scholarly journals A requirement for EDTA in the separation of Photosystems 1 and 2 from the cyanobacterium Chlorogloea fritschii

1981 ◽  
Vol 196 (2) ◽  
pp. 489-493 ◽  
Author(s):  
E H Evans ◽  
C A Pullin
Keyword(s):  
Photosystem 2 ◽  
Photosystems 1 And 2 ◽  
Photosystem 1 ◽  
Molecular Weights ◽  
Kd Values ◽  
Photosystem 2 Activity ◽  
Triton X ◽  
Photosystem 1 And 2

Fractions enriched in Photosystem 1 or Photosystem 2 activity have been isolated from the cyanobacterium Chlorogloea fritschii after extraction of the membranes with digitonin and Triton X-100. Separation of the extract into the two components was achieved by using a Sepharose 6B column, calibration of which gave Kd values of 0.3 for the Photosystem 1 fraction and 0.53 for Photosystem 2. These values corresponded to molecular weights of approx. 500000 and 90000 respectively. The Photosystem 1 particle was shown to aggregate on storage and EDTA was shown to be necessary to separate the Photosystem 1 and 2 fractions.

scholarly journals The photochemical activities and electron carriers of developing barley leaves

1973 ◽  
Vol 136 (3) ◽  
pp. 803-812 ◽  
Author(s):  
Marijana Plesničar ◽  
Derek S. Bendall
Keyword(s):  
Cytochrome B ◽  
Photosystem 2 ◽  
Cytochrome F ◽  
Reaction Centre ◽  
Photosystem 1 ◽  
Photochemical Activities ◽  
Cyclic Photophosphorylation ◽  
Photosystem 2 Activity ◽  
Barley Leaves ◽  
Electron Carriers

The development of photochemical activities in isolated barley plastids during illumination of dark-grown plants has been studied and compared with the behaviour of plastocyanin, cytochromes f, b-559LP, b-563 and b-559HP and pigments P546 (C550) and P700. Electron-transport activity dependent on Photosystem 1 and cyclic photophosphorylation dependent on N-methylphenazonium methosulphate (phenazine methosulphate) were very active relative to the chlorophyll content after only a few minutes of illumination of etiolated leaves, and then rapidly declined during the first few hours of greening. By contrast, Photosystem 2 activity (measured with ferricyanide as electron acceptor) and non-cyclic photophosphorylation were not detectable during the first 2½h of greening, but then increased in total amount in parallel with chlorophyll. The behaviour of the electron carriers suggested their association with either Photosystem 1 or 2 respectively. In the first group were plastocyanin, cytochrome f and cytochrome b-563, whose concentrations in the leaf did not change during greening, and cytochrome b-559LP whose concentration fell to one-half its original value, and in the second group were cytochrome b-559HP and pigment P546, the concentrations of which closely followed the activities of Photosystem 2. Pigment P700 could not be detected during the first hour, during which time some other form of chlorophyll may take its place in the reaction centre of Photosystem 1. The plastids started to develop grana at about the time that Photosystem 2 activity became detectable.

Changes in photosystem 2 activity associated with plant tolerance to lead

1981 ◽  
Vol 21 (3) ◽  
pp. 269-274 ◽  
Author(s):  
J.R. Homer ◽  
R. Cotton ◽  
E.H. Evans
Keyword(s):  
Photosystem 2 ◽  
Plant Tolerance ◽  
Photosystem 2 Activity

The outer membrane of Haemophilus influenzae type b: cell envelope associations of major proteins

1983 ◽  
Vol 29 (2) ◽  
pp. 280-287 ◽  
Author(s):  
J. W. Coulton ◽  
D. T. F. Wan
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Haemophilus Influenzae Type ◽  
Major Protein ◽  
Haemophilus Influenzae Type B ◽  
Type B ◽  
Molecular Weights ◽  
Triton X

Membrane proteins fom the cell envelope of Haemophilus influenzae type b ATCC 9795 were examined by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. When envelopes were extracted with a phosphate-based buffer containing 2% Triton X-100, a major protein of molecular weight 43 000 was detected in fractions containing cytoplasmic membrane proteins. The cell wall material which was Triton X-100 insoluble contained six major proteins of molecular weights 46 000, 40 000, 36 000, 30 000, 27 000, and 16 000. One of these proteins showed a shift in molecular weight from 27 000 to 36 000 when it was heated over a temperature range from 50 °C to 100 °C in buffer containing 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol. This alteration in mobility could be demonstrated either by the membrane-bound form of the protein or by a detergent-soluble form of the protein. Enriched preparations of the 36 000 molecular weight form were obtained by a series of purification steps. Extraction of the Triton X-100 insoluble material with buffer containing 2% Triton X-100, 5.0 mM EDTA yielded chiefly one major protein molecular weight 30 000 and many minor protein species. Pretreatment of the Triton X-100 insoluble fraction with lysozyme followed by extraction with buffer containing 2% Triton X-100, 5.0 mM EDTA released two proteins of molecular weights 16 000 and 27 000 and few minor proteins. By these operational manipulations, the proteins of molecular weights 16 000 and 27 000 may be considered as peptidoglycan-associated proteins.

Isolation and immunochemical characterization of fractions from membranes of Aspergillus fumigatus with protease activity

1990 ◽  
Vol 36 (1) ◽  
pp. 33-41 ◽  
Author(s):  
James E. Piechura ◽  
Viswanth P. Kurup ◽  
Laureen J. Daft
Keyword(s):  
Aspergillus Fumigatus ◽  
Protease Activity ◽  
Analytical Ultracentrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Allergic Bronchopulmonary Aspergillosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Weights ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

Two fractions exhibiting acid protease activity (AFPI and AFPII) were isolated by extraction of membrane vesicles of Aspergillus fumigatus with Triton X-100. These two fractions produced single bands in both polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed apparent molecular weights of 73 000 and 43 000, respectively. Molecular weights determined by gel filtration in the absence and presence of Triton X-100 and sedimentation velocities in analytical ultracentrifugation indicated hydrophobic characteristics, since both fractions readily aggregated and complexed with Triton X-100; both exhibited elevated enzyme activities in the presence of Triton X-100. Carbohydrate content was 93% for AFPI and 85% for AFPII. The enzymatic fractions demonstrated different pH optima in the acid range as well as different temperature stabilities. Both protease fractions cross reacted in double immunodiffusion, while in crossed immunoelectrophoresis both demonstrated five precipitin peaks, each with similar patterns. AFPI demonstrated two additional precipitin peaks in crossed immunoelectrophoresis. As determined by crossed immunoaffinoelectrophoresis, the protease fractions demonstrated galactose and mannose residues. In biotin–avidin enzyme-linked immunosorbent assay both fractions reacted with allergic bronchopulmonary aspergillosis and aspergilloma sera. It can be concluded that the two fractions with protease activity of A. fumigatus reported here may be of significance in Aspergillus-induced diseases. Key words: Aspergillus, membrane, allergens, proteases, aspergillosis.

scholarly journals ISOLATION AND REACTIVATION OF THE AXOSTYLE

1973 ◽  
Vol 56 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Mark S. Mooseker ◽  
Lewis G. Tilney
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Atpase Activity ◽  
Adenosine Triphosphate ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gels ◽  
Molecular Weights ◽  
Dodecyl Sulfate ◽  
Cilia And Flagella ◽  
Triton X

The contractile axostyle is a ribbon-shaped organelle present in certain species of flagellates found in the hindgut of wood eating insects. This organelle propagates an undulatory wave whose motion, like flagella and cilia, is related to microtubules. Unlike the axoneme of cilia and flagella, however, the axostyle is composed of singlet microtubules linked together in parallel rows. Axostyles were isolated from Cryptocercus gut protozoa with Triton X-100. Normal motility of the isolated axostyle could be restored with adenosine triphosphate (ATP); the specific conditions necessary for this reactivation were essentially identical with those reported for the reactivation of isolated flagella or whole sperm. ATPase activity of the isolated axostyle was comparable to the values reported for ciliary or flagellar axonemes. The axostyle was reasonably specific for ATP. Most of the proteins of the isolated axostyle comigrated with proteins of the ciliary axoneme on sodium dodecyl sulfate (SDS) polyacrylamide gels (i e. equivalent molecular weights). These included the following: the higher molecular weight component of dynein, tubulin, linkage protein (nexin), and various secondary proteins. Evidence for dynein in the axostyle is presented and a model proposed to explain how repeated propagated waves can be generated.

scholarly journals Multiple forms of acetylcholinesterase from human erythrocytes

1973 ◽  
Vol 133 (3) ◽  
pp. 521-527 ◽  
Author(s):  
David L. Wright ◽  
David T. Plummer
Keyword(s):  
Enzyme Activity ◽  
Electrophoretic Pattern ◽  
Human Erythrocytes ◽  
Serum Cholinesterase ◽  
Main Peak ◽  
Multiple Forms ◽  
Molecular Weights ◽  
Enzyme Substrate ◽  
Triton X ◽  
Naphthyl Acetate

1. Acetylcholinesterase from human erythrocytes was solubilized with Triton X-100 in strong salt solution and partially purified by (NH4)2SO4 fractionation. This preparation showed three main bands of enzyme activity after electrophoresis on polyacrylamide gel and incubation with either α-naphthyl acetate or acetylthiocholine as enzyme substrate. Two of the multiple forms were completely inhibited by 10μm-eserine and one only partially. Treatment with neuraminidase had no effect on the electrophoretic pattern; therefore sialic acid does not appear to determine or affect the ratios of the acetylcholinesterase multiple forms, unlike those of the serum cholinesterase. 2. Chromatography of the preparation on Sephadex G-200 revealed one major peak of enzyme activity and a suggestion of two minor zones of mol.wt. 546000, 184000 and 93000 (i.e. in the proportion 6:2:1). The main peak was almost completely separated from the Triton X-100 and the overall purification was about 600-fold. Further attempts to purify the enzyme by absorption on calcium phosphate gels were unsuccessful. 3. Electrophoresis of the enzyme preparation on a polyacrylamide gradient for 24h revealed three main bands that corresponded to the three values for molecular weights obtained by column chromatography. After 70h of electrophoresis a further three zones of activity developed making six molecular entities, the molecular weights of which were simple multiples of a monomer, thus resembling the cholinesterase found in serum.

scholarly journals Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilized in detergents

1974 ◽  
Vol 143 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Michelle Letarte-Muirhead ◽  
Ronald T. Acton ◽  
Alan F. Williams
Keyword(s):  
Binding Assay ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Brain Homogenate ◽  
Target Cells ◽  
Rat Tissues ◽  
Molecular Weights ◽  
Ionic Detergents ◽  
Stokes Radius ◽  
Triton X

1. A radioactive binding assay for Thy-1.1 alloantigen which functions in the presence of detergents was established by using glutaraldehyde-fixed thymocytes as target cells. Thy-1.1 activity in detergent extracts was then assayed by measuring inhibition of the binding assay. 2. Solubilization of Thy-1.1 from whole thymocytes, and their membranes by a large number of non-ionic detergents and deoxycholate was studied. In the same extracts Ag-B(4) histocompatibility antigenic activities were measured. With the exception of Nonidet P-40, the detergents did not affect the antigenicity of Thy-1.1, but only Lubrol-PX and deoxycholate gave effective solubilization as measured by activity remaining in the supernatant after centrifugation at 200000g for 40min. With Ag-B(4) antigen, Triton X-100, Triton X-67 and Nonidet P-40 gave effective solubilization as well as Lubrol-PX and deoxycholate. Solubilization of Thy-1.1 activity from leukaemia cells and a brain homogenate was also studied, but none of the non-ionic detergents gave satisfactory results with these tissues. 3. Extracts from thymocyte membranes were further examined by gel filtration and sucrose gradient centrifugation. The Thy-1.1 activity behaved as a single component in deoxycholate with a density similar to that of a globular protein, but in Lubrol-PX the antigen was contained in a low-density complex. In Lubrol-PX extracts Ag-B(4) was also found in aggregates not observed in deoxycholate. 4. The s20,w values for Thy-1.1 and Ag-B(4) antigens in deoxycholate were 2.4 and 4.4, and v̄ values were 0.70 and 0.75 respectively. The Stokes radius observed for Thy-1.1 was 3.1nm and for Ag-B(4) 5.3nm. By using these values the molecular weights for the antigen–detergent complexes were calculated to be 28000 for Thy-1.1 and 100000 for Ag-B(4).

scholarly journals Diaminobenzidine an Electron Donor to Photosystem 1 and to Photosystem 2 in Chloroplasts

1975 ◽  
Vol 52 (1) ◽  
pp. 135-141 ◽  
Author(s):  
Gozal BEN-HAYYIM ◽  
Ziwia DRECHSLER ◽  
Judith GOFFER ◽  
Joseph NEUMANN
Keyword(s):  
Electron Donor ◽  
Photosystem 2 ◽  
Photosystem 1

scholarly journals Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions.

1987 ◽  
Vol 104 (3) ◽  
pp. 537-546 ◽  
Author(s):  
N Carlson ◽  
M Rechsteiner
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Size Distribution ◽  
High Molecular Weight ◽  
Hela Cells ◽  
Insoluble Fraction ◽  
Nuclear Fraction ◽  
Molecular Weights ◽  
Differential Sedimentation ◽  
Triton X

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.

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