scholarly journals Labelling of the cytoplasmic domains of ovine rhodopsin with hydrophilic chemical probes

1984 ◽  
Vol 220 (1) ◽  
pp. 75-84 ◽  
Author(s):  
P L Barclay ◽  
J B C Findlay
Keyword(s):  
Staphylococcus Aureus ◽  
Succinic Anhydride ◽  
Polypeptide Chain ◽  
Chemical Probes ◽  
Disc Membrane ◽  
Radioactive Probe ◽  
Lysine Residues ◽  
Membrane Bound ◽  
Cnbr Cleavage ◽  
Cytoplasmic Loops

The disposition of polypeptide chain of ovine rhodopsin in the photoreceptor disc membrane was investigated by using two hydrophilic reagents, 3,5-di-[125I]iodo-4-diazobenzenesulphonate [( 125I]DDISA) and [14C]succinic anhydride. Both reagents were used to modify rhodopsin in intact disc membranes under conditions where no loss of A500 occurred. Reaction of [125I]DDISA with rhodopsin approached completion after 30 min. Binding was saturated at a 75-fold molar excess of reagent, which gave binding ratios of up to 2 mol/mol of rhodopsin. Proteolysis of rhodopsin, using Staphylococcus aureus V8 proteinase, yielded two membrane-bound fragments, both of which contained bound radioactive probe. Subsequent CNBr cleavage of these fragments produced five radiolabelled peptides which corresponded to the C-terminal region and cytoplasmic loops of rhodopsin. Similar studies with [14C]-succinic anhydride also gave binding ratios of up to 2 mol/mol of rhodopsin. Sequencing of the [14C]succinylated peptides identified the location of the reactive sites as lysine residues 66, 67, 141, 245, 248, 311, 325 and 339 in the polypeptide chain. Non-permeability of both probes was demonstrated by the absence of any radioactivity associated with the intradiscal N-terminal glycopeptide. Sonication of membranes in the presence of [125I]DDISA led to the incorporation of label in this peptide.

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 Inhibitory Spectra and Modes of Antimicrobial Action of Gallotannins from Mango Kernels (Mangifera indicaL.)

2011 ◽  
Vol 77 (7) ◽  
pp. 2215-2223 ◽  
Author(s):  
Christina Engels ◽  
Andreas Schieber ◽  
Michael G. Gänzle
Keyword(s):  
Staphylococcus Aureus ◽  
Wild Type Strain ◽  
Antimicrobial Activities ◽  
Resistance Mechanisms ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Positive Bacteria ◽  
Food Borne ◽  
Membrane Bound ◽  
Bacteria And Fungi

ABSTRACTThis study investigated the antimicrobial activities and modes of action of penta-, hexa-, hepta-, octa-, nona-, and deca-O-galloylglucose (gallotannins) isolated from mango kernels. The MICs and minimum bactericidal concentrations (MBCs) against food-borne bacteria and fungi were determined using a critical dilution assay. Gram-positive bacteria were generally more susceptible to gallotannins than were Gram-negative bacteria. The MICs of gallotannins againstBacillus subtilis,Bacillus cereus,Clostridium botulinum,Campylobacter jejuni,Listeria monocytogenes, andStaphylococcus aureuswere 0.2 g liter−1or less; enterotoxigenicEscherichia coliandSalmonella entericawere inhibited by 0.5 to 1 g liter−1, and lactic acid bacteria were resistant. The use of lipopolysaccharide mutants ofS. entericaindicated that the outer membrane confers resistance toward gallotannins. Supplementation of LB medium with iron eliminated the inhibitory activity of gallotannins againstStaphylococcus aureus, and siderophore-deficient mutants ofS. entericawere less resistant toward gallotannins than was the wild-type strain. Hepta-O-galloylglucose sensitizedLactobacillus plantarumTMW1.460 to hop extract, indicating inactivation of hop resistance mechanisms, e.g., the multidrug resistance (MDR) transporter HorA. Carbohydrate metabolism ofLactococcus lactisMG1363, a conditionally respiring organism, was influenced by hepta-O-galloylglucose when grown under aerobic conditions and in the presence of heme but not under anaerobic conditions, indicating that gallotannins influence the respiratory chain. In conclusion, the inhibitory activities of gallotannins are attributable to their strong affinity for iron and likely additionally relate to the inactivation of membrane-bound proteins.

scholarly journals Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein

1991 ◽  
Vol 273 (3) ◽  
pp. 517-522 ◽  
Author(s):  
L E Grosso ◽  
W C Parks ◽  
L J Wu ◽  
R P Mecham
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Fusion Protein ◽  
Developmental Regulation ◽  
Protein A ◽  
Receptor Binding Site ◽  
Elastin Receptor ◽  
Cnbr Cleavage ◽  
Fibroblast Adhesion ◽  
Nuchal Ligament

A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor.

scholarly journals The enzyme activity of mitochondrial trifunctional protein is not altered by lysine acetylation or lysine succinylation

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0256619
Author(s):  
Yuxun Zhang ◽  
Eric Goetzman
Keyword(s):  
Enzyme Activity ◽  
Bound State ◽  
Loss Of Function ◽  
Substrate Channeling ◽  
Post Translational Modifications ◽  
Lysine Residues ◽  
Membrane Bound ◽  
Mitochondrial Trifunctional Protein ◽  
Long Chain

Mitochondrial trifunctional protein (TFP) is a membrane-associated heterotetramer that catalyzes three of the four reactions needed to chain-shorten long-chain fatty acids inside the mitochondria. TFP is known to be heavily modified by acetyllysine and succinyllysine post-translational modifications (PTMs), many of which are targeted for reversal by the mitochondrial sirtuin deacylases SIRT3 and SIRT5. However, the functional significance of these PTMs is not clear, with some reports showing TFP gain-of-function and some showing loss-of-function upon increased acylation. Here, we mapped the known SIRT3/SIRT5-targeted lysine residues onto the recently solved TFP crystal structure which revealed that many of the target sites are involved in substrate channeling within the TFPα subunit. To test the effects of acylation on substate channeling through TFPα, we enzymatically synthesized the physiological long-chain substrate (2E)-hexadecenoyl-CoA. Assaying TFP in SIRT3 and SIRT5 knockout mouse liver and heart mitochondria with (2E)-hexadecenoyl-CoA revealed no change in enzyme activity. Finally, we investigated the effects of lysine acylation on TFP membrane binding in vitro. Acylation did not alter recombinant TFP binding to cardiolipin-containing liposomes. However, the presence of liposomes strongly abrogated the acylation reaction between succinyl-CoA and TFP lysine residues. Thus, TFP in the membrane-bound state may be protected against lysine acylation.

The cytoplasmic loops of AgrC contribute to the quorum-sensing activity of Staphylococcus aureus

2020 ◽  
Vol 59 (1) ◽  
pp. 92-100
Author(s):  
Qian Huang ◽  
Yihui Xie ◽  
Ziyu Yang ◽  
Danhong Cheng ◽  
Lei He ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Cytoplasmic Loops
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