Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis.


Purification of DNA polymerase from E. coli B has in two cases each time led to the isolation of two separate polymerase activities, enzyme A and enzyme B. Enzyme A was in contrast to enzyme B almost completely devoid of exonuclease activity. Each of the two enzymes yielded a single symmetrical activity peak in gel filtration chromatograms. From the elution volumes the molecular weights were estimated to be about 70,000 for enzyme A and about 150,000 for enzyme B. Treatment of enzyme B with subtilisin led to an increase of about 30 per cent of the polymerase activity while the exonuclease activity almost completely disappeared. The product of the subtilisin treatment (enzyme C) gave rise to a single symmetrical polymerase activity peak in a gel filtration chromatogram. The elution volume was identical to that obtained with enzyme A. It is concluded that enzyme A and enzyme C are formed by limited proteolysis of enzyme B.



Historical Protein Properties (MW, pI, ...), Exonuclease Activity, Source / Purification


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Polymerase Reference Property Result Context
Klenow fragment Klenow H1970 Molecular Weight 7E+04 Dalton Technique: Gel Filtration
Klenow fragment Klenow H1970 Cloned or native Native organism
Klenow fragment Klenow H1970 5-3' Exonuclease No
Klenow fragment Klenow H1970 Tagged No
Klenow fragment Klenow H1970 Specific Activity 4300 units/mg Technique: Polymerase Assay (calf thymus DNA)
Klenow fragment Klenow H1970 Specific Activity 2 units/mg Technique: Exo activity (arbitrary units) (3H poly d(A-T))

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