Expression, purification, and inhibition of in vitro proteolysis of human AMPD2 (isoform L) recombinant enzymes.
AMP deaminase (AMPD) is a multigene family in higher eukaryotes whose three members encode tetrameric isoforms that catalyze the deamination of AMP to IMP. AMPD polypeptides share conserved C-terminal catalytic domains of approximately 550 amino acids, whereas divergent N-terminal domains of approximately 200-330 amino acids may confer isoform-specific properties to each enzyme. However, AMPD polypeptides are subject to limited N-terminal proteolysis during purification and subsequent storage at 4 degrees C. This presents a technical challenge to studies aimed at determining the structural and functional significance of these divergent sequences. This study describes the recombinant overexpression of three naturally occurring human AMPD2 proteins, 1A/2, 1B/2, and 1B/3, that differ by N-terminal extensions of 47-128 amino acids, resulting from the use of multiple promoters and alternative splicing events. A survey of protease inhibitors reveals that E-64 and leupeptin are able to maintain the subunit structure of each AMPD2 protein when they are included in extraction and storage buffers. Gel filtration chromatography of these three purified AMPD2 enzymes comprised of intact subunits reveals that each migrates faster than expected, resulting in observed molecular masses significantly greater than those predicted for native tetrameric structures. However, chemical crosslinking analysis indicates four subunits per AMPD2 molecule, confirming that these enzymes have a native tetrameric structure. These combined results suggest that AMPD2 N-terminal extensions may exist as extended structures in solution.
Haas
Amy Louise
AL
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Sabina
Richard L
RL
D000659:AMP Deaminase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007976:Leupeptins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014162:Transfection
D003432:Cross-Linking Reagents; D007976:Leupeptins; D010455:Peptides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000659:AMP Deaminase; C475566:AMPD2 protein, human; C032854:leupeptin