Formic Acid Cleavage Research Articles

Design and expression of recombinant Xuanwei ham antioxidant peptide in Escherichia coli BL21

Xuanwei Ham Peptide (XHP) is a bioactive compound isolated and extracted from Xuanwei ham. It enhances the oxidative defense system by down-regulating CYP2E1 expression, reducing ROS production, and by activating the Nrf2/HO-1 pathway. In order to solve the problems of low yield, high cost and time-consuming separation process of active peptides from ham. In this study, we designed three peptide sequences derived from Xuanwei ham and expressed them recombinantly. The results showed that the peptides using flexible linker peptide ((GGGGS)2), rigid linker peptide ((EAAAK)2) linkage as well as the head-to-tail tandem linkage were successfully expressed in the E. coli expression system. Peptide monomers were released from the fusion proteins by formic acid cleavage and purified sequentially by Ni-IDA affinity chromatography, membrane filtration and RP-HPLC. The molecular weight of XHP was confirmed to be 717.35 by ESI-MS analysis. The results of the antioxidant activity of XHP showed that the hydroxyl radical scavenging rates of the three recombinant peptides were as high as 79.46%, 87.42%, and 53.60%, respectively. The hydroxyl radical activity of formic acid-cleaved XHP was reduced to 35.31%, but it showed good stability in heat, pH, metal ions and gastrointestinal digestion (GID). The molecular docking results showed that the docking of XHP to Keap1 protein was evaluated by five Molecular dynamics simulation with a maximum binding energy of -8.5 kcal/mol. This study provides references and ideas for the expression of active peptides, which can be widely used in the future can be used in the field of nutraceuticals, functional foods and pharmaceuticals.

Production and Purification of Two Bioactive Antimicrobial Peptides Using a Two-Step Approach Involving an Elastin-Like Fusion Tag.

Antimicrobial resistance is an increasing global threat, demanding new therapeutic biomolecules against multidrug-resistant bacteria. Antimicrobial peptides (AMPs) are promising candidates for a new generation of antibiotics, but their potential application is still in its infancy, mostly due to limitations associated with large-scale production. The use of recombinant DNA technology for the production of AMPs fused with polymer tags presents the advantage of high-yield production and cost-efficient purification processes at high recovery rates. Owing to their unique properties, we explored the use of an elastin-like recombinamer (ELR) as a fusion partner for the production and isolation of two different AMPs (ABP-CM4 and Synoeca-MP), with an interspacing formic acid cleavage site. Recombinant AMP-ELR proteins were overproduced in Escherichia coli and efficiently purified by temperature cycles. The introduction of a formic acid cleavage site allowed the isolation of AMPs, resorting to a two-step methodology involving temperature cycles and a simple size-exclusion purification step. This simple and easy-to-implement purification method was demonstrated to result in high recovery rates of bioactive AMPs. The minimum inhibitory concentration (MIC) of the free AMPs was determined against seven different bacteria of clinical relevance (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and two Burkholderia cenocepacia strains), in accordance with the EUCAST/CLSI antimicrobial susceptibility testing standards. All the bacterial strains (except for Pseudomonas aeruginosa) were demonstrated to be susceptible to ABP-CM4, including a resistant Burkholderia cenocepacia clinical strain. As for Synoeca-MP, although it did not inhibit the growth of Pseudomonas aeruginosa or Klebsiella pneumoniae, it was demonstrated to be highly active against the remaining bacteria. The present work provides the basis for the development of an efficient and up-scalable biotechnological platform for the production and purification of active AMPs against clinically relevant bacteria.

A novel strategy to prepare the precursor peptide of liraglutide

Abstract Glucagon-like peptide-1 (GLP-1) is a natural incretin hormone released from intestinal L-cells in response to hormonal, neural and nutrient stimuli. Liraglutide is derived from human GLP-1 by substituting Lys34 with Arg and conjugating a palmitic acid to Lys26 via a glutamate spacer, which has a long-lasting hypoglycemic effect. The precursor of liraglutide is a K34R mutant of GLP-1 (GLP-1R34) and was produced previously by either yeast protein expression system or peptide solid phase synthesis. However, degradation of secreted proteins limits the effectiveness of yeast protein expression system and peptide solid phase synthesis requires the use of large amounts of organic solvents and expensive condensation agents. In this study, we reported a novel method to prepare GLP-1R34 using E. coli protein expression system. The GLP-1R34 peptide was overexpressed as an MFH-GLP-1R34 fusion protein and was released from the fusion protein through formic acid cleavage. The target peptide was purified by reverse-phase chromatography and ion exchange chromatography. Our work demonstrated a high-productive process for large-scale preparation of the liraglutide precursor with high purity.

High-level expression of angiotensin converting enzyme inhibitory peptide Tuna AI as tandem multimer in Escherichia coli BL21 (DE3)

Food-derived angiotensin converting enzyme inhibitory peptides (ACEIP) show a great promise for prevention and treatment of hypertension. Tuna muscle derived peptide, Tuna AI (PTHIKWGD), is a potent ACEIP with high activity and stability. To improve the expression level of Tuna AI in Escherichia coli, multimer genes of Tuna AI were assembled and expressed as fusion proteins by constructing the vectors of pET30-4nTunaAI (n=1, 2, 4 and 8). The results showed the multimer genes were successfully expressed in the constructed system, and their expression level improved significantly with increasing repeat degree. The E. coli harboring pET30-32TunaAI was selected for production of peptide based on its highest expression level and its fusion protein was suit for urea-washing purification. The peptide monomer was released from the fusion protein by formic acid cleavage, and successively purified by membrane filtration, cation exchange chromatography and size exclusion chromatography, with a final yield of 218.9mg/L. The purified monomeric peptide was confirmed by mass spectrometry analysis, N-terminal sequencing, and activity measurement. This peptide resulted in a significant decrease of systolic blood pressure by 36.5mmHg in rats at 4h. These results suggest our strategy is an economic solution for the mass production of antihypertensive peptides.

Development of elastin-like recombinamer films with antimicrobial activity.

In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.

Design and identification of a high efficient formic acid cleavage site for separation of fusion protein.

The release of target protein with high efficiency and low cost from expressed fusion protein is a key requirement for commercial production of target proteins. To establish such a cleavage system, we have designed four formic acid (FA) cleavage sites C1 (DPDPDP), C2 (DPPDPP), C3 (DDDDPI) and C4 (IVDPNP), which was placed in between the E and G fusion protein. Four expression vectors were individually constructed and expressed in Escherichia coli. Purified proteins were reacted with a series of FA concentrations or under different temperatures followed by SDS-PAGE gel electrophoresis to verify the degree of cleavage efficiency. Results showed that the C2 was the most efficient site compared with the other three. After optimization of cleavage conditions for E-C2-G, the cleavage efficiently could reach as high as 87.3% within 2.5 h in 37% FA at 45 °C. Comparing with previous reports, a significant reduction (26%) of FA concentration at a lower temperature in a short duration of reaction (18 times less) was achieved. We believe the cleavage site of DPPDPP identified in this study can be used in the large-scale production of valuable fusion proteins to save the cost, time and energy.

Chemical Equilibria in Formic Acid/Amine‐CO2 Cycles under Isochoric Conditions using a Ruthenium(II) 1,2‐Bis(diphenylphosphino)ethane Catalyst

AbstractThe equilibrium position in formic acid/amine–CO2 systems has been examined as a function of pressure and temperature under isochoric conditions. The homogeneous ruthenium(II)‐1,2‐bis(diphenylphosphino)ethane catalyst was active in both reactions, that is, in formic acid cleavage producing pure hydrogen and CO2, as well as in carbon dioxide hydrogenation under basic conditions. High yields of formic acid dehydrogenation into H2 and CO2 are favored by low gas pressures and/or high temperatures, and H2 uptake is possible at elevated H2–CO2 pressures. These results take us one step closer to the realization of a practical H2 storage–discharge device.

Hydrogen Storage and Delivery: The Carbon Dioxide – Formic Acid Couple

Carbon dioxide and the carbonates, the available natural C1 sources, can be easily hydrogenated into formic acid and formates in water; the rate of this reduction strongly depends on the pH of the solution. This reaction is catalysed by ruthenium(II) pre-catalyst complexes with a large variety of water-soluble phosphine ligands; high conversions and turnover numbers have been realised. Although ruthenium(II) is predominant in these reactions, the iron(II) - tris[(2-diphenylphosphino)-ethyl]phosphine (PP3) complex is also active, showing a new perspective to use abundant and inexpensive iron-based compounds in the CO2 reduction. In the catalytic hydrogenation cycles the in situ formed metal hydride complexes play a key role, their structures with several other intermediates have been proven by multinuclear NMR spectroscopy. In the other hand safe and convenient hydrogen storage and supply is the fundamental question for the further development of the hydrogen economy; and carbon dioxide has been recognised to be a viable H2 vector. Formic acid--containing 4.4 weight % of H2, that is 53 g hydrogen per litre--is suitable for H2 storage; we have shown that in aqueous solutions it can be selectively decomposed into CO-free (CO < 10 ppm) CO2 and H2. The reaction takes place under mild experimental conditions and it is able to generate high pressure H2 (up to 600 bar). The cleavage of HCOOH is catalysed by several hydrophilic Ru(II) phosphine complexes (meta-trisulfonated triphenylphosphine, mTPPTS, being the most efficient one), either in homogeneous systems or as immobilised catalysts. We have also shown that the iron(II)--hydrido tris[(2-diphenylphosphino)ethyl]phosphine complex catalyses with an exceptionally high rate and efficiency (turnover frequency, TOF = 9425 h(-1)mol(-1); turnover number, TON = 92400) the formic acid cleavage, in environmentally friendly propylene carbonate solution, opening the way to use cheap, non-noble metal based catalysts for this reaction, too.

Studying disulfide bond rearrangement by MALDI-RTOF PSD and MALDI-TOF/RTOF high-energy CID (20 keV) experiments of peptides derived from ammodytoxins

Ammodytoxins (Atxs) are presynaptically neurotoxic phospholipases present in Vipera ammodytes ammodytes snake venom. Atxs show a high sequence homology and contain 14 cysteines which form seven biologically relevant disulfide bridges-connecting non-neighboring cysteines. Formic acid cleavage was performed to confirm protein sequences by MALDI RTOF MS and resulted in 95.6% sequence coverage exhibiting only few formylations. Cysteine-containing peptides showed adjacent signals 2 and/or 4 Da lower (according to the number of cysteines present in the peptide) than the theoretical molecular weight indicating disulfide bridge rearrangement. Post-source decay (PSD) and high-energy collision-induced dissociation (CID) at 20 keV experiments showed fragmentation pattern unique for the reduced, thiol group containing and the oxidized, disulfide bridge harboring peptides. Besides typical low-energy fragment ions observed during PSD experiments (a-, b-, y-type ions), additional high-energy fragment ions (c-, x-, w-, d-type and internal fragments) of significant intensity were generated during fragmentation at 20 keV. In the case of charge directing N- and C-termini, x- and w-type ions were also observed during PSD. Good and up to complete sequence coverage was achieved for all studied peptides from Atxs in the case of high-energy CID, whereas PSD lacked information particularly for larger peptides.

Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3)

The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni(2+)-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC(50) of 3.6 and 1.9 microM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides.

Structure and Function of the c-myc DNA-unwinding Element-binding Protein DUE-B

Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.

Cloning, expression, isotope labeling, and purification of human antimicrobial peptide LL-37 in Escherichia coli for NMR studies

Antimicrobial peptide LL-37 plays an important role in human body’s first line of defense against infection. To better understand the mechanism of action, it is critical to elucidate the three-dimensional structure of LL-37 in complex with bacterial membranes. We present a bacterial expression system that allows the incorporation of 15N and other isotopes into the polypeptide for nuclear magnetic resonance (NMR) analysis. The DNA sequence encoding full-length LL-37 was chemically synthesized and cloned into the pET-32a(+) vector for protein expression in Escherichia coli strain BL21(DE3). The peptide was expressed directly as a His-tagged fusion protein without the inclusion of its precursor sequence. LL-37 was released from the fusion by formic acid cleavage at the AspPro dipeptide bond and separated from the carrier thioredoxin by affinity chromatography and reverse-phase HPLC. The peptide was identified by polyacrylamide gel electrophoresis and further confirmed by mass spectrometry and NMR spectroscopy. Antibacterial activity assays showed that the recombinant LL-37 purified from the bacterial source is as active as that from chemical synthesis. According to the antimicrobial peptide database ( http://aps.unmc.edu/AP/main.html), 111 peptides contain a Met residue, but only 5 contain the AspPro pair, indicating a broader application of formic acid than cyanogen bromide in cleaving fusion proteins. The successful application to the expression of the 66-residue cytoplasmic tail of human MUC1 indicates that the system can be applied to other peptides as well.

Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry

The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W have been recently described, and the primary structure of this gene product was deduced using a bioinformatic approach. In this study, capillary electrophoresis-on-the-chip and mass spectrometry were used to characterize native and overexpressed extracellular lipase protein from S. rimosus . The exact molecular mass of the wild-type and the overexpressed lipase, determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, were in excellent agreement (Deltam=0.11 Da and Deltam=0.26 Da, respectively) with a value of 24165.76 Da calculated from the structure deduced from the nucleotide sequence, considering the mature enzyme with all six cysteines forming disulfide bridges. The primary structure derived from the nucleotide sequence was completely verified using a combination of tryptic digestion and formic acid cleavage of the protein, followed by peptide mass fingerprinting. Selected peptides were further investigated by MALDI low-energy collision-induced dissociation hybrid tandem mass spectrometry, allowing the unambiguous determination of their predicted amino acid sequence. No post-translational modifications of mature S. rimosus lipase were detected. Comparison of the peptide mass fingerprints from the reduced and non-reduced overexpressed enzyme unequivocally revealed three intramolecular disulfide bonds with the following linkages: C27-C52, C93-C101 and C151-C198.

Phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to increase the rate of phosphorylation of Ser(40).

The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser(19) and Ser(40) produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser(19) phosphorylation is less pronounced than that of the Ser(40) phosphorylation. The effect of Ser(19) and Ser(40) phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser(19) and Ser(40) phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser(19) and Ser(40) were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser(40) is approximately 2- to 3-fold higher if Ser(19) is already phosphorylated. These results suggest that phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser(40) to kinases.

The role of carboxy-terminal glycosaminoglycan-binding domain of vitronectin in cytoskeletal organization and migration of endothelial cells.

Vitronectin is a major cell adhesion molecule present in the subendothelial matrix that mediates the attachment and spreading of a variety of cells. The carboxy-terminal end of vitronectin has a consensus sequence for glycosaminoglycan-binding. To define the functional role of this domain, we generated fragments of vitronectin that lack the glycosaminoglycan-binding domain by formic acid cleavage of plasma-derived vitronectin. In addition, we also generated similar recombinant fragments of vitronectin as glutathione S-transferase fusion proteins in E. coli. These fragments were tested for their ability to support the adhesion of human umbilical vein endothelial cells. These fragments promoted endothelial cell adhesion, reaching half maximal activity at 2-5 micrograms/well compared to plasma-derived vitronectin which reached at 0.2 micrograms/well. However, the cells that adhered to these fragments did not develop well-formed focal adhesion plaques and actin stress fibers. In addition, these fragments were poorly chemotactic for endothelial cell migration when compared to intact plasma-derived vitronectin in a modified Boyden chamber assay. The present studies show that carboxy-terminal glycosaminoglycan-binding domain of vitronectin is essential for proper cytoskeletal organization and migration of endothelial cells on vitronectin substratum.

3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin.

Taxol possesses an unusual chemical structure, a unique mechanism of action, and demonstrated activity in human malignancies. It is the only antitumor agent that has a binding site on the microtubule polymer. The interaction of Taxol with the microtubule polymer results in the formation of stable bundles of cellular microtubules that are resistant to depolymerization. Although it has become evident that the microtubule, specifically beta-tubulin, is the target for Taxol, no information is available on the binding site for the drug. In this report, we demonstrate that 3'-(p-azidobenzamido)taxol, an analogue with similar biological activities as Taxol, covalently binds to the N-terminal domain of beta-tubulin after irradiation of the microtubule-drug complex. Taxol competes with [3H]3'-(p-azidobenzamido)-taxol binding, suggesting that the photoaffinity analog and Taxol are binding at the same or overlapping sites. Formic acid cleavage of [3H]3'-(p-azidobenzamido)-taxol-photolabeled beta-tubulin and subsequent protein sequence and mass analyses have identified the N-terminal 31 amino acids as the major site for [3H]3'-(p-azidobenzamido)taxol photoincorporation.

Sequence-selective alkylation and cross-linking induced by mitomycin C upon activation by DT-diaphorase

Aerobic reduction of MMC by DTD, an obligate two-electron reductase, or chemical reduction by sodium borohydride results predominantly in monoalkylation of DNA at the guanine N7 position within 5'-GG-3' and 5'-GTC-3' sequences. The level of guanine N7 alkylation after DTD reduction increased as the pH was decreased from 7.8 and was optimal at pH 6.6. A similar profile of alkylation was obtained when the major metabolite of DTD-mediated MMC metabolism, 2,7-diaminomitosene, was further reduced by DTD. The sequence preference for DNA interstrand cross-linking (ISC) was also determined using singly end-labeled oligonucleotide duplexes. Reduction of MMC by DTD induced DNA cross-links which were resistant to piperidine cleavage. Exposure of cross-linked DNA to dimethyl sulfate or formic acid and subsequent piperidine cleavage displayed a discontinuity in band pattern which suggested a 5'-CG-3' preference for DNA ISC. Major groove alkylation is proposed to occur via generation, and subsequent metabolism by DTD, of 2,7-diaminomitosene. Cross-linking of DNA, at 5'-CG-3' sequences, is proposed to require the formation of either the protonated leucomitomycin C or the leucoaziridinomitosene during DTD-mediated metabolism of MMC.

Two Collagen-binding Domains of Vitronectin.

Vitronectin is a cell-adhesive glycoprotein present in animal blood and extracellular matrix. To establish the molecular basis of vitronectin interactions with extracellular matrix macromolecules, the binding site of vitronectin to collagen has been investigated. Vitronectin fragments obtained by formic acid cleavage were separated by heparin-affinity chromatography followed by gel filtration chromatography. The collagen-binding activity of the fragments was assayed in terms of inhibitory activity on the binding of 125I-vitronectin to immobilized collagen. There were two groups of collagen-binding fragments. One group consisted of 5 heparin-binding fragments with estimated molecular masses of 12 kDa, 14 kDa, 16 kDa, 18 kDa, and 19 kDa in SDS-polyacrylamide gel electrophoresis. The other group consisted of 2 heparin-nonbinding fragments migrating at 18 kDa and 40 kDa. These results indicate that there are two collagen-binding sites in the vitronectin molecule; one located close to the heparin-binding domain in the COOH-terminal half and the other located in the NH2-terminal half of vitronectin.

The complete amino acid sequence of chitinase-c from the seeds of rye (Secale cereal).

The complete amino acid sequence of rye seed chitinase-c (RSC-c) has been analyzed. This was done by first sequencing the tryptic peptides from RCm-RSC-c and then connecting them by analyzing the peptides produced by digestions with lysylendopeptidase and Staphylococcus aureus V8 protease of RCm-RSC-c, and by chymotryptic digestion and formic acid cleavage of S. aureus V8 protease peptides. RSC-c consists of 243 amino acid residues and has a molecular mass of 26,093, and has 92% sequence homology with barley seed basic chitinase which lacks a Cys-rich domain. Cys204 is free and six cysteine residues are linked by disulfide bonds between Cys23 and Cys85, Cys97 and Cys105, and Cys223 and Cys236.

Determination of the epitope for the inhibitory monoclonal antibody 5-B6 on the catalytic subunit of gastric Mg2+-dependent H+-transporting and K+-stimulated ATPase

The monoclonal antibody 5-B6, directed against the alpha-subunit of pig gastric H+,K(+)-ATPase (Mg(2+)-dependent H(+)-transporting and K(+)-stimulated ATPase), was shown to be a potent inhibitor of the K(+)-ATPase activity, thereby binding to the cytoplasmic side of the alpha-subunit of the enzyme [Van Uem, Peters & De Pont (1990) Biochim. Biophys. Acta 1023, 56-62]. In order to define the epitope for 5-B6 on pig gastric H+,K(+)-ATPase more precisely, the alpha-subunit of the enzyme was subjected to limited proteolysis followed by chemical cleavage. Restricted proteolysis with papain followed by sequence analysis yielded an immunoreactive fragment of 27 kDa beginning at Ser379. This fragment was water-soluble and possessed the fluorescein isothiocyanate-reaction site. Limited tryptic digestion in the presence of K+ gave rise to an immunoreactive 56 kDa fragment beginning at Ile456, thus restricting the location of the epitope from Ile456 to the C-terminal end of the 27 kDa fragment (around residue 620). Further degradation of the 27 kDa fragment by means of formic acid cleavage at Asp-Pro bonds resulted initially in the formation of two non-immunoreactive fragments of 17 kDa and 11 kDa, indicating that the epitope for 5-B6 has to be localized around the chemical cleavage sites Asp507 and/or Asp510. Comparison of the primary structure of the alpha-subunits of gastric H+,K(+)-ATPase and non-immunoreactive rat kidney Na+,K(+)-ATPase shows almost no similarity for the sequence containing these formic acid cleavage sites (Thr504-Leu-Glu-Asp-Pro-Arg-Asp-Pro-Arg512), whereas the adjacent sequences are nearly 100% identical. These findings strongly suggest that the epitope for 5-B6 includes (part of) this sequence.