Structural analysis of the S-Layer of Lampropedia hyalina

The two-dimensional (2D) structure of the regularly structured surface layer (S-layer) of the gram-negative eubacterium Lampropedia hyalina has been determined at the molecular level to a nominal resolution of 2.1 nm by transmission electron microscopy and digital image processing. The inner, or “perforate,” layer consists of dimeric block-shaped units located at two-fold symmetry axes. These morphological dimers associate around three-fold symmetry axes to form a continuous layer with p6 symmetry and a lattice constant of 14.6 ± 0.4 nm. Scanning transmission electron microscopy (STEM) yields a mass-per-area (MPA) value for the perforate layer of 3.5 kDa/nm². The outer, or “punctate,” layer is composed of long, roughly cylindrical units centered on six-fold symmetry axes, which are connected by six fine linking arms joining at the three-fold symmetry axes to create a hexagonal layer with a lattice constant of 25.6 ± 0.5 nm. The MPA of the “composite”-i.e., perforate plus punctate—layer is 10.2 kDa/nm².     

Expression of plant chaperonin-60 genes in Escherichia coli.

We have examined the expression in Escherichia coli of genes encoding a plant chloroplast molecular chaperone, chaperonin-60. Purified plant chaperonin-60 is distinct in that it contains two polypeptides, p60cpn-60 alpha and p60cpn-60 beta, which have divergent amino acid sequences (Hemmingsen, S. M., and Ellis, R. J. (1986) Plant Physiol. 80, 269-276; Martel, R., Cloney, L. P., Pelcher, L. E., and Hemmingsen, S. M. (1990) Gene (Amst.) 94, 181-187). The precise polypeptide composition(s) of the active tetradecameric specie(s) (cpn60(14)) has not been determined. Genes encoding the mature forms of the Brassica napus chaperonin polypeptides have been expressed separately and in combination in E. coli to produce three novel strains: alpha, beta, and alpha beta. The plant cpn60 polypeptides accumulated in soluble forms and to similar high levels in each. There was no conclusive evidence that p60cpn-60 alpha assembled into cpn60(14) species in alpha cells. In beta and alpha beta cells, the plant gene products assembled efficiently into cpn60(14) species. Thus, the assembly of p60cpn-60 alpha required the presence of p60cpn-60 beta, whereas the assembly of p60cpn-60 beta could occur in the absence of p60cpn-60 alpha. Significant proportions of the endogenous groEL polypeptides were not assembled into tetradecameric groEL14 in beta and alpha beta cells. Analysis of the tetradecameric species that did form indicated the presence of novel hybrid cpn6014 species that contained both plant and bacterial cpn60 polypeptides.     

Fractionation of desmosomes and comparison of the polypeptide composition of desmosomes prepared from two bovine epithelial tissues

Desmosomes isolated from bovine tongue mucosa or muzzle epidermis appeared identical by ultrastructural analyses but had some differences in their polypeptide compositions as determined by SDS-PAGE. These preparations were extracted in 9 M urea, 10 mM Tris-HCl (pH 9), and 25 mM B-mercaptoethanol and then centrifuged at 240,000g for 30 min. The urea-soluble and insoluble fractions were analyzed by SDS-PAGE. The urea soluble fractions of both tongue and muzzle desmosomes were enriched in polypeptides of 240, 210, 81, and 75 kDa and also polypeptides (40 to 70 kDa) that were keratin-like, as determined by immunoblotting analyses with keratin antisera. The urea insoluble fraction of tongue desmosomes contained glycoproteins of 165, 160, 140, 110, and 100 kDa, while this fraction from muzzle contained glycoproteins of 165, 115, and 105 kDa. Ultrastructural examinations of insoluble pellets obtained from urea extracted tongue and muzzle desmosomes showed that most of the components at the cytoplasmic faces of the desmosomes were removed, while the membrane regions of the desmosomes resisted the treatment. The urea soluble proteins were dialyzed against 10 mM Tris-HCl (pH 7.6), and the resulting preparation was pelleted by centrifugation and examined by electron microscopy. Ultrastructural examination of this material revealed that it had assembled into a fibrillar meshwork, similar to the fibrillar region adjacent to the submembranous plaque of isolated desmosomes. Thus, treatment of isolated desmosomes with 9 M urea allowed the fractionation of membrane-associated desmosomal proteins from cytoplasmic desmosomal proteins. A comparison of these fractions from tongue and muzzle indicated that the polypeptide compositions of the desmosomes varied between tissues, especially with respect to the fractions enriched in either glycoproteins or keratin.     

Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots

The subunit organization of the tonoplast H+-pumping ATPase from oat roots (Avena sativa L. var. Lang) was investigated. Tonoplast vesicles were treated with low ionic strength solutions (0.1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer or 0.1 mM Na EDTA), carbonate, or a chaotropic reagent (KI), and then centrifuged to give a soluble fraction and a pellet. Treatments with low ionic strength solutions or KI resulted in 70-80% reduction in the membrane-associated ATPase activity, but did not affect the K+-stimulated pyrophosphatase activity. Polypeptides of 72, 60, and 41 kDa were solubilized from tonoplast vesicles by these wash treatments. These polypeptides reacted with polyclonal antibodies against the holoenzyme of tonoplast ATPase (anti-ATPase) and copurified with the tonoplast ATPase activity during gel filtration chromatography (Sepharose CL-6B). Mono-specific antibody against the 72- or 60-kDa polypeptide reacted with the solubilized 72- or 60-kDa polypeptide, respectively. However, the N,N-[14C]dicyclohexylcarbodiimide-binding 16-kDa polypeptide and a 13-kDa polypeptide that also reacted with anti-ATPase and copurified with the tonoplast ATPase activity during gel filtration remained in the pellets after the wash treatments. We conclude that the 72- and 60-kDa polypeptides appear to be peripheral subunits of the tonoplast ATPase and that the 16-kDa polypeptide is probably embedded in the membrane bilayer. Additional subunits of the ATPase complex may include a 41-kDa (peripheral) and a 13-kDa (integral) polypeptide. Based on these results, a working model of the tonoplast ATPase analogous to the F1F0-ATPase is proposed.     

Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells

Treatment of bovine tongue mucosa with 1 M KCl induced a split in the lamina densa of the basement membrane zone (BMZ). The epithelium was then separated from the underlying connective tissue. Electron microscopic analysis of the stripped epithelium revealed that hemidesmosomes and their associated intermediate filaments (IF) remain along the basal surface of the epithelium. This surface was solubilized in an SDS/urea-containing buffer. Characterization of components of this protein mixture was undertaken using human autoantibodies from bullous pemphigoid (BP) patients that have been shown to recognize hemidesmosomal plaque elements (Mutasim, D. F., Y. Takahashi, R. S. Labib, G. J. Anhalt, H. P. Patel, and L. A. Diaz. 1985. J. Invest. Dermatol. 84:47-53) and by production of mAbs. Affinity-purified autoantibodies directed against 180- and 240-kD polypeptides present in the protein preparation generated strong immunofluorescence staining patterns along the BMZ of bovine tongue mucosa. Furthermore, immunogold localization revealed that these two polypeptides are associated with the hemidesmosomal plaque. A mAb preparation directed against a 125-kD polypeptide present in this same protein mixture lamina lucida side of the hemidesmosome. Autoantibodies in BP serum samples, affinity- purified 180-kD autoantibodies and the mAb preparation generated a punctate stain along the substratum attached surface of epithelial cells maintained on glass substrata for approximately 1 wk. The spots appeared to be associated with bundles of IF in cultured mouse keratinocytes. These monospecific antibody probes should prove invaluable for the study of hemidesmosome structure, assembly, and function.     

Construction and characterization of mutants impaired in the biosynthesis of the K88ab antigen

Plasmid pFM205 contains the genetic determinant for the K88ab antigen and is composed of a 4.3-megadalton DNA fragment derived from wild-type K88ab plasmid pRI8801 and cloning vehicle pBR322. The K88 NA of pFM205 contains five genes, which code for polypeptides with apparent molecular weights of 17,000, 26,000 (the K88ab subunit), 27,000 27,500, and 81,000. All five polypeptides were synthesized as precursors approximately 2,000 daltons larger than the mature polypeptides, indicating that they are transported across the cytoplasmic membrane by means of a signal sequence. A set of deletion derivatives of pFM205 was constructed, each containing a deletion in one of the five genes. In strains harboring derivatives of pFM205 containing a deletion in the gene for the 17,000- or 81,000-dalton polypeptide, the K88ab subunit was synthesized and transported to the outside of the cell. However, these strains did not adhere to brushborders or guinea pig erythrocytes, suggesting that the K88ab subunits were not assembled into normal fimbriae. Strains harboring plasmids containing a deletion in the gene for the 27,500-dalton polypeptide still adhered to brush borders and guinea pig erythrocytes, although very little K88ab antigen could be detected with an immunological assay. In strains harboring plasmids containing a deletion in the gene for the 27,000-dalton polypeptide, the K88ab subunit was synthesized but was probably subsequently degraded rapidly.     

Studies on the spectrin-like protein from the intestinal brush border, TW 260/240, and characterization of its interaction with the cytoskeleton and actin

The terminal web of the intestinal brush border contains a spectrin- like protein, TW 260/240 (Glenney, J. R., Jr., P. Glenney, M. Osborne, and K. Weber, 1982, Cell, 28:843-854.) that interconnects the "rootlet" ends of microvillar filament bundles in the terminal web (Hirokawa, N., R. E. Cheng, and M. Willard, 1983, Cell, 32:953-965; Glenney J. R., P. Glenney, and K. Weber, 1983, J. Cell Biol., 96:1491-1496). We have investigated further the structural properties of TW 260/240 and the interaction of this protein with actin. Salt extraction of TW 260/240 from isolated brush borders results in a loss of terminal web cross- linkers primarily from the apical zone directly beneath the plasma membrane. Morphological studies on purified TW 260/240 using the rotary shadowing technique confirm earlier results that this protein is spectrin-like and is in the tetrameric state in buffers of low ionic strength. However, examination of TW 260/240 tetramers by negative staining revealed a molecule much straighter and more uniform in diameter than rotary-shadowed molecules. At salt concentrations at (150 mM KCl) and above (300 mM KCl) the physiological range, we observed a partial dissociation of tetramers into dimers that occurred at both 0 degree and 37 degrees C. We also observed (in the presence of 75 mM KCl) a concentration-dependent self-association of TW 260/240 into sedimentable aggregates. We have studied the interaction of TW 260/240 with actin using techniques of co-sedimentation, viscometry, and both light and electron microscopy. We observed that TW 260/240 can bind and cross-link actin filaments and that this interaction is salt- and pH- dependent. Under optimum conditions (25-75 mM KCl, at pH 7.0) TW 260/240 cross-linked F-actin into long, large-diameter bundles. The filaments within these bundles were tightly packed but loosely ordered. At higher pH (7.5) such bundles were not observed, although binding and cross-linking were detectable by co-sedimentation and viscometry. At higher salt (greater than 150 mM KCl), the binding of TW 260/240 to actin was inhibited. The presence of skeletal muscle tropomyosin had no significant effect on the salt-dependent binding of TW 260/240 to F- actin.     

A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta.

On the basis of the complete nucleotide sequence of the single-stranded, covalently closed circular hepatitis delta virus RNA genome (K.-S. Wang, Q.-L. Choo, A. J. Weiner, J.-H. Ou, R. C. Najarian, R. M. Thayer, G. T. Mullenbach, K. J. Denniston, J. L. Gerin, and M. Houghton, Nature [London] 323:508-514, 1986 [Author's correction, 328:456, 1987]), five long open reading frames (ORFs) encoding polypeptides containing a methionine proximal to the amino terminus were expressed in bacteria. Only polypeptides encoded by the antigenomic ORF5 cross-reacted with antisera obtained from patients with hepatitis delta virus infections. Immunological analysis of viral extracts and the recombinant ORF5 polypeptides synthesized in bacteria and yeast cells revealed that ORF5 encodes the immunogenic epitope(s) shared by both hepatitis delta viral polypeptides p27 delta and p24 delta and probably represents the complete structural gene for p27 delta and p24 delta. We also present evidence that ORF5 encodes the hepatitis delta antigen, an antigen originally found in the nuclei of hepatocytes of infected individuals (M. Rizzetto, M. G. Canese, S. Arico, O. Crivelli, F. Bonino, C. G. Trepo, and G. Verme, Gut 18:997-1003, 1977). A comparison of the primary structure of the predicted hepatitis delta antigen polypeptides with that of the core antigen of the hepatitis B virus shows that these polypeptides are very dissimilar.     

The 55K protein on the 5'' termini of adenovirus type 2 DNA is unrelated to virus-coded candidate transformation proteins (E1-53K, E1-40K-50K) and DNA-binding proteins (E2-42K/47K/73K).

A polypeptide of 55,000 daltons (55K) is linked, probably covalently, to the K' termini of adenovirus type 2 DNA. The 55K polypeptide is synthesized during early stages of infection (T. Yamashita, M. Arens, and M. Green, J. Virol. 30: 497-507, 1979) and thus may function in viral DNA replication, gene regulation, or cell transformation. Several virus-coded early polypeptides have been identified that could correspond to the terminal 55K, including the E1-40K-50K and E1-53K candidate transformation polypeptides and the E2-42K/47K/73K single-stranded DNA-binding polypeptide. We show here that two-dimensional tryptic [35S]methionine-peptide maps of the terminal 55K differ completely from [35S]methionine-peptide maps of four related E1-40K-50K polypeptides, the E1-53K, and the related E2-42K, E2-47K, and E2-73K polypeptides. We conclude that the terminal 55K polypeptide does not correspond to any of the known virus-coded early polypeptides.     

Cloning and expression in Escherichia coli of three Rickettsia prowazekii genes, coding outer membrane proteins]

Rickettsia prowazekii (virulent Breinl strain) random genomic DNA fragments were cloned in the lambda gt11 expression vector by using non-palindromic adaptors. Several immunoreactive clones were selected after screening 20,000 individual recombinant plaques with human convalescent serum. Some recombinants synthesized the complete 60 K protein, and others synthesized beta-galactosidase fusion polypeptides containing epitopes of 134 K protein of the R. prowazekii outer membrane. The amplified genomic library was screened with monospecific antibodies directed against abundant 31 K and 29.5 K outer membrane proteins. Several recombinant clones expressing full or part of 29.5 K polypeptide, and none expressing 31 K polypeptide were revealed. The serum of a patient convalescing from epidemic typhus did not react in western blot with recombinant 29.5 K protein.     

Expression of the Bottom-Component RNA of Cowpea Mosaic Virus: Evidence that the 60-Kilodalton VPg Precursor Is Cleaved into Single VPg and a 58-Kilodalton Polypeptide

In cowpea protoplasts infected with cowpea mosaic virus, a bottom-component (B) RNA-encoded 60-kilodalton (60K) polypeptide is synthesized, which is membrane-bound and represents the direct precursor to the genome-bound protein VPg. The relationship between this VPg precursor and other B-RNA-encoded polypeptides was studied. Digestion of the B-RNA-encoded 170K and 84K polypeptides with Staphylococcus aureus protease V8 and subsequent analysis of the generated peptides with antiserum against VPg showed that a VPg sequence resides internally in these polypeptides. Furthermore, a new B-RNA-encoded polypeptide was detected, with a size of 58K, which differed from the 60K polypeptide only in the lack of VPg sequences. A model is presented in which the 60K VPg precursor is generated from the 200K primary translation product from B RNA and further processed to a 58K polypeptide and single VPg.     

Isolation and characterization of two polypeptides that form intermediate filaments in bovine esophageal epithelium

Cells in the stratified squamous epithelium of bovine esophagus contain abundant tonofilaments measuring 6-10 nm in diameter. Two polypeptides, extracted from esophageal epithelium with 0.05 M Tris, pH 7.4, containing 8 M urea and 25 mM beta-mercaptoethanol, comprise 35% of the total extractable protein. These polypeptides have apparent molecular weights of 46,000 and 56,000 daltons and are rich in glutamic acid- glutamine, glycine, and serine. Each polypeptide can be partially purified by DEAE-cellulose chromatography. Mixtures of the purified polypeptides from filaments in vitro that measured 6-10 nm in diameter. Neither polypeptide formed filaments by itself. Filaments formed in vitro give an alpha-keratin type x-ray diffraction pattern.. These data indicate that the tonofilaments in esophageal epithelium are formed primarily from these two polypeptides.     

Cyanobacterial phycobilisomes. Phycocyanin assembly in the rod substructures of anabaena variabilis phycobilisomes

Phycocyanin complexes with "linker" polypeptides (Lundell, D. J., Williams, R. C., and Glazer, A. N. (1981) J. Biol. Chem. 256, 3580-3592) of 27 and 32.5 kilodaltons have been isolated from dissociated Anabaena variabilis phycobilisomes. In 0.05 M phosphate at pH 7.0, these "trimeric" complexes have the molar composition (alpha beta)3 . 27,000 and (alpha beta)3 . 32,500, where alpha and beta are the subunits of phycocyanin and 27,000 and 32,500 denote single copies of the linker polypeptides. The (alpha beta)3 . 27,000 and (alpha beta)3 . 32,500 complexes have lambda max at 638 and 629 nm and fluorescence emission maxima at 651 and 646 nm, respectively. In 0.6 M phosphate at pH 8.0, the (alpha beta)3 . 27,000 complex forms an (alpha beta)6 . 27,000 disc-shaped aggregate as seen in the electron microscope, whereas the (alpha beta)3 . 32,500 complex forms discs, (alpha beta)6 . 32,500, and stacked disc rods of varying lengths. The former material, containing the 27,000 polypeptide, when mixed with the (alpha beta)6 . 32,500 discs, limits their assembly into rods. The spectroscopic properties of the discs and rods assembled in vitro indicate that energy transfer in phycobilisome rod substructures proceeds from (alpha beta)6 . 32,500 discs to the (alpha beta)6 . 27,000 disc proximal to the core and thence to the core.     

Identification of virus-coded nonstructural polypeptides in bunyavirus-infected cells.

Analyses of bunyavirus-infected cell extracts identified at least two virus-induced nonstructural polypeptides. With snowshoe hare (SSH), La Crosse (LAC), and six SSH-LAC reassortant viruses, it was shown that one of these nonstructural polypeptides (NSs, approximate molecular weight, 7.4 X 10(3)) is coded by the SSH small (S)-size viral RNA species. This nonstructural polypeptide was not detected (at least in the same relative abundancies) in LAC virus-infected cells or in cells infected with reassortants having LAC S RNA. For SSH virus, tryptic peptide analyses of either [3H]leucine- or [3H]arginine-labeled NSs indicated that it contains unique sequences not present in the SSH nucleocapsid (N) polypeptide (also coded by the S RNA; J. R. Gentsch and D. H. L. Bishop, J. Virol. 28:417-419, 1978). Analyses of SSH virus-infected cell extracts and extracts of cells infected with SSH-LAC reassortants having SSH medium (M)-size RNA species indicated that a nonstructural polypeptide (NSM; approximate molecular weight, 12 X 10(3)) is coded by the SSH M RNA species. In extracts of LAC virus-infected cells (or cells infected with SSH-LAC reassortants having LAC M RNA), a polypeptide with an electrophoretic mobility slightly faster than that of the SSH NSM polypeptide was observed (approximate molecular weight, 11 X 10(3)); it has been designated LAC NSM. The relationships of the NSM polypeptides to the other M RNA-coded polypeptides (G1 and G2; J. R. Gentsch and D. H. L. Bishop, J. Virol. 30;767-770, 1979) have not been determined. Two additional polypeptides present in both LAC- and SSH-infected cell extracts also appear to be virus induced (one with an approximate molecular weight of 10 X 10(3), p10; the other with an approximate molecular weight of 18 X 10(3), p18). Whether these polypeptides are virus coded has not been determined.     

A modifiedEscherichia coli chaperonin (groEL) polypeptide synthesized in tobacco and targeted to the chloroplasts

The coding region for theEscherichia coli groEL (chaperonin-60) polypeptide was fused downstream of a pea rubisco small subunit transit peptide coding sequence under the control of a tandem 35S CaMV promoter. Transgenic tobacco plants (Nicotiana tabacum cv. Xanthi) containing this modifiedgroEL gene were produced. The modified groEL polypeptide was correctly imported into chloroplasts and accumulated to high or low levels in different plants. The majority of the modified groEL polypeptide was processed correctly to the mature form within the chloroplasts. Approximately 20% of the imported polypeptides retained a portion of the N-terminal transit peptide (TPgroEL). Both groEL and TPgroEL polypeptides assembled into tetradecameric species in the chloroplasts. In plants accumulating high levels of these products, the majority of the plant chaperonin-60 polypeptides in the chloroplast were present in novel hybrid tetradecameric species containing both bacterial and plant chaperonin-60 polypeptides. In plants accumulating low levels of groEL, the predominant species present appeared to be authentic plant cpn60₁₄ and authentic bacterial groEL₁₄. The growth and development of transgenic and control tobacco plants were indistinguishable.Abbreviations cpn60 chaperonin-60 - cpn10 chaperonin-10 - hsp heat shock protein - rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - ssu small subunit - spp stromal processing peptidase - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

Protein-sequence studies on Rh-related polypeptides suggest the presence of at least two groups of proteins which associate in the human red-cell membrane.

The Rh D blood-group antigen forms part of a complex, involving several other polypeptides, that is deficient in the red cells of individuals who lack all the antigens of the Rh blood-group system (Rhnull red cells). These include components recognized by anti-(Rh D) antibodies and the murine monoclonal antibodies R6A and BRIC 125. We have carried out protein-sequence studies on the components immunoprecipitated by these antibodies. Anti-(Rh D) antibodies immunoprecipitate an Mr-30,000-32,000 polypeptide (the D30 polypeptide) and an Mr-45,000-100,000 glycoprotein (D50 polypeptide). Antibody R6A immunoprecipitates two glycoproteins of Mr 31,000-34,000 (R6A32 polypeptide) and Mr 35,000-52,000 (R6A45 polypeptide). The D30 and R6A32 polypeptides were found to have the same N-terminal amino acid sequences, showing that they are closely related proteins. The D50 polypeptide and the R6A45 polypeptide also had indistinguishable N-terminal amino acid sequences that differed from that of the D30 and R6A32 polypeptides. The putative N-terminal membrane-spanning segments of the two groups of proteins showed homology in their amino acid sequence, which may account for the association of each of the pairs of proteins during co-precipitation by the antibodies. Supplementary data related to the protein sequence have been deposited as Supplementary Publication SUP 50417 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.     

DNA-mediated gene transfer of a human cell surface 170-kilodalton glycoprotein. Evidence for association with an endogenous murine protein

We have previously reported the identification and characterization of two related human cell surface protein complexes, very common antigens 1 and 2 (VCA-1, VCA-2) (Kantor, R. R. S., Mattes, M. J., Lloyd, K. O., Old, L. J., and Albino, A. P. (1987) J. Biol. Chem. 262, 15158-15165). We now report the transfection of DNA sequences encoding the 170-kilodalton heterodimer of VCA-2 from human SK-RC-41 renal cancer cells to B78H1 mouse melanoma cells. B78H1 cells were cotransfected with high molecular weight renal cancer DNA and a plasmid vector containing the neomycin resistance gene. Antibiotic-resistant transfectants were screened for the expression of the 170-kDa heterodimer with mouse monoclonal antibody (mAb) J143. Analysis of mAb J143-positive (J143+) transfectants showed that they expressed a 170-kDa heterodimer with an identical molecular weight, isoelectric point, two-dimensional peptide map, and spatial orientation of surface-exposed epitopes to the homologous 170-kDa species seen in human donor cells. The 170-kDa heterodimer in SK-RC-41 cells is associated with a 140-kDa (designated 140(1] polypeptide to form the VCA-2 complex. The 170-kDa complex and the 140(1)-kDa polypeptides are encoded by genes located on different human chromosomes. J143+ transfectants display a molecule of 140 kDa associated with the 170-kDa complex which is biochemically similar, but non-identical, to the human 140(1)-kDa polypeptide on VCA-2. This evidence supports our interpretation that the transfected human 170-kDa heterodimer associates with a murine counterpart of the human 140(1)-kDa polypeptide in J143+ transfectants.     

Identification and characterization of precursors in the biosynthesis of the K88ab fimbria of Escherichia coli

Four genes encoding for polypeptides with apparent molecular weights of 17,000, 26,000 (the fimbrial subunit), 27,000, and 81,000 have been implicated in the biosynthesis of the K88ab fimbria (Mooi et al., J. Bacteriol. 150:512-521, 1982). Escherichia coli mutants with defects in these genes were examined for the presence of fimbrial precursors. An analysis of these mutants revealed that fimbrial subunits accumulated transiently in the periplasmic space before being translocated across the outer membrane. The 81,000-dalton (d) polypeptide is probably involved in translocating fimbrial subunits across the outer membrane, because in the absence of this polypeptide the fimbrial subunits remained in the periplasmic space, where they were found to be associated with the 17,000- and 27,000-d polypeptides. In mutants with a deletion in the gene for the 27,000-d polypeptide, fimbrial precursors were not detected, because the fimbrial subunits were degraded. The 27,000-d polypeptide might be involved in stabilizing a conformation of the fimbrial subunit required to translocate it across the outer membrane. In the absence of the 17,000-d polypeptide, most fimbrial subunits were found in the periplasmic space associated with the 27,000-d polypeptide. However, small amounts of subunits were also translocated across the outer membrane. These extracellular subunits did not adhere to brushborders, suggesting that fimbrial subunits must be modified by the 17,000-d polypeptide to be assembled into functional fimbriae. A model for the biosynthesis of the K88ab fimbria is proposed.     

Soluble proteins from Schistosoma mansoni and japonicum: a comparative biochemical and immunological analysis

1. Soluble proteins were recovered from male Schistosoma mansoni after homogenization in Tris-HCl buffer containing 0.6 M KCl and 1.0% Triton X-100 followed by preparative electrophoresis on SDS-gel. 2. Polyclonal antibodies produced in mice against the soluble fraction were used in comparative analysis of S. mansoni and S. japonicum using immunoblots and immunoprecipitation of in vitro translated polypeptides. 3. Small molecular weight polypeptide (20-22 kdalton), identified by infected mouse serum (IMS) on immunoblots, was predominant in females and was not cross-reactive with heterologous IMS. 4. A 41-43 kdalton polypeptide which appeared as a doublet on immunoblots performed with polyclonal antiserum 4M, was predominant in males of both species although the polypeptides of S. mansoni showed slower electrophoretic mobility, and therefore the larger size (43 kdalton), than that of S. japonicum. 5. Comparison of fluorograms of the immunoprecipitates of in vitro translated polypeptides indicated that IMS of S. mansoni precipitated two, 30 and 94 kdalton, polypeptides while the IMS of S. japonicum identified at 72 kdalton polypeptide. Antisera 1M, 2M and 4M also showed similarities and differences in polypeptides of in vitro translation products of the two species of Schistosoma.     

Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240 000-Mr and 116 000-Mr polypeptides in the presence of a bifunctional thiol probe.

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.