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3 protocols using bovine heart cardiolipin

1

Purification and Characterization of Mutant Cytochrome c

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Horse heart cyt c (type VI, oxidized form), bovine heart cardiolipin (>80% polyunsatured fatty acid content, primary linoleic acid; 98% purity), and laurdan were provided from Sigma Aldrich- Merck KGaA (Darmstadt, Germany) and used without further purification. All reagents were analytical grade.
For mutant cyt c the expression plasmids of horse cyt c (pHCyc) [50 (link)] was subjected to one round of mutagenesis with QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara United States), which introduced Lys72Asn substitution into the horse cyt c gene. Mutant pHCyc plasmids was introduced into E. coli JM 109. Protein expression and purification of the recombinant protein were then conducted as previously described [51 (link)].
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2

Enzymatic Synthesis of 20(OH)D3 and 20,23(OH)2D3

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20(OH)D3 and 20,23(OH)2D3 were synthesised from Vitamin D3 enzymatically using bovine CYP11A1 and were purified by TLC and HPLC, as previously described [10 (link), 17 (link)]. Vitamin D3, dioleoyl phosphaditylcholine, bovine heart cardiolipin, 2-hydroxylpropyl-β-cyclodextrin (cyclodextrin), and glucose-6-phosphate were purchased from Sigma (Sydney, Australia). glucose-6-phosphate dehydrogenase was from Roche (Mannheim, Germany). All solvents were of HPLC grade and were purchased from Merck (Darmstadt, Germany).
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3

Lipid Peroxidation and Membrane Biophysics

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1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (OPC), bovine heart cardiolipin (CL), arachidonic acid (AA), hexane, hexadecane, KCl, HEPES, EDTA, Na2SO4, MES, Tris, adenosine 5-triphosphate (ATP), and guanosine 5-triphosphate (GTP) were purchased from Sigma-Aldrich (Munich, Germany). MPC (1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine) was obtained from Avanti Polar Lipids (Alabaster, AL, USA). Plain silica beads (22-μm diameter) were from Microspheres-Nanospheres (Corpuscular Co., Cold Spring, NY, USA). Chloroform was purchased from Carl Roth (Karlsruhe, Germany). 4-oxo-trans-2-nonenal (4-ONE) and 4-hydroxy-trans-2-nonenal (4-HNE) were purchased from Cayman Chemical (Ann Arbor, MI, USA) or synthesized as previously described [45 (link)].
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