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
| Autor: | Michael Orford, E.David Saggerson |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Male
Biochemistry 5'-nucleotidase Rats Sprague-Dawley Cellular and Molecular Neuroscience chemistry.chemical_compound Adenosine Triphosphate Cytosol Affinity chromatography Adenine nucleotide Nucleotidase Protein purification Animals Phosphatidylinositol Enzyme Inhibitors 5'-Nucleotidase biology Phospholipase C Brain Rats Adenosine Diphosphate Kinetics chemistry Concanavalin A Type C Phospholipases biology.protein |
| Zdroj: | Journal of neurochemistry. 67(2) |
| ISSN: | 0022-3042 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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