Structural aspects of the bovine heart mitochondrial NADH dehydrogenase
Structural aspects of the bovine heart mitochondrial NADH dehydrogenase
Several subunits of Complex I, a preparation of bovine heart mitochondrial NADH-ubiquinone oxidoreductase, (E.C. 1.6.99.3), were isolated using preparative SDS-polyacrylamide gel electrophoresis and an electroelution technique. Antibodies were raised to the flavo-protein fraction (FP) and to its two largest 5lkDa and 24kDa subunits and to the 39kDa and 20kDa hydrophobic protein fraction (HP) subunits. Antibodies to the 42kDa HP fraction subunit were isolated from an antiserum raised to the holoenzyme. Antibodies were also available to the Iron-Protein (IP) fraction's 75kDa, 49kDa, 30kDa and 13kDa subunits and to several mitochondrially encoded subunits of human NADH dehydrogenase. Using the latter antisera three Complex I polypeptides of Mr 30,000, 13,600, and 10,500 were identified as counterparts to the mitochondrially encoded ND-1, ND-3 and 4L products of the human enzyme. A proposal has been made as to the identity of the remaining subunits of the enzyme which have the same genomic origin. The largest of the three aforementioned proteins corresponds to the ND-1 product and its characterisation identified it as the rotenone binding protein. The lipophilic, radiolabelled photoaffinity probe [125I]-TIDS was used to identify the regions of Complex I in direct contact with the membrane. Several subunits, including the previously identified mitochondrially encoded subunits, were labelled and chaotropic fragmentation of the holoenzyme revealed them all to be HP fraction subunits. Subunit interactions within Complex I were analysed by chemical crosslinking using reagents of differential polarity and length (DST, EGS, DMS and DIS). Subunit-specific antisera were used to probe crosslinked Complex I and thereby identify the dimer constituents. By this means, juxtapositioning of the following subunits was established: 75-51kDa, 51-24kDa, 49-30kDa and 49-13kDa, 75-30kDa. Crosslinks were identified between the IP and HP subunits using a dual labelling procedure where TIDS label acted as a marker for several of the HP subunits. The topography of Complex I was studied in situ using the membrane-impermeant reagent, sulphonated-DST, to crosslink isolated mitochondria and submitochondrial particles. It was demonstrated that the 75kDa and 30kDa were predominantly exposed from the matrix and cytoplasm sides. In addition, the transmembranous nature of the 30kDa subunit was established. Evidence for a hydrophobic pocket to accommodate the enzyme's substrate, NADH based on the differential labelling of this binding site by polar and non-polar crosslinking probes is also cited. That such a site is accessible from the matrix side was also established by the in situ crosslinking studies.
University of Southampton
1987
Patel, Salil Dinubhai
(1987)
Structural aspects of the bovine heart mitochondrial NADH dehydrogenase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Several subunits of Complex I, a preparation of bovine heart mitochondrial NADH-ubiquinone oxidoreductase, (E.C. 1.6.99.3), were isolated using preparative SDS-polyacrylamide gel electrophoresis and an electroelution technique. Antibodies were raised to the flavo-protein fraction (FP) and to its two largest 5lkDa and 24kDa subunits and to the 39kDa and 20kDa hydrophobic protein fraction (HP) subunits. Antibodies to the 42kDa HP fraction subunit were isolated from an antiserum raised to the holoenzyme. Antibodies were also available to the Iron-Protein (IP) fraction's 75kDa, 49kDa, 30kDa and 13kDa subunits and to several mitochondrially encoded subunits of human NADH dehydrogenase. Using the latter antisera three Complex I polypeptides of Mr 30,000, 13,600, and 10,500 were identified as counterparts to the mitochondrially encoded ND-1, ND-3 and 4L products of the human enzyme. A proposal has been made as to the identity of the remaining subunits of the enzyme which have the same genomic origin. The largest of the three aforementioned proteins corresponds to the ND-1 product and its characterisation identified it as the rotenone binding protein. The lipophilic, radiolabelled photoaffinity probe [125I]-TIDS was used to identify the regions of Complex I in direct contact with the membrane. Several subunits, including the previously identified mitochondrially encoded subunits, were labelled and chaotropic fragmentation of the holoenzyme revealed them all to be HP fraction subunits. Subunit interactions within Complex I were analysed by chemical crosslinking using reagents of differential polarity and length (DST, EGS, DMS and DIS). Subunit-specific antisera were used to probe crosslinked Complex I and thereby identify the dimer constituents. By this means, juxtapositioning of the following subunits was established: 75-51kDa, 51-24kDa, 49-30kDa and 49-13kDa, 75-30kDa. Crosslinks were identified between the IP and HP subunits using a dual labelling procedure where TIDS label acted as a marker for several of the HP subunits. The topography of Complex I was studied in situ using the membrane-impermeant reagent, sulphonated-DST, to crosslink isolated mitochondria and submitochondrial particles. It was demonstrated that the 75kDa and 30kDa were predominantly exposed from the matrix and cytoplasm sides. In addition, the transmembranous nature of the 30kDa subunit was established. Evidence for a hydrophobic pocket to accommodate the enzyme's substrate, NADH based on the differential labelling of this binding site by polar and non-polar crosslinking probes is also cited. That such a site is accessible from the matrix side was also established by the in situ crosslinking studies.
This record has no associated files available for download.
More information
Published date: 1987
Identifiers
Local EPrints ID: 461323
URI: http://eprints.soton.ac.uk/id/eprint/461323
PURE UUID: 56e6005c-cad9-42a7-b954-827130b3a02d
Catalogue record
Date deposited: 04 Jul 2022 18:43
Last modified: 04 Jul 2022 18:43
Export record
Contributors
Author:
Salil Dinubhai Patel
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