Purification and properties of adenosine 5′-phosphosulphate sulphotransferase from Euglena
| Autor: | Jerome A. Schiff, Jiayang Li |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Euglena gracilis
Dithioerythritol ved/biology.organism_classification_rank.species Biochemistry Euglena Dithiothreitol Substrate Specificity chemistry.chemical_compound medicine Animals Molecular Biology chemistry.chemical_classification Chromatography biology Isoelectric focusing ved/biology Sodium Dodecyl Sulfate Cell Biology Chromatography Ion Exchange biology.organism_classification Adenosine Kinetics Enzyme chemistry Chromatography Gel Thiol Electrophoresis Polyacrylamide Gel Sulfatases Research Article medicine.drug |
| Zdroj: | Biochemical Journal. 274:355-360 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj2740355 |
| Popis: | Adenosine 5′-phosphosulphate sulphotransferase (APSST) was extracted from Euglena gracilis Klebs var. bacillaris mutant W10BSmL by freezing and thawing and was purified about 10,000-fold (to homogeneity) with 10.5% recovery by (NH4)2SO4 precipitation, Sephadex G-100 chromatography, Reactive Blue-agarose, Reactive Dye-agarose, DEAE-cellulose, preparative isoelectric focusing and non-inactivating SDS/PAGE. The active APSST, with a molecular mass of 102 kDa and multiple forms from pI 5.0 to 5.5, is a tetramer held together by covalent (probably disulphide) bonds. An apparent Km of the purified enzyme for adenosine 5′-phosphosulphate (APS) of 0.1 microM is obtained when dithiothreitol is used as the thiol. The enzyme is stimulated by Mg2+, Ca2+ or Ba2+, and uses almost any thiol; dithiothreitol and dithioerythritol give the highest activity. In the absence of APS, the enzyme is inactivated (and is rendered monomeric) by thiols but is protected from thiol inactivation by AMP, adenosine 5′-phosphoramidate (APA) or adenosine 5′-monosulphate (AMS), which also inhibit APSST activity somewhat. The enzyme resists inactivation by SDS in the absence of thiols; SDS stimulates APSST activity at low concentration, but high concentrations are inhibitory. |
| Databáze: | OpenAIRE |
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