N-terminal sequence and distal histidine residues are responsible for pH-regulated cytoplasmic membrane binding of human AMP deaminase isoform E. J Biol Chem 2002 Nov 08;277(45):42654-62
Date
09/06/2002Pubmed ID
12213808DOI
10.1074/jbc.M203473200Scopus ID
2-s2.0-0037044753 (requires institutional sign-in at Scopus site) 13 CitationsAbstract
Mammalian AMP deaminase 3 (AMPD3) enzymes reportedly bind to intracellular membranes, plasma lipid vesicles, and artificial lipid bilayers with associated alterations in enzyme conformation and function. However, proteolytic sensitivity of AMPD polypeptides makes it likely that prior studies were performed with N-truncated enzymes. This study uses erythrocyte ghosts to characterize the reversible cytoplasmic membrane association of human full-sized recombinant isoform E (AMPD3). Membrane-bound isoform E exhibits diminished catalytic activity whereas low micromolar concentrations of the cationic antibiotic, neomycin, disrupt this protein-lipid interaction and relieve catalytic inhibition. The cytoplasmic membrane association of isoform E also displays an inverse correlation with pH in the physiological range. Diethyl pyrocarbonate (DEPC) modification of isoform E nearly abolishes its cytoplasmic membrane binding capacity, and this effect can be reversed by hydroxylamine. Difference spectra reveal that 18 of 29 histidine residues in each isoform E subunit are N-carbethoxylated by DEPC. These combined data demonstrate that protonated imidazole rings of histidine residues mediate a pH-responsive association of isoform E with anionic charges on the surface of the cytoplasmic membrane, possibly phosphatidylinositol 4,5-bisphosphate, a pure noncompetitive inhibitor of the enzyme. Finally, AMPD1 and a series of N-truncated AMPD3 enzymes are used to show that these behaviors are specific to isoform E and require up to 48 N-terminal amino acids, even though this stretch of sequence contains no histidine residues. The pH-responsive cytosol-membrane partitioning of isoform E may be an important mechanism for branch point regulation of adenylate catabolism.
Author List
Mahnke-Zizelman DK, Sabina RLAuthor
Donna K. Mahnke Research Scientist I in the Pediatrics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AMP DeaminaseBinding Sites
Cell Membrane
Histidine
Humans
Hydrogen-Ion Concentration
Hydroxylamine
Isoenzymes
Kinetics
Neomycin
Phosphorylcholine
Recombinant Proteins
