Specificity of subcellular distribution of alkaline phosphatase in Bacillus licheniformis 749/C

1984 ◽  
Vol 30 (1) ◽  
pp. 113-125 ◽  
Author(s):  
Arati Ghosh ◽  
Steve Vallespir ◽  
B. K. Ghosh
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Bacillus Licheniformis ◽  
Diazonium Salt ◽  
Sodium Dodecyl ◽  
Cell Fractionation ◽  
Experimental Approaches ◽  
Intramembrane Proteins

The objective of this investigation was to examine the in vivo characteristics of binding sites for alkaline phosphatase in Bacillus licheniformis cell surface. An attempt was made to correlate the results from several experimental approaches, namely (i) cell fractionation; (ii) ultrastructural cytochemistry; (iii) MgCl2 extraction and sodium dodecyl sulphate – polyacrylamide electrophoresis of the extracted material; (iv) labelling with 125I-labelled diazonium salt to determine the subcellular origin of MgCl2-extracted material. Results show that 40% of the alkaline phosphatase was bound to the plasma membrane, 35% to the cell wall, and 15% was free in the cytosol. The enzyme was present as aggregates in a few discrete sites in the membrane, wall, and cytoplasm. The membrane enzyme was associated with the inside surface. A few aggregates were enclosed in single-layered vesicles which appeared to protrude through the cell wall. The material extracted with magnesium salt consisted of 8–10 proteins including alkaline phosphatase. The majority of the proteins extracted by MgCl2 originated from the outside half of the plasma membrane, whereas, only a few, including alkaline phosphatase, came from the inside half of the plasma membrane. All of these proteins may have formed a complex which was removed by MgCl2 extraction. Patch formation in the membrane indicated specific aggregation of intramembrane proteins after MgCl2 treatment.

Microanatomy of the Periplasm of Bacillus Licheniformis

1971 ◽  
Vol 29 ◽  
pp. 262-263
Author(s):  
B.K. Ghosh
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Bacillus Licheniformis ◽  
External Environment ◽  
Permeability Barrier ◽  
Periplasmic Space ◽  
Modified Technique ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Periplasm of bacteria is the space outside the permeability barrier of plasma membrane but enclosed by the cell wall. The contents of this special milieu exterior could be regulated by the plasma membrane from the internal, and by the cell wall from the external environment of the cell. Unlike the gram-negative organism, the presence of this space in gram-positive bacteria is still controversial because it cannot be clearly demonstrated. We have shown the importance of some periplasmic bodies in the secretion of penicillinase from Bacillus licheniformis.In negatively stained specimens prepared by a modified technique (Figs. 1 and 2), periplasmic space (PS) contained two kinds of structures: (i) fibrils (F, 100 Å) running perpendicular to the cell wall from the protoplast and (ii) an array of vesicles of various sizes (V), which seem to have evaginated from the protoplast.

scholarly journals Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

1988 ◽  
Vol 107 (1) ◽  
pp. 163-175 ◽  
Author(s):  
D J Meyer ◽  
C L Afonso ◽  
D W Galbraith
Keyword(s):  
Monoclonal Antibody ◽  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Solid Phase ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Cell Suspension Cultures ◽  
Isolation And Characterization

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

scholarly journals Profiling theAspergillus fumigatusProteome in Response to Caspofungin

2010 ◽  
Vol 55 (1) ◽  
pp. 146-154 ◽  
Author(s):  
Steven E. Cagas ◽  
Mohit Raja Jain ◽  
Hong Li ◽  
David S. Perlin
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Resistant Strain ◽  
Drug Action ◽  
Susceptible Strain ◽  
Cell Fractionation ◽  
Wild Type ◽  
Subcellular Compartment ◽  
Minimum Effective Concentration ◽  
Potential Biomarkers

ABSTRACTThe proteomic response ofAspergillus fumigatusto caspofungin was evaluated by gel-free isobaric tagging for relative and absolute quantitation (iTRAQ) as a means to determine potential biomarkers of drug action. A cell fractionation approach yielding 4 subcellular compartment fractions was used to enhance the resolution of proteins for proteomic analysis. Using iTRAQ, a total of 471 unique proteins were identified in soluble and cell wall/plasma membrane fractions at 24 and 48 h of growth in rich media in a wild-type drug-susceptible strain. A total of 122 proteins showed at least a 2-fold change in relative abundance following exposure to caspofungin (CSF) at just below the minimum effective concentration (0.12 μg/ml). The largest changes were seen in the mitochondrial hypoxia response domain protein (AFUA_1G12250), the level of which decreased >16-fold in the secreted fraction, and ChiA1, the level of which decreased 12.1-fold in the cell wall/plasma membrane fraction. The level of the major allergen and cytotoxin AspF1 was also shown to decrease by 12.1-fold upon the addition of drug. A subsequent iTRAQ analysis of an echinocandin-resistant strain (fks1-S678P) was used to validate proteins specific to drug action. A total of 103 proteins in the 2 fractions tested by iTRAQ were differentially expressed in the wild-type susceptible strain but not significantly changed in the resistant strain. Of these potential biomarkers, 11 had levels that changed at least 12-fold. Microarray analysis of the susceptible strain was performed to evaluate the correlation between proteomics and genomics, with a total of 117 genes found to be changing at least 2-fold. Of these, a total of 22 proteins with significant changes identified by iTRAQ also showed significant gene expression level changes by microarray. Overall, these data have the potential to identify biomarkers that assess the relative efficacy of echinocandin drug therapy.

scholarly journals Ultrastructural localization of alkaline phosphatase in rat eosinophil leucocytes.

1978 ◽  
Vol 26 (10) ◽  
pp. 862-864 ◽  
Author(s):  
D M Williams ◽  
J E Linder ◽  
M W Hill ◽  
R Gillett
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Electron Microscope ◽  
Outer Surface ◽  
Human Neutrophil ◽  
Osmium Tetroxide ◽  
Diazonium Salt ◽  
Ultrastructural Localization ◽  
Microscope Examination ◽  
Membrane Location

The ultrastructural localization of alkaline phosphatase in eosinophil leucocytes, obtained from experimentally-induced peritoneal exudates in rats, has been studied using an osmiophilic technique with 2-naphthylthiolphosphoryl dichloride as substrate, fast Blue BBN as diazonium salt and postosmication with 1% aqueous osmium tetroxide. With this method identical incubation procedures could be used for both light and electron microscope examination. Eosinophils were the only cells which contained alkaline phosphatase. The enzyme was predominantly associated with the outer surface of the plasma membrane, being present in much lower concentrations in cytoplasmic cisternae. Eosinophil granules only rarely showed reaction product. The plasma membrane location of alkaline phosphatase in eosinophil leucocytes is identical to that recently demonstrated in the human neutrophil.

Purification and characterization of Pseudomonas aeruginosa alkaline phosphatase

1973 ◽  
Vol 19 (10) ◽  
pp. 1225-1233 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Sodium Dodecyl ◽  
Current Theory ◽  
Outer Cell ◽  
Ph Optimum ◽  
Nitrophenyl Phosphate ◽  
A Subunit

Alkaline phosphatase and a subunit form of the enzyme have been isolated from Pseudomonas aeruginosa. The enzyme is pure as judged by molecular-sieve chromatography, sodium dodecyl gel electrophoresis, and ultracentrifugation. The enzyme possesses the following properties: (a) existence of three forms: monomer mol. wt. 39 000, dimer mol. wt. 68 000, and tetramer mol. wt. 139 000; (b) pH optimum 10.5; (c) Michaelis constant Km = 6.6 × 10−5 M p-nitrophenyl phosphate; and (d) energy of activation 5647 cal/mol. Amino acid analysis indicates a protein that is hydrophobic. Its physical behavior in solution supports this conclusion. These results explain the observed association of alkaline phosphatase and lipopolysaccharide and substantiate the current theory that the alkaline phosphatase of P. aeruginosa is bound to the outer cell wall in vivo.

scholarly journals Structural base for the transfer of GPI-anchored glycoproteins into fungal cell walls

2020 ◽  
Vol 117 (36) ◽  
pp. 22061-22067
Author(s):  
Marian Samuel Vogt ◽  
Gesa Felicitas Schmitz ◽  
Daniel Varón Silva ◽  
Hans-Ulrich Mösch ◽  
Lars-Oliver Essen
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Molecular Model ◽  
Md Simulations ◽  
Binding Pocket ◽  
Fungal Cell ◽  
Final Destination ◽  
Fragment Screening

The correct distribution and trafficking of proteins are essential for all organisms. Eukaryotes evolved a sophisticated trafficking system which allows proteins to reach their destination within highly compartmentalized cells. One eukaryotic hallmark is the attachment of a glycosylphosphatidylinositol (GPI) anchor to C-terminal ω-peptides, which are used as a zip code to guide a subset of membrane-anchored proteins through the secretory pathway to the plasma membrane. In fungi, the final destination of many GPI-anchored proteins is their outermost compartment, the cell wall. Enzymes of the Dfg5 subfamily catalyze the essential transfer of GPI-anchored substrates from the plasma membrane to the cell wall and discriminate between plasma membrane-resident GPI-anchored proteins and those transferred to the cell wall (GPI-CWP). We solved the structure of Dfg5 from a filamentous fungus and used in crystallo glycan fragment screening to reassemble the GPI-core glycan in a U-shaped conformation within its binding pocket. The resulting model of the membrane-bound Dfg5•GPI-CWP complex is validated by molecular dynamics (MD) simulations and in vivo mutants in yeast. The latter show that impaired transfer of GPI-CWPs causes distorted cell-wall integrity as indicated by increased chitin levels. The structure of a Dfg5•β1,3-glycoside complex predicts transfer of GPI-CWP toward the nonreducing ends of acceptor glycans in the cell wall. In addition to our molecular model for Dfg5-mediated transglycosylation, we provide a rationale for how GPI-CWPs are specifically sorted toward the cell wall by using GPI-core glycan modifications.

scholarly journals Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan

1975 ◽  
Vol 146 (1) ◽  
pp. 253-267 ◽  
Author(s):  
Z Tynecka ◽  
J B Ward
Keyword(s):  
Cell Wall ◽  
Bacillus Licheniformis ◽  
Cross Linking ◽  
Cross Links ◽  
Increased Sensitivity ◽  
Cross Bridges ◽  
Chain Lengths ◽  
Soluble Material ◽  
Subsequent Addition

The synthesis of peptidoglycan by an autolysin-deficient β-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with β-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.

Cell Wall Particles from E. Coli

1968 ◽  
Vol 26 ◽  
pp. 72-73
Author(s):  
H. S. Slayter ◽  
John H. Nisbet
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Outer Layer ◽  
Similar Material ◽  
E Coli

A number of reports have appeared recently concerning the nature of the cell wall of E. coli (e. g. ref. 1). It has been shown that the region between the outer layer of the cell wall and the inner layer of the plasma membrane consists of strata of protein, lipid, polysaccharide and their complexes. It is also known that the space between the cell wall and the plasma membrane contains substantial activities of various enzymes, such as alkaline phosphatase and acid phosphatase. Micrographs of sections of E. coli often show vesicles which appear to be detached from the cell wall. These range upward from 200Å in diameter. In certain lysine-limited mutants of E. coli a substantial quantity of similar material has been isolated under conditions of lysine deprivation. This material has been characterized chemically (ref. 2).

scholarly journals A formin-mediated cell wall- plasma membrane- cytoskeleton continuum is required for symbiotic infections in Medicago truncatula

2020 ◽  
Author(s):  
Pengbo Liang ◽  
Clara Schmitz ◽  
Beatrice Lace ◽  
Franck Anicet Ditengou ◽  
Jean Keller ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Root Hairs ◽  
Terminal Segment ◽  
Cell Polarization ◽  
Dynamic Responses ◽  
Specific Response ◽  
Dependent Manner ◽  
Nodule Symbiosis

ABSTRACTPlant cell infections are tightly orchestrated by cell wall (CW) alterations, plasma membrane (PM) resident signalling processes and dynamic remodelling of the cytoskeleton. During root nodule symbiosis these processes result in morpho-dynamic responses including root hair swelling and curling, PM invagination and polar growth of a tubular infection structure, the infection thread (IT). However, the molecular details driving and guiding these PM remodelling events remain to be unravelled. Here, we studied a formin protein (SYFO1) in M. truncatula that is specifically induced during rhizobial infection. Phenotypical analysis of syfo1 mutants clearly indicates that the encoded protein is required for efficient rhizobial colonization of root hairs. SYFO1 itself creates a proteinaceous bridge between the CW and the polarized cytoskeleton. It binds to CW components via a proline-rich N-terminal segment, which is indispensable for its function. On the cytoplasmic side of the PM SYFO1 is associated with actin accumulations supporting the hypothesis that it contributes to cell polarization in vivo. This is further sustained by the fact that cell shape changes can be induced in a stimulus-dependent manner in root protoplasts expressing SYFO1. Taken together we provide evidence for the evolutionary re-wiring of a generic cytoskeleton modulator into a symbiosis-specific response.

scholarly journals Effect of cisplatin on the plasma membrane phosphatase activities in ascites sarcoma-180 cells: a cytochemical study.

1983 ◽  
Vol 31 (2) ◽  
pp. 307-317 ◽  
Author(s):  
S K Aggarwal ◽  
I Niroomand-Rad
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Culture Media ◽  
Tumor Burden ◽  
Sarcoma 180 ◽  
Cytochemical Study ◽  
In Vivo Experiments ◽  
Tissue Culture Media

To study the effects of cisplatin [cis-dichlorodiammine-platinum (II)] on tumor cells in the presence or absence of the immune system, animals with ascites sarcoma-180 tumor burden were treated with therapeutic dose levels (9 mg/kg). Similarly, ascites sarcoma-180 cells were maintained in tissue culture media containing the same levels of the drug. Cell samples were taken from the animals at 12-hr intervals for 3 days, whereas samples were drawn from the tissue cultures at 15-, 30-, 45-, and 60-min and at 2-, 3-, 4-, and 5-hr intervals. Treated and untreated cells from in vitro and in vivo experiments, when checked for alkaline phosphatase, 5'-nucleotidase, Ca2+-ATPase, and Na+-K+-ATPase, show a gradual decrease in activity on the plasma membrane. It takes about 60 min for inactivation of any enzyme in vitro, whereas it takes 2 days in in vivo experiments. Quantitative analysis show alkaline phosphatase activity drops from 9.7 to 4.9 nmol in just 15 min, and drops further to 0.79 nmol after 2 hr. Inactivation of various plasma membrane enzymes, resulting in permeability changes, is probably responsible for cell death.

Sign in / Sign up

Export Citation Format

Share Document