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m-Calpain requires DNA for activity on nuclear proteins at low calcium concentrations
R L Mellgren1, K Song, M T Mericle
1Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo 43699.
Abstract:
m-Calpain (calpain II, m-CANP), which normally requires millimolar Ca2+ for activity in vitro, was capable of proteolyzing a number of matrix proteins in isolated rat liver nuclei at Ca2+ concentrations as low as 3 microM (Mellgren, R. L. (1991) J. Biol. Chem. 266, 13920-13924). Treatment of nuclei with deoxyribonuclease I eliminated the activity of m-calpain at low Ca2+ concentrations, while ribonuclease A and phospholipase C had no effect. Addition of DNA to DNase-treated nuclei restored m-calpain activity at low Ca2+. RNA had little if any effect. Eukaryotic and prokaryotic DNA were equally effective, and synthetic polydeoxyribonucleotides were also activators. m-Calpain did not bind to a DNA-cellulose column in the presence of 200 microM Ca2+, and m-calpain preincubated in the presence of DNA and 200 microM Ca2+ was not activated at low Ca2+ concentrations following removal of the DNA. DNA did not alter the Ca2+ requirement for m-calpain-catalyzed cleavage of casein. These results demonstrate that the Ca2+ requirement for proteolysis of nuclear matrix proteins by m-calpain can be dramatically decreased in the presence of DNA. Activation did not seem to be a result of DNA binding directly to calpain but appeared to require interaction of DNA, calpain, and calpain substrates in the nuclear matrix.
Insights
DNA significantly reduces the calcium (Ca2+) requirement for m-calpain activity in nuclear matrix proteolysis. This interaction involves DNA, m-calpain, and nuclear substrates, not direct DNA binding to the enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- m-Calpain (calpain II) typically requires millimolar Ca2+ for enzymatic activity.
- Nuclear matrix proteins are substrates for m-calpain.
Purpose of the Study:
- To investigate the effect of DNA on the Ca2+ requirement of m-calpain for nuclear matrix protein proteolysis.
Main Methods:
- Isolated rat liver nuclei were treated with m-calpain at varying Ca2+ concentrations.
- Enzyme activity was assessed following treatment with DNase I, RNase A, and phospholipase C.
- The effect of DNA and RNA addition on m-calpain activity was evaluated.
Main Results:
- m-Calpain proteolyzed nuclear matrix proteins at Ca2+ concentrations as low as 3 microM in the presence of DNA.
- DNase I treatment abolished low Ca2+ activity, which was restored by DNA addition.
- DNA activation did not involve direct binding to m-calpain but required interaction with substrates in the nuclear matrix.
Conclusions:
- DNA dramatically lowers the Ca2+ requirement for m-calpain-mediated proteolysis of nuclear matrix proteins.
- The activation mechanism involves a complex of DNA, m-calpain, and nuclear substrates.