Subcellular distribution of myocardial 5′-nucleotidase

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The controversial subject of the subcellular location of myocardial adenosine production was studied employing density gradient fractionation of heart muscle combined with a novel method for analyzing distribution profiles based on multiple regression (correlation) analysis. Bungarotoxin binding, N-acetyl-β- d-glucosamidase, cytochrome c oxidase, NADPH-dependent cytochrome c reductase and lactate dehydrogenase were used as markers for the plasma membrane, lysosomes, mitochondria, sarcoplasmic reticulum and cytosol, respectively. The normalized distribution frequencies (fraction of total) of 5′-nucleotidase in mitochondria, lysosomes, plasma membranes, sarcoplasmic reticulum and cytosol in the 50 × g supernatant of total homogenate of heart muscle were found to be 0, 0.25, 0.44, 0.08 and 0.23, respectively. To increase the resolution power of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied, in which the normalized distribution of 5′-nucleotidase in the homogenate was 0, 0.16 and 0.84 in mitochondria, plasma membranes and lysosomes, respectively. This mainly lysosomal 5′-nucleotidase activity was 61% inhibited by the α,β-methylene analog of ADP, indicating that although the latter has been considered specific to the plasma membrane enzyme, it also inhibits the lysosomal enzyme. The intercellular distribution of 5′-nucleotidase was not studied, but the lack of this enzyme in the mitochondria indicate that the adenosine production observed during mitochondrial AMP production, e.g. during acetate oxidation in intact heart muscle, must involve AMP transport out from the mitochondria.