Studies on deoxyribonucleases

Curtis, Peter John (1965) Studies on deoxyribonucleases. PhD thesis, University of Glasgow.

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A nuclease from rat liver has been identified which shows a preference for denatured rather than native DNA as its substrate. It was considered necessary to purify this enzyme before undertaking studies on its properties and specificity. A suitable source of the liver nuclease was shown to be mitochondria. The enzyme was released into solution from isolated mitochondria by treatment with a solution or triton-x-100. The enzyme solution was fractionated firstly with saturated ammonium sulphate taking the precipitate obtained between 35-55% saturation, followed by acetone treatment where the precipitate obtained between 36.9-46.9% acetone concentration was collected. Further purification was achieved by the use of column chromatography. Passage through a column of diethylaminoethyl-cellulose equilibrated with 0.01M tris-HOl buffer pH 8.0 resulted in the removal of 95% of the protein though the majority of the enzyme was not adsorbed. Gel filtration on a column of Sephadex G-75 resulted in a further 4 fold increase in the specific activity. The overall purification obtained was 715 fold. The purified liver nuclease showed neglible acid DNase, phosphate and phosphodiesterase activities, though it hydrolysed both native DNA and RNA as well as denatured DNA. The activities towards these three substrates were eluted together from columns of Sephadex G-75 and G-200 suggesting that they belong to one and the same protein. The purified liver nuclease is rather unstable in that it loses its activities when stored in ice, frozen or freeze-dried. However when stored in the presence of 20% glycerol or 0.01M 2-mercapto-ethanol in ice the DNase and RNase activities were retained for upto 2 weeks. Beyond this period both activities decreased, DNase more rapidly than RNase, suggesting that they belong to separate proteins. The molecular weight of the liver nuclease was estimated to be about 44,000 from its elution profile on a column of Sephadex G-75. The properties of the two activities of the liver nuclease were examined. Both showed a sharp pH optimum about 6.8-7.0 and an absolute requirement for Mg2+ or Mn2+. The optimal concentrations of Mg2++ and Mn2+ were similar and little difference was observed when DNA or RNA were used as substrates. Ca2+ showed no stimulatory effect and in the presence of the optimal concentration of Mg2+, Ca2+ inhibited both activities as did NaCl. The two activities were readily denatured by heating. In all these properties the activities towards denatured DNA and RNA are very similar thus providing further support for the hypothesis that the two activities belong to one and the same protein. The specificity of the nuclease towards DNA and RNA were studied. Both DNase and RNase exhibited an endonucleolytic mode of action since only 8% of the digest consisted of momonucleotides. Experiments were carried out to determine whether the enzyme split the phosphodiester bonds between the 3'-carbon and phosphorus or the 5'-carbon and phosphorus. Treatment of the oligonucleotides produced by the enzyme with snake venom and spleen phosphodiesterase showed that the oligodeoxyribonucleotides and oligoribonucleotides were terminated by 5'-phosphate groups. Finally, the enzyme showed little specificity towards the bases in DNA and RNA. This lack of specificity was demonstrated by determining the proportion of the bases at the 3' and 5' ends of the oligonucleotides produced by the digestion of the DNA and RNA by the enzyme. These results further illustrate the similarity of the two activities, which are endonucleases producing oligonucleotides terminated by 5'-phospate groups.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J N Davidson
Keywords: Biochemistry
Date of Award: 1965
Depositing User: Enlighten Team
Unique ID: glathesis:1965-72683
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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