Properties of 5'-nucleotidase in rat heart sarcolemma.
The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.
D000408:Alamethicin / Q000494:pharmacology; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D004789:Enzyme Activation; D009206:Myocardium / Q000201:enzymology*; D009439:Neuraminidase / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D012508:Sarcolemma / Q000201:enzymology*; D012995:Solubility; D014357:Trypsin / Q000378:metabolism
D000408:Alamethicin; D011092:Polyethylene Glycols; D009708:Nucleotidases; D009439:Neuraminidase; D014357:Trypsin