scholarly journals Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes.

1980 ◽  
Vol 84 (3) ◽  
pp. 717-738 ◽  
Author(s):  
A Adoutte ◽  
R Ramanathan ◽  
R M Lewis ◽  
R R Dute ◽  
K Y Ling ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Membrane Vesicles ◽  
Two Dimensions ◽  
Exponential Growth Phase ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Protein Patterns ◽  
Ciliary Membrane

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.

scholarly journals Characterization of the cilia and ciliary membrane proteins of wild-type Paramecium tetraurelia and a pawn mutant.

1981 ◽  
Vol 89 (2) ◽  
pp. 206-215 ◽  
Author(s):  
S J Merkel ◽  
E S Kaneshiro ◽  
E I Gruenstein
Keyword(s):  
Membrane Proteins ◽  
Mutant Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Blue Staining ◽  
Neutral Sugar ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
One Dimensional ◽  
Coomassie Blue ◽  
Lentil Lectin

Cilia and ciliary membranes were isolated from axenically grown, wild-type Paramecium tetraurelia strain 51s and from the extreme pawn mutant strain, d495, derived from this parental strain. Over 60 protein bands having molecular weights of 15 to greater than 300 kdaltons were detected by Coomassie Blue staining of whole cilia proteins separated by one-dimensional SDS polyacrylamide gel electrophoresis. About 30 of these protein bands were visible in Coomassie Blue-stained membrane separations. About 60 bands were detected by silver staining of one-dimensional gels of membrane proteins. Differences between Coomassie Blue-stained separations of wild-type and pawn mutant strain d495 membrane proteins were seen in the quantity of a band present at 43 kdaltons. Radioiodination of cell surface proteins labeled approximately 15 protein bands in both wild-type and mutant cilia. The major axonemal proteins were unlabeled. Six membrane glycoproteins were identified by staining one-dimensional separations with iodinated concanavalin A and lentil lectin, two lectins that specifically bind both glucose and mannose residues. Two major neutral sugar species present in an acid hydrolysate of the cilia preparation were tentatively identified as glucose and mannose by gas chromatography of the alditol acetate derivatives.

Comparison of the Effect of Various Methods of Dissociation on the Protein Composition of Rat Liver Ribosomal Subunits

1975 ◽  
Vol 53 (9) ◽  
pp. 935-942 ◽  
Author(s):  
Nabil Hanna ◽  
Claude Godin
Keyword(s):  
Rat Liver ◽  
Ribosomal Proteins ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Small Subunit ◽  
Protein Patterns ◽  
Major Loss ◽  
Centrifugation Technique ◽  
High Concentrations

Rat liver ribosomes were dissociated into subunits using EDTA, sodium pyrophosphate, high concentrations of KCl, as well as by incubation with puromycin in presence of 0.5 M KCl. The subunits obtained were analyzed using the density gradient centrifugation technique and their ribosomal proteins were separated by means of two-dimensional polyacrylamide gel electrophoresis. The ribosomal protein patterns of the two subunits isolated using each of the dissociating method were compared to the protein patterns of monosomes prepared by puromycin treatment alone. Our results revealed that the use of chelating agents to dissociate the ribosomes resulted in the loss of some ribosomal proteins from the small subunit. On the other hand, the use of KCl in high concentrations to dissociate the ribsosomes did not appear to cause any major loss of proteins from the ribosomes except for some acidic proteins.

Immuno-proteomics analysis between OMV of vaccine and dominant wild type strains of Bordetella pertussis in Iran

2020 ◽  
Author(s):  
Ali Badamchi ◽  
Fariborz Bahrami ◽  
Alireza Hadizadeh Tasbiti ◽  
Shamsi Yari ◽  
Morvarid Shafiei ◽  
...  
Keyword(s):  
Bordetella Pertussis ◽  
Protein Profile ◽  
Protein Composition ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Cell Vaccine ◽  
Wild Type ◽  
Protein Antigens ◽  
Vaccination Programs ◽  
Type Strains

Background and Objectives: Despite widespread vaccination programs against pertussis, there has been a worldwide re- surgence of the disease in recent years. We aimed to investigate protein composition of outer membrane vesicles (OMV) of Bordetella pertussis (Bp) and to evaluate the immunogenicity of OMV antigens both in the vaccine and the dominant wild type strains in Iran. Materials and Methods: The OMV were purified from both vaccine and wild type strains. The immunoreactivity of the OMVs was investigated by exposing sera taken from the patients and the vaccinated infants. The protein profiles of OMVs were compared using two-dimensional electrophoresis. The LC-MS/MS was used to analyse and identify differentially ex- pressed protein spots. Results: The two type strains showed differences in their 2D gel protein profile. Further analysis of selected proteins from the dominant Iranian strains using LC-MS/MS demonstrated that the identified proteins fell into different functional catego- ries including (i) metabolism, (ii) membrane transport and secretion system, (iii) biosynthesis and degradation, (iv) adaption, adhesion, pathogenicity, conserved hypothetical and protection responses. Moreover, a number of immunogenic proteins were identified including Bp 2434 (serine protease) and Bp 1616 (putative DNA binding protein) from the vaccine and the wild type strains, respectively which could be considered as potential antigens for an OMV vaccine. Conclusion: OMV Bp could be considered as an alternative vaccine against pertussis, containing the bacterium’s protein antigens that can confer equal efficacy compared to a whole bacterial cell vaccine with advantages such as less side effects and lower costs than acellular pertussis vaccines.

scholarly journals Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions.

1983 ◽  
Vol 96 (4) ◽  
pp. 1030-1039 ◽  
Author(s):  
W J Brown ◽  
W A Shannon ◽  
W J Snell
Keyword(s):  
Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Granule Protein ◽  
Isolation And Characterization ◽  
Cytoplasmic Face ◽  
Sds Page ◽  
Granule Membrane ◽  
Granule Membrane Proteins

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co-migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase-catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.

Dissociation and reassociation of trichocyst proteins: biochemical and ultrastructural studies

1987 ◽  
Vol 87 (1) ◽  
pp. 3-25
Author(s):  
J.B. Peterson ◽  
J.E. Heuser ◽  
D.L. Nelson
Keyword(s):  
Protein Concentration ◽  
Morphological Changes ◽  
Protein Composition ◽  
Reducing Agent ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Ultrastructural Studies ◽  
Cylindrical Shaft ◽  
Experimental Protocols ◽  
Acidic Proteins

Trichocysts, the crystalline exocytotic organelles in Paramecium tetraurelia, are composed of small, acidic proteins existing primarily as disulphide-linked dimers. We have disaggregated trichocyst proteins with heat, simultaneously observing the changes in morphology and protein composition. The tip matrix was most heat-labile; its subunits progressively broke away from the distal end. During this process, breakdown of the cylindrical shaft began. Shafts first became flattened and torn lengthwise, yielding smaller, interconnected pieces still having the crystalline arrangement of their 5 nm thick fibres. Ultimately this pattern became disordered, and discrete fibrils of the same thickness disengaged from the meshwork. In freeze-etched preparations these fibrils were composed of thinner filaments in side-by-side association. Disaggregation of the tip sheath began from the distal end before shaft dissociation was complete. Trichocysts broke down to thin fibrils, but probably not to monomeric subunits. At least three proteins were preferentially released in the initial phase of dissociation. Disulphide-reducing agent present during heating increased the rate of dissociation without altering the sequence of morphological changes or the order of release of individual proteins. The rate and extent of heat-induced dissociation were strongly dependent on pH and cation concentration. The stabilizing effects of low pH and of cations were additive. A cooled suspension of fully dissociated trichocysts reassociated into sedimentable aggregates with discernible filamentous order, but without the crystalline structure of intact trichocysts. Reassociation was dependent upon time, temperature and protein concentration. All but one of the trichocyst proteins re-entered the sedimentable aggregate during reassociation. Reassociation was faster and more complete at pH 6 than at pH 8 and was stimulated by Ca2+, Mg2+ and La3+. Trichocyst proteins dissociated in the presence of dithiothreitol did not reassociate, even after removal of the reducing agent. Trichocysts from mutants defective to varying degrees in trichocyst formation were subjected to similar experimental protocols. Heat-dissociated trichocysts of the mutants scc6 and ptA1 reassociated at rates similar to those of wild-type; ftA3 showed slower reassociation, and tam38 showed little or no reassociation. Reassociation of wild-type trichocyst proteins was blocked by the addition of an equal amount of tam38 trichocyst proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Novel Toluene Elimination System in a Toluene-Tolerant Microorganism

2000 ◽  
Vol 182 (22) ◽  
pp. 6451-6455 ◽  
Author(s):  
Hideki Kobayashi ◽  
Katsuyuki Uematsu ◽  
Hisako Hirayama ◽  
Koki Horikoshi
Keyword(s):  
Membrane Proteins ◽  
Cell Membrane ◽  
Outer Membrane ◽  
Parent Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Sensitive Mutant

ABSTRACT In studies of Pseudomonas putida IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture. These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins. The MVs also contained a higher concentration of toluene molecules (0.172 ± 0.012 mol/mol of lipid) than that found in the cell membrane. In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane. The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000.

Application of two-dimensional protein analysis for strain fingerprinting and mutant analysis of Azospirillum species

1989 ◽  
Vol 35 (10) ◽  
pp. 960-967 ◽  
Author(s):  
René De Mot ◽  
Jos Vanderleyden
Keyword(s):  
Gene Dosage ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Analysis ◽  
Resolving Power ◽  
Two Dimensional ◽  
Wild Type ◽  
Protein Patterns ◽  
Gene Dosage Effect ◽  
Chemically Induced ◽  
Mutant Strains

Phenol-extracted total proteins from wild-type and mutant strains of Azospirillum brasilense and A. lipoferum were subjected to two-dimensional polyacrylamide gel electrophoresis. The protein patterns of both species could be readily distinguished by specific configurations of a limited number of spots. The resolving power of the technique allowed its application for strain fingerprinting. Thus, near-identity of A. brasilense strains Sp7 and Cd was demonstrated. In addition, minor changes in protein profiles resulting from spontaneous, chemically induced, or transposon-mediated mutations in the A. brasilense genome were evidenced. For a nitrate reductase negative mutant of strain Sp245 and a Sp7 mutant affected in exopolysaccharide synthesis, production of a truncated protein was involved. In Tn5-generated mutants, a gene dosage effect for the transposon-encoded neomycine phosphotransferase was observed.Key words: Azospirillum, two-dimensional protein analysis, fingerprinting.

Relationships among species of Verticillium: protein composition of spores and mycelium

1971 ◽  
Vol 49 (8) ◽  
pp. 1293-1297 ◽  
Author(s):  
Gilles Pelletier ◽  
Robert Hall
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Morphological Differentiation ◽  
Protein Composition ◽  
Soluble Proteins ◽  
Polyacrylamide Gel ◽  
Close Similarity ◽  
Protein Patterns ◽  
Electrophoresis Of Proteins

Buffer-soluble proteins extracted from six morphologically different isolates of Verticillium were separated by polyacrylamide gel-electrophoresis. Protein patterns from the six isolates were different from one another whether extracts were prepared from conidia, from young colonies composed of mycelium and conidia, or from 6-day-old mycelium. However, the nature of the patterns, and therefore the degree of differences among species patterns, was influenced by the types of cells from which the extracts were prepared.Patterns of proteins from V. tricorpus, V. nigrescens, and an isolate of uncertain identity (isolate 2) which produced chlamydospores and dark mycelium were clearly different from one another whether extracts were prepared from conidia or mycelium. In contrast, conidia of V. albo-atrum, of V. dahliae, and of an isolate which did not produce pigmented structures produced very similar patterns which differed by only a few protein bands. This close similarity of patterns supports the view that V. albo-atrum and V. dahliae are genetically closely related.The protein composition of conidia differed from that of mycelium. In V. albo-atrum, spore extracts contained at least three proteins not detected in mycelium extracts. Differences between spores and mycelium were even greater in V. nigrescens and isolate 2. Analysis of V. dahliae showed differences between spores, 3-day-old mycelium, and 6-day-old mycelium.Our results support the view that gel-electrophoresis of proteins is useful as a taxonomic tool provided attention is given to the degree of morphological differentiation of the materials to be compared.

Analysis of two-dimensional protein patterns from developmental stages of the potato cyst-nematode, Globodera rostochiensis

Parasitology ◽  
1992 ◽  
Vol 105 (3) ◽  
pp. 461-474 ◽  
Author(s):  
J. M. De Boer ◽  
H. A. Overmars ◽  
J. Bakker ◽  
F. J. Gommers
Keyword(s):  
Developmental Stages ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Globodera Rostochiensis ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Silver Stain ◽  
Molecular Weights

SUMMARYTwo-dimensional polyacrylamide gel electrophoresis was used to examine the differences in total protein composition between two motile stages and two sedentary stages of the potato cyst-nematode, Globodera rostochiensis. Using a sensitive silver stain, 542 reproducible protein spots were distinguished. A list of these spots is presented, showing their apparent molecular weights, estimated isoelectric points, and occurrences in the different developmental stages. When the protein patterns were compared, 401 spots were found to change their presence or size in one or more of the four developmental stages. It is therefore estimated that during the post-embryonic development of G. rostochiensis, 74% of the polypeptides undergo modulation of their synthesis, or are affected by protein degradation or modification. In the motile stages several abundant proteins were present, which disappeared or decreased in concentration in the sedentary stages. Some of these proteins are presumably muscle proteins, and their modulation may illustrate the degeneration of body-wall musculature in the sedentary stages. It is concluded that the potato cyst-nematode has a very dynamic protein metabolism.

scholarly journals Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins.

1983 ◽  
Vol 97 (5) ◽  
pp. 1412-1420 ◽  
Author(s):  
L Eisenbach ◽  
R Ramanathan ◽  
D L Nelson
Keyword(s):  
Membrane Proteins ◽  
Cell Immobilization ◽  
Excitable Membrane ◽  
Cross Linking ◽  
Paramecium Tetraurelia ◽  
Electron Microscopic ◽  
Ciliary Membrane ◽  
Electrophysiological Properties ◽  
Immobilization Antigen ◽  
Major Antigen

The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross-reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i-antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential.

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