The Orientation of Cytochrome c Oxidase in Coupled Phospholipid Membrane Vesicles—A Spin-Label Study

1975 ◽  
Vol 53 (3) ◽  
pp. 364-370 ◽  
Author(s):  
J. A. Kornblatt ◽  
W. L. Chen ◽  
J. C. Hsia ◽  
G. R. Williams
Keyword(s):  
Molecular Weight ◽  
Binding Site ◽  
Cytochrome Oxidase ◽  
Spin Label ◽  
Resonance Spectrum ◽  
Membrane Vesicles ◽  
Phospholipid Membrane ◽  
Vesicle Membrane ◽  
Exterior Surface ◽  
Label Study

Cytochrome oxidase, an enzyme containing six different subunits, has been shown to span the inner mitochrondrial membrane. The arrangement of the subunits within the membrane is unknown. We have specifically labeled the 25 000 molecular weight subunit with a spin-label derivative of N-ethylmaleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (NEM-SL(5)). NEM-SL(5)-labeled cytochrome oxidase can be incorporated into phospholipid membranes to form coupled vesicles of the Hinkle, Kim &Racker ((1972) J. Biol. Chem. 247, 1338–1339) type. The resonance spectrum of NEM-SL(5) is similar in both soluble and vesicular cytochrome oxidase. Since ascorbate has been shown to reduce only spin label that is exposed to the exterior surface of a closed vesicle, we have used ascorbate to determine the NEM-SL(5)-binding site in the coupled vesicles. NEM-SL(5)-labeled cytochrome oxidase vesicles are reduced by 10 mM ascorbate with [Formula: see text] of 1 min at 22 °C. The rate of reduction is relatively independent of temperature. We conclude that (1) cytochrome oxidase is unidirectionally or preferentially oriented in the vesicle membrane, and (2) the NEM-SL(5)-binding site on the 25 000 molecular weight subunit is exposed to the external aqueous medium.

Spin-label study of energy-coupled active transport in Escherichia coli membrane vesicles

Biochemistry ◽  
1974 ◽  
Vol 13 (25) ◽  
pp. 5210-5214 ◽  
Author(s):  
Joseph J. Baldassare ◽  
Dan E. Robertson ◽  
Alice G. McAfee ◽  
Chien Ho
Keyword(s):  
Escherichia Coli ◽  
Active Transport ◽  
Spin Label ◽  
Membrane Vesicles ◽  
Label Study

A spin label study of E. coli membrane vesicles

1973 ◽  
Vol 53 (2) ◽  
pp. 617-623 ◽  
Author(s):  
Joseph J. Baldassare ◽  
Alice Gerke McAfee ◽  
Chien Ho
Keyword(s):  
Spin Label ◽  
Membrane Vesicles ◽  
Label Study ◽  
E Coli

scholarly journals Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes.

1988 ◽  
Vol 106 (1) ◽  
pp. 39-50 ◽  
Author(s):  
D M Virshup ◽  
V Bennett
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Binding Site ◽  
Sodium Hydroxide ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Dependent Manner ◽  
Vesicle Membrane ◽  
Brain Membranes

The assembly polypeptides are an integral component of coated vesicles and may mediate the linkage of clathrin to the vesicle membrane. We have purified assembly polypeptides in milligram quantities from bovine brain by an improved procedure. Hydrodynamic and chemical crosslinking studies indicate that the protein is an asymmetric heterotetramer with a molecular weight of 252,000, containing two subunits of Mr 98,000-115,000, one subunit of 52,000, and one subunit of 16,000. Two-dimensional peptide maps of the subunits show that the 16- and 52-kD polypeptides are not derived from the higher molecular weight species, and that the group of bands at 98-115 kD are related. Electron microscopic visualization shows an essentially globular protein with one or two knob-like tails. We demonstrate a specific membrane protein binding site for 125I-labeled assembly polypeptides in 0.1 N sodium hydroxide-extracted bovine brain membranes based on the following criteria: (a) binding is displaceable by unlabeled ligand, (b) the binding site is destroyed by protease treatment of the membranes, and (c) the distribution of binding between vesicle-depleted membranes and coated vesicle membranes parallels the in vivo localization of assembly polypeptides and clathrin. This binding site is likely to be an integral membrane protein because (a) it is enriched in the sodium hydroxide-extracted membranes stripped of most of their peripheral membrane proteins, and (b) the binding site is partially extracted by 0.5% Triton X-100. A similar binding site appears to be present in coated vesicles. Clathrin binds to the hydroxide-stripped membranes in an assembly polypeptides dependent manner, and this binding is diminished by Triton extraction of the membranes. This assay may aid in identification of the membrane receptor for the assembly polypeptides.

scholarly journals A spin label study of the thyroid hormone-binding sites in human plasma thyroxine transport proteins.

1981 ◽  
Vol 256 (2) ◽  
pp. 831-836
Author(s):  
S.Y. Cheng ◽  
G. Rakhit ◽  
F. Erard ◽  
J. Robbins ◽  
C.F. Chignell
Keyword(s):  
Thyroid Hormone ◽  
Human Plasma ◽  
Spin Label ◽  
Binding Sites ◽  
Transport Proteins ◽  
Hormone Binding ◽  
Label Study

scholarly journals Analysis of extracellular vesicle mRNA derived from plasma using the nCounter platform

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jillian W. P. Bracht ◽  
Ana Gimenez-Capitan ◽  
Chung-Ying Huang ◽  
Nicolas Potie ◽  
Carlos Pedraz-Valdunciel ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Rna Extraction ◽  
Membrane Vesicles ◽  
Gene Signature ◽  
Extracellular Vesicle ◽  
Probe Design ◽  
Phospholipid Membrane ◽  
Lower Plasma ◽  
Mrna Targets ◽  
Dnase Treatment

AbstractExtracellular vesicles (EVs) are double-layered phospholipid membrane vesicles that are released by most cells and can mediate intercellular communication through their RNA cargo. In this study, we tested if the NanoString nCounter platform can be used for the analysis of EV-mRNA. We developed and optimized a methodology for EV enrichment, EV-RNA extraction and nCounter analysis. Then, we demonstrated the validity of our workflow by analyzing EV-RNA profiles from the plasma of 19 cancer patients and 10 controls and developing a gene signature to differentiate cancer versus control samples. TRI reagent outperformed automated RNA extraction and, although lower plasma input is feasible, 500 μL provided highest total counts and number of transcripts detected. A 10-cycle pre-amplification followed by DNase treatment yielded reproducible mRNA target detection. However, appropriate probe design to prevent genomic DNA binding is preferred. A gene signature, created using a bioinformatic algorithm, was able to distinguish between control and cancer EV-mRNA profiles with an area under the ROC curve of 0.99. Hence, the nCounter platform can be used to detect mRNA targets and develop gene signatures from plasma-derived EVs.

Apparent molecular weight of the substance P binding site in rat brain

1988 ◽  
Vol 152 (1-2) ◽  
pp. 171-174 ◽  
Author(s):  
Yoshihiro Nakata ◽  
Chie Hiraoka ◽  
Tomio Segawa
Keyword(s):  
Molecular Weight ◽  
Substance P ◽  
Binding Site ◽  
Rat Brain ◽  
Apparent Molecular Weight

scholarly journals Spin-label study of hemoglobin conformations in solution.

1967 ◽  
Vol 58 (1) ◽  
pp. 19-26 ◽  
Author(s):  
S. Ogawa ◽  
H. M. McConnell
Keyword(s):  
Spin Label ◽  
Label Study
Sign in / Sign up

Export Citation Format

Share Document