Abstract
As purified from the hepatopancreas of Nephrops norvegicus, the 16-kDa proton channel proteolipid is found to contain an endogenous divalent ion binding site that is occupied by Cu 2+. The EPR spectrum has g-values and hyperfine splittings that are characteristic of type 2 Cu 2+. The copper may be removed by extensive washing with EDTA. Titration with Ni 2+ then induces spin–spin interactions with nitroxyl spin labels that are attached either to the unique Cys 54, or to fatty acids intercalated in the membrane. Paramagnetic relaxation enhancement by the fast-relaxing Ni 2+ is used to characterise the binding and to estimate distances from the dipolar interactions. The Ni 2+-binding site on the protein is situated around 14–18 Å from the spin label on Cys 54, and is at a similar distance from a lipid chain spin-labelled on the 5 C-atom, but is more remote from the C-9 and C-14 positions of the lipid chains.
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Schedule a callMore From: Biochimica et Biophysica Acta (BBA) - Biomembranes
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