Purification of novel peptide antibiotics from human milk

A strategy was established for the identification of novel antimicrobial peptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avoid the purification of the known abundant antimicrobial milk protein lysozyme, it was identified in HPLC fractions by its enzymatic activity and by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS). On condition that lysozyme was not detectable and antibacterial activity of HPLC fractions was caused by a peptide, which was confirmed by proteolytic cleavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. The new antimicrobial peptide resembles a proteolytic fragment of human casein-κ (residues 63–117) and inhibits the growth of Gram-positive, Gram-negative bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolysis and may play an important role in the host defense system of the newborn.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.     

Human hemoglobin-derived peptides exhibit antimicrobial activity: a class of host defense peptides

Hemoglobin is a known source of biologically active peptides with various functions. In the present study, we report for the first time the existence of natural processed hemoglobin fragments exhibiting antimicrobial activity in humans. Two antimicrobial hemoglobin-derived peptides were purified from a human placental peptide library by consecutive chromatographic steps tracking the maximum growth inhibitory activity against Escherichia coli BL21. These peptides, consisting of 17 and 36 amino acid residues, were identified as being C-terminal fragments of gamma-hemoglobin and beta-hemoglobin, respectively. The antimicrobial beta-hemoglobin fragment was also purified from lysed erythrocytes, demonstrating that proteolytic degradation of hemoglobin into small bioactive peptides already starts inside erythrocytes. The identified peptides inhibit the growth of Gram-positive and Gram-negative bacteria and yeasts in micromolar concentrations. Moreover, by LPS-binding, the beta-hemoglobin fragment reduces biological activity of endotoxins. In contrast, even at high concentrations, the identified antimicrobial hemoglobin peptides do not exhibit toxic activity on human primary blood cells. We conclude that antimicrobial hemoglobin-derived peptides could be important effectors of the innate immune response killing microbial invaders.     

The identification of epitopic sites in human alpha 1-proteinase inhibitor.

Human alpha 1-proteinase inhibitor (alpha 1-PI) yielded nine fragments on cleavage with CNBr. The amino acid sequences of these fragments were determined. Three of these CNBr-cleavage fragments, namely fragment I (residues 64-220), fragment II (residues 243-351) and fragment III (residues 1-63), were found to bind rabbit polyclonal antibodies against chemically oxidized alpha 1-PI and mouse polyclonal antibodies against native alpha 1-PI by the Bio-Dot method (enzyme-linked immunosorbent assay on nitrocellulose). These fragments, I, II and III, inhibited by 60%, 25% and 5% respectively the binding between alpha 1-PI and the rabbit antibodies. Fragments I, II and III were subjected to proteolytic digestion, and 15, ten and five peptides were obtained from these fragments respectively. Only four of these peptides showed binding to the mouse antibodies against native alpha 1-PI. These were residues 40-63, 79-86, 176-206 and 299-323. A panel of monoclonal antibodies was prepared by conventional hybridoma technology, with chemically oxidized alpha 1-PI as the antigen. The ability of the monoclonal antibodies to bind native alpha 1-PI and CNBr-cleavage fragments I-III was determined. The monoclonal antibodies fell into three categories. Most (over 90%) belonged to group I, which was capable of binding alpha 1-PI and only fragment I. Antibodies in groups II and III bound alpha 1-PI and either fragment II or fragment III respectively. The ability of the peptides derived from proteolytic digestion of fragments I, II and III to bind three monoclonal antibodies representing each of the three groups was determined. Among all the peptides tested, only one (residues 176-206) derived from fragment I showed binding to the antibodies from group I, one (residues 299-323) derived from fragment II showed binding to the antibodies from group II, and one (residues 40-63) from fragment III showed binding to the antibodies from group III. Each of these three peptides also inhibited the binding between alpha 1-PI and the corresponding monoclonal antibodies. From these data we concluded that at least four epitopic regions (residues 40-63, 79-86, 176-206 and 299-323) were present in alpha 1-PI. Specific monoclonal antibodies to three of these sites were obtained.     

Primary structure of tyrosinase from Neurospora crassa. I. Purification and amino acid sequence of the cyanogen bromide fragments

Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionylthreonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on peptides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.     

Isolation of products and intermediates of pancreatic prosomatostatin processing: use of fast atom bombardment mass spectrometry as an aid in analysis of prohormone processing

Major products and an intermediate in the proteolytic processing pathway of preprosomatostatin I from anglerfish (Lophius americanus) were purified and characterized. Proteolytic mapping by fast atom bombardment mass spectrometry was used to rapidly locate regions of the peptides whose masses deviated from those deduced from the cDNA sequence. Amino acid analysis and partial Edman sequencing were also used to confirm the structures. The protein structural data indicate a Glu for Gly substitution at position 83 of preprosomatostatin I (aPPSS-I, numbering from the initiator Met) relative to the cDNA sequence. Two of the peptides isolated, aPPSS-I (26-52) (7.5 nmol X g-1) and aPPSS-I (26-92) (49.5 nmol X g-1), define signal cleavage as occurring between Cys-25 and Ser-26. A partial sequence was obtained from fragment ions in the mass spectrum of a peptide corresponding to aPPSS-I (94-105) (58 nmol X g-1). The 14-residue somatostatin [SS-14 corresponding to aPPSS-I (108-121)] has previously been isolated [Noe, B. D., Spiess, J., Rivier, J. E., & Vale, W. (1979) Endocrinology (Baltimore) 105, 1410-1415]. Taken together, these peptides suggest a pathway for prosomatostatin I processing in which the residues corresponding to SS-14 and the immediately preceding 14 residues are cleaved from the prohormone via endoproteolysis (order of cleavage not determined). The fragment aPPSS-I (94-105) was isolated in lower yield than SS-14 and may represent a secondary site of cleavage. Subsequent cleavage at arginine-53 results in the minor peptide aPPSS-I (26-52).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of monoclonal antibodies against ascorbate peroxidase isoenzymes: purification and epitope-mapping using immunoaffinity column chromatography

We have developed three monoclonal antibodies against spinach chloroplastic (chl-mAb3 and chl-mAb6) and cytosolic (cyt-mAb1) ascorbate peroxidase (APX) isoenzymes to analyze the cross-reactivity and the structure of the epitopes for each monoclonal antibody. All three antibodies reacted specifically with their respective isoenzymes, but none cross-reacted with the others. Immunoreactive fragments in proteolytic recombinant APX isoenzymes were detected by means of the absorption on the corresponding immunoaffinity column. The cyt-mAb1 reacted with a peptide fragment containing the distal His region obtained by the lysyl endopeptidase digestion. The chl-mAb6 was capable of binding to the fragment, D-I-K-E-K-R, which is consistent with an inherent region of chloroplastic isoenzymes. No fragments reacting to the chl-mAb3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, which may be destroyed by the enzymatic cleavage. We concluded that the peptides identified as epitopes are characteristic evidence of monoclonal antibodies.     

Identification of discontinuous von Willebrand factor sequences involved in complex formation with botrocetin. A model for the regulation of von Willebrand factor binding to platelet glycoprotein Ib

We have used proteolytic fragments and overlapping synthetic peptides to define the domain of von Willebrand factor (vWF) that forms a complex with botrocetin and modulates binding to platelet glycoprotein (GP) Ib. Both functions were inhibited by the dimeric 116-kDa tryptic fragment and by its constituent 52/48-kDa subunit, comprising residues 449-728 of mature vWF, but not by the dimeric fragment III-T2 which lacks amino acid residues 512-673. Three synthetic peptides, representing discrete discontinuous sequences within the region lacking in fragment III-T2, inhibited vWF-botrocetin complex formation; they corresponded to residues 539-553, 569-583, and 629-643. The 116-kDa domain, with intact disulfide bonds, exhibited greater affinity for botrocetin than did the reduced and alkylated 52/48-kDa molecule, and both fragments had significantly greater affinity than any of the inhibitory peptides. Thus, conformational attributes, though not strictly required for the interaction, contribute to the optimal functional assembly of the botrocetin-binding site. Accordingly, 125I-labeled botrocetin bound to vWF and to the 116-kDa fragment immobilized onto nitrocellulose but not to equivalent amounts of the reduced and alkylated 52/48-kDa fragment; it also bound to the peptide 539-553, but only when the peptide was immobilized onto nitrocellulose at a much greater concentration than vWF or the proteolytic fragments. These studies demonstrate that vWF interaction with GP Ib may be modulated by botrocetin binding to a discontinuous site located within residues 539-643. The finding that single point mutations in Type IIB von Willebrand disease are located in the same region of the molecule supports the concept that this domain may contain regulatory elements that modulate vWF affinity for platelets at sites of vascular injury.     

Amino acid sequence of a cyanogen bromide fragment containing the two tryptophanyl residues of lobster arginine kinase (Homarus vulgaris).

Lobster arginine kinase [EC 2.7.3.3] contains 2 tryptophanyl residues and 9 methionyl residues. The whole carboxymethylated protein was first subjected to CNBr cleavage and the resulting fragments were isolated by gel filtration and other experimental approaches. One fragment, CB5, which contains 60 residues including the two tryptophanyl residues and two of the five cysteinyl residues of the protein, was characterized and the results are reported inthis paper. The overall strategy for the establishment of the complete sequence of this fragment was based on the use of three types of peptides: (a) whole cyanogen bromide peptide CB5 which was partially characterized by automatic Edman degradation using a sequencer: 42 steps were performed out of 60 residues, (b) tryptic peptides of CB5, (c) peptides formed by cleavage of S-carboxymethylated arginine kinase (whole protein) at the two tryptophanyl residues with BNPS-skatole. The complete amino acid sequence of the CNBr polypeptide (CB5) which contains the two tryptophanyl residues of the whole protein was established.     

Identification and characterization of a phospholipid-binding site of bovine factor Va

Coagulation factor Va is a cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. The phospholipid-binding domain of bovine factor Va has been reported to be located on the light chain of the molecule and more precisely on a fragment of Mr = 30,000 which is obtained after digestion of factor Va light chain by factor Xa. This proteolytic fragment is located in the NH2-terminal part of factor Va light chain (residues 1564-1765). In order to further characterize the lipid-binding domain of bovine factor Va, isolated bovine light chain was preincubated with synthetic phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine) and digested with trypsin, chymotrypsin, and elastase. Two peptide regions protected from proteolytic cleavage were identified and characterized from each proteolytic digestion. A comparison of the NH2-terminal sequence and amino acid composition of the two tryptic peptides with the deduced sequence of human factor V indicates a match with residues 1657-1791 of the light chain of human factor V for one peptide and residues 1546-1656 for the other peptide. When chymotrypsin or elastase were used for digestion, the NH2-terminal sequence of one peptide showed a match with residues 1667-1797 of the light chain, while the other peptide presented an NH2-terminal sequence identical with the previously described for the bovine factor Va light chain. When these peptides were assayed for direct binding to phospholipid vesicles, only the tryptic and the chymotryptic peptides covering the middle region of the A3 domain of the bovine factor Va light chain demonstrated an ability to interact with phospholipid vesicles. Thus, knowing that the factor Xa cleavage site on the factor Va light chain is located between residues 1765 and 1766 of the light chain this lipid-binding region of the bovine factor Va is further localized to amino acid residues 1667-1765.     

A kinetic model of vertebrate 20S proteasome accounting for the generation of major proteolytic fragments from oligomeric peptide substrates

There is now convincing evidence that the proteasome contributes to the generation of most of the peptides presented by major histocompatibility complex class I molecules. Here we present a model-based kinetic analysis of fragment patterns generated by the 20S proteasome from 20 to 40 residues long oligomeric substrates. The model consists of ordinary first-order differential equations describing the time evolution of the average probabilities with which fragments can be generated from a given initial substrate. First-order rate laws are used to describe the cleavage of peptide bonds and the release of peptides from the interior of the proteasome to the external space. Numerical estimates for the 27 unknown model parameters are determined across a set of five different proteins with known cleavage patterns. Testing the validity of the model by a jack knife procedure, about 80% of the observed fragments can be correctly identified, whereas the abundance of false-positive classifications is below 10%. From our theoretical approach, it is inferred that double-cleavage fragments of length 7-13 are predominantly cut out in "C-N-order" in that the C-terminus is generated first. This is due to striking differences in the further processing of the two fragments generated by the first cleavage. The upstream fragment exhibits a pronounced tendency to escape from second cleavage as indicated by a large release rate and a monotone exponential decline of peptide bond accessibility with increasing distance from the first scissile bond. In contrast, the release rate of the downstream fragment is about four orders of magnitude lower and the accessibility of peptide bonds shows a sharp peak in a distance of about nine residues from the first scissile bond. This finding strongly supports the idea that generation of fragments with well-defined lengths is favored in that temporary immobilization of the downstream fragment after the first cleavage renders it susceptible for a second cleavage.     

Isolation, characterization and N-terminal sequences of the CNBr-cleavage peptides from human complement Factor B. Localization of a free thiol group and a sequence defining the site cleaved by factor D.

Nine CNBr-cleavage peptides from Factor B (a component of the alternative pathway of complement) were isolated. Each was characterized by amino acid analysis and automated Edman degradation. One peptide contained a methionyl bond resistant to cleavage by CNBr. The number of CNBr-cleavage peptides is in agreement with the results of amino acid analysis of Factor B and the fragments Ba and Bb. A total of 358 unique residues were identified from the N-terminal sequences of the CNBr-cleavage peptides. These represent approx. 50% and 60% of the total residues of Factor B and fragment Bb respectively. Alignment of two CNBr-cleavage peptides (CB-VIc and CB-IV) provided a continuous segment of 140 residues. This sequence contained the site cleaved by Factor D to generate the Ba and Bb fragments during the activation of complement. Peptide CB-IV contained a free thiol group at a position corresponding to residue 33 of fragment Bb. Amino sugar analyses of Factor B and of fragments Bb and Ba indicated that all the carbohydrate structures of factor B are N-linked to asparagine through N-acetylglucosamine. The two carbohydrate-attachment sites of the Bb fragment were identified.     

Serratia protease. Amino acid sequences of both termini, the 53 residues in the middle region containing the sole methionine residue, and a probable zinc-binding region

Reexamination of the molecular mass and the amino acid composition of Serratia protease revealed the presence of 1 mol of methionine per mol of protein (about 46K daltons), and this was confirmed by BrCN cleavage followed by separation of the two fragments. The sole methionine residue was located near the middle region of the molecule. The amino(N)-terminal sequence was determined by Edman degradation of the protein and studies of several proteolytic peptides, establishing a sequence of 18 residues with a heterogeneous N-terminus. The carboxyl(C)-terminal sequence was determined by carboxypeptidase A digestion and tritium-labeling of the citraconylated C-terminal half segment to be -Phe-Ile-Val. The sequences of a total of 53 residues containing the methionine residue and a total of 38 residues containing two histidine residues were established by the application of various conventional methods to a BrCN peptide and several proteolytic peptides. The segment containing the histidine residues was homologous with that containing the two histidine residues chelating the zinc atom of thermolysin. The 38-residue segment may be directly connected to the 53-residue segment.     

The interaction site for tamoxifen aziridine with the bovine estrogen receptor

Calf uterine estrogen receptor was covalently labeled with [3H]tamoxifen aziridine during affinity chromatography purification. After carboxymethylation, affinity labeled receptor was digested with trypsin under limit conditions and the labeled peptides were fractionated by reversed-phase high performance liquid chromatography into one major and two minor components. Sequence analysis of the dominant labeled fragment indicated the facile cleavage of label during Edman degradation but identified two peptides, both derived from the extreme carboxyl terminus of the steroid-binding domain. The 17 residues of one peptide were fully conserved in all estrogen receptors. This fragment contained five nucleophilic amino acids and was considered as the more favored interaction site for tamoxifen aziridine. A corresponding region of the glucocorticoid receptor has recently been identified as one of three major contact sites for glucocorticoids (Carlstedt-Duke, J., Str?mstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and J?rnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). A comparison of amino acid physical characteristics in the hormone-binding domains of human estrogen and glucocorticoid receptors demonstrated an excellent structural correlation between the two regions and delineated elements in the estrogen receptor which may be directly involved in estradiol binding.     

Amino acid sequence of an active fragment of potato proteinase inhibitor IIa.

The complete amino acid sequence of an active fragment of potato proteinase inhibitor IIa has been established by the Edman degradation procedure and the carboxypeptidase technique. Sequence analyses were carried out on the reduced and carboxymethylated active fragment and its tryptic peptides. To aid in the alignment of some tryptic peptides, the partial sequences of two fragments obtained by selective tryptic cleavage of the reactive site peptide bond of inhibitor IIa at acidic pH, with subsequent reduction and carboxymethylation, were also analyzed. The active fragment consisted of 45 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by subtilisin [EC 3.4.21.14.] at pH 6.5. yielded 3 cystine-containing peptides. Sequence analyses of these peptides revealed that the 3 disulfide linkages were located between Cys(10) and Cys(24), Cys(14) and Cys(35), and Cys(20) and Cys(43). The reactive site peptide bond of inhibitor IIa, a Lys-Ser bond, was located between positions 32 and 33 of the active fragment. The overall sequence of the active fragment was quite different from those of potato chymotrypsin inhibitor I (subunit A) and potato carboxypeptidase inhibitor.     

Determination of a cAMP-dependent protein kinase phosphorylation site in the C-terminal region of human endothelial actin-binding protein

Three different C-terminal regions of human endothelial actin-binding protein-280 (ABP-280 or ABP; nonmuscle filamin) were subcloned and efficiently expressed in the Escherichia coli BL21 (DE3) system as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As predicted by the aminoacid sequence one of the fragments, a 109-kDa peptide (residues 1671-2647), contained a calpain cleavage site and two potential cAMP-dependent protein kinase (PKA) phosphorylation sites (serine 2152 and threonine 2336). A second fragment, a 74-kDa peptide (residues 1671-2331), contained a calpain cleavage site and one of the three presumptive PKA phosphorylation sites (serine 2152). The third fragment, a 48-kDa peptide (residues 2223-2647), contained only one of the PKA sites (threonine 2336). Phosphorylation of these truncated peptides indicated that only the fragments containing serine 2152 incorporated phosphate after PKA treatment. Site-directed mutagenesis analysis confirmed that serine 2152 is the unique substrate for PKA in the C-terminal region of ABP. The functional significance of phosphorylation of this residue, which belongs to a serine-proline motif, is discussed.     

Amino acid sequence of the Bb fragment from complement Factor B. Sequence of the major cyanogen bromide-cleavage peptide (CB-II) and completion of the sequence of the Bb fragment.

The amino acid sequence of peptide CB-II, the major product (mol.wt. 30 000) of CNBr cleavage of fragment Bb from human complement Factor B, is given. The sequence was obtained from peptides derived by trypsin cleavage of peptide CB-II and clostripain digestion of fragment Bb. Cleavage of two Asn-Gly bonds in peptide CB-II was also found useful. These results, along with those presented in the preceding paper [Gagnon & Christie (1983) Biochem. J. 209, 51-60], yield the complete sequence of the 505 amino acid residues of fragment Bb. The C-terminal half of the molecule shows strong homology of sequence with serine proteinases. Factor B has a catalytic chain (fragment Bb) with a molecular weight twice that of proteinases previously described, suggesting that it is a novel type of serine proteinase, probably with a different activation mechanism.     

Generation of peptides by human erythrocytes: facts and artifacts

Previously reported data on peptide composition of human erythrocyte lysate were obtained under conditions that did not exclude proteolytic degradation of hemoglobin in the process of peptide isolation. Comparative chromatographic analysis of the diluted erythrocyte lysate incubated in acidic conditions with or without proteolytic enzyme inhibitors showed that several peptides earlier identified as intraerythrocyte ones in fact result from hemoglobin degradation by erythrocyte acidic protease(s) during incubation of the lysate. A rational scheme excluding postlysis proteolysis was developed for isolation of peptide fraction. Further analysis resulted in determination of structure and content of about 50 endogenous intraerythrocyte hemoglobin fragments. A primary endopeptidase splitting of alpha- and beta-globin chains followed by consecutive exopeptidase trimming of primary fragments is suggested as a degradation mechanism. The intraerythrocyte peptides were shown to differ from peptides excreted by the erythrocytes to the extracellular medium in the primary culture. It was also found that intraerythrocyte peptides cannot play the role of precursors of hemoglobin fragments present in tissue extracts.     

Partial cleavage mapping of the cytoskeletal protein vinculin. Antibody and talin binding sites.

Vinculin is a 1066-amino acid protein found at several types of actin-membrane junction. To locate sites of interest in the primary structure, a map was derived using partial cleavage reactions. Of several different types of cleavage tested, the most useful was the 5-5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) reaction which cuts at cysteine residues. About 30 well defined fragments were obtained from vinculin, and several methods were used to locate these products in the sequence. Comparison of the peptides generated from whole vinculin with those from the 90-kDa amino-terminal proteolytic fragment revealed which originated there. The use of [14C]cyanide in conjunction with DTNB showed which peptides contained the original amino terminus. Secondary cleavage with N-chlorosuccinimide, a tryptophan-specific reagent, helped locate fragments, although it led to apparent increases in molecular weight of the products. These experiments revealed the location of 10 of the major DTNB fragments on the sequence. This map was used to locate binding sites. The site of interaction between vinculin and the focal contact protein talin was mapped by binding labeled talin to the separated fragments. The binding site was found to be in the amino-terminal 325 amino acids. The binding site of a commercially obtained monoclonal antivinculin antibody was mapped using Western blotting of cleaved vinculin. It proved to bind in the central area of the molecule between amino acid residues 545 and 737. Thus the cysteine cleavage reaction products provide a map of general utility for locating features on the vinculin molecule.