DNA binding proteins of rat thigh muscle: purification and characterization of an endonuclease

Rajakumar, A. R. Augustine ; Shanmugam, G. (1986) DNA binding proteins of rat thigh muscle: purification and characterization of an endonuclease Journal of Biosciences, 10 (1). pp. 85-94. ISSN 0250-5991

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Official URL: http://www.ias.ac.in/jarch/jbiosci/10/85-94.pdf

Related URL: http://dx.doi.org/10.1007/BF02702843

Abstract

Two major DNA binding proteins of molecular weights 34,000 and 38,000 have been identified in the 30,000 g supernatant (S-30) fraction of rat thigh muscle extracts. The presence of 38 KD DNA binding protein in the muscle S-30 could be demonstrated only if Triton X-100 treated extracts were used for Afinity chromatography suggesting that this protein may be a membrane associated DNA binding protein. The 38 KD DNA binding protein differed from the 34 KD DNA binding protein also in its chromatographic behaviour in DE-52 columns in which the 38 KD protein was retained, while the 34 KD protein came out in the flow-through in an electrophoretically pure form. The 34 KD DNA binding protein can also be purified by precipitation with MgCl2. Incubation of 0 · 15 M NaCl eluates (containing the 38 KD and/or 34 KD DNA binding protein) in the presence of 100 mM Mg2+ resulted in the specific precipitation of the 34 KD protein. Prolonged incubation (30 days) of the 0 · 15 Ì NaCl eluates containing the two DNA binding proteins at 4°C led to the preferential degradation of the 34 KD DNA binding protein. Nitrocellulose filter binding assays indicated selective binding of purified 34 KD protein to ss DNA. Purified 34 KD DNA binding protein cleaved pBR 322 supercoiled DNA, and electrophoresis of the cleavage products in agarose gels revealed a major DNA band corresponding to the circular form of DNA.

Item Type:Article
Source:Copyright of this article belongs to Indian Academy of Sciences.
Keywords:Afinity Chromatography; DNA Binding Protein; Endonuclease Cleavage of pBR 322 DNA
ID Code:52749
Deposited On:04 Aug 2011 08:11
Last Modified:18 May 2016 06:10

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