Oxidative modulation and inactivation of rabbit cardiac adenylate deaminase.
Oxidative stress and adenine nucleotide catabolism occur concomitantly in several disease states, such as cardiac ischaemia-reperfusion, and may act as synergistic determinants of tissue injury. However, the mechanisms underlying this potential interaction remain ill-defined. We examined the influence of oxidative stress on the molecular, kinetic and regulatory properties of a ubiquitous AMP-catabolizing enzyme, adenylate deaminase (AMPD) (EC 3.5.4.6). To this intent, rabbit heart AMPD and an H2O2/ascorbate/iron oxidation system were employed. Enzyme exposure to the complete oxidation system acutely impaired its catalytic activity, lowered the Vmax. by 7-fold within 5 min, and rendered the enzyme unresponsive to nucleotide effectors. Irreversible AMPD inactivation resulted within about 15 min of oxidative insult and was not prevented by free-radical scavengers. Oxidative stress did not affect the molecular mass, tetrameric nature, Km, immunoreactivity or trypsinolytic pattern of the enzyme; nor did it induce carbonyl formation, Zn2+ release from the holoenzyme or net AMPD S-thiolation. This injury pattern is inconsistent with a radical-fragmentation mechanism as the basis for the oxidative AMPD inactivation observed. Rather, the sensitivity of the enzyme to both S-thiolation and thiol alkylation and the significant (3 of 9/mol of denatured enzyme) net loss of DTNB-reactive thiols on exposure to oxidant strongly implicate the conversion of essential thiol moieties into stable higher-oxidation states in the oxidative inactivation of cardiac AMPD. The altered thiol status of the enzyme on oxidative insult may prohibit a catalytically permissible conformation and, in so doing, increase AMP availability to 5'-nucleotidase in vivo.
Janero
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DR
Research Department, Ciba Pharmaceuticals, Summit, NJ 07901.
Yarwood
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D015720:5'-Nucleotidase / Q000378:metabolism; D000659:AMP Deaminase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000478:Alkylation; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002384:Catalysis; D002712:Chlorides; D004228:Dithionitrobenzoic Acid; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D018384:Oxidative Stress; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D002712:Chlorides; D005290:Ferric Compounds; D005296:Ferrous Compounds; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D006861:Hydrogen Peroxide; D015720:5'-Nucleotidase; D014357:Trypsin; D000659:AMP Deaminase; D015032:Zinc; D001205:Ascorbic Acid; C029451:ferrous chloride; C024555:ferric chloride