Effect of crosslinking on mitochondrial structure and function

Abstract

Rat liver mitochondria were treated with ethylacetimidate and methylbutyrimidate, monofunctional imidates, and with dimethylsuberimidate, a bifunctional imidate, and the effects on structure and function studied. Mitochondria treated with 5 mM dimethylsuberimidate or greater did not respond osmotically when placed in deionized water. Sodium dodecylsulfate‐polyacrylamide gel electrophoresis revealed that at concentrations > 5 mM dimethylsuberimidate nearly all mitochondrial polypeptides failed to enter 6% gels, indicating crosslinking of both membrane and soluble proteins. Extensive amidination by ethylacetimidate and methylbutyrimidate had little effect on ascorbate‐tetramethylphenylenediamine oxidase while extensive inhibition resulted from dimethylsuberimidate treatment. The possible involvement of molecular motion in electron transport is discussed.

Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites

“…Chemical cross-linkers are useful tools in the study of near-neighbor relationships in biological structures with defined protein arrangements. They were first used (7) to establish the stoichiometric relationship between oligomers of soluble proteins and have since been fruitfully applied to several complex systems including ribosomes (e.g., references 14,35), red cell membranes (e.g., references 36,41), mitochondria (39) and chromatin (26). If, in the native microsomal membranes, ribophorins are indeed associated with the binding sites either as constituents of these sites or as part of larger complexes containing ribosome receptors, then it should be possible to detect a close spatial relationship by covalently linking the ribophorins to the ribosomes.…”

Can multiple low-density immunoadsorbents and ‘negative’ distance matrices help predict the three-dimensional structure of any globular protein?

“…Conventional cross-linking methods have been most useful in studying 'nearest-neighbour' interactions in subcellular organelles (Bickle et al, 1972;Steck, 1972;Thomas & Kornberg, 1975;Tinberg et al, 1975). Although useful information regarding inter-residue distances in single-chain globular proteins (Hartmann & Wold, 1967) and the subunit stoichiometry (Davies & Stark, 1970) and orientation of protomers (D'Albis & Gratzer, 1976) in oligomeric proteins has occasionally been obtained, the information from 'in-solution' cross-linking methods regarding the tertiary structure of proteins is too limited to be of significant value in predictive studies of protein topology.…”