Basic Charges on Mammalian R bonuclease Molecules and the Ability to Attack Double‐Stranded. RNA

Abstract

A ribonuclease recently isolated from the pancreas of the lesser rorqual or pike whale (Baluenoptera acutorostrata), definitely more basic than bovine ribonuclease A, degrades very efficiently doublestranded RNA under conditions where the action of bovine pancreatic ribonuclease is minimal.On the contrary, a ribonuclease from the pancreas of reindeer, definitely less basic than bovine ribonuclease A, is also significantly less active than the bovine enzyme at degrading the doublehelical RNA.Two ribonucleases (from the pancreas of guinea pig, the A component, and red deer), with a net charge and a number of basic amino acids similar to those of bovine pancreatic ribonuclease, show an activity on double-stranded RNA which is indistinguishable from that of bovine ribonuclease A.Finally, a ribonuclease from the pancreas of rat, having a net charge equal to that of bovine ribonuclease A, but three basic residues more than the latter enzyme, shows a modest activity towards the double-helical RNA.These results are compatible with the idea that the action on secondary structures of RNA by ribonucleases non-specific for double-stranded RNA may be related to the number (and possibly the position) of basic charges on the protein molecules. Double-stranded RNA's are specifically degraded by ribonuclease 111 from Escherichiu coli [l -41, whereas the action on them of bovine pancreatic ribonuclease A is negligible, provided the ionic strength is high enough to maintain the secondary structure of the nucleic acids [5,6]. Resistance of RNA to digestion by ribonuclease A under standard conditions [7] is generally assumed, in fact, as one of the criteria to assess that the RNA has a stable secondary structure.Other nucleases have been described as capable of degrading double-stranded as well as single-stranded RNA [S, 91. Among them, bovine seminal ribonuclease (ribonuclease BS-l), a basic (PI = 10.3), dimeric protein [lo-121, in many respects similar to ribonuclease A [lo, 113, definitely degrades double-helical polyribonucleotides [13].Abbreviations. NaCl/Cit, 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7; poly(A), polyadenylate; poly(A) . poly(U) and poly(G) . poly(C), the complexes formed between polyadenylate and polyuridylate, and between polyguanylate and polycytidylate, respectively; poly(rUj . poly(dA), the complex formed by polyribouridylate and polydeoxyriboadenylate.Enzymes. Ribonuclease TI (EC 3.1.4.8); bovine pancreatic ribonuclease A (EC 3.1.4.22); ribonuclease 111 (EC 3.1.4.24). ~-Artificial dimerization [14-161 of bovine ribonuclease A makes this enzyme also capable of significantly depolymerizing double-stranded RNA's [13, 15-171. The dimeric nature of a ribonuclease active on double-stranded RNA, with the simultaneous availability of two active sites on its molecule, was consequently thought to be responsible for the activity towards double-helical polyribonucleotides [ 3 8,191. However, recent results (degradation of DNA . RNA hybrids by ribonuclease , and by aggregates of ribonuclease A [21], the main...

Dimerization of Deoxyribonuclease I, Lysozyme and Papain

“…On the basis of the ideas discussed above, all the present and many of the past observations can be explained, in particular those concerning ribonucleases or ribonuclease derivatives having a higher number of basic charges than bovine RNAase A, all of which appear to be more efficient than RNAase A at degrading double-stranded RNAs at high ionic strengths [l, 3,10,35,38].…”

“…(a) When the number of positively charged groups of an enzyme molecule increases (dimeric versus monomeric seminal RNAase, dimeric lysozyme and papain, but also bovine RNAase A dimers [10,34] or derivatives like polysperminc-ribonuclease [35] versus native RNAase A, as well as very basic RNAases [3]), the optimum of enzyme activity on a polyanionic substrate is shifted to higher ionic strength values (Fig. 3,4, and [l]).…”

“…The interaction between single-stranded or doublestranded RNAs and ribonucleases of the bovine pancreatic type is influenced by several interdependent variables, such as ionic strength and pH of the medium [l], stability of the secondary structure of the nucleic acid duplex [ 2 ] , charge characteristics of the enzyme proteins [3], and the extent of glycosylation of the enzyme molecule [4], all of which affect the efficiency of enzyme action.…”

Biological actions of the oligomers of ribonuclease A

“…Moreover, within each pair of conformational isomers, the more basic conformer is more active than the less basic one. A hypothesis, repeatedly confirmed [5,6,49,50], was advanced years ago [49 -51] to explain the action that ribonucleases more basic than RNase A show against dsRNA. It can also apply to the RNase A multimers.…”

The Solution Structure and Dynamics of Human Pancreatic Ribonuclease Determined by NMR Spectroscopy Provide Insight into Its Remarkable Biological Activities and Inhibition

“…These enhanced activities depend on positively charged residues near but not in the active site. 12,14,15 RNase 1 not only is a digestive enzyme but also plays crucial biological roles; it has been reported to inactivate HIV and might protect the fetus against HIV infection during pregnancy. 16 RNase 1 has been implicated in the regulation of vascular homeostasis 17 and triggers the development of immature dendrites as well as stimulates their production of cytokines.…”