Studies of 5'-nucleotidase in the perfused rat heart. Including measurements of the enzyme in perfused skeletal muscle and liver.

Perfused rat hearts catalyze the hydrolysis of AMP added to the perfusion fluid at a rate of 35 mumol/g dry weight/min. The activity is specific for 5'-nucleoside monophosphates, little activity being observed with 2' and 3'-AMP. The enzyme exhibits Michaelis-Menten kinetics in situ and is inhibited competitively by adenosine-5'-alpha, beta-methylene diphosphonate (Ki = 13 muM). This, as well as the nucleotide specificity, confirms that the hydrolysis is catalyzed by 5'-nucleotidase. The maximum activity of 5'-nucleotidase in perfused hearts is equal to or greater than that found in heart homogenates; thus, all of the enzyme is accessible to AMP added externally. Hydrolysis of endogenous AMP was studied in the perfused heart. Under aerobic conditions hearts contain very low amounts of purine nucleosides, and little or no nucleoside is found in the effluent perfusate. Under anaerobic conditions hearts accumulate adenosine, inosine, and hypoxanthine and release all three substances into the perfusate. Hydrolysis of externally added AMP was also observed in perfused skeletal muscle and liver, at rates of 10 and 17 mumol/g dry weight/min, respectively.