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.

Phospholipid polar head specificity of D-3-hydroxybutyrate dehydrogenase activation studied by new synthetic phospholipids and analogues.

D-3-hydroxybutyrate dehydrogenase, an inner-mitochondrial enzyme responsible for the interconversion of two ketone bodies, is a well known phospholipid dependent enzyme. Newly synthesized phospholipid analogues were used to study the structural requirement for lipid activation of the purified enzyme. A positive charge on the polar head is required but must be at the surface of lipid vesicles. In contrast the maximum velocity and the Michaelis constant values are not strongly dependent on the nature of the zwitterionic phospholipid polar head.

[Effects of synthetic bioactive lipids on the activity of D-beta-hydroxybutyrate dehydrogenase, a membrane enzyme]. / Effets de lipides bioactifs synthétiques sur l'activité de la D-beta-hydroxybutyrate déshydrogénase, une enzyme membranaire.

The structural requirements of lecithins analogs for purified D-beta-hydroxybutyrate dehydrogenase activation have been studied with chemically defined phospholipids. It appears that the trimethylamine group of choline can be changed by a pyridinium group. On the other hand, the decrease of density of the positive charges on the liposomes surface obtained by dilution of such bearing molecules with negative or non charged phospholipids increases the enzyme reactivation. Finally, the PAF acether, a lipid mediator, is able to reactivate the enzyme in similar conditions as these obtained with mitochondrial phosphatidylcholines.

Photolabelling of D-beta-hydroxybutyrate apodehydrogenase with azidoaryl phospholipids.

Two synthetic photoactive azidoarylphosphatidylcholines were used to investigate the level of interaction between D-beta-hydroxybutyrate apodehydrogenase (apoBDH), an amphipathic membrane protein, with the hydrophobic domain of phospholipids. The two synthetic lecithins, PL I (1-myristoyl-2-12-N-(4-azido-2-nitrophenyl) aminododecanoyl-sn-glycero-3-phosphocholine) and PL II (1-myristoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-phosphocholine), are able to reactivate the non-active purified apoBDH as well as the non-photoactive homologs, indicating that the photoreactive chemical groups are without effect on the cofactor properties of phosphatidylcholine. Photoirradiation of reconstituted complexes between phospholipid containing azidoaryllecithin and apoBDH leads to a covalent binding of some synthetic lecithin molecules on the protein. The labelling, about 3 times higher with PL II than with PL I, suggests that the area of interacting domain of BDH with the hydrophobic moiety of phospholipid is more important at or near the surface of the lipid bilayer than in the inner part. This approach is further demonstration that BDH is an integral protein.