Outer (cell wall) membrane proteins of Pseudomonas aeruginosa

1973 ◽  
Vol 19 (12) ◽  
pp. 1469-1471 ◽  
Author(s):  
J. D. Stinnett ◽  
R. G. Eagon
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Cell Envelope ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Outer Cell ◽  
Sucrose Density Gradient Centrifugation ◽  
Protein Components ◽  
Envelope Membranes ◽  
Outer Cell Wall

Cell envelope membranes were isolated from Pseudomonas aeruginosa. These membranes were resolved into cytoplasmic membrane rich and outer (cell wall) membrane rich fractions by discontinuous sucrose density gradient centrifugation. The resolution was based on the separation of enzyme activities and 2-keto-3-deoxyoctonate. Analysis by gel electrophoresis revealed that two of the three major cell envelope protein components were found in the fraction rich in outer (cell wall) membrane. These two protein components were previously shown to occur in a protein–lipopolysaccharide complex in this microorganism.

scholarly journals Localization of ferrochelatase and of newly synthesized haem in membrane fractions from Rhodopseudomonas spheroides

1978 ◽  
Vol 174 (1) ◽  
pp. 277-281 ◽  
Author(s):  
J Barrett ◽  
O T G Jones
Keyword(s):  
Cell Envelope ◽  
Gradient Centrifugation ◽  
Specific Radioactivity ◽  
Sucrose Density Gradient ◽  
Particulate Fraction ◽  
Outer Face ◽  
Sucrose Density Gradient Centrifugation ◽  
Intact Cells ◽  
Rhodopseudomonas Spheroides ◽  
Cytochrome Content

Cells of Rhodopseudomonas spheroides, strains R-26 or GVP, were grown photosynthetically, disrupted and two particulate fractions separated by sucrose-density-gradient centrifugation. The upper particulate fraction, enriched in bacteriochlorophyll, was identified as containing the chromatophores; the lower particulate fraction had the characteristics of the cell envelope. The two fractions differed in cytochrome content and cytochrome spectra. Ferrochelatase was found almost exclusively in the chromatophore fraction and was located on the outer face of the chromatophores, i.e. in contact with the cytosol in intact cells. The addition of 59FeCl3 to cells growing in low-iron media resulted in labelling of the protohaem fraction (probably arising from cytochrome b) of the membranes. The specific radioactivity of the haem of the chromatophores rose more rapidly than that of the envelope fraction and then after 2 h declined to approximately the same value, suggesting that haems of the chromatophore may act as precursors of haem of the envelope.

In vitro studies of an alkaline phosphatase – cell wall complex from Pseudomonas aeruginosa

1975 ◽  
Vol 21 (1) ◽  
pp. 9-16 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Complex Formation ◽  
Wall Material ◽  
Outer Cell ◽  
Whole Cells ◽  
Wall Components ◽  
Outer Cell Wall

Alkaline phosphatase (APase) of Pseudomonas aeruginosa exists primarily in the periplasmic region of the cell, i.e., between the cytoplasmic membrane and the outer tripartite layer. The enzyme is also found in the culture filtrate or associated with the outer layer of the cell wall. APase forms a complex with released outer cell wall material, and lipopolysaccharide (LPS) is associated with the complex. Since the enzyme was purified to homogeneity, it became desirable to determine whether complex formation with LPS, or the outer cell wall, affected any properties of the purified phosphatase. The ratio of activities of purified APase with p-nitrophenylphosphate and β-glycerolphosphate as substrates is about 4:1. The ratio of activities with enzyme complexed with LPS is about 1:1. The energy of activation of sucrose or magnesium released enzyme is 9500 cal/mol whereas the values for purified enzyme plus LPS, purified enzyme, purified enzyme plus phosphatidylethanolamine (PE), and purified enzyme plus LPS plus PE range from 3400 to 8700 cal/mol. These changes occur in the physiological temperature range, 27 to 39C, of this organism. Sucrose-released enzyme in the presence of substrate is inactivated at 47C whereas pure enzyme plus substrate is affected at 41C. The addition of LPS, PE, or a combination of both increases the temperature of inactivation from 45 to 51C. The results suggest that certain properties of the purified enzyme differ from those of the enzyme released from whole cells by either sucrose or magnesium resuspension. The addition of cell wall components such as LPS and PE to purified APase restores these properties. The evidence suggests that artificial complex formation changes the environment of the enzyme protein such that the environment now resembles that which exists within the whole cell wall.

The Outer Cell-Wall Membrane of Pseudomonas aeruginosa

1974 ◽  
Vol 130 (Supplement) ◽  
pp. S81-S93 ◽  
Author(s):  
J. C. Sadoff ◽  
M. S. Artenstein
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Outer Cell ◽  
Outer Cell Wall

The effect of complement and other cell wall reagents on tetracycline and streptomycin resistance in Pseudomonas aeruginosa

1974 ◽  
Vol 20 (8) ◽  
pp. 1101-1107 ◽  
Author(s):  
J. T. Tseng ◽  
L. E. Bryan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Wall ◽  
Cell Membrane ◽  
Resistant Strain ◽  
Streptomycin Resistance ◽  
Outer Cell ◽  
Similar Magnitude ◽  
Peptidoglycan Layer ◽  
Outer Cell Wall

Lysozyme-free antiserum and complement treatment of strain 1885 of Pseudomonas aeruginosa was observed to destroy the penetration barrier of the outer cell wall to lysozyme but not to induce leakage of acid-soluble nucleotides through the cell membrane. The same treatment did not produce a significant increase in uptake of 3H-tetracycline or 3H-streptomycin by the resistant strain 1885 in spite of the destruction of the penetration barrier to lysozyme. A significant increase in both streptomycin and tetracycline uptake occurred in carbenicillin-treated strains but the increase was similar for both susceptible and resistant (to tetracycline and streptomycin) strains of P. aeruginosa. These data suggest (1) the outer cell wall is not a significant penetration barrier to these drugs; (2) the peptidoglycan layer does function as a penetration barrier of similar magnitude in resistant and susceptible cells; (3) the resistance of the strains is a property of the cell membrane or materials intimately associated with the cell membrane. The latter conclusion was further supported by the differential uptake of streptomycin in NaCl-lysozyme-induced spheroplasts of strains 1885 and 2379.

Alkaline Phosphatase Suhunits in the Culture Filtrate of Pseudomonas aeruginosa

1972 ◽  
Vol 50 (3) ◽  
pp. 268-276 ◽  
Author(s):  
K.-J. Cheng ◽  
D. F. Day ◽  
J. W. Costerton ◽  
J. M. Ingram
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Culture Filtrate ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Growth Cycle ◽  
Active Species ◽  
Acid Dissociation ◽  
Sucrose Density Gradient Centrifugation

Growth of Pseudomonas aeruginosa at pH 6.8 for 14 h under inorganic phosphate limiting conditions is accompanied by the appearance of cell-associated alkaline phosphatase, and no active phosphatase is detected in the culture filtrate. Analysis of cells and the culture filtrate during the growth cycle showed that active phosphatase accumulated simultaneously in both the cells and the filtrate during the early log phase but as growth proceeded beyond 10 h the activity in the filtrate disappeared and the pH dropped from 6.8 to 4.7. Dialysis against 0.01 M Tris and 0.001 M MgCl2 had no effect upon the phosphatase levels of early growth phase (i.e. up to 10 h) culture filtrates. However, dialysis of filtrates obtained after 10 h of growth restored the decreased phosphatase activity to the level present in 10 h filtrates. Treatment of a 14 h filtrate with either trypsin or pepsin prevented the recovery of activity by dialysis whereas such treatments had no effect upon partially purified phosphatase. Sucrose density gradient centrifugation of the 14 h culture filtrate revealed the presence of activity, after dialysis of each fraction against 0.01 M Tris and 0.001 M MgCl2, which corresponded to a molecular weight of 60 000 as compared to a molecular weight of 125 000 for active culture filtrate or partially purified enzyme. Partially purified phosphatase is inactivated and dissociated after treatment at pH 4.2 and the enzyme is reactivated and reassociated after dialysis or dilution into Tris and MgCl2. The results suggest that active phosphatase, molecular weight 125 000, is secreted to the culture filtrate during the early stages of growth and the activity disappears coincidently with the appearance of an inactive species of molecular weight 60 000. The accumulation of the inactive species in the culture filtrate is the result of acid dissociation of the active species as the initial pH of the filtrate decreases from pH 6.8 to pH 4.7.

scholarly journals Fractionation of the proteins of plant microbodies

1974 ◽  
Vol 144 (3) ◽  
pp. 559-566 ◽  
Author(s):  
R H Brown ◽  
J M Lord ◽  
M J Merrett
Keyword(s):  
Membrane Proteins ◽  
Ricinus Communis ◽  
Osmotic Shock ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Weight One ◽  
Protein Components

1. Glyoxysomes and peroxisomes have been isolated from dark- and light-grown seedlings of pumpkin (Cucurbita pepo) by sucrose-density-gradient centrifugation. 2. Pumpkin microbodies and castor-bean (Ricinus communis) glyoxysomes may be fractionated, by a combination of osmotic shock and treatment with KCl, into three distinct groups of proteins: readily soluble (matrix enzymes), solubilized in the presence of KCl (membrane-bound enzymes) and relatively insoluble (membrane ‘ghost’ proteins). 3. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of ‘ghost’ fractions indicated that the membrane proteins were generally of low molecular weight; one gel band (mol.wt. 27000–28000) was common to all three microbodies. 4. Although there were major differences in the soluble protein components of pumpkin glyoxysomes and peroxisomes, electrophoresis of the pumpkin microbody ‘ghosts’ indicated that the membrane proteins were similar, four main components being common to each class of microbody (monomer molecular weights 42000, 34000, 27000 and 17000).

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

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