N-terminal Sequence and Distal Histidine Residues Are Responsible for pH-regulated Cytoplasmic Membrane Binding of Human AMP Deaminase Isoform E

Abstract

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 cytosolmembrane partitioning of isoform E may be an important mechanism for branch point regulation of adenylate catabolism.AMP deaminase (AMPD 1 ; EC 3.5.4.6) is a highly regulated enzyme catalyzing a branch point reaction in the adenylate catabolic pathway, and its expression in mammalian tissues and cells is characterized by a multigene family (1-5). In humans, the AMPD1 gene encodes isoform M (6), AMPD2 encodes isoform L (7), and AMPD3 encodes isoform E (8, 9). Primary amino acid sequence alignments identify divergent N-terminal and conserved C-terminal domains across human AMPD isoforms (7,8). In addition, all three human AMPD genes produce multiple transcripts that encode additional variation at or near, the N terminus of each isoform (8, 10, 11).Human AMPD isoforms have been purified and characterized from endogenous sources (12-15). However, extreme N-terminal regions in mammalian AMPD polypeptides are highly sensitive to proteolysis during purification and subsequent storage of the enzyme at 4°C (16 -19). Recombinant technology provides a means to overexpress AMPD enzymes that can be purified with subunits predominantly intact, although these are also subject to proteolysis during storage at 4°C (20). The availability of purified AMPD enzymes with subunits predominantly intact has stimulated interest in the structural and functional significance of extreme N-terminal sequences that were...

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage

“…Mechanistically ( Figure 8 ), we propose that refrigerated RBC storage promotes, among many cascades of events, 2 – 5 the activation of AMPD3 (the RBC-specific isoform of AMPD, and the only one identified here using deep proteomics). AMPD3 can be activated by increases in intracellular ROS 44 and calcium, 60 , 61 along with decreases in intracellular pH; 62 , 63 in contrast, increases in 2,3-DPG downregulate AMPD3 activity. 63 Notably, refrigerated RBC storage, especially after storage day 14, leads to increases in ROS and intracellular calcium, and decreases in pH and 2,3-DPG, 2 , 6 all of which can combine to activate AMPD3, as our steady state and metabolic flux results suggest.…”

Differential transcription of the bovine AMPD3 gene in single blastocysts cultured under various oxygen tensions

“…These purine nucleotide catabolic and interconverting enzymes ( Sims et al. 1999) have been linked to the cellular pH‐responses ( Mahnke‐Zizelman and Sabina 2002). Ogasawara et al.…”

Proteinuria in AMPD2‐deficient mice

“…1997; Korzeniewski 2006). However, the precise functional relevance of AMPD2 in kidney tissues has not been clearly established (Nowak & Kaletha 1992; Mahnke‐Zizelman & Sabina 2002). To further study the function of AMPD2, we established AMPD2‐deficient mice and detected very low levels of AMPD activity in liver, kidney, and brain samples.…”