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Two ribonucleases H from cultured plant cells
Journal of Biochemistry
|May 1, 1979
Summary
Researchers purified two distinct ribonuclease H (RNase H) enzymes from carrot cells. These enzymes, one dependent on manganese (Mn2+) and the other on magnesium (Mg2+), degrade RNA within RNA-DNA hybrid structures.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Ribonuclease H (RNase H) enzymes play a crucial role in nucleic acid metabolism.
- Understanding the specific properties of different RNase H forms is essential for various biotechnological applications.
Purpose of the Study:
- To partially purify and characterize two distinct RNase H enzymes from cultured carrot cells (strain GD-2).
- To investigate the cofactor dependency (Mn2+ vs. Mg2+), optimal pH, and substrate specificity of these enzymes.
Main Methods:
- Partial purification of enzymes using phosphocellulose and blue Sepharose chromatography.
- Enzyme activity assays to determine optimal pH, metal ion concentration, and cofactor requirements.
- Gel filtration chromatography for molecular weight estimation.
Main Results:
- Two RNase H forms were identified: one Mn2+-dependent and one Mg2+-dependent.
- The Mg2+-dependent RNase H showed optimal activity at pH 9.0 with 10-15 mM Mg2+, while the Mn2+-dependent enzyme was optimal at pH 8.0 with 0.4 mM Mn2+.
- Both enzymes require sulfhydryl reagents for maximal activity and degrade RNA in RNA-DNA hybrids, releasing oligonucleotides with 5'-phosphate and 3'-hydroxyl termini.
Conclusions:
- Carrot cells contain at least two distinct RNase H enzymes with differential metal ion dependencies.
- These enzymes exhibit specific biochemical properties that distinguish them, suggesting specialized roles in cellular processes.