LABELING OF THE MITOCHONDRIAL-MEMBRANE D-3-HYDROXYBUTYRATE DEHYDROGENASE (BDH) WITH NEW BIFUNCTIONAL PHOSPHOLIPID ANALOGS

D-3-Hydroxybutyrate dehydrogenase (BDH), an inner mitochondrial protei n, is a well-known phospholipid dependent enzyme. It is a primary dehy drogenase of the oxidative phosphorylation system and is involved in t he redox balance of the NAD+/NADH pool. The preparation of fluorescent phospholipids and newly synthesized bifunctional phospholipid analogu es (fluorescent and photoactivatable) allowed us to study the structur al requirement for lipid activation of the purified enzyme. This paper reports the chemical synthesis protocols to prepare these new phospho lipids and their characterization. Illumination experiments of complex es between bifunctional phospholipids and BDH which lead to a cross-li nked polypeptide indicate that both the polar head and the hydrophobic moiety of phospholipids interact with BDH. The bifunctional phospholi pids were also tested on other lipid-binding proteins, i.e., horse cyt ochrome c and bovine serum albumin, and demonstrated the promising pot ential of this new type of photoactivatable molecules which can be fol lowed merely by fluorescence without radioactive labeling.