The effect of polyamines on the poly(adenylic acid)-induced inhibition of ribonuclease activity

Abstract

Segments of poly(A) at the 3'-termini of 5 S rRNA inhibit the activities of ribonucleases from Citrobacter, Enterobacter, bovine pancreas, human spleen and human plasma. Certain polyamines, or compounds containing polyamine substructures, mediate reversal of this inhibition. Effective compounds contain three amino groups, at least two of which are charged and are separated from the others by no less than three carbon atoms. Spermidine and 9-aminoacridines, which contain substituted propyl- or butylamino moieties at the 9-amino position and which bear two positive charges per molecule, are efficacious at low concentrations (5 microM). A decrease in effectiveness is associated with the removal of one aromatic ring from the 9-aminoacridines. However, the resulting 4-aminoquinolines, unlike the acridines, do not inhibit enzyme activity when present in concentrations above 30 microM. Relocating the diamino side chain from the 4- to the 8-position of the quinoline nucleus causes a decrease in charge density to +1, with the result that such compounds are ineffective. The orders of polyamine efficacy of reversal of inhibition were similar for enzymes from Citrobacter, bovine pancreas, and human plasma, and paralleled the order of binding of polyamines to either poly(A) or 5 S rRNA. This was not the case with Enterobacter and human spleen RNAases, indicating that the identity of the most effective polyamines depends on the RNAase studied. The combination of variable 3'-terminal poly(A) segment length and polyamine identity and concentration constitutes a system by which RNAase activities, and, therefore, substrate-degradation rates, may be easily varied.

Polyamines bound to nucleic acids during dormancy and activation of tuber cells of Helianthus tuberosus

“…Data published bL evy's group, however, implicated polyamines as factor reversing the in vitro inhibition of RNases by poly(A sequences (e.g. Levy et al 1975, Karpetsky et al 1981 Moreover, spermine prevented in vitro polyadenylation by binding to the primer (various polynucleotides) a-; shown by Rose and Jacob (1976). The order of poiyamine efficacy both in protection from and in stimulation of the activity of several RNases paralleled their order of binding to the different RNAs (spermine > spermidine > putrescine).…”

Poly(adenylic acid) in small amounts, free or covalently linked to substrate, protects RNA from hydrolysis by ribonuclease

“…These results are of particular interest, because, previously, from the results of Tm ('melting' temperature) measurements, differences were found in 1981 the structures of 5 S [3H]rRNA.(A). as n was increased (Karpetsky et al, 1980a). The implication of the present data is that, for the activity of RNAase A, the differences among these substrate molecules are not particularly important.…”

“…Also, for one polynucleotide, the polyamine may abolish any sensitivity of enzyme activity to poly(A) segment length, whereas for another substrate, activity will decline in the presence of polyamine as longer poly(A) tracts are used. These differences in polyamine effects prompted an investigation, reported in the following paper (Karpetsky et al, 1980b), of variation in inhibition reversal with changes in enzyme identity for a number of polyamine analogues.…”

“…That the effect of poly(A) was specific was indicated by the finding that substitution of tracts of poly(C) for the polypurine did not prevent the subsequent enzymic degradation of the RNA to which the tracts were linked (Hieter et al, 1976). Further studies indicated that covalent linkage of poly(A) to RNA yields a molecule having a structure distinct from that expected based on the simple sum of its component parts (Karpetsky et al, 1980a). With some ribonucleases, under certain reaction conditions, profound differences in enzyme activity were found on comparing free homopolymeric poly(A) with tracts of the polypurine covalently linked to substrate.…”

“…And, finally, at the molecular level, what types of mechanisms control the inhibition of RNAases mediated by poly(A) and, therefore, the ambient concentrations of substrate? These questions are addressed in this and the following paper (Karpetsky et al, 1980b), wherein the mechanisms of poly(A) inhibition of RNAase activity are considered along with ways to control this inhibition without changing the concentrations of enzyme, RNA, or poly(A).…”