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After a short introduction with some examples of cytotoxic ribonucleases, the importance of natural or artificial dimerization (oligomerization) as a way for a ribonuclease to acquire novel functional properties has been pointed out. In particular, the role of the three dimensional domain swapping mechanism in bovine pancreatic ribonuclease A oligomerization, as well as its impact for the acquisition of novel biological functions (among which a remarkable antitumor action) by the enzyme protein in oligomeric form have been discussed. Finally, the structural and functional features that could explain why oligomeric ribonuclease A becomes able to display a cytotoxic activity, and the possible use and limits of the three dimensional domain-swapped oligomers of ribonuclease A as anticancer therapeutic agents have been described and discussed.

After a short introduction with some examples of cytotoxic ribonucleases, the importance of natural or artificial dimerization (oligomerization) as a way for a ribonuclease to acquire novel functional properties has been pointed out. In particular, the role of the three dimensional domain swapping mechanism in bovine pancreatic ribonuclease A oligomerization, as well as its impact for the acquisition of novel biological functions (among which a remarkable antitumor action) by the enzyme protein in oligomeric form have been discussed. Finally, the structural and functional features that could explain why oligomeric ribonuclease A becomes able to display a cytotoxic activity, and the possible use and limits of the three dimensional domain-swapped oligomers of ribonuclease A as anticancer therapeutic agents have been described and discussed.

AbstractTo establish the mechanism of dsRNA degradation by mammalian single-stranded-preferring ribonucleases, and, in particular, the influence of their positively charged non-catalytic amino acid residies, we have studied the kinetic parameters of the depolimerization of single- and double-stranded polyribonucleotides such as poly(U), poly(U)·poly(A), poly(C) and poly(C)·poly(I) by the action of human seminal RNase, bovine seminal RNase and ox pancreas RNase A. While the activities of these RNases on poly(I)·poly(C) were definitely lower than those on poly(C), the activities of human seminal and bovine seminal RNases on poly(U)·poly(A) and poly(U) were of the same order of magnitude under physiological salt conditions. The ratio of the RNase A degrading activities towards poly(U) and poly(U)·poly(A) at I = 0.16 M is ten times higher than the corresponding ratios determined with bovine seminal and human seminal ribonucleases. The high activities of these two RNases towards poly(U)·poly(A) are discussed on the basis of their efficient destabilizing action on this double-helical nucleic acid due to their high affinity for poly(A). The destabilizing action of human seminal RNase and bovine seminal RNase on the poly(U)·poly(A) duplex is higher than that measurable with bovine RNase A because of the higher number of positive charges present on those enzyme molecules. This may therefore explain why human seminal and bovine seminal ribonucleases are more efficient than RNase A in the depolymerization of poly(U)·poly(A) at physiological ionic strength.

The catalytic properties and substrate preference of several highly purified human ribonucleases from different organs and body fluids have been examined in detail using various low-molecular-weight compounds and single- or double-stranded polyribonucleotides as substrates. All single-stranded polyribonucleotides were degraded by nonpancreatic-type (npt) RNases at a slower rate than by pancreatic-type (pt) enzymes: ptRNases were 20 times more active on RNA and poly(U) substrates and more than 6000 times more active on poly(C). Pancreatic-type RNases degraded poly(C) faster than RNA, showing a strong preference for poly(C) over poly(U) with the following activity ratios: RNA/poly(C), 0.44; RNA/poly(U), 12; poly(C)/poly(U), 27, In contrast, nptRNases cleaved RNA more rapidly than synthetic homopolymers, preferring poly(U) over poly(C) with the following ratios: RNA/poly(C), 130; RNA/poly(U), 10; poly(C)/poly(U), 0.08. Human ptRNases degraded poly(A) and double-stranded polyribonucleotides about 100 and 400 times faster, respectively, than bovine RNase A. However, no measurable activity could be detected on these substrates with nptRNases. The activities of ptRNases on dinucleoside phosphates (CpN and UpN) or uridine and cytidine 2',3'-cyclic phosphates were similar to those of bovine RNase A; nptRNases, instead, cleaved only CpA and UpA at an appreciable rate. The effects of pH, ionic strength, and divalent cations on the activity of these ribonucleases were also investigated using yeast RNA as a substrate.Copyright 1994, 1999 Academic Press, Inc.

Ohne Zusammenfassung:

The occurrence and some properties of the relatively copious uterine and cervical fluid which is formed following mating or an ovulating dose of gonadotrophin, and the scanty and viscous endometrial secretion produced in the progestational phase of pregnancy in the rabbit, have been reported earlier (Lutwak-Mann, 1962).In the present study the activity was examined of 5-nucleotidase (Heppel & Hilmoe, 1951), adenosinetriphosphatase (ATPase) (Heppel & Hilmoe, 1953), and purine nucleosidase (Price, Otley & Plesner, 1955). The experimental materials were obtained from the animals at autopsy. In each case the hormonal treatment listed below is expressed as dose per animal.Materials referred to as uterine and cervical fluid were obtained from rabbits at: (i) 8–12 hr. after either copulation or i.v. injection of 25 i.u. gonadotrophin (Pregnyl, Organon); (ii) the same stage and from identically treated animals which, however, had been pre-treated 30 hr. earlier with 2–4 mg. of either norethynodrel

Ohne Zusammenfassung:

Although pancreatic ribonucleases are extensively studied proteins, little information is available on nucleic acids coding for these enzymes. Here, for the first time, the structure of a gene coding for such an enzyme, the well known bovine pancreatic ribonuclease, is reported. The coding region of this gene is devoid of introns, whereas the 5′ untranslated sequence of the pancreatic transcript contains an intron of 735 nucleotides. This intervening sequence is endowed with signals (CAAT and TATA boxes) which might act as regulatory elements. The structural organization of this gene suggests that the sequence coding for the bovine pancreatic ribonuclease might be expressed under the control of two different promoters.