scholarly journals Sequence variability in the retinal-attachment domain of mammalian rhodopsins

1984 ◽  
Vol 217 (3) ◽  
pp. 605-613 ◽  
Author(s):  
D J C Pappin ◽  
J B C Findlay
Keyword(s):  
Staphylococcus Aureus ◽  
Visual Pigment ◽  
Gel Filtration ◽  
Aldehyde Group ◽  
Marked Variation ◽  
Sequence Variability ◽  
Membrane Bound ◽  
Covalently Linked ◽  
Sepharose 4B

Ovine rhodopsin was regenerated with 11-cis-[15-3H]retinal and cleaved in situ by Staphylococcus aureus V8 proteinase to give two membrane-bound fragments of Mr 27 000 (V8-L) and 12 000 (V8-S). After purification of the proteolysed complex by affinity chromatography with concanavalin A-Sepharose 4B, [3H]retinal was covalently linked to the protein by reduction with borohydride. The purified [3H]-retinyl V8-S fragment was cleaved with CNBr and trifluoroacetic acid, the resulting peptides resolved by gel filtration and the [3H]retinyl peptide sequenced. The protocol developed for the isolation and sequencing of this region of the ovine protein was applied directly, and reproducibly, to bleached and unregenerated porcine and equine opsins. Comparisons of the primary structures of the fragments reveals marked variation in the sequence immediately after the lysine residue shown in the ovine protein to be the attachment point for the aldehyde group of the chromophore. Mutable positions are localized in regions previously predicted as adopting nonregular or distorted conformations and hint at structural arrangements that may provide a better understanding of the spectral and functional properties of the visual pigment.

scholarly journals Isolation and characterization of the CNBr peptides from the proteolytically derived N-terminal fragment of ovine opsin

1983 ◽  
Vol 211 (3) ◽  
pp. 661-670 ◽  
Author(s):  
M Brett ◽  
J B C Findlay
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Permeation Chromatography ◽  
Isolation And Characterization ◽  
Peptide Mixture ◽  
Gel Permeation ◽  
Membrane Bound ◽  
Sepharose 4B ◽  
Differential Solubility

Ovine rhodopsin may be cleaved in situ by Staphylococcus aureus V8 proteinase into two membrane-bound fragments designated V8-L (27 000 mol.wt.) and V8-S (12 000 mol.wt.). After purification of the proteolysed complex by affinity chromatography in detergent using concanavalin A immobilized on Sepharose 4B, the two polypeptide fragments may be separated by gel-permeation chromatography on Sephadex LH-60. Digestion of the N-terminal-derived V8-L fragment with CNBr in 70% (v/v) trifluoroacetic acid resulted in a peptide mixture that could be fractionated by procedures involving gel-permeation chromatography in organic and aqueous solvents and the use of differential solubility. The complete or partial sequences of all ten peptides are reported.

scholarly journals Identification of the sites in opsin modified by photoactivated azido[125I]iodobenzene

1986 ◽  
Vol 236 (2) ◽  
pp. 389-395 ◽  
Author(s):  
M D Davison ◽  
J B C Findlay
Keyword(s):  
Staphylococcus Aureus ◽  
Polypeptide Chain ◽  
Phospholipid Bilayer ◽  
Alpha Helices ◽  
Transmembrane Segments ◽  
Lysine Residues ◽  
Membrane Bound ◽  
Derivatives Of

Opsin labelled with photoactivated 1-azido-4-[125I]iodobenzene was proteolysed in situ with Staphylococcus aureus V8 proteinase to yield two radioactive membrane-bound fragments. These were separated, cleaved with CNBr and the resultant peptides sequenced in order to locate the radiolabelled residues. In the whole molecule, there was clear evidence for modification of at least 20 sites, identified as derivatives of cysteine, tryptophan, tyrosine, histidine and lysine residues. The probe primary reacted, therefore, with nucleophilic substituents. The positions of the modified sites relative to the confines of the phospholipid bilayer were consistent with all other studies on the disposition of the polypeptide chain. The location of these sites substantiated an earlier suggestion that not all the transmembrane segments should be regarded as continuous regular alpha-helices.

scholarly journals EssC is a specificity determinant for Staphylococcus aureus type VII secretion

2018 ◽  
Author(s):  
Franziska Jäger ◽  
Holger Kneuper ◽  
Tracy Palmer
Keyword(s):  
Staphylococcus Aureus ◽  
Protein Secretion ◽  
Secretion System ◽  
Sequence Variability ◽  
Display Sequence ◽  
Type Vii Secretion ◽  
Substrate Protein ◽  
Protein Secretion System ◽  
Membrane Bound ◽  
Substrate Proteins

ABSTRACTThe Type VII protein secretion system (T7SS) is found in actinobacteria and firmicutes, and plays important roles in virulence and interbacterial competition. A membrane-bound ATPase protein, EssC in Staphylococcus aureus, lies at the heart of the secretion machinery. The EssC protein from S. aureus strains can be grouped into four variants (EssC1-EssC4) that display sequence variability in the C-terminal region. Here we show that the EssC2, EssC3 and EssC4 variants can be produced in a strain deleted for essC1 and that they are able to mediate secretion of EsxA, an essential component of the secretion apparatus. They are, however, unable to support secretion of the substrate protein EsxC, which is encoded only in essC1-specific strains. This finding indicates that EssC is a specificity determinant for T7 protein secretion. Our results support a model where the C-terminal domain of EssC interacts with substrate proteins whereas EsxA interacts elsewhere.

scholarly journals Nondissociating cationic immune complexes can deposit in glomerular basement membrane.

1983 ◽  
Vol 158 (5) ◽  
pp. 1561-1572 ◽  
Author(s):  
T Caulin-Glaser ◽  
G R Gallo ◽  
M E Lamm
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Immune Complexes ◽  
Gel Filtration ◽  
Lamina Densa ◽  
Net Charge ◽  
The Subject ◽  
Covalently Linked ◽  
Circulating Antibody

The mechanisms by which immune complexes deposit in the glomerular basement membrane have been the subject of much debate, with the relative importance of direct deposition of circulating immune complexes (IC) vs. formation of IC in situ from the binding of circulating antibody to structural or exogenous planted antigen being at issue. In order to determine whether intact IC can deposit as such, covalently linked IC were prepared by a two-step reaction involving the bifunctional reagent toluene-2,4-diisocyanate (TDI), the antigen bovine gamma globulin (BGG), and rabbit anti-BGG antibody. Antigen and antibody were covalently cross-linked, with little self-linkage of antigen or antibody, and IC were purified by gel filtration. The net charge of the complexes was varied by chemical means, either before or after IC formation. When cationic IC were injected intravenously into mice, there was codeposition of antigen and antibody diffusely in the glomerular basement membrane (GBM), and deposits were observed ultrastructurally in the laminae rarae, interna and externa, and the lamina densa. Thus, under conditions of restricted appropriate charge, intact IC can cross the glomerular basement membrane and deposit in subepithelial sites without being excluded by size alone.

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

scholarly journals Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

2014 ◽  
Vol 81 (3) ◽  
pp. 1024-1031 ◽  
Author(s):  
Bhagyalakshmi Kalidass ◽  
Muhammad Farhan Ul-Haque ◽  
Bipin S. Baral ◽  
Alan A. DiSpirito ◽  
Jeremy D. Semrau
Keyword(s):  
Methane Monooxygenase ◽  
High Specificity ◽  
Copper Uptake ◽  
Content Type ◽  
Soluble Methane Monooxygenase ◽  
Sds Page ◽  
Genes Encoding ◽  
Key Factor ◽  
Membrane Bound

ABSTRACTIt is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that inMethylosinus trichosporiumOB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced byM. trichosporiumOB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and activein situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin.

scholarly journals Characterization of the binding of plasminogen activators to plasma membranes from human liver

1992 ◽  
Vol 287 (3) ◽  
pp. 911-915 ◽  
Author(s):  
G Nguyen ◽  
S J Self ◽  
C Camani ◽  
E K O Kruithof
Keyword(s):  
Human Liver ◽  
Plasma Membranes ◽  
Gel Filtration ◽  
Mannose Receptor ◽  
Hepatoma Cells ◽  
Tissue Type ◽  
High Affinity ◽  
Liver Membranes ◽  
Membrane Bound ◽  
Pai 1

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.

scholarly journals In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Hyun Ok Ham ◽  
Zheng Qu ◽  
Carolyn A. Haller ◽  
Brent M. Dorr ◽  
Erbin Dai ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sortase A ◽  
Bioactive Coatings ◽  
In Situ Regeneration
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