Purification and regulation of an AMP-specific cytosolic 5'-nucleotidase from dog heart.
The major enzyme responsible for adenosine production during myocardial hypoxia or ischemia is 5'-nucleotidase. We purified an AMP-specific 5'-nucleotidase to homogeneity from the 150,000-g supernatant of dog heart homogenate using phosphocellulose, DEAE-cellulose, and ADP-agarose affinity chromatography. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of the purified enzyme yielded a single protein band of 43 kDa. The molecular mass of the holoenzyme, determined by gel filtration and sucrose density-gradient centrifugation, was approximately 166 kDa, suggesting a tetrameric structure. Dog heart cytosolic 5'-nucleotidase was active at physiological pH (6.8-7.8) and demonstrated a preference for AMP over IMP as substrate. The enzyme exhibited sigmoidal saturation kinetics, with half-maximal activity at 2.6 mM AMP in the absence of ADP. ADP (0-250 microM) activated cytosolic 5'-nucleotidase by increasing maximal velocity and affinity for AMP. The enzyme was inhibited by 4 mM ATP, but 5'-nucleotidase activity increased as [ATP] was reduced. Mg2+ was required for activity, with maximal activation at approximately 3.5 mM free Mg2+. These data suggest that the regulation of AMP-specific cytosolic 5'-nucleotidase by adenine nucleotides and free Mg2+ may be important in the production of adenosine during conditions promoting ATP hydrolysis, such as myocardial hypoxia or ischemia.
Department of Physiology and Biophysics, Medical College of Ohio, Toledo 43699-0008.
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Darvish
P J
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Metting
D015720:5'-Nucleotidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D004285:Dogs; D007477:Ions; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D007477:Ions; D008670:Metals; D000249:Adenosine Monophosphate; D015720:5'-Nucleotidase