Binding of laminin and fibronectin by the trypsin-resistant major structural domain of the crystalline virulence surface array protein of Aeromonas salmonicida.

The surface of Aeromonas salmonicida is covered by a tetragonal paracrystalline array (A-layer) composed of a single protein (A-protein, Mr = 50,778). This array is a virulence factor. Cells containing A-layer and isolated A-layer sheets specifically bound laminin and fibronectin with high affinity. Binding by cells was inactivated by selective removal of A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor tetragonal paracrystalline array producing Aeromonas hydrophila and Aeromonas sobria strains bound either matrix protein. Laminin binding was by a single class of high affinity interactions (cell Kd = 1.52 nM), whereas fibronectin bound via two classes of interactions, one being similar to that of laminin (cell Class 2 interaction Kd = 6.6 nM). This interaction with both proteins was partly hydrophobic. The Class 1 fibronectin interaction was of lower affinity (cell Kd = 218 nM) and distinct. Purified A-protein inhibited binding of both matrix proteins to A-layer, and trypsin cleavage localized the matrix-protein binding region to the N-terminal major trypsin-resistant structural domain of A-protein. Monoclonal antibody inhibition studies showed that A-protein was folded such that Fabs of only one of two antibodies with epitopes mapping C-terminal to this trypsin-resistant peptide was capable of blocking binding.     

Novel structural patterns in divalent cation-depleted surface layers of Aeromonas salmonicida.

The fish pathogen Aeromonas salmonicida possesses a regular surface layer (or A-layer) which is an important virulence determinant. The A-protein, a single bilobed protein organized in a p4 lattice of M4C4 arrangement with two morphological domains, comprises this layer. The role of divalent cations in the A-layer structure was studied to better understand A-protein subunit interactions affecting structural flexibility and function. Divalent cation bridges were found to be involved in the integrity of the A-layer. Two novel A-layer patterns were formed as the result of growth under calcium limitation or by chelation of divalent cations with EDTA or EGTA, thereby constituting the first reported case of formation of distinct regular arrays upon divalent cation depletion. Furthermore, under these conditions A-protein was sometimes released as tetrameric units, rather than in monomeric form. The formation of the two novel patterns is best explained by a sequence of structural rearrangements, following disruption of only one of the two A-layer morphological units, that is, those held together by divalent cation bridges. The free tetrameric units represent four A-protein subunits clustered around the unaffected four-fold axis.     

Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis

Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced into Aeromonas salmonicida 449 which produces a surface protein array known as the A-layer. Kanamycin-resistant exconjugants of 449 with altered ability to produce the A-layer were selected by virtue of their altered colonial morphology and color on medium containing the dye Congo red. Analysis of culture supernatants, periplasmic shock fluid, outer membranes, and whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to A-protein revealed five classes of single-insertion mutations that affected the ability of cells to produce and export A-protein and to assemble the A-layer. These studies suggest that A-protein is produced from a single chromosomal gene. The subunits subsequently pass through the periplasm and across the outer membrane. At least one gene product is required for this export. Assembly of A-layer on the cell surface then requires the presence of O polysaccharide chains on the lipopolysaccharide. In one case, insertion of Tn5 resulted in loss of ability to produce both A-protein and lipopolysaccharide with O polysaccharide chains, suggesting that synthesis of A-protein and synthesis of lipopolysaccharide may involve coordinate regulation.     

Novel structural patterns in divalent cation-depleted surface layers of Aeromonas salmonicida

The fish pathogen Aeromonas salmonicida possesses a regular surface layer (or A-layer) which is an important virulence determinant. The A-protein, a single bilobed protein organized in a p4 lattice of M₄C₄ arrangement with two morphological domains, comprises this layer. The role of divalent cations in the A-layer structure was studied to better understand A-protein subunit interactions affecting structural flexibility and function. Divalent cation bridges were found to be involved in the integrity of the A-layer. Two novel A-layer patterns were formed as the result of growth under calcium limitation or by chelation of divalent cations with EDTA or EGTA, thereby constituting the first reported case of formation of distinct regular arrays upon divalent cation depletion. Furthermore, under these conditions A-protein was sometimes released as tetrameric units, rather than in monomeric form. The formation of the two novel patterns is best explained by a sequence of structural rearrangements, following disruption of only one of the two A-layer morphological units, that is, those held together by divalent cation bridges. The free tetrameric units represent four A-protein subunits clustered around the unaffected four-fold axis.     

Congo red agar, a differential medium for Aeromonas salmonicida, detects the presence of the cell surface protein array involved in virulence.

Strains of the fish pathogen Aeromonas salmonicida which possess the cell surface protein array known as the A-layer (A+) involved in virulence formed deep red colonies on tryptic soy agar containing 30 micrograms of Congo red per ml. These were readily distinguished from colorless or light orange colonies of avirulent mutants lacking A-layer (A-). The utility of Congo red agar for quantifying A+ and A- cells in the routine assessment of culture virulence was demonstrated. Intact A+ cells adsorbed Congo red, whereas A- mutants did not bind Congo red unless first permeabilized with EDTA. The dye-binding component of A+ cells was shown to be the 50,000-Mr A-protein component of the surface array. Purified A-protein avidly bound Congo red at a dye-to-protein molar ratio of about 30 by a nonspecific hydrophobic mechanism enhanced by high salt concentrations. Neither A+ nor A- cells adsorbed to Congo red-Sepharose columns at low salt concentrations. On the other hand, A+ (but not A-) cells were avidly bound at high salt concentrations.     

Characterization of Aeromonas salmonicida variants with altered cell surfaces and their use in studying surface protein assembly

Aeromonas salmonicida variants were characterized for alterations in their cell surface structure and used to examine reconstitution of the surface protein layer (A-layer). Variants lacking outer membrane O-polysaccharide were devoid of A-layer and excreted stainable floret-like material of the surface protein (A-protein). One variant, showing partial loss of O-polysaccharide, was associated with a disrupted A-layer and excretion of some A-protein. Variants lacking A-protein but possessing O-polysaccharide rapidly absorbed and concentrated sufficient excreted A-protein at the cell surface to coat the cells with a single confluent layer. Although differences in electrophoretic mobilities of A-proteins and O-polysaccharides from typical and atypical strains were evident, the different A-proteins and A-protein-deficient variants were interchangeable for reconstitution of a surface protein layer. No association of A-protein with cell surfaces of unrelated gram-negative bacteria was observed.Abbreviations A-layer additional surface protein layer - A-protein surface protein - Ast Aeromonas salmonicida typical - Asa Aeromonas salmonicida atypical - A^- phenotypically A-protein-negative variant - O^- phenotypically O-polysaccharide-negative variant - O^wk phenotypically O-polysaccharide weak variant - BHI brain heart infusion - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TEM transmission electron microscopy     

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.     

High-affinity binding of the basement membrane protein collagen type IV to the crystalline virulence surface protein array of Aeromonas salmonicida

The surface of the fish pathogen Aeromonas salmonicida is covered by a paracrystalline array (the A-layer) which is a virulence factor for the organism. Quantification of the ability of A. salmonicida cells to bind collagen types I and IV in a ¹²⁵I-radiolabelled liquid-phase assay showed that A-layer-positive cells bound high levels of collagen type IV, but significantly lower levels of collagen type I. Collagen type IV binding was confirmed using non-radiolabelled enzyme-linked immunosorbent assays. ¹²⁵I-Collagen type IV binding was rapid, specific, saturable, high affinity, and essentially irreversible by unlabelled collagen type IV. The A-layer was responsible for collagen type IV binding because binding was inactivated by selective removal of the A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor strains of Aeromonas hydrophila possessing a morphologically similar paracrystalline array bound this basement membrane protein.     

Physical and functional S-layer reconstitution in Aeromonas salmonicida.

The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identified by their conspicuous absence in S-layer-defective mutants. As a different approach to establish the multifunctional nature of this S-layer, we established methods for reconstitution of the S-layer of A. salmonicida. Then we investigated the functional competence of the reconstituted S-layer. S-layers were reconstituted in different systems: on inert membranes or immobilized lipopolysaccharide (LPS) from purified S-layer protein (A-protein) or on viable cells from either A-protein or preassembled S-layer sheets. In the absence of divalent cations and LPS, purified A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltration, into macroscopic, semicrystalline sheets formed by oligomers loosely organized in a tetragonal arrangement. In the presence of Ca2+, purified A-protein assembled into normal tetragonal arrays of interlocked subunits. A-protein bound with high affinity (Kd, 1.55 x 10(-7) M) and specificity to high-molecular-weight LPS from A. salmonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A. salmonicida cells which contained LPS, and Ca2+ affected both a regular tetragonal organization of the reattached A-protein and an enhanced reattachment of the A-protein to the cell surface. The reconstitution of preformed S-layer sheets (produced by an S-layer-secreting mutant) to an S-layer-negative mutant occurred consistently and efficiently when the two mutant strains were cocultured on calcium-replete solid media. Reattached A-protein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer-specific phage but largely lacked regular organization, as judged by its inability to bind immunoglobulins. Reattached S-layer sheets were regularly organized and imparted the properties of porphyrin binding, hydrophobicity, autoaggregation, adherence to and invasion of fish macrophages and epithelial cells, and resistance to macrophage cytotoxicity. However, cells with reconstituted S-layers were still sensitive to complement and insensitive to the antibiotics streptonigrin and chloramphenicol, indicating incomplete functional reconstitution.     

Vaccine efficacy in spotted wolffish Anarhichas minor: relationship to molecular variation in A-layer protein of atypical Aeromonas salmonicida

Atypical Aeromonas salmonicida strains comprise a heterogeneous group in terms of molecular and phenotypic characteristics. They cause various conditions of ulcer diseases or atypical furunculosis and are being isolated in increasing number from various fish species and geographical areas. Several marine fish species susceptible to atypical A. salmonicida, including spotted wolffish Anarhichas minor O., are now being farmed and new vaccines may be needed. A commercial furunculosis vaccine for salmon is reported to protect wolffish poorly against experimental challenge with atypical A. salmonicida. The protective antigen(s) in furunculosis vaccines is still unclear, but in oil-adjuvanted vaccine for Atlantic salmon Salmo salar L., the surface A-layer was shown to be important for protection. In spotted wolffish, the efficacy of atypical furunculosis vaccines seems to vary with the atypical A. salmonicida strains used as bacterin in the vaccine. In the present study we investigated whether differences in the A-layer protein among atypical strains might be responsible for the observed variation in vaccine efficacy. Atypical A. salmonicida strains from 16 fish species in 11 countries were compared by genome polymorphism analysis using amplified fragment length polymorphism (AFLP) fingerprinting and by comparative sequencing of the vapA genes encoding the A-protein. The A-protein sequences appeared to be highly conserved except for a variable region between Residues 90 to 170. Surprisingly, the grouping of strains based on AFLP- or A-protein sequence similarities was consistent. In addition, serological differences in the A-protein among the strains were demonstrated by an A-protein-specific monoclonal antibody. Vaccines based on atypical A. salmonicida strains possessing genetically and serologically different A-layer proteins were shown to result in significantly different protection in spotted wolffish.     

Association of NASP with HSP90 in mouse spermatogenic cells: stimulation of ATPase activity and transport of linker histones into nuclei

NASP (nuclear autoantigenic sperm protein) is a linker histone-binding protein found in all dividing cells that is regulated by the cell cycle (Richardson, R. T., Batova, I. N., Widgren, E. E., Zheng, L. X., Whitfield, M., Marzluff, W. F., and O'Rand, M. G. (2000) J. Biol. Chem. 275, 30378-30386), and in the nucleus linker histones not bound to DNA are bound to NASP (Alekseev, O. M., Bencic, D. C., Richardson R. T., Widgren E. E., and O'Rand, M. G. (2003) J. Biol. Chem. 278, 8846-8852). In mouse spermatogenic cells tNASP binds the testis-specific linker histone H1t. Utilizing a cross-linker, 3,3'-dithiobissulfosuccinimidyl propionate, and mass spectrometry, we have identified HSP90 as a testis/embryo form of NASP (tNASP)-binding partner. In vitro assays demonstrate that the association of tNASP with HSP90 stimulated the ATPase activity of HSP90 and increased the binding of H1t to tNASP. HSP90 and tNASP are present in both nuclear and cytoplasmic fractions of mouse spermatogenic cells; however, HSP90 bound to NASP only in the cytoplasm. In vitro nuclear import assays on permeabilized HeLa cells demonstrate that tNASP, in the absence of any other cytoplasmic factors, transports linker histones into the nucleus in an energy and nuclear localization signal-dependent manner. Consequently we hypothesize that in the cytoplasm linker histones are bound to a complex containing NASP and HSP90 whose ATPase activity is stimulated by binding NASP. NASP-H1 is subsequently released from the complex and translocates to the nucleus where the H1 is released for binding to the DNA.     

Distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) from Aeromonas salmonicida in Atlantic salmon, Salmo salar L.

The distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) purified from the outer surface of the fish pathogen Aeromonas salmonicida, was studied in Atlantic salmon. Radiolabelling was achieved by conjugating the antigens to tyramine cellobiose (TC) or fluorescein isothiocyanate (FITC) which were radioiodinated either before or after conjugation. Since both TC and FITC are trapped intralysosomally at the cellular site of uptake, the ligands are advantageous in studies on tissue distribution of antigens. Injection of TC-A-layer protein and TC-LPS resulted in high specific radioactivity (cpm g⁻¹tissue) in both head kidney and trunk kidney. In contrast, only low specific radioactivity was recovered in spleen, heart and liver. Surprisingly, use of FITC-LPS as the antigen changed the uptake to be high in both spleen and head kidney. Radiolabelled (¹²⁵I-TC-) LPS and A-protein, administered by a dorsal aorta catheterisation technique, were cleared from the blood within 24 h. In immunised fish, the antibody activity against the A-layer protein was diminished even within 10 min after administration, in contrast to the level of anti-LPS antibodies which remained high. These results suggest that immune-complex formation took place at least with the A-layer protein, but the uptake of A-layer protein in the various tissues did not differ significantly in vaccinated (A. salmonicida bacterin) and non-vaccinated fish.     

A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization.

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques. All forms of A-protein were found to activate the secretion of tumor necrosis factor alpha from murine macrophage. To date, this represents the first large-scale production of biologically active recombinant A-protein.     

A temperate bacteriophage specific for strains ofAeromonas salmonicida possessing A-layer,a cell surface virulence factor

A temperate bacteriophage designated TP446 was isolated from culture supernatants ofAeromonas salmonicida strain A446. Phage TP446 adsorbed to all of the typical and atypical strains ofA. salmonicida tested that possessed A-layer, the surface protein array that represents the primary virulence factor of this fish pathogen. In contrast, TP446 failed to adsorb to mutants lacking A-layer. These results indicate that the A-layer is a component of the receptor for phage TP446.     

Screening microorganisms for insulin binding reveals binding by Burkholderia multivorans and Burkholderia cenocepacia and novel attachment of insulin to Aeromonas salmonicida via the A-layer

Exposure to microorganisms is considered an environmental factor that can contribute to Type 1 diabetes. Insulin-binding proteins (IBPs) on microorganisms may induce production of antibodies that can react with the human insulin receptor (HIR) with possible consequences in developing a diabetic autoimmune response against HIR and insulin. The interaction of insulin with microorganisms was studied by screening 45 microbial species for their ability to bind insulin. Binding assays were performed using labelled insulin to identify insulin-binding components on the microorganisms. Burkholderia multivorans and Burkholderia cenocepacia isolated from patients with cystic fibrosis (CF) and the fish pathogen Aeromonas salmonicida were the only strains of those tested, which showed insulin-binding components on their cell surfaces. Further work with A.?salmonicida suggested that the insulin-binding activity of A.?salmonicida is due to the A-layer. A mutant of A.?salmonicida lacking the A-layer showed binding, but at a much reduced rate suggesting another insulin-binding component in addition to the high affinity of the A-protein. Soluble protein lysates were subjected to Western ligand blotting using peroxidase-labelled insulin to detect IBPs. Two positive IBPs were apparent at approximately 30 and 20?kDa in lysates from Burkholderia strains, but no IBP was detected in A.?salmonicida lysates.     

A-Protein from Achromogenic Atypical Aeromonas salmonicida: Molecular Cloning, Expression, Purification, and Characterization

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS–PAGE and gel filtration chromatography. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques. All forms of A-protein were found to activate the secretion of tumor necrosis factor α from murine macrophage. To date, this represents the first large-scale production of biologically active recombinant A-protein.     

Genetic diversity among A-proteins of atypical strains of Aeromonas salmonicida

The virulence array protein gene A (vapA) encoding the A-protein subunit of the surface layer of 23 typical and atypical strains of Aeromonas salmonicida from salmonids and marine fish species were sequenced, and the deduced A-protein sequences compared. The A-proteins of the typical A. salmonicida ssp. salmonicida strains were shown to be identical, while amino acid variability was revealed among A-proteins of atypical strains. The highest amino acid variability appears to be in a predicted surface exposed region and is believed to result in antigenic differences among the atypical strains of A. salmonicida.     

Different binding of human interferon alpha 1 and alpha 2 to common receptors on human and bovine cells. Studies with recombination interferons produced in Escherichia coli

Minicells from Escherichia coli DS410 harboring cDNA for human interferon (IFN) alpha 1 or alpha 2 were metabolically labeled with [3H]leucine and the radioactive IFN was purified to homogeneity by immune precipitation with anti-IFN-alpha serum. These preparations of radioactive IFN-alpha 1 and -alpha 2 were used to study the binding on two human (FL and Daudi) and one bovine (MDBK) cell lines. IFN-alpha 2 specifically bound well to both human and bovine cells, while IFN-alpha 1 bound very poorly to human cells but well to bovine cells. Specific binding of radioactive IFN-alpha 2 to these cell lines was completely inhibited by not only nonradioactive IFN-alpha 2 but also IFN-alpha 1, and binding of IFN-alpha 1 to bovine cell was also competed by IFN-alpha 2 as well as IFN-alpha 1, indicating that the receptors for both IFNs are identical. However, 50-100-fold (on human cells) or 4-fold (on bovine cell) more nonradioactive IFN-alpha 1 than -alpha 2 was required to inhibit the binding of radioactive IFN-alpha 2 to the receptors. Scatchard analysis showed that IFN-alpha 1 and -alpha 2 bind to the receptors on human cells with an apparent Kd of greater than 6 X 10(-10) and 3 X 10(-11) M, respectively, while on bovine cells with a Kd of 4.2 X 10(-11) and 1.6 X 10(-11) M, respectively. These results show that the different target cell specificity of IFN-alpha 1 and -alpha 2 in regard to antiviral activity (Streuli, M., Hall, A., Boll, W., Stewart, W. E., II, Nagata, S., and Weissmann, C. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2848-2852) is due to the different binding activity of IFN-alpha molecules to their common receptors.     

Localization of binding sites within human von Willebrand factor for monomeric type III collagen

Purified human plasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III collagen in a saturable (KD = 1 X 10(-8) M), specific, and rapid manner with a stoichiometry of approximately 1:15 [vWf subunit (Mr 270,000):collagen trimer (Mr 300,000)]. Two reduced and alkylated CNBr peptides of vWf, termed M11 residues 542-622 and M20 residues 948-998 [Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., & Fujikawa, K. (1986) Biochemistry 25, 3171-3184], inhibited vWf binding to collagen. With 125I-vWf (2 X 10(-9) M) as ligand, M11, M20, fragment III (a dimeric, V8 protease, NH2-terminal fragment, Mr 320,000 referenced above), and unlabeled vWf inhibited binding to collagen with EC50 values of 4.8 X 10(-7), 9.4 X 10(-7), 1.1 X 10(-7), and 0.2 X 10(-7) M, respectively. M11 and M20 bind to collagen directly when 125I-labeled peptides are used as ligands. Other CNBr fragments of vWf were less effective as inhibitors (5-fold or less) and bound less avidly to collagen (5-fold or less) compared to M11 and M20. A murine anti-human vWf monoclonal antibody (MR5), which blocks the binding of vWf to collagen, bound selectively to both M11 and M20 when tested in an enzyme-linked immunoadsorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Additional binding sites for the pyruvate dehydrogenase kinase but not for protein X in the assembled core of the mammalian pyruvate dehydrogenase complex: binding region for the kinase.

A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of approximately 60 dihydrolipoyl transacetylase (E2) subunits, approximately 6 protein X subunits, and approximately 2 pyruvate dehydrogenase kinase heterodimers (KcKb). Using a preparation of resolved kinase in which Kc much greater than Kb, E2-X-KcKb subcomplex additionally bound at least 15 catalytic subunits of the kinase (Kc) and a much lower level of Kb. The binding of Kc to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-KcKb subcomplex. This pattern of binding Kc but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the Kc subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the kinase (M. Rahmatullah et al., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugation yielded an E2L-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2.