The essential cationic charge of phospholipid polar head in the reactivation of d-β-hydroxybutyrate apodehydrogenase revealed by cationic surfactants

Wild Type and Mutant Human Heart (R)-3-Hydroxybutyrate Dehydrogenase Expressed in Insect Cells

“…We have shown that carboxypeptidase cleavage of only a few (e14) C-terminal amino acids from BH-BDH prevents activation by PC (Adami et al, 1993). Further, for BDH reconstituted in bilayer lipids containing PC, the C-terminus is protected from enzymatic digestion with carboxypeptidase, an effect not observed in the absence of PC in the bilayer (Berrez et al, 1984;McIntyre et al, 1990;Adami et al, 1993). These data suggest that in the presence of activating PC, the C-terminus of BDH interacts with the lipid.…”

“…For BDH, the mechanism of lipid activation has remained elusive despite detailed characterization of the nature of the lipid requirement and its effect on activity [albeit see Gazzotti et al (1974)]. We postulate, on the basis of sequence analysis (Marks et al, 1992) and proteolysis studies (Berrez et al, 1984;Maurer et al, 1975;Adami et al, 1993), that the C-terminal domain of BDH is involved in lipid binding and probably the activation by PC. By contrast, the activation of protein kinase C by phosphatidylserine appears to require the N-terminal domain for binding to the lipid.…”

Wild Type and Mutant Human Heart (R)-3-Hydroxybutyrate Dehydrogenase Expressed in Insect Cells

“…We have shown that carboxypeptidase cleavage of only a few (e14) C-terminal amino acids from BH-BDH prevents activation by PC (Adami et al, 1993). Further, for BDH reconstituted in bilayer lipids containing PC, the C-terminus is protected from enzymatic digestion with carboxypeptidase, an effect not observed in the absence of PC in the bilayer (Berrez et al, 1984;McIntyre et al, 1990;Adami et al, 1993). These data suggest that in the presence of activating PC, the C-terminus of BDH interacts with the lipid.…”

“…For BDH, the mechanism of lipid activation has remained elusive despite detailed characterization of the nature of the lipid requirement and its effect on activity [albeit see Gazzotti et al (1974)]. We postulate, on the basis of sequence analysis (Marks et al, 1992) and proteolysis studies (Berrez et al, 1984;Maurer et al, 1975;Adami et al, 1993), that the C-terminal domain of BDH is involved in lipid binding and probably the activation by PC. By contrast, the activation of protein kinase C by phosphatidylserine appears to require the N-terminal domain for binding to the lipid.…”

Distance estimate of the active center of D-.beta.-hydroxybutyrate dehydrogenase from the membrane surface

“…With the nitroxide 17 A from the membrane surface, the reactive sulfhydryl moiety could be located between 9 and 25 A from the surface, depending upon the angle at which the MSL molecule is positioned with respect to the transverse plane of the bilayer (Figure 10). Location of the nitroxide above the membrane surface is unlikely since the reconstituted enzyme is relatively inaccessible to proteolysis (Berrez et al, 1984;Maurer et al, 1985), indicating that the enzyme is largely immersed within the phospholipid bilayer. The orientation of the nitroxide with respect to the transverse plane has previously been evaluated by simulation of the saturation-transfer EPR spectra of [15N]MSL-enzyme in MPL vesicles (McIntyre et al, 1984b) using an anisotropic diffusion model (Robinson & Dalton, 1980).…”

“…The enzyme is an amphipathic protein which is inserted unidirectionally into the bilayer (McIntyre et al, 1979) but does not traverse the bilayer to the polar region at the inner face as shown by proton NMR studies (Deese et al, 1986). Yet, the enzyme in the membrane is protected from proteolysis as compared with the apoenzyme in aqueous buffer (Berrez et al, 1984;Maurer et al, 1985), suggesting that the protein is immersed to an appreciable extent within the bilayer. The organization of the enzyme in the bilayer (MPL) is tetrameric (McIntyre et al, 1983).…”

Amino acid sequence of the nucleotide-binding site of D-(-)-.beta.-hydroxybutyrate dehydrogenase labeled with arylazido-.beta.-[3-3H]alanylnicotinamide adenine dinucleotide

“…Tryptic Digestion of BDH. The phospholipid-reconstituted BDH has been shown to be resistant to tryptic digestion (Berrez et al, 1984;Maurer et al, 1985). Therefore, phospholipid was extracted from the lyophilized, [3H]N3-NAD- treated BDH by addition of 3 mL of chloroform/methanol (2:1).…”

Wild Type and Mutant Human Heart (R)-3-Hydroxybutyrate Dehydrogenase Expressed in Insect Cells

“…We have shown that carboxypeptidase cleavage of only a few (e14) C-terminal amino acids from BH-BDH prevents activation by PC (Adami et al, 1993). Further, for BDH reconstituted in bilayer lipids containing PC, the C-terminus is protected from enzymatic digestion with carboxypeptidase, an effect not observed in the absence of PC in the bilayer (Berrez et al, 1984;McIntyre et al, 1990;Adami et al, 1993). These data suggest that in the presence of activating PC, the C-terminus of BDH interacts with the lipid.…”

“…For BDH, the mechanism of lipid activation has remained elusive despite detailed characterization of the nature of the lipid requirement and its effect on activity [albeit see Gazzotti et al (1974)]. We postulate, on the basis of sequence analysis (Marks et al, 1992) and proteolysis studies (Berrez et al, 1984;Maurer et al, 1975;Adami et al, 1993), that the C-terminal domain of BDH is involved in lipid binding and probably the activation by PC. By contrast, the activation of protein kinase C by phosphatidylserine appears to require the N-terminal domain for binding to the lipid.…”

Distance estimate of the active center of D-.beta.-hydroxybutyrate dehydrogenase from the membrane surface

“…With the nitroxide 17 A from the membrane surface, the reactive sulfhydryl moiety could be located between 9 and 25 A from the surface, depending upon the angle at which the MSL molecule is positioned with respect to the transverse plane of the bilayer (Figure 10). Location of the nitroxide above the membrane surface is unlikely since the reconstituted enzyme is relatively inaccessible to proteolysis (Berrez et al, 1984;Maurer et al, 1985), indicating that the enzyme is largely immersed within the phospholipid bilayer. The orientation of the nitroxide with respect to the transverse plane has previously been evaluated by simulation of the saturation-transfer EPR spectra of [15N]MSL-enzyme in MPL vesicles (McIntyre et al, 1984b) using an anisotropic diffusion model (Robinson & Dalton, 1980).…”

“…The enzyme is an amphipathic protein which is inserted unidirectionally into the bilayer (McIntyre et al, 1979) but does not traverse the bilayer to the polar region at the inner face as shown by proton NMR studies (Deese et al, 1986). Yet, the enzyme in the membrane is protected from proteolysis as compared with the apoenzyme in aqueous buffer (Berrez et al, 1984;Maurer et al, 1985), suggesting that the protein is immersed to an appreciable extent within the bilayer. The organization of the enzyme in the bilayer (MPL) is tetrameric (McIntyre et al, 1983).…”

Amino acid sequence of the nucleotide-binding site of D-(-)-.beta.-hydroxybutyrate dehydrogenase labeled with arylazido-.beta.-[3-3H]alanylnicotinamide adenine dinucleotide

“…Tryptic Digestion of BDH. The phospholipid-reconstituted BDH has been shown to be resistant to tryptic digestion (Berrez et al, 1984;Maurer et al, 1985). Therefore, phospholipid was extracted from the lyophilized, [3H]N3-NAD- treated BDH by addition of 3 mL of chloroform/methanol (2:1).…”

Wild Type and Mutant Human Heart (R)-3-Hydroxybutyrate Dehydrogenase Expressed in Insect Cells

“…We have shown that carboxypeptidase cleavage of only a few (e14) C-terminal amino acids from BH-BDH prevents activation by PC (Adami et al, 1993). Further, for BDH reconstituted in bilayer lipids containing PC, the C-terminus is protected from enzymatic digestion with carboxypeptidase, an effect not observed in the absence of PC in the bilayer (Berrez et al, 1984;McIntyre et al, 1990;Adami et al, 1993). These data suggest that in the presence of activating PC, the C-terminus of BDH interacts with the lipid.…”

“…For BDH, the mechanism of lipid activation has remained elusive despite detailed characterization of the nature of the lipid requirement and its effect on activity [albeit see Gazzotti et al (1974)]. We postulate, on the basis of sequence analysis (Marks et al, 1992) and proteolysis studies (Berrez et al, 1984;Maurer et al, 1975;Adami et al, 1993), that the C-terminal domain of BDH is involved in lipid binding and probably the activation by PC. By contrast, the activation of protein kinase C by phosphatidylserine appears to require the N-terminal domain for binding to the lipid.…”

Distance estimate of the active center of D-.beta.-hydroxybutyrate dehydrogenase from the membrane surface

“…With the nitroxide 17 A from the membrane surface, the reactive sulfhydryl moiety could be located between 9 and 25 A from the surface, depending upon the angle at which the MSL molecule is positioned with respect to the transverse plane of the bilayer (Figure 10). Location of the nitroxide above the membrane surface is unlikely since the reconstituted enzyme is relatively inaccessible to proteolysis (Berrez et al, 1984;Maurer et al, 1985), indicating that the enzyme is largely immersed within the phospholipid bilayer. The orientation of the nitroxide with respect to the transverse plane has previously been evaluated by simulation of the saturation-transfer EPR spectra of [15N]MSL-enzyme in MPL vesicles (McIntyre et al, 1984b) using an anisotropic diffusion model (Robinson & Dalton, 1980).…”

“…The enzyme is an amphipathic protein which is inserted unidirectionally into the bilayer (McIntyre et al, 1979) but does not traverse the bilayer to the polar region at the inner face as shown by proton NMR studies (Deese et al, 1986). Yet, the enzyme in the membrane is protected from proteolysis as compared with the apoenzyme in aqueous buffer (Berrez et al, 1984;Maurer et al, 1985), suggesting that the protein is immersed to an appreciable extent within the bilayer. The organization of the enzyme in the bilayer (MPL) is tetrameric (McIntyre et al, 1983).…”

Amino acid sequence of the nucleotide-binding site of D-(-)-.beta.-hydroxybutyrate dehydrogenase labeled with arylazido-.beta.-[3-3H]alanylnicotinamide adenine dinucleotide

“…Tryptic Digestion of BDH. The phospholipid-reconstituted BDH has been shown to be resistant to tryptic digestion (Berrez et al, 1984;Maurer et al, 1985). Therefore, phospholipid was extracted from the lyophilized, [3H]N3-NAD- treated BDH by addition of 3 mL of chloroform/methanol (2:1).…”