Regulation of rat AMP deaminase 3 (isoform C) by development and skeletal muscle fibre type

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AMP deaminase isoforms purified from endogenous sources display smaller than predicted subunit molecular masses, whereas baculoviral expression of human AMPD1 (isoform M) and AMPD3 (isoform E) cDNAs produces full-sized recombinant enzymes. However, nearly 100 N-terminal amino acid residues are cleaved from each recombinant polypeptide during storage at 4°C. Expression of N-truncated cDNAs (⌬L96AMPD1 and ⌬M90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E. Conversely, wild type recombinant isoforms display significantly higher K m(app) values in the absence of ATP. Gel filtration analysis demonstrates native tetrameric structures for all recombinant proteins, except the wild type AMPD1 enzyme, which forms aggregates of tetramers that disperse upon cleavage of N-terminal residues at 4°C. These data: 1) confirm that available literature on AMP deaminase is likely derived from N-truncated enzymes and 2) are inconsistent with a new model proposing native trimeric structure of an N-truncated rabbit skeletal muscle AMP deaminase (Ranieri-Raggi, M., Montali, U., Ronca, F., Sabbatini, A., Brown, P. E., Moir, A. J. G., and Raggi, A. (1997) Biochem. J. 326, 641-648). N-terminal residues also influence actomyosin-binding properties of the enzyme, which are enhanced and suppressed by AMPD1 and AMPD3 sequences, respectively. Finally, co-expression of AMPD1 and AMPD3 recombinant polypeptides produces tetrameric enzymes with either isoform-specific or mixed subunits, and also reveals that tetramer assembly is driven by relative polypeptide abundance with no apparent preference for like subunits.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Novel Aspects of Tetramer Assembly and N-terminal Domain Structure and Function Are Revealed by Recombinant Expression of Human AMP Deaminase Isoforms

Mahnke-Zizelman¹,

Tullson²,

Sabina³

1998

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“…12). The AMPD1 gene encodes human isoform M and rat isoform A (13); the AMPD2 gene encodes the human isoform L and rat isoform B (14,15); and the AMPD3 gene encodes the human isoform E and the rat isoform C (16,17). A single AMPD gene has also been identified in yeast (18).…”

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confidence: 99%

Regulation of AMP Deaminase by Phosphoinositides

Sims

Mahnke-Zizelman

Profit

et al. 1999

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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...

“…AMPD1 levels are high in all fibers but more so in glycolytic muscle, whereas AMPD3 is primarily restricted to oxidative fibers (24,49,50). The AMPD1 isoform exhibits a high contractile protein binding capacity that is, in part, attributable to N-terminal sequence (20,21).…”

Section: Figmentioning

confidence: 99%

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

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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...