Purification of an Endonuclease from Adenovirus‐Infected KB Cells

Abstract

This report describes the purification of an endonuclease from extracts of adenovirus-type-2-infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus-infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus-infected cells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated.The endonuclease in crude extracts from uninfected or adenovirus-infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus-type-2-infected KB cells.A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.In previous work an endonuclease specific for double-stranded DNA has been described in association with the penton subunit of adenovirus type 2 [l], in extracts of KB cells infected with adenovirus types 2 and 12 and in association with purified

Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2-infected KB cells

“…The enzyme was purified as described previously (1). Recently, we have modified the purification procedures (Stabel and Doerfler, unpublished), and for some of the experiments endonuclease isolated according to the modified technique was used.…”

“…The endonuclease was eluted with a gradient of 0 -1.0 M KC1 in the same buffer. The fractions containing endonuclease were pooled and concentrated by vacuum dialysis against 0.5 M NaCl, 0.05 M Tris-acetate, pH 8.0, 0.02% NaN Subsequently, the endonuclease was further purified by gel filtration on Bio-gel A-0.5 as previously described (1). Again, the fractions containing endonuclease activity were pooled, dialyzed against 0.05 M Na-acetate, pH 4.0 and chromatographed on a column of phosphocellulose (Pll, Whatman; 23 cm x 1.5 cm diameter) equilibrated with the same buffer.…”

“…Recently, Reif et al (1,2) reported the purification and identification of two endonucleases from adenovirus (Ad) type 2 infected KB cells. These activities were also found to be present in uninfected human KB cells.…”

An Adenovirus Cytocidal Function Related to the Control of a Cellular pH 4 Endonuclease Activity

“…When H2WT-or mock-infected cell extracts were treated with protease, a net stimulation of the acidic endonuclease was observed, and an inactivation of the neutral endonuclease (Reif et al, 1977b). H2WT-, H2cytl41-and mock-infected cell extracts were treated with proteinase K (0-05 mg/ml, for 30 min at 37 °C) and tested for pH 4 endonuclease activity.…”

“…Cell extracts were prepared as described by Reif et al (1977b). In brief, KB cells were infected by H2WT, H2tsl 11, or interserotypic recombinant Q2, i.e.…”