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1.
The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20-30 kdalton range. The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60-80 kdalton region and in increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.  相似文献   

2.
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.  相似文献   

3.
The light-harvesting chlorophyll ab-protein complex has been isolated from barley thylakoids by a rapid, single-step procedure involving adsorption chromatography on controlled-pore glass columns. The Triton X-100-solubilized complex contains a polypeptide of apparent molecular weight, 26,000; the 0.25% Triton X-100 light-harvesting chlorophyll ab-protein has spectral characteristics consistent with its assumed in vivo state. On the same column free chlorophyll and carotenoids have been separated from chlorophyll-protein complex 1, but this complex contained many polypeptides other than those associated with chlorophyll. This method is potentially suitable for the isolation of other thylakoid membrane proteins. It may also be generally applicable for fractionation of intrinsic membrane proteins from other sources and for separation of mixed Triton X-100-lipid micelles.  相似文献   

4.
Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus. Dialysis of the axonemes against 1 mm Tris-HCl buffer (pH 8.3)-0.1 mm EDTA-0.1 mm dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas.  相似文献   

5.
After solubilization of polyhedra of Autographa californica, Lymantria dispar, and Mamestra brassicae nuclear polyhedrosis viruses, PAGE showed at least eight distinct polyhedral polypeptide bands. Whereas the molecular weights of the major polypeptide were similar for the three NPVs (28.0–30.0 kdalton), characteristic differences between the species were found for the minor polypeptides having molecular weights in the range from 12.4 to 62.0 kdalton. It is assumed that these polypeptides are not generated by polyhedral alkaline protease since they are detected after protease inactivation. The data demonstrate that different baculoviruses can be distinguished from each other by SDS-PAGE of their polyhedral polypeptides.  相似文献   

6.
The chlorophyll-protein and polypeptide composition of manganese deficient and control sugar beet thylakoids was examined using three different detergent-electrophoresis systems. On a per chlorophyll basis, manganese deficiency reduced the amounts of CPa complex (separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis), and CP 47 and CP 43 complexes (separated by octylglucoside/SDS-polyacrylamide gel electrophoresis) without decreasing the amounts of light harvesting complexes. Lithium dodecylsulfate/Triton X-100 polyacrylamide gel electrophoresis showed that manganese deficiency decreased several thylakoid polypeptides, including a chlorophyll b containing 30 kilodalton chlorophyll-protein complex, but did not decrease the amounts of 28 and 29 kilodalton light-harvesting chlorophyll b-containing polypeptides.  相似文献   

7.
《FEBS letters》1985,188(1):68-72
A photochemically active reaction centre complex has been isolated from photosystem II preparations of spinach chloroplasts by Triton X-100 solubilisation and sucrose gradient fractionation. Electrophoresis of the complex revealed 5 bands indicating polypeptides of apparent molecular masses of 47, 43, 33, 30 and 10 kDa. Lipid analyses showed that polar, as well as neutral, lipids are associated with the complex. For approx. 40 chlorophyll a molecules there were 3.4 plastoquinone-9, 3.3 pheophytin a, 2.9 β-carotene, 29.3 monogalactosyldiacylglycerol and 12.4 sulphoquinovosyldiacylglycerol molecules. These results suggest that this photosystem II reaction centre complex, which most likely represents a minimum photochemically active unit, is a lipoprotein complex. A striking feature of the associated polar lipids is their very low degree of unsaturation.  相似文献   

8.
Gliadin was fractionated into three fractions; ω-gliadin, Fraction III (γ-gliadin) and Fraction IV (α- and β-gliadin). The determination of the molecular weights (MW) of the three fractions was performed by both SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and sedimentation equilibrium. In SDS–PAGE, ω-gliadin gave three bands (MW 50,000, 54,000 and 64,000), Fraction III two bands (MW 38,000 and 46,000) and Fraction IV two bands (MW 33,000 and 38,000), The sedimentation analysis showed that each fraction was fairly homogeneous relative to molecular weight. The molecular weights obtained by sedimentation were 28,000 for Fraction III and 27,000 for both Fraction IV and ω-gliadin. The disagreement in molecular weight between sedimentation and gel electrophoresis was discussed.  相似文献   

9.
Isolated photosystem I (PSI)-110 particles, prepared using a minimal concentration of Triton X-100 [J. E. Mullet, J. J. Burke, and C. J. Arntzen (1980) Plant Physiol. 65, 814-822] and further subjected to short-term solubilization with sodium dodecyl sulfate (SDS), were resolved into four pigment-containing bands on polyacrylamide gel electrophoresis (PAGE). We have identified these in order of increasing electrophoretic mobility as being (a) CPIa, (b) CPI, (c) the light-harvesting complex of photosystem I (LHC-I), and (d) a free pigment-zone. LHC-I had an absorption maximum in the red at 668-669 nm and a shoulder at 650 nm, which was resolved by its first-derivative spectrum to indicate the presence of chlorophyll b. LHC-I exhibited a 77 degrees K fluorescence emission maximum at 729-730 nm. The 77 degrees K fluorescence emission maxima of CPIa and CPI, excised from the gel, were at 729 and 722 nm, respectively. The LHC-I band, excised from the gel and rerun on dissociating SDS-PAGE, was resolved into two polypeptide doublets of 24-22.5 and 21-20.5 kDa. The CPIa band under similar conditions was resolved into polypeptides of 68, 24, 22.5, 21, 20.5, 19, 15, and 14 kDa; on the contrary, CPI contained only the 68-kDa polypeptide. When intact thylakoids were subjected to "nondenaturing" SDS-PAGE, LHC-I comigrated with an oligomeric form (dimer) of the light-harvesting chlorophyll a/b pigment-protein that preferentially serves photosystem II (LHCP-II). When this combined LHC-I/LHCP-II pigment-protein band was prepared by SDS-PAGE from isolated stroma lamellae, it exhibited a long-wavelength fluorescence band near 730 nm at 77 degrees K. When a similar preparation was obtained from sucrose density gradients containing SDS [J. Argyroudi-Akoyunoglou and H. Thomou (1981) FEBS Lett. 135, 171-181], it was found to be enriched in a 21-kDa polypeptide. The data suggest that the 21-kDa polypeptide of LHC-I is the chlorophyll-containing polypeptide responsible for the long-wavelength fluorescence of LHC-I; other polypeptides in the complex (20.5, 22.5, and 24 kDa) presumably bind chlorophyll and also serve an antennae function.  相似文献   

10.
The P700 chlorophyll α-protein was purified by preparative sodium dodecyl sulfate (SDS) gel electrophoresis from SDS-solubilized barley (Hordeum vulgare L., cv Himalaya) chloroplast membranes. After elution from the gel in the presence of 0.05 to 0.1% Triton X-100, the recovered protein had a chlorophyll/P700 ratio of 50 to 60/1 and contained no chlorophyll b or cytochromes. Analysis of the polypeptide composition of the chlorophyll-protein revealed a 58 to 62 kilodalton (kD) polypeptide component but no lower molecular weight polypeptides. The 58 to 62 kD component was further resolved into two distinct polypeptide bands which were subsequently mapped by partial cyanogen bromide digestion and Staphylococcus aureus proteolysis. Based on results from the mapping experiments and other data, we suggest that the two components are conformational variants of a single polypeptide. Measurement of the chlorophyll to protein ratio by quantitative amino acid analysis and consideration of the yield of P700 in the protein isolate suggest that, contrary to previous models (Bengis and Nelson, 1975, 1977), P700in vivo is associated with a minimum of four subunits of approximately 60 kD.

Antibodies raised against the photochemically active chlorophyll-protein complex from barley reacted specifically with the 58 to 62 kD apoprotein. The same preparative electrophoresis procedure was used to isolate photochemically active P700 chlorophyll a-protein from soybean (Glycine max L.), tobacco (Nicotiana tobacum L.), petunia (Petunia × hybrida), tomato (Lycopersicum esculentum), and Chlamydomonas reinhardti. The isolated complex from all species exhibited identical polypeptide compositions and chlorophyll/P700 ratios. Antibodies to the barley protein cross reacted with all species tested demonstrating the highly conserved structure of the apoprotein.

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11.
Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant clathrin, had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.NRCC No. 23142  相似文献   

12.
《The Journal of cell biology》1988,107(6):2679-2688
Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.  相似文献   

13.
A fractionation of Chromatium vinosum into an outer layer (cell wall) and three intracellular membrane fractions by isopycnic sucrose density centrifugation of a total membrane fraction obtained by lysis of lysozyme-EDTA spheroplasts is decribed. The three intracellular fractions (I, II, and III) have apparent densities of 1.11, 1.14, and 1.16, respectively, and contain the bulk of the photosynthetic pigments. Fraction II is enriched in bacteriochlorophyll and contains about 49% of the total membrane protein and 60% of the membrane bacteriochlorophyll. The outer membrane fraction (IV, cell wall) has a density of 1.23 and contains 5% of the membrane protein and 0.8% of the bacteriochlorophyll. Fraction I is enriched in lipids and phosphorus and has only a trace of diaminopimelate (DAP). Fractions II and III both contain a significant content of DAP. Fraction IV has no DAP, but has a fatty acid composition similar to that of the envelope fraction. Electrophoresis of the fractions on sodium dodecylsulfate-containing gels yielded from 8–13 bands of protein. Fractions I, II, and III contained the same series of unique proteins, while fraction IV contained another group of unique proteins. In fraction IV the bulk of the proteins traveled in one band with a molecular weight of 41,500. Examination of the fractions and whole spheroplasts in the electron microscope showed that fractions I, II and III were composed of large membrane structures in the form of membrane reticulum with bud-like appendages, and elongated flattened tubes. Fraction IV was composed of large ovoid structures which were seen to lie on the outer surface of the whole spheroplasts. These results suggest that the normal in vivo state of the intracellular membranes is that of an interconnected series of tubules and vesicles extending throughout the cell cytoplasm.  相似文献   

14.
Electrophoretic analysis by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that the light-harvesting chlorophyll ab-protein complex of barley thylakoids contains only one polypeptide of apparent molecular weight 26 000. The barley mutant, deficient in chlorophyll b and this light-harvesting complex, lacks this polypeptide.The addition of a nonionic detergent, Triton X-100, to the sodium dodecyl solubilization buffer prior to SDS polyacrylamide tube gel electrophoresis, allowed separation of a relatively stable complex, characterized as an oligomeric form of the light-harvesting complex. The oligomer also contained a polypeptide with an apparent molecular weight of 26 000. The absorption and fluorescence spectral properties of the oligomer are similar to those of the monomer. It is suggested that the oligomer of the light-harvesting chlorophyll ab-protein is closer to the in vivo form rather than the monomer.  相似文献   

15.
Band 3 proteins are the predominant polypeptide components of the human erythrocyte membrane and have been implicated in various transport activities. Following extraction of membrane ghosts with dimethylmaleic anhydride to remove two polypeptides (Bands 4.2 and 6) associated with Band 3, Band 3 proteins were solubilized along with the other major membrane glycoproteins (PAS 1–3) with Triton X-100 under nondenaturing conditions. Band 3 proteins were then purified (>95%) on a large scale by chromatography via thioldisulfide interchange on activated thiol-Sepharose 4B [agarose-(glutathione-2-pyridyl disulfide) conjugate]. This procedure allows the preparation of 20 to 25 mg of purified Band 3 proteins in high yield (>80%) from ghosts in a soluble form suitable for physical, chemical, and functional characterization.  相似文献   

16.
Band 3 is the predominant approximately 90,000-dalton polypeptide component of the human erythrocyte membrane. It was solubilized selectively, along with the other major glycoproteins, by extracting membrane ghosts with Triton X-100 under nondenaturing conditions. Two major polypeptides remained associated with Band 3 under these conditions; however one (Band 6) could be dissociated at an ionic strength of 0.15 and the other (Band 4.2) by treatment with p-chloromercuribenzoate. Band 3 was then purified (greater than or equal to 97%) by aminoethyl cellulose ion exchange chromatography. The isolated protein was free of phospholipid and was moderately enriched in apolar amino acid residues; it contained galactose and glucosamine but very little sialic acid and galactosamine. When Band 3 was labeled by treatment of ghosts with galactose oxidase plus KB3H4 and then purified, the electrophoretic mobility of its radioactivity lagged slightly behing that of its Coomassie blue staining profile. Variation in glycosylation could therefore cause the diffuse trailing zone characteristically observed for Band 3 on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The ultraviolet circular dichroism of Band 3 was stable in nonionic detergent and suggested an alpha helix content of 43%, a value close to that estimated for this polypeptide in the membrane.  相似文献   

17.
The thylakoid polypeptides of the cyanobacterium Anacystis nidulans R2 were analyzed by Triton X-114 phase fractionation [C. Bordier (1981) J. Biol. Chem.256, 1604–1607, as adapted for photosynthetic membranes by T. M. Bricker and L. A. Sherman (1982) FEBS Lett.149, 197–202]. In this procedure, polypeptides with extensive hydrophobic regions (i.e., intrinsic proteins) form mixed micelles with Triton X-114, and are separated from extrinsic proteins by temperature-mediated precipitation of the mixed Triton X-114-intrinsic protein micelles. The polypeptide pattern after phase fractionation was highly complementary, with 62 of the observed 110 polypeptide components partitioning into the Triton X-114-enriched fraction. Identified polypeptides fractionating into the Triton X-114 phase included the apoproteins for Photosystems I and II, cytochromes f and b6, and the herbicide-binding protein. Identified polypeptides fractioning into the Triton X-114-depleted (aqueous) phase included the large and small subunits of RuBp carboxylase, cytochromes c550 and c554, and ferredoxin. Enzymatic radioiodination of the photosynthetic membranes followed by Triton X-114 phase fractionation allowed direct identification of intrinsic polypeptide components which possess surface-exposed regions susceptible to radioiodination. The most prominent of these polypeptides was a 34-kDa component which was associated with photosystem II. This phase partitioning procedure has been particularly helpful in the clarification of the identity of the membrane-associated cytochromes, and of photosystem II components. When coupled with surface-probing techniques, this procedure is very useful in identifying intrinsic proteins which possess surface-exposed domains. Phase fractionation, in conjunction with the isolation of specific membrane components and complexes, has allowed the identification of many of the important intrinsic thylakoid membrane proteins of A. nidulans R2.  相似文献   

18.
The chemical composition and presence of immunogenic components in the lysates of the cell walls of group A Streptococcus, type M29, were studied. The lysates were prepared with the use of muramidase. Fc-Receptors were detected in the lysates. Within the first 30 minutes of cell wall lysis by muramidase, 4 times higher amounts of the protein reacting with fibrinogen excreted than in the subsequent 4 hours. The lysates contained immunogenic proteins. Fraction III isolated by chromatography of the 30-minute lysate on DEAE-trisacryl formed a single precipitation band with lysate antiserum. The lysate Fraction IV forming three precipitation bands contained a protein not specific of the type. The protein was identical to the protein antigen from Triton X-100 extracts of group A Streptococcus, types M1, M12 and M29. The group-specific polysaccharide was detected in the lysate Fraction I and Fraction II of the 4-hour lysate.  相似文献   

19.
The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35 000 is a porin, responsible for the size-dependent permeability of the outer membrane.  相似文献   

20.
J. Hladík  P. Pančoška  D. Sofrová 《BBA》1982,681(2):263-272
Thylakoid membranes of the cyanobacterium Plectonema boryanum solubilized with Triton X-100 can be resolved into three fractions of pigment-protein complexes (Hladík, J. and Sofrová, D. (1981) Photosynthetica 15, 490–503). Fraction I contained relatively the highest amount of carotenoids as well as monomeric forms of chlorophyll a, Fractions II and III contained chlorophyll-protein complexes with a characteristic exciton-split circular dichroism in the red region. It has been shown that fraction III is an oligomeric form of the chlorophyll-protein complex of fraction II. Circular dichroism spectra indicate that, different from fraction II, fraction III contains specifically oriented and space-fixed molecules of carotenoids. Thermal dissociation of fracion III to fraction II is accompanied by disappearance of the positive circular dichroism effect of carotenoids in the 500–550 nm region, thus causing deorganization of the carotenoids, proceeding in parallel to the geometrical rearrangement of chlorophyll molecules. Extraction of the carotenoids of fraction III with heptane is acompanied by dissociation of fraction III. We assume that the observed effects are due to binding of the two pigments to the protein component of the complex and that carotenoids can mediate a part of the interactions which stabilize the structure of pigment-protein complexes. Thus, besides the light-harvesting and protective functions, carotenoids can also play a structural role.  相似文献   

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