scholarly journals pH-Dependent Interaction between C-Peptide and Phospholipid Bicelles

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Sofia Unnerståle ◽  
Lena Mäler
Keyword(s):  
Ph Dependence ◽  
Structural Rearrangement ◽  
Low Ph ◽  
Resonance Spectroscopy ◽  
Aggregation State ◽  
C Peptide ◽  
Ph Dependent ◽  
Lipid Interaction ◽  
Dependent Interaction

C-peptide is the connecting peptide between the A and B chains of insulin in proinsulin. In this paper, we investigate the interaction between C-peptide and phospholipid bicelles, by circular dichroism and nuclear magnetic resonance spectroscopy, and in particular the pH dependence of this interaction. The results demonstrate that C-peptide is largely unstructured independent of pH, but that a weak structural induction towards a short stretch of β-sheet is induced at low pH, corresponding to the isoelectric point of the peptide. Furthermore, it is demonstrated that C-peptide associates with neutral phospholipid bicelles as well as acidic phospholipid bicelles at this low pH. C-peptide does not undergo a large structural rearrangement as a consequence of lipid interaction, which indicates that the folding and binding are uncoupled. In vivo, local variations in environment, including pH, may cause C-peptide to associate with lipids, which may affect the aggregation state of the peptide.

scholarly journals pH-dependent Perforation of Macrophage Phagosomes by Listeriolysin O from Listeria monocytogenes

1997 ◽  
Vol 186 (7) ◽  
pp. 1159-1163 ◽  
Author(s):  
Kathryn E. Beauregard ◽  
Kyung-Dall Lee ◽  
R. John Collier ◽  
Joel A. Swanson
Keyword(s):  
Listeria Monocytogenes ◽  
Ph Dependence ◽  
Mouse Bone Marrow ◽  
Listeriolysin O ◽  
Bafilomycin A1 ◽  
Wild Type ◽  
Major Virulence Factor ◽  
Ph Dependent ◽  
Vacuolar Ph

The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow–derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9–6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.

scholarly journals pH-dependence of the phospholipid interaction of diphtheria-toxin fragments

1985 ◽  
Vol 231 (1) ◽  
pp. 123-128 ◽  
Author(s):  
C Montecucco ◽  
G Schiavo ◽  
M Tomasi
Keyword(s):  
Diphtheria Toxin ◽  
Ph Dependence ◽  
Low Ph ◽  
Membrane Insertion ◽  
Ph Values ◽  
Egg Lecithin ◽  
A Chain ◽  
Lipid Interaction

Photoreactive phospholipids have been used to probe the lipid interaction of diphtheria toxin. Low pH values induce the membrane insertion of both the binding and enzymic fragments of the toxin. The efficiency of this process is much higher with asolectin than with egg lecithin (phosphatidylcholine)/cholesterol liposomes. The low-pH-induced interaction of the toxin fragments with the membrane hydrocarbon phase is more evident for the enzymic A-chain than for the binding B-chain, and it is fully reversed by returning the pH to neutrality.

scholarly journals Opening of Size-Selective Pores in Endosomes during Human Rhinovirus Serotype 2 In Vivo Uncoating Monitored by Single-Organelle Flow Analysis

2005 ◽  
Vol 79 (2) ◽  
pp. 1008-1016 ◽  
Author(s):  
Marianne Brabec ◽  
Daniela Schober ◽  
Ernst Wagner ◽  
Nora Bayer ◽  
Robert F. Murphy ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Flow Analysis ◽  
Fluid Phase ◽  
Fluorescein Isothiocyanate ◽  
Low Ph ◽  
Human Rhinovirus ◽  
Endosomal Escape ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor

ABSTRACT The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.

scholarly journals pH-Dependent Expression of Periplasmic Proteins and Amino Acid Catabolism in Escherichia coli

2002 ◽  
Vol 184 (15) ◽  
pp. 4246-4258 ◽  
Author(s):  
Lauren M. Stancik ◽  
Dawn M. Stancik ◽  
Brian Schmidt ◽  
D. Michael Barnhart ◽  
Yuliya N. Yoncheva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Ph Dependence ◽  
Outer Membrane Proteins ◽  
Stress Protein ◽  
Low Ph ◽  
Wide Range ◽  
Periplasmic Proteins ◽  
Ph Dependent ◽  
External Ph

ABSTRACT Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in which E. coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.

Spectroscopic imaging of human disease

1996 ◽  
Vol 54 ◽  
pp. 884-885
Author(s):  
D.J. Meyerhoff
Keyword(s):  
Magnetic Field ◽  
Magnetic Resonance ◽  
Field Gradient ◽  
Human Disease ◽  
Morphological Changes ◽  
Magnetic Field Gradient ◽  
Spectroscopic Imaging ◽  
Resonance Spectroscopy ◽  
Tissue Water

Magnetic Resonance Imaging (MRI) observes tissue water in the presence of a magnetic field gradient to study morphological changes such as tissue volume loss and signal hyperintensities in human disease. These changes are mostly non-specific and do not appear to be correlated with the range of severity of a certain disease. In contrast, Magnetic Resonance Spectroscopy (MRS), which measures many different chemicals and tissue metabolites in the millimolar concentration range in the absence of a magnetic field gradient, has been shown to reveal characteristic metabolite patterns which are often correlated with the severity of a disease. In-vivo MRS studies are performed on widely available MRI scanners without any “sample preparation” or invasive procedures and are therefore widely used in clinical research. Hydrogen (H) MRS and MR Spectroscopic Imaging (MRSI, conceptionally a combination of MRI and MRS) measure N-acetylaspartate (a putative marker of neurons), creatine-containing metabolites (involved in energy processes in the cell), choline-containing metabolites (involved in membrane metabolism and, possibly, inflammatory processes),

Rapid Catalyst Capture Enables Metal-Free Parahydrogen-Based Hyperpolarized Contrast Agents

2018 ◽  
Author(s):  
Danila Barskiy ◽  
Lucia Ke ◽  
Xingyang Li ◽  
Vincent Stevenson ◽  
Nevin Widarman ◽  
...  
Keyword(s):  
Magnetic Resonance ◽  
Contrast Agents ◽  
Inductively Coupled Plasma ◽  
Organometallic Compounds ◽  
Atomic Emission ◽  
Platinum Group Metals ◽  
Resonance Spectroscopy ◽  
Plasma Atomic Emission ◽  
Inductively Coupled

<p>Hyperpolarization techniques based on the use of parahydrogen provide orders of magnitude signal enhancement for magnetic resonance spectroscopy and imaging. The main drawback limiting widespread applicability of parahydrogen-based techniques in biomedicine is the presence of organometallic compounds (the polarization transfer catalysts) in solution with hyperpolarized contrast agents. These catalysts are typically complexes of platinum-group metals and their administration in vivo should be avoided.</p> <p><br></p><p>Herein, we show how extraction of a hyperpolarized compound from an organic phase to an aqueous phase combined with a rapid (less than 10 seconds) Ir-based catalyst capture by metal scavenging agents can produce pure parahydrogen-based hyperpolarized contrast agents as demonstrated by high-resolution nuclear magnetic resonance (NMR) spectroscopy and inductively coupled plasma atomic emission spectroscopy (ICP-AES). The presented methodology enables fast and efficient means of producing pure hyperpolarized aqueous solutions for biomedical and other uses.</p>

Rapid Catalyst Capture Enables Metal-Free Parahydrogen-Based Hyperpolarized Contrast Agents

2018 ◽  
Author(s):  
Danila Barskiy ◽  
Lucia Ke ◽  
Xingyang Li ◽  
Vincent Stevenson ◽  
Nevin Widarman ◽  
...  
Keyword(s):  
Magnetic Resonance ◽  
Contrast Agents ◽  
Inductively Coupled Plasma ◽  
Organometallic Compounds ◽  
Atomic Emission ◽  
Platinum Group Metals ◽  
Resonance Spectroscopy ◽  
Plasma Atomic Emission ◽  
Inductively Coupled

<p>Hyperpolarization techniques based on the use of parahydrogen provide orders of magnitude signal enhancement for magnetic resonance spectroscopy and imaging. The main drawback limiting widespread applicability of parahydrogen-based techniques in biomedicine is the presence of organometallic compounds (the polarization transfer catalysts) in solution with hyperpolarized contrast agents. These catalysts are typically complexes of platinum-group metals and their administration in vivo should be avoided.</p> <p><br></p><p>Herein, we show how extraction of a hyperpolarized compound from an organic phase to an aqueous phase combined with a rapid (less than 10 seconds) Ir-based catalyst capture by metal scavenging agents can produce pure parahydrogen-based hyperpolarized contrast agents as demonstrated by high-resolution nuclear magnetic resonance (NMR) spectroscopy and inductively coupled plasma atomic emission spectroscopy (ICP-AES). The presented methodology enables fast and efficient means of producing pure hyperpolarized aqueous solutions for biomedical and other uses.</p>

Rationalizing the pH-Activity Response for Escherichia Coli’s Glycinamide Ribonucleotidetransformylase Through Computational Methods

2019 ◽  
Author(s):  
Adrian Roitberg ◽  
Pancham Lal Gupta
Keyword(s):  
Transfer Reaction ◽  
Catalytic Mechanism ◽  
Ph Dependence ◽  
Ph Effects ◽  
Dependent Manner ◽  
E Coli ◽  
Gar Tfase ◽  
Activity Curve ◽  
Ph Dependent ◽  
Formyl Transfer

<div>Human Glycinamide ribonucleotide transformylase (GAR Tfase), a regulatory enzyme in the de novo purine biosynthesis pathway, has been established as an anti-cancer target. GAR Tfase catalyzes the formyl transfer reaction from the folate cofactor to the GAR ligand. In the present work, we study E. coli GAR Tfase, which has high sequence similarity with the human GAR Tfase with most functional residues conserved. E. coli GAR Tfase exhibits structural changes and the binding of ligands that varies with pH which leads to change the rate of the formyl transfer reaction in a pH-dependent manner. Thus, the inclusion of pH becomes essential for the study of its catalytic mechanism. Experimentally, the pH-dependence of the kinetic parameter kcat is measured to evaluate the pH-range of enzymatic activity. However, insufficient information about residues governing the pH-effects on the catalytic activity leads to ambiguous assignments of the general acid and base catalysts and consequently its catalytic mechanism. In the present work, we use pH-replica exchange molecular dynamics (pH-REMD) simulations to study the effects of pH on E. coli GAR Tfase enzyme. We identify the titratable residues governing the pH-dependent conformational changes in the system. Furthermore, we filter out the protonation states which are essential in maintaining the structural integrity, keeping the ligands bound and assisting the catalysis. We reproduce the experimental pH-activity curve by computing the population of key protonation states. Moreover, we provide a detailed description of residues governing the acidic and basic limbs of the pH-activity curve.</div>

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