Alkaline phosphatase localization and spheroplast formation of Pseudomonas aeruginosa

1970 ◽  
Vol 16 (12) ◽  
pp. 1319-1324 ◽  
Author(s):  
K. -J. Cheng ◽  
J. M. Ingram ◽  
J. W. Costerton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Electron Microscopic ◽  
Erroneous Conclusion ◽  
Lead Salts ◽  
Phosphatase System ◽  
Low Levels ◽  
Cell Wall Deposition ◽  
Periplasmic Enzyme

Pseudomonas aeruginosa cells grown in the presence of low levels of inorganic phosphate contain an inducible alkaline phosphatase system. The enzyme has been localized by electron microscopic techniques in the region between the cytoplasmic membrane and the tripartite layer of the cell wall, i.e. the periplasmic space. No deposits of lead salts are observed upon examination of either uninduced cells or cells in which the enzyme has been completely removed by 0.2 M magnesium washing. Samples of cells which were treated with glutaraldehyde before enzyme localization studies show cell wall deposition of lead salts which could give rise to the erroneous conclusion that the alkaline phosphatase was located in the tripartite layer. Cytochemical and biochemical studies are presented which show that discontinuities within the cell wall are insufficient to account for the release of this periplasmic enzyme and that dissociation by a divalent metal, increased pH, or both is required. As a consequence of this study it was possible to prepare true spheroplasts of P. aeruginosa.

Alkaline phosphatase of Pseudomonas aeruginosa: the mechanism of secretion and release of the enzyme from whole cells

1973 ◽  
Vol 19 (11) ◽  
pp. 1407-1415 ◽  
Author(s):  
J. M. Ingram ◽  
K. -J. Cheng ◽  
J. W. Costerton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Culture Filtrate ◽  
Outer Wall ◽  
Outer Cell ◽  
Periplasmic Space ◽  
Electron Microscopic ◽  
Whole Cells ◽  
Variable Quantity

The release of alkaline phosphatase from whole cells of Pseudomonas aeruginosa as a function of the MgCl2 concentration is proportional to the release of lipopolysaccharide from the cells. Cells grown under conditions where APase is almost completely secreted to the culture filtrate, i.e. growth at pH 7.6, also secrete lipopolysaccharide. Twenty percent sucrose releases a variable quantity of whole cell phosphatase. Localization of this portion of enzyme by biochemical and electron-microscopic techniques showed that it is located on the cell surface exterior to the outer tripartite layer. Phosphatase, which is not released by sucrose, but which is released by MgCl2, is located in the periplasmic space. Phosphatase is located in three areas; the culture filtrate, the outer cell wall surface, and the periplasmic space. The results suggest that A Pase is associated with, and bound to, a cell wall fraction which contains lipopolysaccharide and that the enzyme is "transported" through the outer wall in complex with this fraction. Liberation of the complex from the outer wall may be accomplished by the mechanical shearing forces developed during growth or during the sucrose suspension procedure.

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.

scholarly journals Interactions of Alkaline Phosphatase and the Cell Wall of Pseudomonas aeruginosa

1971 ◽  
Vol 107 (1) ◽  
pp. 325-336 ◽  
Author(s):  
K.-J. Cheng ◽  
J. M. Ingram ◽  
J. W. Costerton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Cell Wall

Release of rhodanese from Pseudomonas aeruginosa by cold shock and its localization within the cell

1979 ◽  
Vol 25 (3) ◽  
pp. 340-351 ◽  
Author(s):  
R. W. Ryan ◽  
M. P. Gourlie ◽  
R. C. Tilton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Outer Membrane ◽  
Electron Micrographs ◽  
Cold Shock ◽  
Cytoplasmic Membrane ◽  
Lactic Dehydrogenase ◽  
Periplasmic Space ◽  
Whole Cells ◽  
Periplasmic Enzyme

Whole cells of Pseudomonas aeruginosa possess rhodanese activity. The enzyme can be released by rapidly resuspending the cells in cold Tris–HCl buffer. Approximately 95% of the rhodanese activity is released by cold shock. Release of the enzyme can be inhibited either by preincubating the cells with Mg2+ or by incorporating Mg2+ into the shocking buffer. The effect of Mg2+ can be reversed by washing the cells twice with buffer prior to cold shock. While rhodanese can be released from P. aeruginosa by cold shock, lactic dehydrogenase, a cytoplasmic enzyme, remains within the cell. Diazo-7-amino-1,3-naphthalenedisulfonic acid, a compound which does not penetrate the cytoplasmic membrane, completely inactivated rhodanese and alkaline phosphatase, a periplasmic enzyme, whereas lactic dehydrogenase retained its full activity. These data suggest that rhodanese in P. aeruginosa, like alkaline phosphatase, is located distal to the cytoplasmic membrane in the periplasmic space. Electron micrographs also show that portions of the lipopolysaccharide outer membrane are shed from the cell during cold shock, while cells preincubated with Mg2+ did not release segments of their outer membrane.

Freeze-Fracture Replication in the Ultrastructure of Blue-Green Algae

1967 ◽  
Vol 25 ◽  
pp. 74-75
Author(s):  
L. V. Leak
Keyword(s):  
Cell Wall ◽  
Electron Microscope ◽  
Green Algae ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Electron Microscopic ◽  
Photosynthetic Membranes ◽  
Blue Green Algae ◽  
Cell Biol ◽  
Cellular Components

Electron microscopic observations of freeze-fracture replicas of Anabaena cells obtained by the procedures described by Bullivant and Ames (J. Cell Biol., 1966) indicate that the frozen cells are fractured in many different planes. This fracturing or cleaving along various planes allows one to gain a three dimensional relation of the cellular components as a result of such a manipulation. When replicas that are obtained by the freeze-fracture method are observed in the electron microscope, cross fractures of the cell wall and membranes that comprise the photosynthetic lamellae are apparent as demonstrated in Figures 1 & 2.A large portion of the Anabaena cell is composed of undulating layers of cytoplasm that are bounded by unit membranes that comprise the photosynthetic membranes. The adjoining layers of cytoplasm are closely apposed to each other to form the photosynthetic lamellae. Occassionally the adjacent layers of cytoplasm are separated by an interspace that may vary in widths of up to several 100 mu to form intralamellar vesicles.

Cell Wall Ultrastructure in Three Strains of a Cariogenic Streptococcus

1971 ◽  
Vol 29 ◽  
pp. 338-339
Author(s):  
M. J. Kramer ◽  
Alan L. Coykendall
Keyword(s):  
Cell Wall ◽  
Dental Caries ◽  
Streptococcus Mutans ◽  
Microscopic Study ◽  
Electron Microscopic Study ◽  
Genetic Heterogeneity ◽  
Morphological Characteristics ◽  
Electron Microscopic ◽  
Dna Reassociation ◽  
Wall Ultrastructure

During the almost 50 years since Streptococcus mutans was first suggested as a factor in the etiology of dental caries, a multitude of studies have confirmed the cariogenic potential of this organism. Streptococci have been isolated from human and animal caries on numerous occasions and, with few exceptions, they are not typable by the Lancefield technique but are relatively homogeneous in their biochemical reactions. An analysis of the guanine-cytosine (G-C) composition of the DNA from strains K-1-R, NCTC 10449, and FA-1 by one of us (ALC) revealed significant differences and DNA-DNA reassociation experiments indicated that genetic heterogeneity existed among the three strains. The present electron microscopic study had as its objective the elucidation of any distinguishing morphological characteristics which might further characterize the respective strains.

Molecular Structure of the Outermost Cell Wall Component of Spirillum Serpens

1977 ◽  
Vol 35 ◽  
pp. 342-343
Author(s):  
Wah Chiu ◽  
David Grano
Keyword(s):  
Molecular Structure ◽  
Cell Wall ◽  
Outer Membrane ◽  
Gel Electrophoresis ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Cell Wall Component ◽  
Protein Components ◽  
Exposure Technique ◽  
Structural Aspects

The periodic structure external to the outer membrane of Spirillum serpens VHA has been isolated by similar procedures to those used by Buckmire and Murray (1). From SDS gel electrophoresis, we have found that the isolated fragments contain several protein components, and that the crystalline structure is composed of a glycoprotein component with a molecular weight of ∽ 140,000 daltons (2). Under an electron microscopic examination, we have visualized the hexagonally-packed glycoprotein subunits, as well as the bilayer profile of the outer membrane. In this paper, we will discuss some structural aspects of the crystalline glycoproteins, based on computer-reconstructed images of the external cell wall fragments.The specimens were prepared for electron microscopy in two ways: negatively stained with 1% PTA, and maintained in a frozen-hydrated state (3). The micrographs were taken with a JEM-100B electron microscope with a field emission gun. The minimum exposure technique was essential for imaging the frozen- hydrated specimens.

Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment

1989 ◽  
Vol 47 ◽  
pp. 912-913
Author(s):  
S.K. Aggarwal
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Kidney Tissue ◽  
Membrane Glycoproteins ◽  
Electron Microscopic ◽  
Before And After ◽  
Interstrand Cross Links ◽  
Cross Links

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.

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