Reconstitution studies on sarcoplasmic reticulum vesicles
Reconstitution studies on sarcoplasmic reticulum vesicles
The functions of the various proteins found in the membrane of sarcoplasmic reticulum (SR) of skeletal muscle have been investigated by comparing the properties of different fractions of SR and by reconstitution of solubilised SR under a variety of conditions which alter the composition of the reconstituted system. It is possible to fractionate SR into two distinct populations of vesicles: heavy SR and light SR. These populations are derived from different regions of the SR in muscle and have different protein compositions. The Ca2+ uptake and release properties of these two populations have been characterised and support the view that Ca2+ release can be mediated via proteins other than the Ca2+ release channels of SR. A comparison is made between the properties of vesicles solubilised and reconstituted in low K+ buffer and those solubilised and reconstituted in high K+ buffer which have a lower fractional stimulation by the Ca2+-ionophore A23187 and show lower levels of Ca2+ uptake. These observations are discussed in terms of the different protein composition of the two populations of vesicles and in terms of the greater degree of disruption caused by detergent solubilisation in high ionic strength buffers.Purified(Ca2++Mg2+)-ATPase has been reconstituted into lipid vesicles and the effects of androstenol and cholesterol on Ca2+ uptake investigated. The increase in Ca2+ uptake seen in vesicles containing the sterols is likely to be due to their interactions with the ATPase rather than to effects on the general permeability of the membrane. A comparison is made between the use of the three metallochromic dyes murexide, antipyrylazo III and arsenazo III used to monitor Ca2+ fluxes. It is shown that arsenazo III interacts with the (Ca2++Mg2+)-ATPase and probably with the phosphate transporter and that antipyrylazo III is the dye of choice.
University of Southampton
1990
Grimes, Eileen Ann
(1990)
Reconstitution studies on sarcoplasmic reticulum vesicles.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The functions of the various proteins found in the membrane of sarcoplasmic reticulum (SR) of skeletal muscle have been investigated by comparing the properties of different fractions of SR and by reconstitution of solubilised SR under a variety of conditions which alter the composition of the reconstituted system. It is possible to fractionate SR into two distinct populations of vesicles: heavy SR and light SR. These populations are derived from different regions of the SR in muscle and have different protein compositions. The Ca2+ uptake and release properties of these two populations have been characterised and support the view that Ca2+ release can be mediated via proteins other than the Ca2+ release channels of SR. A comparison is made between the properties of vesicles solubilised and reconstituted in low K+ buffer and those solubilised and reconstituted in high K+ buffer which have a lower fractional stimulation by the Ca2+-ionophore A23187 and show lower levels of Ca2+ uptake. These observations are discussed in terms of the different protein composition of the two populations of vesicles and in terms of the greater degree of disruption caused by detergent solubilisation in high ionic strength buffers.Purified(Ca2++Mg2+)-ATPase has been reconstituted into lipid vesicles and the effects of androstenol and cholesterol on Ca2+ uptake investigated. The increase in Ca2+ uptake seen in vesicles containing the sterols is likely to be due to their interactions with the ATPase rather than to effects on the general permeability of the membrane. A comparison is made between the use of the three metallochromic dyes murexide, antipyrylazo III and arsenazo III used to monitor Ca2+ fluxes. It is shown that arsenazo III interacts with the (Ca2++Mg2+)-ATPase and probably with the phosphate transporter and that antipyrylazo III is the dye of choice.
This record has no associated files available for download.
More information
Published date: 1990
Identifiers
Local EPrints ID: 458308
URI: http://eprints.soton.ac.uk/id/eprint/458308
PURE UUID: c0deb2c6-c26a-42c9-842d-52b28bb22fd9
Catalogue record
Date deposited: 04 Jul 2022 16:46
Last modified: 04 Jul 2022 16:46
Export record
Contributors
Author:
Eileen Ann Grimes
Download statistics
Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.
View more statistics