth and digestive maturation, the establishment of symbiotic microflora, and the development of gut-associated lymphoid tissues. The number, the potency, and the importance of bioactive compounds in milk and especially in fermented milk products are probably greater than previously thought. They include certain vitamins, specific proteins, bioactive peptides, oligosaccharides, organic (including fatty) acids. Some of them are normal milk components, others emerge during digestive or fermentation processes. Fermented dairy products and probiotic bacteria decrease the absorption of cholesterol. Whey proteins, medium-chain fatty acids and in particular calcium and other minerals may contribute to the beneficial effect of dairy food on body fat and body mass. There has been growing evidence of the role that dairy proteins play in the regulation of satiety, food intake and obesity-related metabolic disorders. Milk proteins, peptides, probiotic lactic acid bacteria, calcium and other minerals can significantly reduce blood pressure. Milk fat contains a number of components having functional properties. Sphingolipids and their active metabolites may exert antimicrobial effects either directly or upon digestion.     

Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study

Poly(ADP ribose) polymerase (EC 2.4.2.30) was studied using monoclonal antibodies for three different epitopes on the enzyme. The epitopes were mapped in relation to the functional domains of the protein and the inhibitory properties of the antibodies. The intranuclear and interspecies immunoreactivity of the enzyme was also investigated. The epitope of antibody 2 was mapped to the 17 kDa fragment generated by chymotryptic digestion of the C-terminal 54 kDa NAD-binding domain. Antibody 9 binds to the N-terminal 29 kDa fragment of the DNA binding domain and inhibits the enzyme activity by 80%. This antibody was used to purify poly(ADP ribose) polymerase by immunoaffinity chromatography. The third antibody binds to a central 36 kDa fragment that possesses part of the DNA-binding domain and the automodification domain. This antibody increases the enzymatic activity by 30%. An analysis of the species cross-reactivity of the antibodies was carried out by immunoblot analysis of nuclear proteins. Antibody 10 binding was detected in rat FR3T3 cells, Chinese hamster ovary cells (CHO) and epidermoid carcinoma lung human cells (CALU-1). The other two antibodies are specific for the human and bovine enzymes. Western blot analysis showed the association of poly(ADP ribose) polymerase with residual nuclear material obtained after nuclease treatment and high-salt extraction. Immunofluorescence studies with the three different monoclonals demonstrated that accessibility of the epitopes varies in the nucleus.     

Immunochromatography on insolubilized antibodies of very low affinity: application to immunoadsorbence of bovine alpha-fetoprotein.

Cross-reactive antibodies were utilized to prepare immunoadsorbents possessing a very low affinity to bovine alpha-fetoprotein (AFP). A goat anti-human AFP serum cross-reactive with bovine AFP was first depleted of antibodies reactive with bovine AFP in immunodiffusion. The remaining antibodies from this serum and gamma-globulin from a sheep antiserum against rabbit AFP, without prior absorption, were coupled to Sepharose. Chromatography of fetal calf serum on these adsorbents resulted in retardation of bovine AFP relative to other proteins. A major part of the AFP eluted from the columns with phosphate-buffered saline. The rest eluted as a sharp peak with a small quantity of 4 or 6 M urea. The elution of AFP with the initial column buffer has made it possible to prepare pure AFP that has not been subjected to the chaotropic elution buffers usually employed in affinity chromatography. Elimination of the washing step and the ease of elution has allowed purification of gram amounts of AFP. The fact that immunoadsorbents prepared from antibodies with no detectable reactivity in immunodiffusion still caused delayed elution in chromatography suggests that this procedure may be useful in search of proteins cross-reactive with a known protein.     

Epitope mapping of a monoclonal antibody specific to bovine dry milk: involvement of residues 66-76 of strand D in thermal denatured beta-lactoglobulin

beta-Lactoglobulin (beta-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured beta-LG. In the present study, we show a dramatic increase in beta-LG immunoreactivity when heating raw milk between 70 and 80 degrees C. To map out the specific epitope of beta-LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66-76) of beta-LG. Circular dichroic spectral analysis shows that carboxymethylation on beta-LG not only resulted in a substantial loss of beta-configuration but also exerted a 10 times increase in immunoreactivity as compared with heated beta-LG. The result suggests that a further disordered structure occurred in beta-LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated beta-LG and its binding to retinol or palmitic acid. Most interestingly, pH 9-10, which neutralizes the Lys groups of beta-LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of beta-LG participated in the thermal denaturation between 70 and 80 degrees C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.     

Specific polyclonal antibodies to the carboxyl terminus of [Met]enkephalin-Arg6-Gly7-Leu8

The octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu was recently isolated from bovine adrenal chromaffin granules and serves as a marker for proenkephalin from which it is derived. Polyclonal antisera which are highly specific for the carboxyl terminus have been raised against the synthetic peptide. The only significant cross-reactivity was with the 18.2-k Da and 5.3-k Da enkephalin-containing peptides (EC peptides) which contain the octapeptide at their carboxyl termini and the [des-Tyr] and [des-Tyr-Gly] congeners of the octapeptide. Extracts of bovine adrenal medulla and rat spinal cord were shown to contain significant amounts of the octapeptide, the two larger EC peptides, and the two smaller congeners.     

Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme

The selectivity of immobilized metal affinity chromatography (IMAC) systems for the purification of phosphopeptides is poor. This is particularly a problem with tryptic digests of proteins where a large number of acidic peptides are produced that also bind during IMAC. The hypothesis examined in this work was that the selectivity of IMAC columns for phosphopeptides could be increased by using endoproteinase glu-C (glu-C) for protein digestion. Glu-C cleaves proteins at acidic residues and should reduce the number of acidic residues in peptides. This method was successfully applied to a mixture of model proteins and bovine milk. The percentage of phosphorylated peptides selected from proteolytic digests of the milk sample was increased from 40% with trypsin to 70% with glu-C. Additionally, this method was coupled with stable isotope coding methods to quantitatively compare the concentration of phosphoproteins between samples.