Labeling of the mitochondrial membrane D-3-hydroxybutyrate dehydrogenase (BDH) with new bifunctional phospholipid analogues.
D-3-Hydroxybutyrate dehydrogenase (BDH), an inner mitochondrial protein, is a well-known phospholipid dependent enzyme. It is a primary dehydrogenase of the oxidative phosphorylation system and is involved in the redox balance of the NAD+/NADH pool. The preparation of fluorescent phospholipids and newly synthesized bifunctional phospholipid analogues (fluorescent and photoactivatable) allowed us to study the structural requirement for lipid activation of the purified enzyme. This paper reports the chemical synthesis protocols to prepare these new phospholipids and their characterization. Illumination experiments of complexes between bifunctional phospholipids and BDH which lead to a cross-linked polypeptide indicate that both the polar head and the hydrophobic moiety of phospholipids interact with BDH. The bifunctional phospholipids were also tested on other lipid-binding proteins, i.e., horse cytochrome c and bovine serum albumin, and demonstrated the promising potential of this new type of photoactivatable molecules which can be followed merely by fluorescence without radioactive labeling.
Laboratoire de Biologie Moléculaire et Cellulaire (LBMC), Université de Bourgogne, Faculté des Sciences, Dijon, France.
B
B
Nasser
C
C
Morpain
J
J
Zirkel
M
M
Seiter
B
B
Laude
W E
WE
Trommer
N
N
Latruffe
D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology*; D008051:Lipid Bilayers / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D010743:Phospholipids / Q000138:chemical synthesis* / Q000378:metabolism* / Q000494:pharmacology; D010777:Photochemistry; D013329:Structure-Activity Relationship
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D010743:Phospholipids; D006884:Hydroxybutyrate Dehydrogenase