Rickettsia prowazekii polypeptide composition and protective antigens

Serologically active preparations of R. prowazekii membranes were obtained by the lysis of purified R. prowazekii with ether and by differential and gradient centrifugation. Purified R. prowazekii and their membranes were analyzed by the method of electrophoresis in acrylamide gel. The former contained not less than 30 proteins with molecular weights of 10 000-169 000 daltons, while the membrane preparations contained 5 main polypeptides with molecular weights of 12400, 21500, 29600, 34000 and 133600 daltons. Antisera obtained after the immunization of rabbits with the membrane preparation were found to contain antibodies reacting in the complement fixation test and neutralizing rickettsial toxin.     

Biochemical and immunochemical study of antigen preparations of Rickettsia prowazekii

The biochemical and immunochemical study of the qualitative composition of R. prowazekii antigenic preparations, isolated and purified by different methods, indicates that these preparations contain high-molecular polypeptide 3 (133600 D), morphologically linked with the cell membrane of R. prowazekii. The method of adsorption chromatography on calcium phosphate permits obtaining specific rickettsial antigens with a greater degree of purification from ballast admixtures than the methods of acid precipitation and gel filtration on Sephadex G-200.     

The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplasts

A positively charged amino acid sequence, located on the NH2 terminus of the polypeptides of the chlorophyll a/b light harvesting complex, stabilizes thylakoid membrane adhesion. Threonine residues in this segment are the site of light-induced, reversible phosphorylation; this covalent modification results in changes in excitation-energy distribution in chloroplast membranes. Removal of the positively charged peptide by treatment with trypsin or chemical modification of amino acids in the sequence disrupts thylakoid adhesion and inhibits regulation of excitation-energy distribution. Purified preparations of the chlorophyll a/b light harvesting complex consist of 2 major polypeptides of 27 and 26 kDa and 2 minor polypeptides of 29 and 25 kDa (based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Trypsin treatment of the isolated chlorophyll proteins decreases the apparent molecular mass of the 27- and 26-kDa polypeptides by 1-1.5 kDa and releases 3 peptides; [Lys, Arg], Ser-Ala-Thr-Thr-Lys-Lys, and Ser-Ala-Thr-Thr-Lys. These peptides probably form the overlap sequence, [Lys, Arg]-Ser-Ala-Thr-Thr-Lys-Lys. The polypeptides of the chlorophyll a/b light-harvesting complex were separated by isoelectric focusing into 5 chlorophyll protein fractions which had isoelectric points between 4.0 and 4.55. The 27-kDa polypeptides had an isoelectric point of 4.3, and bound 11 chlorophyll molecules/polypeptide.     

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

A collection of 17 monoclonal antibodies elicited against the light-harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC-II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b.     

Biological properties of antibiotic-resistant mutants of the mildly pathogenic Rickettsia prowazekii E strain

The use of R. prowazekii strain E with low pathogenicity as live vaccine against exanthematous typhus is limited by its high specific reactogenicity, which is probably due to the reversion of the virulence of the strain. One of the approaches to the stabilization of the avirulent properties of strain E is obtaining its mutants with stable decreased pathogenic properties. The article presents the data on the infectious properties of R. prowazekii antibiotic-resistant strain E mutants obtained in earlier experiments, in respect of chick embryos and laboratory animals, as well as the capacity of this strain for producing immunity to challenge with R. prowazekii virulent strain in guinea pigs. The study has revealed that the erythromycin-resistant mutant of R. prowazekii strain E, induced by nitrosoguanidine (NG), has lower infective capacity for chick embryos, guinea pigs, cotton rats and white mice. The infective capacity of the NG-induced rifampicin-resistant and spontaneous erythromycin-resistant mutants of R. prowazekii strain E is similar to the infective capacity of the initial strain. The rifampicin-resistant and spontaneous erythromycin-resistant mutants of R. prowazekii strain E possess immunogenicity similar to that of the initial strain E, and the NG-induced erythromycin-resistant mutant possesses lower, but sufficiently pronounced immunogenicity despite its decreased infective capacity for guinea pigs.     

Purification of membrane polypeptides of rat liver peroxisomes

Peroxisomes were obtained by sucrose density gradient centrifugation from the livers of di(2-ethylhexyl)phthalate-fed rats, and the membranes were prepared by carbonate extraction (Fujiki, Y., Fowler, S., Shio, H., Hubbard, A.L., & Lazarow, P.B. (1982) J. Cell Biol. 93, 103-110). The integrated membrane polypeptides were solubilized with sodium dodecyl sulfate, and purified by repeated polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Separation of 70 and 68 kDa polypeptides was not attempted in the present study because of their close migration in polyacrylamide gel electrophoresis. Other polypeptides with apparent molecular masses of 41, 27, 26, and 22 kDa were purified to near homogeneity. Antibodies were raised against these purified preparations. The 68 kDa polypeptide is suggested to be produced by the proteolytic modification of 70 kDa polypeptide, since the former increased concomitantly with decrease of the latter when the liver homogenate was incubated, and this change was prevented in the presence of leupeptin during the incubation. The 41 kDa polypeptide was a minor component. The 70 and 68 kDa polypeptides and 41 kDa polypeptide and their antibodies were cross-reactive, but the relation of these polypeptides was not clear. The 27 and 26 kDa polypeptides seemed to be another species of membrane polypeptides, although the relationship of these two polypeptides remains to be clarified. The 22 kDa polypeptide is not related to other membrane polypeptides. The results of immunoblot analysis of subcellular fractions of the liver and an electron microscopic immunocytochemical study to locate the antigenic sites with protein A-gold complex suggest that all of these polypeptides are localized on peroxisomal membranes. On proliferation of rat liver peroxisomes by administration of di(2-ethylhexyl)phthalate, a peroxisome proliferator, all of these polypeptides were markedly increased.     

Proteins of Epstein-Barr Virus. II. Electrophoretic analysis of the polypeptides of the nucleocapsid and the glucosamine- and polysaccharide-containing components of enveloped virus.

Two series of experiments were undertaken to identify the topological location of the structural polypeptides of Epstein-Barr virus. In the first series of experiments, nucleocapsids prepared by detergent treatment of enveloped virus with Nonidet P-40 and sodium deoxycholate were found to be composed of seven polypeptides, VP2, 6, 7.5, 24, 27, 31, ANd 33, which ranged in molecular weight from over 200 X 10(3) to 28 X 10(3). Nine other polypeptides, VP 4, 7, 8, 10, 15, 16, 23, 28, and 29, could be identified in preparations of Epstein-Barr virus nucleocapsids, but the relative amount of this second group of polypeptides was less in preparations of nucleocapsids than in preparations of enveloped virus. The incomplete removal of these polypeptides from enveloped virus by detergent treatment suggests that some of these polypeptides may be components of the envelope or tegument that lie in close proximity to the outer surface of the nucleocapsid In the second series of experiments periodic acid-Schiff-staining and glucosamine-containing components were identified with similar electrophoretic mobility to several of the polypeptides of enveloped virus (VP 5, 8, 9, 11, 12, 13, 14, 15, 16, 17, 28, and 29) that were completely or incompletely removed from purified virus preparations by detergent treatment. The similarity between the polypeptide composition of the nucleocapsids of Epstein-Barr virus and herpes simplex virus was in contrast to the dissimilarity between the nonnucleocapsid polypeptides of Epstein-Barr virus and herpes simplex virus.     

Purification of peroxisomes from livers of normal and clofibrate-treated mice

A method for the isolation of peroxisomes from livers of normal and clofibrate-treated mice is described. The method utilizes glutaraldehyde to stabilize peroxisomal membranes, and isopycnic centrifugation of a light mitochondrial fraction through a linear metrizamide gradient to achieve optimal resolution from other organelles. On the basis of the biochemical and morphological data, the peroxisomal preparations are indicated as of high purity: contamination by mitochondria, lysosomes, and plasma membranes is negligible, and the level of contaminating microsomes is around 5% for normal peroxisomes and 8% for peroxisomes from clofibrate-treated mice. Peroxisomal membranes prepared by carbonate extraction contain two major polypeptides of approximately 70,000 Da, and show 2 and 8% contamination by microsomal membrane protein for the preparations from normal and clofibrate-treated mice, respectively.     

Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates.

To determine whether certain outer membrane proteins are associated with growth of Bacteroides thetaiotaomicron on polysaccharides, we developed a procedure for separating outer membranes from inner membranes by sucrose density centrifugation. Cell extracts in 10% (wt/vol) sucrose-10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4) were separated into two fractions on a two-step (37 and 70% [wt/vol]) sucrose gradient. These fractions were further resolved into outer membranes (p = 1.21 g/cm3) and inner membranes (p = 1.14 g/cm3) on sucrose gradients. About 20 to 26% of the total 3-hydroxy fatty acids from lipopolysaccharide and 2 to 3% of the total cellular succinate dehydrogenase activity were recovered in the outer membrane preparation. The inner membrane preparation contained 22 to 49% of the total succinate dehydrogenase activity and 2 to 3% of the total 3-hydroxy fatty acids from lipopolysaccharide. Outer membranes contained a lower concentration of protein (0.34 mg/mg [dry weight]) than did the inner membranes (0.68 mg/mg [dry weight]). Molecular weights of inner membrane polypeptides ranged from 11,000 to 133,000. The most prominent polypeptides had molecular weights ranging from 11,000 to 26,000. In contrast, the molecular weights of outer membrane polypeptides ranged from 17,000 to 117,000. The most prominent polypeptides had molecular weights ranging from 42,000 to 117,000. There were several polypeptides in the outer membranes of bacteria grown on polysaccharides (chondroitin sulfate, arabinogalactan, or polygalacturonic acid) which were not detected or were not as prominent in outer membranes of bacteria grown on monosaccharide components of these polysaccharides.     

Location of an F-pilin pool in the inner membrane.

Polyacrylamide gel analysis of [35S]methionine-labeled membrane preparations from Escherichia coli has revealed the presence of five polypeptides present only in the membranes of cells containing the conjugative plasmid F. In addition to the previously reported product of traT, polypeptides migrating with apparent molecular weights of 100,000, 23,500, 12,000, and 7,000 were resolved. Membrane preparations from F traJ mutants lacked these polypeptides, indicating that all of these proteins are tra gene products. The 7,000-molecular-weight polypeptide comigrated with unlabeled purified F-pilin protein. About 4 to 5% of the total radioactive label in whole membrane preparations was present in this polypeptide, indicating the existence of a substantial pool of membrane-associated F-pilin. The polypeptide could be extracted from whole membrane preparations with Triton X-100 and was found in the inner membrane fraction of membranes separated by sucrose density centrifugation.     

Preparation and polypeptide composition of chlorophyll-free plasma membranes from leaves of light-grown spinach and barley

Chlorophyf l-free preparations of plasma membranes from leaves of barley (Hordeum vulgare L. cv. Kristina) and spinach (Spinada oleracea L. cv. Viking II) were obtained by partition in an aqueous dextran-polyethylene glycol two-phase system. CJlu-can synthetase II (EC 2.4,1.34), a marker for the plasma membrane, was highly enriched in both preparations. Silicotungstic acid, a specific stain for the plasma membrane, indicated a purity close to 100% for the barley preparation. Both plasma membrane preparations contained a light-reducible b-cytochrome, as shown by low temperature spectroscopy. The plasma membranes had a tow protein content compared to the bulk of intracellular membranes. The polypeptide composition of the barley and spinach plasma membranes showed striking similarities, with.the most prominent polypeptides in the 49-58 kdalton region, and some further prominent bands in the 30 kcialton region. Some high molecular weight polypeptides in the 73-110 kdalton region were also typical for the plasma membranes compared to the microsomal fractions.     

Structure of a bacterial photosynthetic membrane. Isolation, polypeptide composition, and selective proteolysis

A procedure for the isolation of highly purified bacterial photosynthetic membranes from Rhodopseudomonas viridis is described. The purity of the final membrane fraction has been confirmed by electron microscopy. Seven major polypeptide bands are associated with the photosynthetic membranes, and all seven are resistant to solubilization in Triton X-100 detergent. Two pigmented bands with apparent molecular weights of 44K and 41K are thought to be cytochromes. The three polypeptides with apparent molecular weights of 38K, 32K, and 28K have been reported in reaction center preparations of other laboratories. Two low-molecular-weight (16K and 11K) bands bind bacteriochlorophyll b and may represent light-harvesting bacteriochlorophyll-protein complexes. The structures that were isolated seem to represent complete photosynthetic membranes, consisting of reaction center, electron transport, and light-harvesting components, all arranged in the regular lattice characteristic of viridis. Selective proteolysis of these membranes indicates that all membrane components are accessible to digestion by trypsin and pronase, except for the light-harvesting complexes.     

Similarities in the polypeptide composition of glyoxysomal and endoplasmic-reticulum membranes from castor-bean endosperm.

Microsomal fractions, glyoxysomes and mitochondria were isolated from homogenates of germinating castor-bean (Ricinus communis) endosperm by sucrose-density-gradient centrifugation. Washed membrane preparations from these cellular fractions were examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. At corresponding developmental stages the endoplasmic-reticulum and glyoxysomal membranes were strikingly similar in polypeptide composition, at least 16 polypeptides being present in membranes isolated from 3-day-old tissue. Supplying [35S]methionine to intact endosperm tissue resulted in the labelling of all membrane polypeptides, the specific radioactivity in the endoplasmic reticulum being greater than for equivalent polypeptides of the glyoxysomal membrane. Washing these membranes with sodium deoxycholate solution extensively solubilized protein components, with the exception of a predominant polypeptide of mol.wt. 55000. Mitochondrial membrane preparations differed from those of the endoplasmic reticulum and glyoxysomes in polypeptide molecular-weight distribution and the [35S]methionine-labelling pattern. The similarity in polypeptide composition between endoplasmic-reticulum and glyoxysomal membranes is discussed in relation to glyoxysome biogenesis.     

Phosphorylation of chlamydomonas reinhardi chloroplast membrane proteins in vivo and in vitro

Phosphorylation of thylakoid membrane proteins in the chloroplast of wild-type and mutant strains of Chlamydomonas reinhardi has been studied in vivo and in vitro. Intact cells or purified membranes were labeled with [32P]orthophosphate or [gamma-32P]ATP, respectively, and the presence of phosphorylated polypeptides was detected by autoradiography after membrane fractionation by SDS PAGE. The 32P was esterified to serine and threonine residues. At least six polypeptides were phosphorylated in vitro and in vivo, and corresponded to components of the photosystem II complex contributing to the formation of the light-harvesting-chlorophyll (LHC) a,b-protein complex, the DCMU binding site (32-35 kdaltons), and the reaction center (26 kdaltons). In agreement with previous reports (Alfonzo, et al., 1979, Plant Physiol., 65:730-734; and Bennett, 1979, FEBS (Fed. Eur. Biochem. Soc.) Lett., 103:342-344), the membrane-bound protein kinase was markedly stimulated by light in vitro via a mechanism requiring photosystem II activity. Phosphorylation of thylakoid membrane polypeptides in vivo was, however, completely independent of illumination. Similar amounts of phosphate were incorporated into the photosynthetic membranes of cells incubated in the dark, in white light with or without 3-(3,4- dichlorophenyl-1,1-dimethyl urea (DCMU), or in red or far-red light. Different turnovers of the phosphate were observed in the light and dark, and a phosphoprotein phosphatase involved in this turnover process was also associated with the membrane. Comparison of the amount of esterified phosphate per protein in vivo and the maximum incorporation in isolated membranes revealed that only a small fraction of the available sites could be phosphorylated in vitro. In contrast to the DCMU binding site, the LHC and 26-kdalton polypeptide were not phosphorylated in vivo when the reaction center II polypeptides of 44- 54 kdaltons were missing. The finding that all the phosphoproteins appear to be components of the photosystem II complex and are only partially dephosphorylated in vivo suggests strongly that protein phosphorylation might play an important role in the maintenance of the organizational integrity of this complex. The observation that the LHC is not phosphorylated in the absence of the reaction center lends support to this idea.     

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.     

Monoclonal antibodies to the species-specific and group antigens of Rickettsia prowazekii]

A number of hybridomas to different R. prowazekii determinants were obtained by the hybridization of spleen cells of BALB/c mice immunized with R. prowazekii corpuscular and soluble antigens. Some of the monoclonal antibodies (McAb) reacted with R. prowazekii thermolabile species-specific protein and did not react with R. typhi antigens (McAb of batches B4/4 and A-D3). McAb C5/2 and A3/2 reacted with the group thermostable antigen, common for R. prowazekii and R. typhi. McAb to the species-specific thermolabile antigen belonged to IgG2a. The McAb thus obtained permit the identification of R. prowazekii and R. typhi and the solution of the problem of the intragroup differentiation of rickettsiae belonging to the typhus group.     

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.     

Relations between plasma membrane and lysosomal membrane. 1. Fate of covalently labelled plasma membrane protein

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4 degrees C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37 degrees C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell-associated labels) was rapidly released (half-life less than 1 h), and most label (65%) was released slowly (half-life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37 degrees C, the patterns of 125I-polypeptides quantified by autoradiography after SDS-PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides. The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2-4% of cell-associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H-labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell-associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.     

Immunochemical relationship of the major outer membrane protein of Rhodopseudomonas sphaeroides 2.4.1 to proteins of other photosynthetic bacteria.

Immunoblots of sodium dodecyl sulfate-polyacrylamide gels derived from outer membrane preparations of various strains of Rhodopseudomonas sphaeroides revealed polypeptides which cross-reacted with antibody directed against the major outer membrane protein of R. sphaeroides 2.4.1. Immunochemical quantitation of the major outer membrane protein of strain 2.4.1 showed approximately 5.5 x 10(4) molecules per cell whether cells were grown chemoheterotrophically or photoheterotrophically. Rhodospirillum rubrum outer membranes contained a cross-reactive protein, whereas the outer membranes derived from Rhodopseudomonas capsulata and Paracoccus denitrificans showed no cross-reaction with the antibody prepared against the major outer membrane protein from R. sphaeroides 2.4.1.     

Structure of fumarate reductase on the cytoplasmic membrane of Escherichia coli.

The terminal electron transfer enzyme fumarate reductase has been shown to be composed of a membrane-extrinsic catalytic dimer of 69- and 27-kilodalton (kd) subunits and a membrane-intrinsic anchor portion of 15- and 13-kd subunits. We prepared inverted membrane vesicles from a strain carrying the frd operon on a multicopy plasmid. When grown anaerobically on fumarate-containing medium, the membranes of this strain are highly enriched in fumarate reductase. When negatively stained preparations of these vesicles were examined with an electron microscope, they appeared to be covered with knob-like structures about 4 nm in diameter attached to the membrane by short stalks. Treatment of the membranes with chymotrypsin destroyed the 69-kd subunit, leaving the 27-, 15-, and 13-kd subunits bound to the membrane; these membranes appeared to retain remnants of the structure. Treatment of the membranes with 6 M urea removed the 69- and 27-kd subunits, leaving the anchor polypeptides intact. These vesicles appeared smooth and structureless. A functional four-subunit enzyme and the knob-like structure could be reconstituted by the addition of soluble catalytic subunits to the urea-stripped membranes. In addition to the vesicular structures, we observed unusual tubular structures which were covered with a helical array of fumarate reductase knobs.