Regulation of the interaction of purified human erythrocyte AMP deaminase and the human erythrocyte membrane.

Pipoly, G M; Nathans, Gene R.; Chang, Donald; Deuel, Thomas F.

doi:10.1172/jci109376

Cited by 14 publications

(10 citation statements)

References 19 publications

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“…However, the interaction with lipids may prove to be physiologically relevant. Whereas hydropathy analysis suggests that AMPD isoforms do not contain putative transmem-brane spanning domains, the AMPD3 enzyme can associate with erythrocyte membrane fractions (40,41). In this report, we provide several independent lines of evidence that the specific, high affinity interaction between the AMPD3 isoform and phosphoinositides constitutes an important mechanism for enzyme regulation and localization.…”

mentioning

confidence: 80%

Regulation of AMP Deaminase by Phosphoinositides

Sims

Mahnke-Zizelman

Profit

et al. 1999

Journal of Biological Chemistry

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AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a K i of 110 nM. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/ K)X n (R/K)XKK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo.Phosphoinositides and inositol polyphosphates (referred to collectively as inositides) are components of many pathways in eukaryotic cells, functioning in second messenger cascades, acting as regulators of many proteins, and operating as membrane localization signals (1-3). Numerous protein and lipid kinases, adaptor proteins, ion channels, phospholipases, modulators of small GTPases, and actin-binding proteins are regulated by inositides (1-3). To identify novel targets for inositides, our laboratories and others have used purification schemes employing affinity resins that contain tethered inositol polyphosphate head groups (3-9). These affinity purifications were successful in the identification of inositide binding in the clathrin adaptor/assembly protein AP-2 (6), centaurin ␣ (7), a centaurin ␣ orthologue (8), and a phospholipase C (PLC) 1 -related protein (9). In addition to AP-2 and centaurin ␣, we isolated several other proteins from rat brain, one of which was approximately 80 kDa (5). Published reports show that inositol polyphosphates modulate the activity of the enzyme AMP deaminase (EC 3.5.4.6) (AMPD) (10, 11), an enzyme family whose endogenous, purified subunit molecular masses are between 66 and 88 kDa. Therefore, we hypothesized that AMPD could be the 80-kDa protein isolated using the inositide affinity resin.AMPD is a diverse and highly regulated enzyme located at a branchpoint in the adenine nucleotide catabolic pathway and is important in regulating nucleotide pools. AMPD is also a component of the purine nucleotide cycle, an energy-generating pathway reportedly operative in many animal tissues (reviewed in Ref. 12). The AMPD1 gene encodes human isoform M and rat iso...

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confidence: 80%

Regulation of AMP Deaminase by Phosphoinositides

Sims

Mahnke-Zizelman

Profit

et al. 1999

Journal of Biological Chemistry

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“…E1b is one of three identified AMPD3 spliceoforms, and the ⌬M90 variant is modeled after the primary N-terminal proteolytic product generated from this protein during purification and extended storage at 4°C (20). ⌬M90 was typically included in most experiments to simulate proteolyzed endogenous enzyme likely used in a previous study that examined the membrane association of human erythrocyte AMPD (26). Fig.…”

Section: Association Of Ampd3 Enzymes With Erythrocyte Ghosts-mentioning

confidence: 99%

“…Substrate-induced Dissociation of AMPD3 Enzymes from Erythrocyte Ghosts-Previous work has shown that EG association of purified human erythrocyte AMPD results in catalytic inhibition of the enzyme, although substrate concentrations greater than 100 M can disrupt this interaction (26). Therefore, it was necessary to determine a substrate concentration where the EG dissociation of the E1b and ⌬M90 proteins was essentially complete in order that enzyme assay could be used as a reliable quantitative index following their partitioning into EG pellets.…”

Section: Association Of Ampd3 Enzymes With Erythrocyte Ghosts-mentioning

confidence: 99%

“…This was initially revealed by the observation that human erythrocyte membrane preparations contained AMPD activity (25). Subsequent work using purified human erythrocyte AMPD (isoform E) demonstrated an association with the cytoplasmic face of erythrocyte ghost (EG) membranes that was also accompanied by reduced catalytic activity (26). Moreover, membrane association and the related inhibition of catalytic activity could be reversed in the presence of small molecule effectors, including substrate (26).…”

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confidence: 99%

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

Mahnke-Zizelman

Sabina

2002

Journal of Biological Chemistry

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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...

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“…It was shown that the purified human erythrocyte AMP deaminase binds specifi cally to the cytoplasmic surface of the eryth rocyte membrane and the stability of AMP deaminase is markedly improved by interac tion with the membrane [22], The ability of binding of AMP deaminase to other proteins was also demonstrated [7,21]. Shiraki et al [26] showed that AMP deaminase from rat skeletal muscles binds myosin, and that this binding might modify the regulatory proper ties of AMP deaminase.…”

Section: Distributionmentioning

confidence: 99%

Intracellular Distribution of AMP Deaminase in the Pig Thyroid Gland

Jaroszewicz¹,

Stelmach²

1984

Enzyme

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AMP deaminase and adenosine deaminase activities were assayed in the subcellular fractions of pig thyroid gland. AMP deaminase is localized in the cytosolic fraction; however, this enzyme showed remarkable tendency to bind with the subcellular particulate fractions. Adenosine deaminase is also localized in the cytosolic fraction but in contradistinction to AMP deaminase, adenosine deaminase under the same experimental conditions has no tendency of binding to subcellular particulate fractions. The significance of AMP deaminase binding to subcellular particulate fractions is discussed.

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Regulation of the interaction of purified human erythrocyte AMP deaminase and the human erythrocyte membrane.

Abstract: A B S T R A C T

Cited by 14 publications

References 19 publications

Regulation of AMP Deaminase by Phosphoinositides

Regulation of AMP Deaminase by Phosphoinositides

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

Intracellular Distribution of AMP Deaminase in the Pig Thyroid Gland

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