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Affinity labelling and characterization of the ppp(A2'p)nA-dependent endoribonuclease from different mammalian
Abstract:
The ppp(A2'p)nA-dependent endoribonucleases from a number of different mammalian sources have been investigated. The enzyme from reticulocyte lysates shows optimal activity of 50-150 mM KCl and requires the presence of Mg2+. Whilst the enzyme is inactivated after passage of reticulocyte lysates through Sephadex columns in the absence of ATP, it retains full activity provided ATP is included in the column buffer. The activity of the partially purified nuclease was unaffected by the addition of reticulocyte RNase inhibitor, which, in contrast, effectively inhibited other endogenous endonucleases. The ppp(A2'p)nA-dependent Rnase co-purified with a ppp(A2'p)nA-binding protein and with a protein which could be specifically covalently labelled with an oxidised radioactive analogue of ppp(A2'p)nA. This covalent labelling could be carried out either with the partially purified RNase or in crude extracts from rabbit reticulocytes, mouse Krebs and Ehrlich ascites tumour cells and human lymphoblastoid (Daudi) or HeLa cells. In each case the affinity labelled protein migrated to a position corresponding to a apparent molecular weight of about 85 000 on electrophoresis on dodecylsulphate/polyacrylamide gels. In all cases labelling could be prevented by the addition of an excess of unlabelled ppp(A2'p)nA but not, for example, by a similar excess of the biologically inactive dimer ppp(A2'p)'A. It is concluded that the RNase and ppp(A2'p)nA binding activities are likely to reside in the same molecule.
Insights
Mammalian cells contain ppp(A2'p)nA-dependent endoribonucleases. These enzymes, crucial for RNA degradation, were found to co-purify with a specific binding protein, suggesting a single molecule performs both functions.
Area of Science:
- Molecular Biology
- Biochemistry
- Enzymology
Background:
- Mammalian cells utilize ppp(A2'p)nA-dependent endoribonucleases for RNA processing and degradation.
- Investigating the properties and molecular characteristics of these enzymes is crucial for understanding cellular RNA homeostasis.
Purpose of the Study:
- To characterize the ppp(A2'p)nA-dependent endoribonucleases from various mammalian sources.
- To elucidate the molecular association of the nuclease activity with ppp(A2'p)nA binding.
Main Methods:
- Enzyme activity assays using reticulocyte lysates and varying KCl concentrations.
- Investigating enzyme stability during purification with and without ATP.
- Affinity labeling using an oxidized radioactive analogue of ppp(A2'p)nA.
- SDS-PAGE to determine the molecular weight of labeled proteins.
Main Results:
- The enzyme exhibits optimal activity in the presence of 50-150 mM KCl and Mg2+.
- Enzyme stability during purification is maintained by ATP.
- The nuclease activity co-purified with a ppp(A2'p)nA-binding protein.
- Affinity labeling identified a protein of approximately 85,000 apparent molecular weight, specifically binding ppp(A2'p)nA.
Conclusions:
- The ppp(A2'p)nA-dependent endoribonuclease activity and the ppp(A2'p)nA binding activity are likely located on the same molecular entity.
- This finding provides insights into the mechanism of RNA degradation mediated by these enzymes.