Immunological relationship between different types of bovine intermediate filaments

In order to examine the relationship between the intermediate filaments from Purkinje fibres of the cow heart conduction system and five proposed subclasses of mammalian intermediate filaments, the gel electrophoresis-derived enzyme-linked immunosorbent assay (GEDELISA) has been used to examine the specificity and crossreactivity of our antibodies against the Purkinje fibre intermediate filament protein, skeletin. Bovine tissues known to contain intermediate filaments of the five main subclasses were examined with antiskeletin and with preimmune serum and the specific antiserum absorbed with pure skeletin as controls. The antibodies raised against Purkinje fibre skeletin reacted with all three polypeptides of the "neurofilament triplet", with glial fibrillary acidic protein (GFAP), with smooth muscle desmin and also slightly with some prekeratin subunits and with endothelial vimentin. From studies with monoclonal antibodies and amino acid sequencing, certain regions of all intermediate filaments are suggested to be structurally related. Here we show that Purkinje fibre skeletin seems to share antigenic determinants with the proposed five main classes of intermediate filaments. Our antibody is the first carefully controlled experimentally induced antibody having such properties. This might be due to the special attributes of the intermediate filament system in Purkinje fibres, which themselves have unique properties.     

Microtubule-associated protein tau. A component of Alzheimer paired helical filaments

Microtubule-associated protein tau was purified from bovine brain microtubules by either (1) phosphocellulose chromatography, (2) heat treatment at pH 6.4, (3) heat treatment at pH 2.7, (4) heat treatment at pH 2.7 followed by extraction with perchloric acid and precipitation with glycerol, or (5) by precipitation with ammonium sulfate followed by extraction with perchloric acid. All of these tau preparations reacted specifically with antibodies to Alzheimer paired helical filaments. Affinity purified antibodies to tau labeled both Alzheimer neurofibrillary tangles and plaque neurites but not amyloid in Alzheimer brain tissue sections and labeled paired helical filament polypeptides on Western blots. Human brain tau and paired helical filament polypeptides co-migrated on sodium dodecyl sulfate-polyacrylamide gels. These results suggest that tau is a major component of Alzheimer paired helical filaments.     

Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium- potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.     

The conduction system in the human heart at midgestation-immunohistochemical demonstration of the intermediate filament protein skeletin

Summary The intermediate filaments have during recent years increasingly attracted attention and new information on the distribution of the subunits of the filaments has become available by the use of specific antibodies. In the present study human fetal hearts at midgestation were studied with immunofluorescence microscopy for a demonstration of the intermediate filament subunit skeletin. In the ordinary ventricular and atrial myocytes the fluorescence was moderate, mainly concentrated to the Z disk levels. The proximal parts of the conduction system also showed a moderate fluorescence, while the fluorescence was intense in the cells in the peripheral parts. The shift from moderate to intense fluorescence occurred in the very proximal part of the left bundle branch and in the distal part of the intramural right bundle branch. We conclude that the conduction cells attain Purkinje fibre-like characteristics at these levels, as evidenced by the content of skeletin. Furthermore, the human fetal Purkinje fibres are more easily distinguished with the technique used herein than with conventional histological techniques. The observed differences in content of skeletin are discussed in terms of the cytoskeletal function of intermediate filaments and the embryology of the conduction system.     

A study of the effect of sodium dodecyl sulfate on bovine β-casein self-association

The effect of low concentrations of sodium dodecyl sulfate on the self-association of β-casein in solution has been reinvestigated at neutral pH by using instrinsic fluorescence measurements, analytical ultracentrifugation, gel filtration chromatography, and the fluorescent properties of the probe, anilinonaphthalene sulfonate. Sodium dodecyl sulfate was found to interact with the protein so that the normal equilibrium between monomers and micellelike polymers was displaced toward polymer formation. At higher concentrations of sodium dodecyl sulfate, the β-casein polymers became smaller while the monomer-polymer equilibrium remained displaced toward polymer formation. It seems likely that there is a limited number of sites on the β-casein molecule that bind sodium dodecyl sulfate strongly. As a consequence of this binding, the balance of electrostatic and hydrophobic forces is altered to increase the degree of self-association at low concentrations of sodium dodecyl sulfate, despite the increase in net negative charge per protein monomer.     

Purification and Characterization of Axial Filaments from Treponema phagedenis Biotype reiterii (the Reiter Treponeme)

Axial filaments have been purified from Treponema phagedenis biotype reiterii (the Reiter treponeme) and partially characterized chemically. The preparations consist largely of protein but also contain small amounts of hexose (3%). Filaments dissociate to subunits in acid, alkali, urea, guanidine, and various detergents. Amino acid analyses show an overall resemblance to other spirochetal axial filaments and to bacterial flagella. Dissociated filaments migrate as a single band upon acrylamide gel electrophoresis at pH 4.3 (in 4 M urea and 10 (3) M ethylenediaminetetraacetate) and at pH 12, but in sodium dodecyl sulfate gels, three bands are obtained under a wide variety of conditions. Two of these bands migrate very close together, with molecular weights of 33,000 +/- 500. The other band has a molecular weight of 36,500 +/- 500. Analysis of axial filaments by the dansyl chloride method yields both methionine and glutamic acid as amino terminal end groups. Sedimentation equilibrium measurements on dissociated axial filaments in 7 M guanidine hydrochloride yield plots of log C against varkappa(2) which vary with the speed and initial protein concentration used. Molecular weight values calculated from these plots are consistent with a model in which axial filament subunits are heterogeneous with respect to molecular weight in the approximate range of 32,000 to 36,000.     

Electrophoretic isolation and partial characterization of a glycoprotein of the submandibular glands of the mouse

A protein has been isolated from the water-extract of the submandibular glands of the mouse, after Biogel P-300 column passage, followed by preparative polyacrylamide gel electrophoresis at pH 4.3 and subsequently at pH 8.9, designated the AM1 protein. The isolated protein was electrophoretically pure in 7.5, 10 and 15% polyacrylamide gels both at pH 4.3 and at pH 8.9. Likewise, by electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel only one protein band could be detected. Of the total amount of the water-extractable submandibular proteins the recovery of this protein component comprised 3 to 5 per cent. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 28 000, both in 7.5 and 15% gel. The isoelectric point was determined by isoelectric focusing in 4.8% polyacrylamide slabgel to be 4.85. The amino acid analysis showed that the ratio of acidic amino acids (Glx plus Asx) to basic amino acids (Lys plus Arg) is 2.3. The glycoprotein consists of protein for 77.4 per cent and of carbohydrate for 22.6 per cent. The molar ratio of the carbohydrates was GlcNH2:GalNH2:Man:Gal:Glc:Fuc:sialic acid = 22.0:1.3:3.0:1.7:10.0:2.6:0.3. The glycoprotein was not secreted from the submandibular glands by stimulation with cholinergic (acetylcholin) or adrenergic (noradrenalin) drugs both in vitro and in vivo. So, it appeared that this glycoprotein could be characterized as a cellular, non-secretory component of these salivary glands.     

Reassembly of a fimbrial hemagglutinin from Pseudomonas solanacearum after purification of the subunit by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Distilled water homogenates of Pseudomonas solanacearum B1, a highly fimbriated strain, strongly agglutinated human group A erythrocytes. The fimbriae and hemagglutinating activity were precipitated from the crude extract with 1% acetic acid, redissolved at pH 10, and precipitated again with 20 mM CaCl2 at pH 6.9. Ca2+, Mg2+, and Zn2+ had similar ability to precipitate the fimbrial hemagglutinin, but Na+ and K+ were much less effective. The fimbrial protein in the precipitate was purified to homogeneity by preparative gel electrophoresis in sodium dodecyl sulfate. The major protein band was eluted, and sodium dodecyl sulfate was removed by chromatography on ion retardation resin (AG 11A8) in 6 M urea. After dialysis against 10 mM sodium acetate (pH 4.5) to remove the urea, the protein reassembled to yield long fibers. These fibers were identical to fimbriae in the crude extract in diameter (6 nm) and in their ability to cause hemagglutination. The purified fimbriae contained no carbohydrates and wee similar to other bacterial fimbriae in amino acid composition, with hydrophobic amino acids comprising 41.8% of the total.     

Neurospora crassa mitochondria contain two forms of a 4'-phosphopantetheine-modified protein

When Neurospora crassa was labeled with [14C]pantothenic acid during growth, the mitochondrial fraction contained two bands of radioactivity of Mr 19,000 and 22,000 by sodium dodecyl sulfate gel electrophoresis. The 19-kDa band was converted to the 22-kDa band by four treatments which are characteristic of the cleavage of a thioester bond: dithiothreitol and 2-mercaptoethanol at basic but not neutral pH, alkaline methanolysis, sodium borohydride in tetrahydrofuran, and hydroxylamine at neutral pH. Mitochondrial subfractionation indicated that the 22-kDa form was preferentially associated with the soluble fraction while the 19-kDa form was found in all fractions. Several properties of the mitochondrial protein were similar to the Escherichia coli acyl carrier protein: Mr on sodium dodecyl sulfate gels, decreased electrophoretic mobility under deacylating conditions, isoelectric point, and covalent attachment of 4'-phosphopantetheine. The 19- and 22-kDa bands may therefore represent acylated and deacylated forms of a mitochondrial acyl carrier protein.     

A comparison of the dodecyl sulfate-induced precipitation of the myelin basic protein with other water-soluble proteins

The interactions of sodium dodecyl sulfate with a number of proteins were examined at a variety of pH values ranging from 4.8 to 11.6 The dodecyl sulfate-induced precipitation of some of these proteins was observed within a relatively limited range of total dodecyl sulfate concentration. Most of the basic proteins precipitated at low pH but as the isoelectric point of the protein was approached the amount of protein that precipitated decreased. Bovine myelin basic protein was unique in that it precipitated at all pH values examined both above and below its isoelectric point. Thus, the dodecyl sulfate-induced precipitation of myelin basic protein appears to be different from the dodecyl sulfate-induced precipitation of most proteins. A comparison of protein precipitation at equivalent dodecyl sulfate: protein molar or weight ratios revealed very little difference in the precipitation behavior of the proteins studied. When the bovine myelin basic protein was cleaved at its single tryptophan residue, the N-terminal fragment (1–115) formed insoluble dodecyl sulfate complexes at pH values ranging from 4.8 to 9.2. The C-terminal fragment (116–169) precipitated almost completely at pH 4.8 but to a lesser extent at pH 7.4 and 9.2 Equimolar mixtures of the N- and C-terminal fragments precipitated in the presence of dodecyl sulfate at pH 7.4 and 9.2 to an extent greater than the C-terminal fragment alone but comparable to the N-terminal fragment alone or the whole basic protein. These results suggest: (a) that the mechanism by which dodecyl sulfate induces the precipitation of myelin basic protein may be unique compared to other proteins and (b) that the intact myelin basic protein is not necessary for its precipitation by dodecyl sulfate.     

Subunit structure of Mycobacterium smegmatis fatty acid synthetase. Evidence for identical multifunctional polypeptide chains

Fatty acid synthetase from Mycobacterium smegmatis has been purified to near homogeneity as judged by a variety of electrophoretic criteria under both native and dissociating conditions. A single protein band was obtained on gel electrophoresis in sodium dodecyl sulfate or 8 M urea at various pH values and on isoelectric focusing in 8 M urea. A subunit molecular weight of about 290,000 was found by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or by sedimentation equilibrium ultracentrifugation in 6 M guanidine HCl. Quantitative Quantitative determination of pantetheine, of flavin, and of the number of fatty acids synthesized during a single enzyme turnover all yield values corresponding to a stoichiometry of about 1 mol per mol of subunit, providing strong evidence that M. smegmatis fatty acid synthetase is an oligomer of identical, multifunctional polypeptide chains.     

The effect of carboxymethylating a single methionine residue on the subunit interactions of glycophorin A.

Human red cell glycophorin A shows an equilibrium between dimeric and monomeric forms which have been disignated PAS-1 and PAS-2, respectively. This equilibrium, which is dependent upon protein concentration is achieved by incubation in sodium dodecyl sulfate solutions at elevated temperatures and is assayed by sodium dodecyl sulfate gel electrophoresis. Carboxymethylation of glycophorin A in guanidine hydrochloride or urea alters the interactions between polypeptide chains so that the lower molecular weight form (PAS-2) is obtained much more readily. If the carboxymethylation is performed at pH 3.0 the reaction is limited to the two methionine residues of glycophorin A which are located at positions 8 and 81 in the sequence. In the presence of sodium dodecyl sulfate, only one of the two methionine residues is carboxymethylated, and glycoprotein modified under these conditions does not exhibit the change in electrophoretic mobility. Experiments with [1-14C]iodoacetic acid demonstrated that Met-81, located in the hydrophobic domain of the protein, is the residue protected by sodium dodecyl sulfate. Modification of Met-81 destabilizes the dimeric form relative to the monomer by weakening the interactions between polypeptide chains. The experiments described in this paper confirm that the hydrophobic domain of glycophorin A is involved in subunit interactions and that Met-81 plays a critical role in those interactions.     

Chemical cross-linking of hemoglobin H. A possible approach to introduce cooperativity and modification of its oxygen transport properties.

Native and reconstituted hemoglobin H molecules were cross-linked with glutaraldehyde at pH values close to the physiological. The Schiff base adducts were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after reduction with sodium borohydride. The major component had a molecular weight of about 31 000 which corresponded to the dimeric species of the beta subunit. In contrast to the native protein, which has very high oxygen affinity and no heme-heme interaction or 2,3-diphosphoglyceric acid effect, the modified hemoglobin H molecules showed cooperative oxygen binding, decreased oxygen affinity and a noticeable 2,3-diphosphoglyceric acid effect.     

Characterization of fructose 1,6-bisphosphatase from bakers' yeast

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.     

Protease activity of CND41, a chloroplast nucleoid DNA-binding protein, isolated from cultured tobacco cells

CND41 is a 41 kDa DNA-binding protein isolated from chloroplast nucleoids of cultured tobacco cells. The presence of the active domain of aspartic protease in the deduced amino acid sequence of CND41 suggests that it has proteolytic activity. To confirm this, CND41 was highly purified from cultured tobacco cells and its proteolytic activity was characterized with fluorescein isothiocyanate-labeled hemoglobin as the substrate. The purified CND41 had strong proteolytic activity at an acidic pH (pH 2-4). This activity was inhibited by various chemicals, including the nucleoside triphosphates, NADPH, Fe(3+) and sodium dodecyl sulfate.     

Reversible inactivation of pancreatic deoxyribonuclease A by sodium dodecyl sulfate. Removal of COOH-terminal residues from the denatured protein by carboxypeptidase A.

In the course of experiments on the role of the COOH-terminal residues in pancreatic deoxyribonuclease, we undertook to ascertain whether the presence of sodium dodecyl sulfate would render the normally unavailable terminus susceptible to hydrolysis by carboxypeptidase A. When DNase A is dissolved in 0.005% sodium dodecyl sulfate the protein becomes enzymically inactive when assayed against DNA in the same sodium dodecyl sulfate concentration. The loss of activity caused by treatment with sodium dodecyl sulfate for 1 hour at 45 degrees can be fully restored if the detergent-containing solution is diluted 10-fold into 6 M guanidinium chloride and then 10-fold into a pH 7.0 buffer, 10 mM in CaCl2, prior to a 100-fold dilution for assay. The presence of Ca2+ is essential for the refolding process. If the same degree of dilution is made into sodium dodecyl sulfate-free buffer without the guanidinium chloride step, there is very little reversal of the inactivation. An almost complete loss of regenerable activity is caused by 1 hour of digestion by carboxypeptidase at 45 degrees in the presence of 0.03% sodium dodecyl sulfate. Although up to 6 amino acid residues can be removed from the COOH terminus, the loss of activity can be correlated with the removal of either 1 or 2 amino acid residues (-Leu-Thr) from the COOH-terminal sequence. Thus, DNase A is one of the several enzymes in which residues at the COOH terminus are essential to the active conformation. If the enzyme minus 2 to 6 terminal residues was mixed with a 15-residue COOH-terminal peptide (obtained by cyanogen bromide cleavage), only about 2% activity could be regenerated.     

Mycoplasma Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System: Purification and Characterization of Enzyme I

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella.

Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.