Elongation mechanism and substrate specificity of 2',5'-oligoadenylate synthetase.

Abstract

2',5'1Oligoadenylate synthetase has been purified from a rabbit reticulocyte lysate to a high degree of purity. The enzyme contained no detectable interfering activities that could degrade the nucleoside triphosphate substrate or the oligomeric products. Two basic properties of this enzyme have been examined: the elongation mechanism for the synthesis of oligoadenylates and the substrate specificity for nucleotides. Kinetic studies on the formation of different o igomeric intermediates show that the dimer pppA2'p5'A is the first product to accumulate in predominant proportion during the first period of reaction; the trimer and other longer oligomers appear after a lag phase. The amount of the trimer increases at the expense of the dimer. Preformed dimers and trimers added to the incubation mixture were readily incorporated into higher oligomers, suggesting the free access of these dimers and trimers to the active center after the onset of polymerization of ATP. The results indicate clearly that the enzyme catalyzes the de no-vo synthesis of the oligonucleotide from ATP and that the mechanism of elongation of the 2',5'-oligonucleotides catalyzed by the enzyme is not processive. Polymerization of a mixture of ATP and another nucleoside triphosphate shows that the enzyme is not only an ATP polymerase. The 2',5'-oligoadenylate synthetase is in fact a 2',5'-nucleotidyltransferase a catalyzes the formation of co-oligonucleotides. However, the purified reticulocyte enzyme catalyzed only the addition of one unit of GMP, UMP, CMP, 2'1dAMP, 3'-dAMP, dCMP, dGMP, or TMP to the 2'.OH end of a preformed oligoadenylate. A procedure for the separation of 2',5'-oligonucleotides with or without the 5'-triphosphate end also is described. (7) indicates that this enzyme also has a function independent of interferon.In this paper, we describe the reaction mechanism of the enzyme purified from rabbit reticulocytes and the synthesis of 2',5'-co-oligonucleotides. MATERIALS AND METHODSMaterials. All radioactive nucleotides were obtained from Amersham. All chemicals were the purest obtainable. Poly(I). poly(C) and poly(I)-poly(C)agarose were from Choay Institute (Paris, France). Calf intestine alkaline phosphatase was from Boehringer. Polyethyleneimine (Polymin P) was a gift from BASF. Cellulose MN 300 was from Macherey Nagel (Dfiren, Germany). Nuclease P1 was from Yamasa Shoyo Co (Tokyo, Japan).Purification of Oligo(A) Synthetase. Phenylhydrazine was injected into two rabbits for 5 days, according to Borsook et al. (19), and the blood was collected 2 days later, and the reticulocytes were isolated. The lysate (85 ml; 160 mg of protein per ml) obtained by osmotic shock was passed through a DEAEcellulose (DE-52) column (50 ml) equilibrated in 20 mM TrisHCl, pH 8.0/5 mM Mg(OAc)2/25 mM KCl/1 mM dithiothreitol/10% (vol/vol) glycerol (buffer A). The oligo(A) synthetase activity was recovered with the proteins not retained on the column (105 ml, 32 mg of protein per ml), and this filtrate was applied to a poly(I)-poly(C)-agarose colum...

69‐kDa and 100‐kDa Isoforms of Interferon‐Induced (2′‐5′)Oligoadenylate Synthetase Exhibit Differential Catalytic Parameters

“…Besides its capacity to synthesize oligomers of 2-5A from ATP, the (2'-5')oligoadenylate synthetase has been previously reported to manifest a nucleotidyltransferase activity on other nucleoside triphosphates (Ball and White, 1979;Justesen et al, 1980). This latter activity was further studied here by using purified preparations of p69 and p100.…”

“…In addition to the synthesis of 2-5A molecules, partially purified (2'-5')oligoadenylate synthetase preparations have been shown to catalyze in vitro the transfer of a nucleotide monophosphate moiety to the 2'-OH end of a preformed 2-5A molecule or to a nucleotide with the structure RpA like NAD', adenosine-(S')tetraphospho(2')adenosine (A,,A) and tRNA (Ball and White, 1979;Justesen et al, 1980;Cayley and Kerr, 1982;Justesen et al, 1985). However, biological relevance of these latter modified nucleotides is not yet clear.…”

Identification of the ATP Binding Domain of Recombinant Human 40-kDa 2′,5′-Oligoadenylate Synthetase by Photoaffinity Labeling with 8-Azido-[α-32P]ATP

“…The donor site binds ATP, 2-5A, NAD ϩ , or other nucleotides with AMP moieties (10 -12). The acceptor site binds ATP, thereby providing the substrate for 2Ј-adenylation (10,11). Kinetic studies suggest a high affinity (K d ϭ 9 M) and a lower affinity (K d ϭ 1000 M) binding site (12).…”

“…2-5A synthetase is a nonprocessive enzyme, as evidenced by the formation of a series of 2Ј,5Ј-oligoadenylates of the formula, 2Ј,5Ј-A(pA) n in which n Ն 1 (10,11). Two nucleotide binding sites on 2-5A synthetase have been suggested as requirements for formation of 2-5A, i.e.…”

Similar biological effects of different low doses of interferon alpha in cancer patients

“…Differential white cell counts were performed using May- (Aguet & Blanchard, 1981). The 2-SA synthetase activity was measured by an assay described earlier (Justesen et al, 1980). Antiviral state was evaluated by an adaptation of the technique of Levin & Hahn (1981 The number of receptor sites/cell decreased markedly without affinity changes within 24 h after the first dose of 0.3 x 106 IU, resulting in a receptor down-regulation of about 70% at 48 h. This latter was then reversed, resulting in a complete recovery of the initial number of receptors.…”