Phospholipid protection against proteolysis of D-beta-hydroxybutyrate dehydrogenase, a lecithin-requiring enzyme.
D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme which is localized on the inner face of the mitochondrial inner membrane. The apodehydrogenase, i.e. the purified enzyme devoid of lipid, has been purified from beef heart mitochondria and as such is inactive. It can be reactivated by insertion into phospholipid vesicles containing lecithin. Proteolytic digestion with different proteases has been carried out to obtain insight into the orientation of the enzyme in the membrane and to assess the extent of immersion of the protein into the phospholipid bilayer. Digestion of the apodehydrogenase with either trypsin, chymotrypsin, Staphylococcus aureus protease, thermolysin, carboxypeptidases A and Y, or Pronase (from Streptomyces griseus) leads to loss of activity, as assayed with phospholipid. Limited digestion with carboxypeptidase results in complete inactivation. Of the proteases tested, only Pronase and chymotrypsin cleave and inactivate the enzyme inserted into phospholipid vesicles (enzyme-phospholipid complex). For the enzyme-phospholipid complex, the loss of activity with Pronase digestion follows a single exponential decay to less than 10% of the initial activity. With chymotrypsin digestion, the staining intensity of the original approximately 31,500-dalton polypeptide decreases more rapidly than the loss of enzymic activity. The enzyme-phospholipid complex, after limited cleavage with chymotrypsin, retains enzymic activity and resonance energy transfer from protein to bound NADH and an approximately 26,000-dalton polypeptide is observed. Phospholipid alters the cleavage pattern with both chymotrypsin and Pronase, and the rate of inactivation of the enzyme-phospholipid complex is slowed in the presence of NAD(H). Moreover, the rate of inactivation of the apodehydrogenase with chymotrypsin is diminished approximately 3-fold in the presence of NAD+. Digestion of submitochondrial vesicles with either trypsin, chymotrypsin, or Pronase rapidly inactivates D-beta-hydroxybutyrate dehydrogenase; the addition of NAD+ or NADH, together with dithiothreitol and increased salt (to 50 mM), decreases the rate of inactivation, and with trypsin, virtually eliminates inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006884:Hydroxybutyrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008666:Metalloendopeptidases; D008929:Mitochondria, Heart / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology*; D010743:Phospholipids / Q000378:metabolism*; D011402:Pronase / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D001051:Apoenzymes; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010743:Phospholipids; D009243:NAD; D006884:Hydroxybutyrate Dehydrogenase; D002268:Carboxypeptidases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D043422:Carboxypeptidases A; D002918:Chymotrypsin; D014357:Trypsin; D008666:Metalloendopeptidases; D011402:Pronase; D013820:Thermolysin; C022653:auR protein, Staphylococcus aureus