Monomers of the catalytic domain of human neuropathy target esterase are active in the presence of phospholipid.
NEST is a hydrophobic recombinant polypeptide comprising the catalytic domain (residues 727-1216) of neuropathy target esterase. NEST in bacterial lysates has potent esterase activity, which is lost after its solubilization and purification in detergent-containing solutions. Activity in purified NEST preparations was restored by the addition of phospholipids before the removal of detergent by dialysis. The pattern of digestion by proteinase K of NEST-phospholipid complexes suggested that NEST might incorporate in a topologically random fashion into nascent liposomes and that the bulk of each NEST molecule might be exposed either to the liposome lumen or the external medium. Significant quantities of NEST were liberated from NEST-phospholipid complexes by treatment with dilute acid or alkali, suggesting that charge interactions might contribute to the association; however, NEST was irreversibly denatured at these pH values. Treatment of NEST-phospholipid complexes with glutaraldehyde afforded some protection against the inactivation of esterase activity by detergent but the pattern of cross-linked forms of NEST generated did not indicate pre-existing oligomers. Similarly, the inactivation of esterase activity in NEST-phospholipid complexes by radiation indicated that NEST monomers are catalytically active. The foregoing observations are not compatible with structural algorithms predicting that the catalytic serine residue lies at the centre of one of three transmembrane helices in NEST.
Atkins
Jane
J
MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester LE1 9HN, U.K.
Luthjens
Lee H
LH
Hom
Marinus L
ML
Glynn
Paul
P
D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002793:Cholic Acids / Q000494:pharmacology; D003432:Cross-Linking Reagents; D003902:Detergents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005976:Glutaral; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes; D010743:Phospholipids / Q000378:metabolism*; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002793:Cholic Acids; D003432:Cross-Linking Reagents; D003902:Detergents; D004791:Enzyme Inhibitors; D008081:Liposomes; D010743:Phospholipids; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D005976:Glutaral