Interactions between apo-(D-beta-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence.
Interactions of D-beta-hydroxybutyrate dehydrogenase with phospholipids were investigated by both intrinsic- and extrinsic-fluorescence approaches. The intrinsic fluorescence, mainly caused by tryptophan residues, increased upon re-activation in the presence of phospholipids bearing a positive charge, i.e. phosphatidylcholine, but decreased in the presence of non-re-activating phospholipids with a negative charge. This indicates either that the environment of tryptophan residues is affected by charges rather than by hydrophobic chains of phospholipids, or that the enzyme undergoes different conformational changes depending on the nature of the phospholipids. On the other hand, the graph of the temperature-dependence of the fluorescence intensities of the enzyme embedded in dimyristoylphosphatidylcholine liposomes exhibits a break around 21 degrees C. This indicates either that at least one tryptophan residue is closely in contact with the hydrophobic chains of phospholipids or that there is a change in the environment of tryptophan residues owing to the physical state of the phospholipids. The addition of D-beta-hydroxybutyrate apo-dehydrogenase to phospholipid liposomes containing diphenylhexatriene (a fluorescent probe) increased the diphenylhexatriene fluorescence polarization. Moreover, there was a partial fluorescence energy transfer from tryptophan to diphenylhexatriene. These results strongly favour the possibility that there is a portion of the enzyme polypeptide chain inserted into the phospholipid hydrophobic region. All these results demonstrate that D-beta-hydroxybutyrate apo-dehydrogenase interacts with both polar and hydrophobic parts of phospholipids and leads to small, but essential, conformational changes of the enzyme.
D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001665:Binding Sites; D004161:Diphenylhexatriene; D005454:Fluorescence Polarization; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000032:analysis
D001051:Apoenzymes; D001059:Apoproteins; D010743:Phospholipids; D009243:NAD; D004161:Diphenylhexatriene; D014364:Tryptophan; D006884:Hydroxybutyrate Dehydrogenase