Abstract
(2'-5')(A)n synthetase is one of the mediators of interferon action. If activated by double-stranded RNA it converts ATP into pyrophosphate and (2'-5')(A)n. In turn, (2'-5')(A)n activates a latent endoribonuclease (RNase L) which cleaves single-stranded RNA. We report here the isolation and characterization of a homogeneous human (2'-5')(A)n synthetase. The enzyme was purified from interferon-treated HeLA S3 cells by chromatography of a ribosomal salt wash fraction on DEAE-cellulose, poly(I) . poly(C) agarose, and CM-cellulose. The purified (2'-5')(A)n synthetase can convert over 90% of ATP into (2'-5')(A)n. The enzyme is unstable but can be stabilized by certain nonionic detergents (e.g. Triton X-100). Its apparent Mr = 100,000, as determined by gel electrophoresis in sodium dodecyl sulfate, and about 80,000, as determined by centrifugation through a glycerol gradient. The human (2'-5')(A)n synthetase is similar to the corresponding enzyme from mouse Ehrlich ascites tumor cells, but differs from the latter in size (100,000 versus 105,000 daltons) and in ionic conditions required for maximal activity.
Highlights
(2‘-5’)(A)“synthetase is one of the mediators of inter- (2’-5’)(A),synthetase has been detecteind mouseEAT and feron action
The resulting supernatant fraction was passed through a 12-mlDEAE-cellulose column (Whatman DE-52) equilibrated with buffer B (10 mM Tris-C1, 30 mM P-mercaptoethanol, 4 mM Mg(OAc)*,1 mM EDTA, 10% (v/v) glycerol, 10 p~ phenylmethylsulcould induce (2’-5’)(A)n synthetase in HeLa cells [32]
The enzyme is present in the high salt wash. This is passed through a DEAE-cellulose columnandthe resulting flow through fraction is further purified by affinity chromatography on poly(I).poly(C) agarose (Table I)
Summary
A mouse EAT interferon fraction eluting from controlled pore glass at pH 2 and from phosphocellulose with 400 mM sodium phosphate (pH 6.2) was prepared according to published procedures [31]. The specific activity of the preparation is 10’ NIH mouse reference standard interferon units/mg of protein. Isolation of Human (2‘-5Y(A),,Synthetase tralizing units/l interferon unit) did not decrease the activity of the preparation. Ml the culture was supplemented with 500 mouse NIH reference standard units of interferon/ml; 24 h later the cells were harvested, disintegrated, and a low speed supernatant fraction ( S 30) was prepared following procedures described for preparing S 30 from EAT cells [13].Fifty ml ofthe S 30 obtained from 40 liters of cell suspension at 0 “C for 35 min, 25p1of 0.2 M glycine in 0.1 M sodium borate buffer (pH 8.5) were added. The pellet fraction (ribosomes) was suspended in 8 ml of buffer A
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