Analytical ultracentrifugation with fluorescence detection and biosafety containment and its application to the prion protein

2008 ◽  
pp. 72-76 ◽  
Author(s):  
M. Pitschke ◽  
K. Post ◽  
D. Riesner
Keyword(s):  
Prion Protein ◽  
Fluorescence Detection ◽  
Analytical Ultracentrifugation

scholarly journals Interaction of the cellular prion protein with raft-like lipid membranes

2007 ◽  
Vol 388 (1) ◽  
pp. 79-89 ◽  
Author(s):  
Kerstin Elfrink ◽  
Luitgard Nagel-Steger ◽  
Detlev Riesner
Keyword(s):  
Prion Protein ◽  
Lipid Bilayers ◽  
Analytical Ultracentrifugation ◽  
Chinese Hamster ◽  
Lipid Vesicles ◽  
Cellular Prion Protein ◽  
Model Membranes ◽  
Degree Of Saturation ◽  
Insertion Reaction ◽  
Chip Surface

Abstract Conversion of the cellular isoform of the prion protein (PrPC) into the disease-associated isoform (PrPSc) plays a key role in the development of prion diseases. Within its cellular pathway, PrPC undergoes several posttranslational modifications, i.e., the attachment of two N-linked glycans and a glycosyl phosphatidyl inositol (GPI) anchor, by which it is linked to the plasma membrane on the exterior cell surface. To study the interaction of PrPC with model membranes, we purified posttranslationally modified PrPC from transgenic Chinese hamster ovary (CHO) cells. The mono-, di- and oligomeric states of PrPC free in solution were analyzed by analytical ultracentrifugation. The interaction of PrPC with model membranes was studied using both lipid vesicles in solution and lipid bilayers bound to a chip surface. The equilibrium and mechanism of PrPC association with the model membranes were analyzed by surface plasmon resonance. Depending on the degree of saturation of binding sites, the concentration of PrPC released from the membrane into aqueous solution was estimated at between 10-9 and 10-7 M. This corresponds to a free energy of the insertion reaction of -48 kJ/mol. Consequences for the conversion of PrPC to PrPSc are discussed.

On the utility of fluorescence-detection analytical ultracentrifugation in probing biomolecular interactions in complex solutions: a case study in milk

2020 ◽  
Vol 49 (8) ◽  
pp. 677-685 ◽  
Author(s):  
Jennifer M. Crowther ◽  
Marita Broadhurst ◽  
Thomas M. Laue ◽  
Geoffrey B. Jameson ◽  
Alison J. Hodgkinson ◽  
...  
Keyword(s):  
Fluorescence Detection ◽  
Analytical Ultracentrifugation ◽  
Biomolecular Interactions ◽  
Complex Solutions

scholarly journals Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex

2012 ◽  
Vol 21 (9) ◽  
pp. 1253-1268 ◽  
Author(s):  
Xin Wang ◽  
Chongxu Zhang ◽  
Yueh-Chin Chiang ◽  
Shaun Toomey ◽  
Matthew P. Power ◽  
...  
Keyword(s):  
Fluorescence Detection ◽  
Analytical Ultracentrifugation ◽  
Detection System ◽  
The Novel ◽  
Novel Technique ◽  
Fluorescence Detection System

The Nature of Rapidly Extracted Phycocyanin from the Blue-Green Alga Plectonema Boryanum

1971 ◽  
Vol 29 ◽  
pp. 366-367
Author(s):  
M. Kessel ◽  
R. MacColl
Keyword(s):  
Self Assembly ◽  
Analytical Ultracentrifugation ◽  
Uranyl Acetate ◽  
The Self ◽  
Major Protein ◽  
Subunit Structure ◽  
Blue Green Alga ◽  
Thin Sections ◽  
Blue Green Algae ◽  
Cacodylate Buffer

The major protein of the blue-green algae is the biliprotein, C-phycocyanin (Amax = 620 nm), which is presumed to exist in the cell in the form of distinct aggregates called phycobilisomes. The self-assembly of C-phycocyanin from monomer to hexamer has been extensively studied, but the proposed next step in the assembly of a phycobilisome, the formation of 19s subunits, is completely unknown. We have used electron microscopy and analytical ultracentrifugation in combination with a method for rapid and gentle extraction of phycocyanin to study its subunit structure and assembly.To establish the existence of phycobilisomes, cells of P. boryanum in the log phase of growth, growing at a light intensity of 200 foot candles, were fixed in 2% glutaraldehyde in 0.1M cacodylate buffer, pH 7.0, for 3 hours at 4°C. The cells were post-fixed in 1% OsO4 in the same buffer overnight. Material was stained for 1 hour in uranyl acetate (1%), dehydrated and embedded in araldite and examined in thin sections.

Metallic prions

2004 ◽  
Vol 71 ◽  
pp. 193-202 ◽  
Author(s):  
David R Brown
Keyword(s):  
Prion Protein ◽  
Binding Capacity ◽  
Normal Function ◽  
Transmissible Spongiform Encephalopathies ◽  
Published Data ◽  
Normal Brain ◽  
Copper Binding ◽  
Loss Of Function ◽  
Spongiform Encephalopathies ◽  
The Brain

Prion diseases, also referred to as transmissible spongiform encephalopathies, are characterized by the deposition of an abnormal isoform of the prion protein in the brain. However, this aggregated, fibrillar, amyloid protein, termed PrPSc, is an altered conformer of a normal brain glycoprotein, PrPc. Understanding the nature of the normal cellular isoform of the prion protein is considered essential to understanding the conversion process that generates PrPSc. To this end much work has focused on elucidation of the normal function and activity of PrPc. Substantial evidence supports the notion that PrPc is a copper-binding protein. In conversion to the abnormal isoform, this Cu-binding activity is lost. Instead, there are some suggestions that the protein might bind other metals such as Mn or Zn. PrPc functions currently under investigation include the possibility that the protein is involved in signal transduction, cell adhesion, Cu transport and resistance to oxidative stress. Of these possibilities, only a role in Cu transport and its action as an antioxidant take into consideration PrPc's Cu-binding capacity. There are also more published data supporting these two functions. There is strong evidence that during the course of prion disease, there is a loss of function of the prion protein. This manifests as a change in metal balance in the brain and other organs and substantial oxidative damage throughout the brain. Thus prions and metals have become tightly linked in the quest to understand the nature of transmissible spongiform encephalopathies.

Sign in / Sign up

Export Citation Format

Share Document