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1.
Identification and purification of a soluble species of gp120 released by zymolyase treatment of Pneumocystis carinii. 总被引:2,自引:0,他引:2
Purified zymolyase containing beta-glucanase activity releases a soluble species of the gp120 component of the high molecular weight surface antigen complex of rat- and human-derived Pneumocystis carinii. We have purified the soluble gp120 from rat-derived P. carinii by concanavalin A-affinity- and hydrophobic-interaction liquid chromatography. A single band was detected in this fraction by silver staining and immunoblotting. We have also partially purified a soluble form of the corresponding high molecular weight surface antigen from human-derived P. carinii. Identification and purification of a nondenatured soluble species of gp120 will assist in the characterization of its interactions within the surface antigen complex and with host molecules. 相似文献
2.
The membrane-anchor of Paramecium temperature-specific surface antigens is a glycosylinositol phospholipid 总被引:3,自引:0,他引:3
Y Capdeville M L Cardoso de Almeida C Deregnaucourt 《Biochemical and biophysical research communications》1987,147(3):1219-1225
The temperature-specific G surface antigen of Paramecium primaurelia strain 156 was biosynthetically labeled by [3H]myristic acid in its membrane-bound form, but not in its soluble form. It could be cleaved by a phosphatidylinositol-specific phospholipase C from Trypanosoma brucei or from Bacillus cereus which released its soluble form with the unmasking of a particular glycosidic immunodeterminant called the crossreacting determinant. The Paramecium enzyme, capable of converting its membrane-bound form into the soluble one, was inhibited by a sulphydril reagent in the same way as the trypanosomal lipase. From this evidence we propose that the Paramecium temperature-specific surface antigens are anchored in the plasma membrane via a glycophospholipid, and that an endogenous phospholipase C may be involved in the antigenic variation process. 相似文献
3.
Immunological evidence of a common structure between Paramecium surface antigens and Trypanosoma variant surface glycoproteins 总被引:2,自引:0,他引:2
The surface antigens (SAgs) of Paramecium and the variant surface antigens (VSGs) of Trypanosoma can be purified in two distinct molecular forms: a soluble form (solubilized in dilute ethanolic solution in the case of Paramecium, or in water for Trypanosoma) and a membranal form, amphiphile (solubilized in SDS). In trypanosomes, the enzymatic conversion of the membrane form into the soluble form is accompanied by the unmasking of a particular immunological determinant, called cross-reacting determinant (CRD), which is located in the COOH-terminal phospho-ethanolamine glycopeptide. We demonstrate immunological homologies between Paramecium SAgs and Trypanosoma VSGs. A determinant corresponding to the CRD of VSGs is borne by the ethanol-soluble form of the SAgs and by two cross-reacting light chains also present in ethanolic cellular extracts (together with the soluble form), and not by the membranal form of SAgs. Furthermore, we show that the membranal form of Paramecium SAgs can be converted into soluble form and that this enzymatic conversion also yields cross-reacting light chains. We also demonstrate that the membranal form is the physiological form in paramecia stably expressing a given SAg. 相似文献
4.
Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens. 总被引:8,自引:1,他引:7
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We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship. 相似文献
5.
Capdeville Y 《Protist》2000,151(2):161-169
In Paramecium primaurelia, the two major classes of cell surface proteins, the surface antigen (SAg) and the surface GPI proteins (SGPs), are linked to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. In the present study, we have characterized the expression of the SGPs in several geographical strains of P. primaurelia and P. tetraurelia at different temperatures, 23 degrees C and 32 degrees C. The identification of the expressed SGPs was performed on purified cilia, by establishing the SGP SDS-PAGE profiles under four different conditions: with or without their anchoring lipid, cleaved with a Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), and either in a reduced or in an unreduced state. This screening revealed the existence of specific sets of ciliary SGPs, as a function of temperature and the geographical origin of the strains. The SGPs the most abundant at 23 degrees C and 32 degrees C displayed a rapid turnover. We also looked for the presence of PI-PLC releasable proteins in purified cortices. In addition to the SAg and SGPs, the cortical fraction was shown to contain other PI-PLC releasable proteins, not found in the ciliary fraction, thus localized exclusively in the interciliary region. 相似文献
6.
A new class of Paramecium surface proteins anchored in the plasma membrane by a glycosylinositol phospholipid. Membrane anchor of Paramecium cross-reacting glycoproteins.
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Treatment of paramecia with ethanol or Triton X-100 solubilizes a major membrane protein, namely the surface antigen (SAg), and a set of glycopeptides in the range 40-60 kDa, which cross-react with the SAg. We demonstrate that these glycopeptides, called 'cross-reacting glycoproteins' (CRGs), are distinct molecules from the SAg. First, after purification of CRGs from ethanolic extracts of Paramecium primaurelia expressing the 156G SAg, the amino acid composition of a given CRG was found to be different from, and incompatible with, that of the 156G SAg. Secondly, we showed that the CRGs, although not immunologically detectable, are present in fractions containing the myristoylated form of the 156G SAg. The treatment of these fractions by phosphatidylinositol-specific phospholipases C enables us to reveal the CRGs through the unmasking of two distinct epitopes. One is the 'cross-reacting determinant' (CRD), initially described for the variant surface glycoproteins (VSGs) of Trypanosoma; the other determinant, called 'det-2355', is specific to the SAg and to the CRGs. Our results suggest that (1) phosphatidylinositol is covalently linked to the CRGs and (2) the CRD and the det-2355 are localized in the same region of the CRGs. We propose that the CRGs are a new set of surface proteins anchored in the cell membrane of Paramecium via a glycosylinositol phospholipid, in the same way as the SAgs. 相似文献
7.
Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli. 总被引:2,自引:0,他引:2
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Antigen 43 has been identified as a unique protein complex in the outer membrane of Escherichia coli. The complex contains two different polypeptides, alpha (Mr, 60,000) and beta (Mr, 53,000), in equal stoichiometry (P. Owen, P. Caffrey, and L.-G. Josefsson, J. Bacteriol. 169:3770-3777, 1987). The alpha subunit was released in a water-soluble form upon heating of outer membranes to 60 degrees C and was purified to apparent homogeneity by gel filtration and ion-exchange chromatography. The purified protein was acidic (pI 4.6) and had a polarity of 49.2%. The N-terminal sequence showed homology with the N termini of certain enterobacterial fimbrial subunits. In addition, antigen 43 underwent a reversible phase variation similar to that of type 1 fimbriae. By use of subunit-specific antisera, it was shown that the purified alpha subunit was capable of reassociating with the beta polypeptide. However, electron microscopic examination indicated that antigen 43 does not form a recognizable surface structure. The available evidence supports the view that antigen 43 is a complex consisting of a peripheral membrane protein (alpha) anchored to a subunit (beta) that is integral to the outer membrane. 相似文献
8.
cAMP levels in Paramecium increased dose dependently after a step increase of [Ca] or [Sr] in the incubation, provided K was present. Two mM Ca or Sr tripled cAMP concentrations within 3 s and induced an increase in forward swimming speed. The increase in cAMP formation was strictly dependent on the Donnan ratio [K]: square root [Ca]. Na, Li, or tetraethylammonium could not replace K. The data provide evidence for regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the K: Ca ratio. 相似文献
9.
Müller A Klöppel C Smith-Valentine M Van Houten J Simon M 《Biochimica et biophysica acta》2012,1818(1):117-124
Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content. 相似文献
10.
N Azzouz Y Capdeville 《Biology of the cell / under the auspices of the European Cell Biology Organization》1992,75(3):217-223
Biosynthetic labelling experiments performed on P primaurelia strain 156, expressing the temperature-specific G surface antigen, 156G SAg, demonstrated that the purified 156G SAg contained the components characteristic of a GPI-anchor. [3H]ethanolamine, [3H]myo-inositol, [32P]phosphoric acid and [3H]myristic acid could all be incorporated into the surface antigen. Myristic acid labelling was lost after treatment in vitro with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). After complete digestion by pronase, a fragment containing the intact GPI-anchor of 156G surface antigen was isolated. This fragment was shown to be hydrophobic and glycosylated and to possess an epitope found specifically in the GPI component of GPI-anchored proteins. The role of the GPI-tail in anchoring the 156G surface antigen into the membrane was assessed by determining that purified 156G molecules with the GPI-anchor could be incorporated into lipid vesicles and red cell ghosts whereas the 156G molecules lacking the GPI-anchor, as result of treatment with B thuringiensis PI-PLC, could not. It has also been shown that the membrane-bound form and the soluble form, obtained after cleavage of the 156G SAg lipid moiety either by an endogenous PI-PLC or by a bacterial PI-PLC, displayed identical circular dichroic spectra. 相似文献
11.
Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei. 总被引:20,自引:5,他引:15
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D Gilley J R Preer Jr K J Aufderheide B Polisky 《Molecular and cellular biology》1988,8(11):4765-4772
Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of paramecium-type telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences. 相似文献
12.
Cecilia Zazueta Gabriela Zafra Gabriela Vera César Sánchez Edmundo Chávez 《Journal of bioenergetics and biomembranes》1998,30(5):489-498
For many years the calcium uniporter has eluded attempts of purification, partly because of the difficulties inherent in the purification of low-abundance hydrophobic proteins (Reed and Bygrave, 1974). Liquid-phase preparative isoelectric focusing improved the fractionation of mitochondrial membrane proteins. A single 6-h run resulted in a 90-fold increase in specific activity of pooled active fractions over a semipurified fraction, allowing for enrichment of the calcium transport function in cytochrome oxidase vesicles. An additional powerful tool in the isolation of the uniporter was the use of the labeled inhibitor 103Ru360 as an affinity ligand; by following this procedure a protein of 18 kDa was purified in nondenatured, but rather inactive, form. The labeled protein corresponds to the protein that showed Ca2+ transport activity. 相似文献
13.
Characterization of a tubular basement membrane component reactive with autoantibodies associated with tubulointerstitial nephritis 总被引:3,自引:0,他引:3
R J Butkowski J P Langeveld J Wieslander J R Brentjens G A Andres 《The Journal of biological chemistry》1990,265(34):21091-21098
A kidney tubular basement membrane (TBM) component that is bound by antibodies from individuals with anti-TBM antibody-associated tubulointerstitial nephritis (TIN) was purified and characterized (TIN antigen). TIN antigen was prepared from rabbit TBM by extraction with guanidine and purified by ion-exchange, gel filtration, and reversed-phase chromatography. Based upon yields of protein and antibody reactivity, TIN antigen accounts for about 9% of the mass of TBM and thus is a major component of this basement membrane. A predominant 58-kDa form comprises about 90% of purified TIN antigen, and a 50-kDa form accounts for the remainder. The two forms share the amino-terminal sequence Ser-Ile-Phe-Gln-Gly-Gln-Tyr-X-Arg-Ser-Phe-Gly- and give similar tryptic peptide maps, indicating that they are structurally related. Their amino acid compositions overall are similar to laminin and entactin/nidogen. The absence of hydroxyproline and hydroxylysine and the low levels of glycine in TIN antigen indicate that it is noncollagenous. No similarities were found between other known proteins and sequences of tryptic peptides and the amino terminus of TIN antigen, suggesting that it is distinct from other characterized basement membrane components. A goat polyclonal antibody toward rabbit TIN antigen showed the same kidney distribution as human antibodies and was completely inhibited in enzyme-linked immunosorbent assay by purified TIN antigen. These data further support the idea that TIN antigen is the primary target for anti-TBM antibodies associated with TIN. This research presents methods to prepare TIN antigen for biochemical studies and investigations of its role in anti-TBM autoimmune TIN. 相似文献
14.
F Mercier H Reggio G Devilliers D Bataille P Mangeat 《Biology of the cell / under the auspices of the European Cell Biology Organization》1989,65(1):7-20
A monoclonal antibody (mab 146.14) marker of the movement of acid-secreting membranes in rat gastric parital cells has been produced and characterized. Mab 146.14 recognized a 95-kD major component of a purified membrane fraction of rat gastric mucosa, the protein composition of which was similar to that of well characterized porcine H+ -K+ ATPase-enriched membranes, and that presented the characteristic shift of density depending on whether it was purified from resting or stimulated tissues. Further biochemical analysis characterized the antigen as a membranous protein that might be in its native form, part of a higher multimolecular complex. Immunocytochemical localization of the antigen demonstrated that only membranes related to acid secretion in parietal cells expressed the 95-kD antigen. In resting conditions, the 95-kD antigen was diffusely distributed in the cell cytoplasm associated with inactive tubulovesicles. In stimulated cells, by contrast, all the antigen was recovered associated with secretory active microvilli formed by the apical insertion of the previously resting internal tubulovesicles. In conclusion, the 95-kD antigen, presumably a part of the rat gastric proton pump, is a marker of acid-secreting membranes in rat parietal cells. The translocation of antigen and membranes, observed by both light and electron microscopy supports the fusion model of membrane insertion from a cytoplasmic storage pool to the apical surface upon stimulation of acid secretion. 相似文献
15.
In Paramecium primaurelia, the two major classes of cell surface proteins, the surface antigen (SAg) and the surface GPI proteins (SGPs), are linked to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. In the present study, we have characterized the expression of the SGPs in several geographical strains of P. primaurelia and P. tetraurelia at different temperatures, 23 °C and 32 °C. The identification of the expressed SGPs was performed on purified cilia, by establishing the SGP SDS-PAGE profiles under four different conditions: with or without their anchoring lipid, cleaved with a Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), and either in a reduced or in an unreduced state. This screening revealed the existence of specific sets of ciliary SGPs, as a function of temperature and the geographical origin of the strains. The SGPs the most abundant at 23 °C and 32 °C displayed a rapid turnover. We also looked for the presence of PI-PLC releasable proteins in purified cortices. In addition to the SAg and SGPs, the cortical fraction was shown to contain other PI-PLC releasable proteins, not found in the ciliary fraction, thus localized exclusively in the interciliary region. 相似文献
16.
An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE. Sequence comparisons uncovered a low but significant homology with a Schizosaccharomyces pombe protein and the C-terminal half of the beta-fructofuranosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes. Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation. 相似文献
17.
Fok AK Yamauchi K Ishihara A Aihara MS Ishida M Allen RD 《The Journal of eukaryotic microbiology》2002,49(3):185-196
Previous studies have shown that the vacuolar-ATPase (V-ATPase) of the contractile vacuole complexes (CVCs) in Paramecium multimicronucleatum is necessary for fluid segregation and osmoregulation. In the current study, immunofluorescence showed that the development of a new CVC begins with the formation of a new pore around which the collecting canals form. The decorated membranes are then deposited around the newly formed collecting canals. Quick-freeze deep-etch techniques reveal that six 10-nm-wide V-ATPase V, sectors, tightly packed into a 20 x 30-nm rectangle, form two rows of these compacted sectors that helically wrap around the cytosolic side of decorated membrane tubules. During new CVC formation, packing of decorated tubules around mature CVCs was temporarily disrupted so that some of these decorated tubules became transformed into decorated vesicles. Freeze-fracturing of these decorated vesicles revealed a highly pitted E-face and a particulate P-face. The V-ATPase was purified for the first time in any ciliated protozoan and shown to contain, as in other cells, the V1 subunits A to E, and four 14-20 kDa polypeptides. The B subunit was cloned and found to be encoded by one gene containing four short introns. This subunit has 510 amino acid residues with a predicted molecular weight of 56.8 kDa, a value similar to B subunits of other organisms. Except for the N- and C-termini, it has a 75% sequence identity with other B subunits, suggesting that the B subunits in Paramecium, like other species, have been conserved and that the entire surface of this subunit may be important in interacting with other subunits. 相似文献
18.
Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins 总被引:1,自引:1,他引:0
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《The Journal of cell biology》1983,97(5):1412-1420
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. 相似文献
19.
20.
By use of techniques described recently for lac permease [Roepe, P.D., & Kaback, H.R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6087], the melibiose permease from Escherichia coli, another polytopic integral plasma membrane protein, has been purified in a metastable soluble form after overexpression of the melB gene via the T7 RNA polymerase system. As demonstrated with lac permease, soluble melibiose permease is dissociated from the membrane with 5.0 M urea and appears to remain soluble in phosphate buffer at neutral pH after removal of urea by dialysis, although the protein aggregates in a time- and concentration-dependent fashion. Moreover, soluble melibiose permease behaves as a monomer during purification by size exclusion chromatography in the presence of urea. Circular dichroism of purified soluble melibiose permease reveals that the protein is highly helical in potassium phosphate buffer and that secondary structure is disrupted in 5.0 M urea. Finally, purified melibiose permease can be reconstituted into proteoliposomes, and the preparations catalyze membrane potential driven H+/melibiose or Na+/methyl 1-thio-beta,D-galactopyranoside symport. The results provide further support for the notion that hydrophobic transmembrane proteins may be able to assume a nondenatured conformation in aqueous solution and extend the implication that the approach described may represent a general method for rapid isolation and reconstitution of this class of membrane proteins. 相似文献