A low-Km 5'-nucleotidase from rat brain cytosolic fraction: purification, kinetic properties, and description of regulation by a novel factor that increases sensitivity to inhibition by ATP and ADP.
A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000-g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low Km for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by alpha,beta-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of Mr = 54,300 +/- 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- Km "soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000-g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.
Orford
M R
MR
Department of Biochemistry and Molecular Biology, University College London, England.
Saggerson
E D
ED
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D010738:Type C Phospholipases / Q000378:metabolism
D004791:Enzyme Inhibitors; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D015720:5'-Nucleotidase; D010738:Type C Phospholipases