The degradation of 2ʹ, 5ʹ-linked oligoadenylates by metals

Author

Alice Marie George

Date of Award

8-1984

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Walter R. Farkas

Committee Members

Jeff Becker, Gary Sayler

Abstract

Cells treated with interferon produce a family of 2¹,5¹ linked oligoadenylates, pppA(2¹p5¹A)_n, which inhibit protein synthesis via activation of a latent nuclease and which have been implicated in the antiviral action of interferon. The most abundant of the oligoadenylates is the trimer which must be phosphorylated at the 5¹ end to be biologically active. Since it was previously shown in this laboratory that Pb²⁺ caused the depolymerization of RNA, experiments were conducted to determine if Pb²⁺ would degrade this 2¹,5¹ (A) as well. If this were the case, elevated levels of Pb²⁺ or other metals in vivo might interfere with the antiviral action of interferon. The reaction was followed using reverse-phase high performance liquid chromatography. A number of metals, including Pb²⁺ and Fe³⁺ were found to degrade the nonphosphorylated trimer "core," 2¹ApApA at a concentration of 0.001 M when incubated at 37°C. The compound 2¹,3¹ cyclic AMP was detected among the degradation products indicating that the reaction proceeded via a cyclic intermediate.

Phosphorylation of the tri(A) at the 5¹ end greatly increased the rate of its degradation by Pb²⁺. The rate constant for the degradation of the core compound was 0.01 Hr^-1 while the rate constants for 2¹pApApA and 2¹pppApApA were 1.2 and 1.6 Hr^-1 respectively. With Fe³⁺, the rate of degradation was also affected by 5¹ phosphorylation. The core was degraded by Fe³⁺ with a rate constant of 0.01 Hr^-1 while the monophosphate was degraded very rapidly with a rate constant of 2.5 Hr^-1. Unlike with Pb²⁺, Fe³⁺ degraded the triphosphate at a rate slower than that with the monophosphate; the rate constant for the degradation of the triphosphate by Fe³⁺ was 0.06 Hr^-1 . The other metal tested, Nd³⁺ , degraded the core faster than either Pb²⁺ or Fe³⁺ (rate constant 0.05 Hr^-1), but the rate of degradation of the phosphorylated derivatives by Nd³⁺ was slower than the core with both 2¹pApApA and 2¹pppApApA having rate constants of 0.01 Hr^-1.

In addition to tri(A), the susceptibility of 2¹,5¹ and 3¹,5¹ triinosinic acid to Pb²⁺ and Fe³⁺ was also tested. The rate constants for the degradation of 2¹ and 3¹ tri(I) by Pb²⁺ were 0.04 and 0.03 Hr^-1 respectively, compared to a rate constant of 0.01 Hr^-1 for the degradation of tri(A) by Pb²⁺. The rate constants for the 3+ degradation of all three of these trimers by Fe³⁺ were the same, 0.01 Hr^-1.

The extent to which Fe³⁺ degraded the tri(A) was susprising since under experimental conditions, Fe³⁺ was not in solution, but was present as a colloidal suspension of solid ferric hydroxide. The degradation of the tri(A) by Fe³⁺ was due to solid phase catalysis since once the solid Fe(0H)₃ was removed by centrifugation, the supernatant did not degrade the tri(A).

When incubated with tRNA and polyadenylic acid, Pb²⁺ caused extensive degradation after only 1 hour at 37°C. Fe³⁺ on the other hand, did not degrade tRNA or poly (A), even when incubated with poly(A) for 96 hours at 37°C.

Recommended Citation

George, Alice Marie, "The degradation of 2ʹ, 5ʹ-linked oligoadenylates by metals. " Master's Thesis, University of Tennessee, 1984.
https://trace.tennessee.edu/utk_gradthes/14629