The ordered macromolecular surface of polyester inclusion bodies inPseudomonas oleovotans

1995 ◽  
Vol 41 (13) ◽  
pp. 84-93 ◽  
Author(s):  
Elizabeth S. Stuart ◽  
R. Clinton Fuller ◽  
Robert W. Lenz
Keyword(s):  
Inclusion Bodies ◽  
Amorphous State ◽  
Immunogold Labelling ◽  
Boundary Region ◽  
Pseudomonas Oleovorans ◽  
Sds Page ◽  
Degradative Enzymes ◽  
Layer Chromatography ◽  
Intracellular Inclusion

Intracellular inclusion bodies of poly(β-hydroxyalkanoates) (PHAs) have been studied in various microorganisms since Lemoigne's discovery of PHAs in 1925. Recently, the research in several laboratories, including our own, has addressed the role of proteins, lipids, and water associated with these accumulations. Our research has examined the boundary of polymer inclusion bodies. Electron microscopy demonstrated that the polymer is encompassed by two paracrystalline arrays. SDS-PAGE, Western blot, or immunogold labelling demonstrated that both contain a 43-kDa protein as a major component. Immunogold labelling also demonstrated that 55- and 59-kDa proteins are located, exclusively, on the array associated with the accumulating polymer. Results from microelemental analysis and preliminary thin-layer chromatography of released lipids were consistent with the suggestion that phospholipids also had a role in this organized assembly. A model has been suggested, aimed at focusing attention on this organized boundary region. It is consistent with maintenance of the amorphous state of the polymer both intracellularly and after isolation, provides sites for biosynthetic and degradative enzymes, and accounts for the polyester, protein, and lipid components known to be present. Interestingly, the anti-43-kDa antibody also recognized a 43-kDa species released from the outer surface of this microbe. The research presented here and the model developed from it, suggest that microbial synthesis, containment, and degradation of polyester are carried out in association with a highly organized and complex intracellular assembly that may provide, within the bacterial cytosol, a unique microenvironment for biochemical activities.Key words: polyester, inclusion granule, Pseudomonas oleovorans.

scholarly journals Characterization of a Dye-Decolorizing Peroxidase from Irpex lacteus Expressed in Escherichia coli: An Enzyme with Wide Substrate Specificity Able to Transform Lignosulfonates

2021 ◽  
Vol 7 (5) ◽  
pp. 325
Author(s):  
Laura Isabel de de Eugenio ◽  
Rosa Peces-Pérez ◽  
Dolores Linde ◽  
Alicia Prieto ◽  
Jorge Barriuso ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Veratryl Alcohol ◽  
Dye Decolorization ◽  
Irpex Lacteus ◽  
Type D ◽  
Lignin Peroxidases

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

scholarly journals THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

1974 ◽  
Vol 63 (2) ◽  
pp. 587-598 ◽  
Author(s):  
Izhak Nir ◽  
Michael O. Hall
Keyword(s):  
Lipid Extraction ◽  
Fatty Acid Analysis ◽  
Major Protein ◽  
Rod Photoreceptor ◽  
Thin Sections ◽  
Retinal Rod ◽  
Layer Chromatography ◽  
Chloroform Methanol ◽  
Photoreceptor Membranes

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO4 with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO4. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals cAMP induces a rapid and reversible modification of the chemotactic receptor in Dictyostelium discoideum.

1985 ◽  
Vol 100 (3) ◽  
pp. 715-720 ◽  
Author(s):  
C Klein ◽  
J Lubs-Haukeness ◽  
S Simons
Keyword(s):  
Dictyostelium Discoideum ◽  
Concentration Dependence ◽  
Time Course ◽  
Camp Synthesis ◽  
Sds Page ◽  
Reversible Modification ◽  
Camp Receptor ◽  
Camp Stimulation ◽  
Stimulation Of

Stimulation, within 1 min after cAMP stimulation, of aggregation-competent Dictyostelium discoideum amebae was found to cause a rapid (within 1 min) modification of the cell's surface cAMP receptor. The modified receptor migrated on SDS PAGE as a 47,000-mol-wt protein, as opposed to a 45,000-mol-wt protein labeled on unstimulated cells. The length of time this modified receptor could be detected depended upon the strength of the cAMP stimulus: 3-4 min after treatment with 10(-7) M cAMP, cells no longer possessed the 47,000-mol-wt form of the cAMP receptor. Instead, the 45,000-mol-wt form was present. Stimulation of cells with 10(-5) M cAMP, however, resulted in the persistent (over 15 min) expression of the modified receptor. The time course, concentration dependence, and specificity of stimulus for this cAMP-induced shift in the cAMP receptor were found to parallel the cAMP-stimulated phosphorylation of a 47,000-mol-wt protein. In addition, both phenomena were shown to occur in the absence of endogenous cAMP synthesis. The possibility that the cAMP receptor is phosphorylated in response to cAMP stimulation, and the role of this event in cell desensitization, are discussed.

scholarly journals Role of maltase in the utilization of sucrose by Candida albicans

1993 ◽  
Vol 291 (3) ◽  
pp. 765-771 ◽  
Author(s):  
P R Williamson ◽  
M A Huber ◽  
J E Bennett
Keyword(s):  
Candida Albicans ◽  
Intracellular Localization ◽  
Cross Reactivity ◽  
Neutral Sugar ◽  
Sequence Specificity ◽  
Western Blots ◽  
Sds Page ◽  
Molecular Masses ◽  
Kinetics Of

Two isoenzymes of maltase (EC 3.2.1.20) were purified to homogeneity from Candida albicans. Isoenzymes I and II were found to have apparent molecular masses of 63 and 66 kDa on SDS/PAGE with isoelectric points of 5.0 and 4.6 respectively. Both isoenzymes resembled each other in similar N-terminal sequence, specificity for the alpha(1-−>4) glycosidic linkage and immune cross-reactivity on Western blots using a maltase II antigen-purified rabbit antibody. Maltase was induced by growth on sucrose whereas beta-fructofuranosidase activity could not be detected under similar conditions. Maltase I and II were shown to be unglycosylated enzymes by neutral sugar assay, and more than 90% of alpha-glucosidase activity was recoverable from spheroplasts. These data, in combination with other results from this laboratory [Geber, Williamson, Rex, Sweeney and Bennett (1992) J. Bacteriol. 174, 6992-6996] showing lack of a plausible leader sequence in genomic or mRNA transcripts, suggest an intracellular localization of the enzyme. To establish further the mechanism of sucrose assimilation by maltase, the existence of a sucrose-inducible H+/sucrose syn-transporter was demonstrated by (1) the kinetics of sucrose-induced [14C]sucrose uptake, (2) recovery of intact [14C]sucrose from ground cells by t.l.c. and (3) transport of 0.83 mol of H+/mol of [14C]sucrose. In total, the above is consistent with a mechanism whereby sucrose is transported into C. albicans to be hydrolysed by an intracellular maltase.

scholarly journals Pyrrolnitrin Biosynthesis From Rhizospheric Serratia Spp. With Antifungal Activity and Binding Interactions of PrnF With Ligands

2020 ◽  
Author(s):  
Shraddha P. Pawar ◽  
Ambalal B. Chaudhari
Keyword(s):  
Antifungal Activity ◽  
Phytopathogenic Fungi ◽  
Bacterial Strains ◽  
Rrna Sequence ◽  
16S Rrna Sequence ◽  
Protein Model ◽  
Stenotrophomonas Rhizophila ◽  
K 12 ◽  
Layer Chromatography

Abstract Pyrrolnitrin (PRN) from rhizobacteria displays a key role in biocontrol of phytopathogenic fungi in rhizospheric soil. Therefore, different rhizospheric soils were investigated for the prevalence of PRN producer in minimal salt (MS) medium containing tryptophan (0.2 M NaCl; pH 8) using three successive enrichments. Of 12% isolates, only five bacterial strains had shown PRN secretion, screened with Thin Layer Chromatography (Rf 0.8) and antifungal activity (27 mm) against phytopathogen. The phenetic and 16S rRNA sequence revealed the close affiliation of isolates (KMB, M-2, M-11, TW3, and TO2) to Stenotrophomonas rhizophila (KY800458), Enterobacter spp. (KY800455), Brevibacillus parabrevis (KY800454), Serratia marcescens (KY800456) and Serratia nemtodiphila (KY800457). Purified compound from isolates was characterised using UV, IR, HPLC, LCMS and GCMS as PRN. However, BLASTn hit of prn gene sequences from both Serratia species showed 99% similarity with NADPH dependent FMN reductase component (prnF). The homology protein model of prnF was developed from translated sequence of S. marcescens TW3 with chromate reductase of Escherichia coli K-12. Docking with FMN and NADPH was performed. The study demonstrated the possible role of prnF NADPH dependent FMN reductases in prnD for supply of reduced flavin in rhizobacterial strain of Serratia spp. which may pave a way to understand PRN production.

Slip, twinning, and fracture at a grain boundary in the L12 ordered structure—AΣ = 9 tilt boundary

1988 ◽  
Vol 3 (5) ◽  
pp. 848-855 ◽  
Author(s):  
M. H. Yoo ◽  
A. H. King
Keyword(s):  
Intergranular Fracture ◽  
Local Stress ◽  
Boundary Region ◽  
Anisotropic Elasticity ◽  
Tilt Boundary ◽  
Microplastic Deformation ◽  
Ordered Structure ◽  
Local Stress Concentration ◽  
Boron Segregation

The role of interaction between slip dislocations and a Σ = 9 tilt boundary in localized microplastic deformation, cleavage, or intergranular fracture in the Li2 ordered structure has been analyzed by using the anisotropic elasticity theory of dislocations and fracture. Screw superpartials cross slip easily at the boundary onto the (1$\overline 1$1) and the (001) planes at low and high temperatures, respectively. Transmission of primary slip dislocations onto the conjugate slip system occurs with a certain degree of difficulty, which is eased by localized disordering. When the transmission is impeded, cleavage fracture on the ($\overline 1$11) plane is predicted to occur, not intergranular fracture, unless a symmetric double pileup occurs simultaneously. Absorption (or emission) of superpartials occurs only when the boundary region is disordered. Slip initiation from pre-existing sources near the boundary can occur under the local stress concentration. Implications of the present result on the inherent brittleness of grain boundaries in Ni3 Al and its improvement by boron segregation are discussed.

On the role of superoxide in reactions catalyzed by rubredoxin of Pseudomonas oleovorans

1973 ◽  
Vol 54 (4) ◽  
pp. 1540-1545 ◽  
Author(s):  
Sheldon W. May ◽  
Bernard J. Abbott ◽  
Andre Felix
Keyword(s):  
Pseudomonas Oleovorans

Intracellular Inclusion Bodies of Broad Bean Wilt Virus

1974 ◽  
Vol 80 (2) ◽  
pp. 127-135 ◽  
Author(s):  
D. Miličić ◽  
Mercedes Wrischer ◽  
N. Juretić
Keyword(s):  
Inclusion Bodies ◽  
Broad Bean ◽  
Wilt Virus ◽  
Broad Bean Wilt Virus ◽  
Intracellular Inclusion
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