The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

Correlative immunolocalization with high-resolution light microscopy and Electron Microscopy using London Resin Gold embedded tissue

1995 ◽  
Vol 53 ◽  
pp. 702-703
Author(s):  
M. L. Grove ◽  
B. A. Evans ◽  
D. N. Misra ◽  
J. Zhao ◽  
D. H. Alpers ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Polyclonal Antibodies ◽  
Intracellular Localization ◽  
Intrinsic Factor ◽  
Gastric Epithelium ◽  
Electron Microscopic Level ◽  
Immunoperoxidase Staining ◽  
Rat Tissues ◽  
Electron Microscopic ◽  
Thin Sections

Immunoelectron microscopy specimens are often embedded in hydrophilic resins, like London Resin Gold(LRG), which permit antibody staining without etching (or deplasticizing) the sections. This characteristic of hydrophilic resins also allows for immunohistochemistry at the light level on semi thin sections (0.5μm - 1μm). The ability to do immunohistochemistry at both the light and electron microscopic level on the same tissue block allows focused ultrastructural study. Immunohistochemistry on semi-thin sections displays cellular localization of macromolecules, permitting more specificity in the selection of areas for studying intracellular localization ultrastructurally. We have developed a method for immunoperoxidase staining of LRG embedded tissues, utilizing anti-human polyclonal antibodies directed against intrinsic factor (Fig. 1). Intrinsic factor (IF), a cobalamin binding protein, is known to be produced in the stomach, pancreas and salivary glands of most mammals. We are interested in distribution of IF in gastric epithelium, small intestine (ileum) and supporting tissues in both gastrointestinal tract sites. Previously, we have described the cellular localization of IF in human and rat tissues.

scholarly journals Xylanase Attachment to the Cell Wall of the Hyperthermophilic Bacterium Thermotoga maritima

2007 ◽  
Vol 190 (4) ◽  
pp. 1350-1358 ◽  
Author(s):  
Wolfgang Liebl ◽  
Christoph Winterhalter ◽  
Wolfgang Baumeister ◽  
Martin Armbrecht ◽  
Michael Valdez
Keyword(s):  
Outer Membrane ◽  
Signal Peptide ◽  
Cellular Localization ◽  
Culture Supernatant ◽  
Thermotoga Maritima ◽  
Surrounding Medium ◽  
Hydrophobic Core ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Amino Terminal

ABSTRACT The cellular localization and processing of the endo-xylanases (1,4-β-d-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane (“toga”) of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.

Intracellular Localization of Two Betaine Lipids by Cell Fractionation and Immunomicroscopy

1997 ◽  
Vol 52 (7-8) ◽  
pp. 487-495 ◽  
Author(s):  
K. Department of Chemistry and Biochem ◽  
W. Department of Chemistry and Biochem ◽  
A. Faculty of Biology, University of B
Keyword(s):  
Fluorescence Microscopy ◽  
Cellular Localization ◽  
Gradient Centrifugation ◽  
Intracellular Localization ◽  
Rabbit Serum ◽  
Cell Fractionation ◽  
Thin Sections ◽  
Ochromonas Danica ◽  
Betaine Lipids ◽  
Cyt C

Abstract The cellular localization of the betaine lipids diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) was investi­ gated by a) chemical analysis of subcellular fractions and b) immunochemical methods using specific antisera and either fluorescence microscopy or electron microscopy for detection of the label. A homogenate of Lycopodium annotinum (Pteridophyta) was fractionated by differential and density gradient centrifugation. The particulate fractions obtained were analyzed for chlorophyll, cyt c oxidase, NADH-cyt c reductase and DGTS. Non-plastidial fractions were enriched in DGTS and only minor amounts of this lipid could be attributed to chloroplasts. Anti-DGTS and anti-DGTA sera were produced by immunization of rabbits. The monospecificity of the antisera was examined with cells of Chlamydomonas reinhardtii (Chlorophyceae) containing DGTS, Pavlova lutheri (Haptophyceae) containing DGTA and Ochromonas danica (Chrysophyceae) containing both DGTS and DGTA. Euglena gracilis which is free of betaine lipids, was used as a control. For the test, a FITC-coupled goat anti-rabbit antibody was used and detected by fluorescence microscopy. Thin sections of Ochromonas and Pavlova were incubated first with the anti-lipid sera and subsequently with a gold-coupled anti-rabbit serum and then examined in the electron microscope. With O chro­ monas, anti-DGTS as well as anti-DGTA sera gave an accumulation of gold label in the cytoplasmic space but not in the chloroplasts. Similar results were obtained with Pavlova using anti-DGTA serum. These results describe for the first time the cytochemical localiza­ tion of DGTS and DGTA strongly suggesting both these lipids to be associated mainly with non-plastidial structures.

scholarly journals Parathyroid hormone receptor in intact embryonic chicken bone: characterization and cellular localization.

1982 ◽  
Vol 94 (2) ◽  
pp. 379-386 ◽  
Author(s):  
C M Silve ◽  
G T Hradek ◽  
A L Jones ◽  
C D Arnaud
Keyword(s):  
Parathyroid Hormone ◽  
Cellular Localization ◽  
Adenosine Monophosphate ◽  
Biologically Active ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Parathyroid Hormone Receptor ◽  
Specific Localization ◽  
Grain Distribution

The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.

Comparative ultrastructure of methanogenic bacteria

1975 ◽  
Vol 21 (2) ◽  
pp. 121-129 ◽  
Author(s):  
J. G. Zeikus ◽  
V. G. Bowen
Keyword(s):  
Morphological Characteristics ◽  
Cell Types ◽  
Methanogenic Bacteria ◽  
Diverse Group ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Intracytoplasmic Membranes ◽  
Microscopic Studies ◽  
Different Cell Types ◽  
Comparative Ultrastructure

Electron-microscopic studies using thin sections revealed that methane-producing bacteria were an ultrastructurally diverse group. Fine structure and morphological characteristics separated these bacteria into four discrete cell types. Methanogenic bacteria displayed a gram-positive cell wall that varied considerably among different cell types. Differences in granular inclusions, reserve materials, and intracytoplasmic membranes were observed. Unique ultrastructural features were not shared by all methanogenic species studied.

scholarly journals Intracellular localization of thymosin alpha 1 by immunoelectron microscopy using a monoclonal antibody.

1987 ◽  
Vol 35 (2) ◽  
pp. 181-187 ◽  
Author(s):  
C Auger ◽  
C Stahli ◽  
N Fabien ◽  
J C Monier
Keyword(s):  
Monoclonal Antibody ◽  
Epithelial Cells ◽  
Intracellular Localization ◽  
Immunofluorescence Assay ◽  
Secretory Process ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Thymic Hormone ◽  
Thin Sections ◽  
Thymosin Alpha 1

Distribution of thymosin alpha 1 in normal mice (OF1) or autoimmune mice (NZB) was investigated using immunocytochemical techniques on sections of GMA- and Epon-embedded mouse thymuses. A monoclonal antibody directed against synthetic thymosin alpha 1 was used. With the immunofluorescence assay, patchy staining of thymosin alpha 1 was found in the cytoplasm of epithelial cells of the subcapsullary and medullary zones of OF1 thymus. In NZB thymus, the fluorescent pattern was less precisely localized. At the electron microscopic level, immunolabeling of Epon-embedded ultra-thin sections revealed ferritin in some vacuoles of epithelial cells. Ferritin labeling in OF1 thymus was found in several small vacuoles of the same cell, but was present in large, dense vacuoles in NZB thymus. These differences might reflect differences in the secretory process of thymic hormone.

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