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Ribonuclease A digestion by proteinase K
Zeitschrift Fur Naturforschung. Section C, Biosciences
|September 1, 1978
Summary
Proteinase K digestion of ribonuclease A produces ribonuclease K, a catalytically active fragment. Combined with trypsin, this aids in messenger RNA (mRNA) isolation from polysomes.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Ribonuclease A (RNase A) is a crucial enzyme in molecular biology.
- Understanding its degradation products and inactivation is important for biochemical applications.
Purpose of the Study:
- To characterize the major degradation product of Ribonuclease A (RNase A) digested by Proteinase K.
- To investigate the combined effect of Proteinase K and trypsin on RNase A inactivation.
- To explore the utility of these enzymatic actions in messenger RNA (mRNA) isolation.
Main Methods:
- Enzymatic digestion of RNase A using Proteinase K.
- Electrophoretical and immunological analysis of degradation products.
- Enzymatic digestion using combined Proteinase K and trypsin.
- Assessment of enzyme inactivation rates.
Main Results:
- Proteinase K digestion yielded a single major product, identified as ribonuclease K (RNase K).
- RNase K, comprising residues (1--20/21--124) of RNase A, retained full catalytic activity.
- Combined action of Proteinase K and trypsin significantly accelerated RNase A inactivation.
Conclusions:
- Ribonuclease K is a stable, active fragment of RNase A generated by Proteinase K.
- The accelerated inactivation of RNase A by Proteinase K and trypsin offers a potential method for mRNA isolation from polysomes.