Lyophilization of bovine ribonuclease A (RNase A; Sigma, type XII-A) from 40% acetic acid solutions leads to the formation of < 14 aggregated species that can be separated by ion-exchange chromatography. Several aggregates were identified, including two variously deamidated dimeric subspecies, two distinct trimeric and two distinct tetrameric RNase A conformers, besides the two forms of dimer characterized previously [Gotte, G. & Libonati, M. (1998) Two different forms of aggregated dimers of ribonuclease A. Biochim. Biophys. Acta 1386, 106±112]. We also have possible evidence for the existence of two forms of pentameric RNase A. The two forms of trimers and tetramers are characterized by: (a) slightly different gel filtration patterns; (b) different retention times in ion-exchange chromatography; and (c) different mobilities in cathodic gel electrophoresis under nondenaturing conditions. Therefore, they appear to have distinct structural organizations responsible for a different availability of their positively charged amino acid residues. All RNase A oligomers, in particular the two distinct trimeric and tetrameric conformers, degrade poly(A)´poly(U), viral double-stranded RNA and polyadenylate with a catalytic efficiency that is in general higher for the more basic species. On the contrary, the activity of the RNase A oligomers, from dimer to pentamer, on yeast RNA and poly(C) (Kunitz assay) is lower than that of monomeric RNase A.

Bovine pancreatic RNase A (ribonuclease A) aggregates to form various types of catalytically active oligomers during lyophilization from aqueous acetic acid solutions. Each oligomeric species is present in at least two conformational isomers. The structures of two dimers and one of the two trimers have been solved, while plausible models have been proposed for the structures of a second trimer and two tetrameric conformers. In this review, these structures, as well as the general conditions for RNase A oligomerization, based on the well known 3D (three-dimensional) domain-swapping mechanism, are described and discussed. Attention is also focused on some functional properties of the RNase A oligomers. Their enzymic activities, particularly their ability to degrade double-stranded RNAs and polyadenylate, are summarized and discussed. The same is true for the remarkable antitumour activity of the oligomers, displayed in vitro and in vivo, in contrast with monomeric RNase A, which lacks these activities. The RNase A multimers also show an aspermatogenic action, but lack any detectable embryotoxicity. The fact that both activity against double-stranded RNA and the antitumour action increase with the size of the oligomer suggests that these activities may share a common structural requirement, such as a high number or density of positive charges present on the RNase A oligomers.

Bovine pancreatic ribonuclease (RNase A) forms two types of dimers (a major and a minor component) upon concentration in mild acid. These two dimers exhibit different biophysical and biochemical properties. Earlier we reported that the minor dimer forms by swapping its N-terminal alpha-helix with that of an identical molecule. Here we find that the major dimer forms by swapping its C-terminal beta-strand, thus revealing the first example of three-dimensional (3D) domain swapping taking place in different parts of the same protein. This feature permits RNase A to form tightly bonded higher oligomers. The hinge loop of the major dimer, connecting the swapped beta-strand to the protein core, resembles a short segment of the polar zipper proposed by Perutz and suggests a model for aggregate formation by 3D domain swapping with a polar zipper.

Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) · poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) · poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.Keywords: RNase A oligomers; trimers and tetramers of RNase A; properties of trimeric and tetrameric RNase A; RNase A aggregates higher than dimersThe study of the manner in which proteins aggregate in vitro can help in understanding the process of protein aggregation in vivo, and, therefore, also the origin of pathologic proteins responsible for several severe diseases.Although it has recently been reported that native ribonuclease A can dimerize at neutral pH (Park and Raines 2000), it is known that by lyophilization from 30-50% acetic acid solutions RNase A gives rise to oligomers (Crestfield et al. 1962) ranging from dimers to pentamers and possibly higher aggregates, each oligomeric species existing in the form of at least two conformational isomers (Gotte et al. 1999).Dimeric RNase A obtained by lyophilization consists of a minor and a major component that are in the ratio of about 1:3-1:4. Their crystal structures have been determined (Liu et al. 1998(Liu et al. , 2001, and the two dimeric conformers are 3D domain-swapping dimers formed by the exchange of the N-terminal ␣-helix of each monomeric subunit in the case of the minor dimer, of the C-terminal ␤-strand, instead, for the major RNase A dimer. This unique behavior of RNase A, in the dimeric aggregates of which the two known mechanisms of protein aggregation by domain swapping coexist, expands the range of possibi...

When concentrated in mildly acidic solutions, bovine pancreatic ribonuclease (RNase A) forms long-lived oligomers including two types of dimer, two types of trimer, and higher oligomers. In previous crystallographic work, we found that the major dimeric component forms by a swapping of the C-terminal ␤-strands between the monomers, and that the minor dimeric component forms by swapping the N-terminal ␣-helices of the monomers. On the basis of these structures, we proposed that a linear RNase A trimer can form from a central molecule that simultaneously swaps its N-terminal helix with a second RNase A molecule and its C-terminal strand with a third molecule. Studies by dissociation are consistent with this model for the major trimeric component: the major trimer dissociates into both the major and the minor dimers, as well as monomers. In contrast, the minor trimer component dissociates into the monomer and the major dimer. This suggests that the minor trimer is cyclic, formed from three monomers that swap their C-terminal ␤-strands into identical molecules. These conclusions are supported by cross-linking of lysyl residues, showing that the major trimer swaps its N-terminal helix, and the minor trimer does not. We verified by X-ray crystallography the proposed cyclic structure for the minor trimer, with swapping of the C-terminal ␤-strands. This study thus expands the variety of domain-swapped oligomers by revealing the first example of a protein that can form both a linear and a cyclic domain-swapped oligomer. These structures permit interpretation of the enzymatic activities of the RNase A oligomers on double-stranded RNA.Keywords: 3D domain swapping; bovine pancreatic ribonuclease; protein oligomerization; enzyme activity on double stranded RNA; cross-linking 3D domain swapping is a mechanism for protein oligomerization, in which two or more molecules of the same protein form an oligomer by exchanging identical domains. In a 3D domain-swapped dimer, one subunit lends a domain to replace the identical domain of the other subunit, and vice versa. The interface that exists between the swapped domain and the rest of the protein in both the monomer and the domain-swapped oligomer is termed the closed interface. The interface that exists only in the domain-swapped oligomer is termed the open interface. The loop presenting different conformations in the monomer and the domainswapped oligomer is termed the hinge loop. Since 3D domain swapping was found in the dimeric structure of diphtheria toxin (Bennett et al. 1994), more than 30 proteins have been reported to be domain swapped. 3D domain swapping is a possible mechanism for the formation of protein aggregates including amyloids (Klafki et al. 1993;Bennett et al. 1995;Schlunegger et al. 1997;Cohen and Prusiner 1998;Liu et al. 1998Liu et al. , 2001. 3D domain swapping also endows proteins with additional properties, for exReprint requests to: David Eisenberg, University of California, Laboratory of Structural Biology and Molecular Medicine, Department of Che...

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.Bovine ribonuclease A oligomerizes in the forms of dimers (1), trimers, tetramers, and higher order oligomers (2) during lyophilization from 40% acetic acid solutions. Each oligomer consists of two conformational isomers, which can be separated by cation exchange chromatography into a less basic and a more basic species (2, 3). The molecular structures of the two dimers have been solved (4, 5). They form by a three-dimensional domain-swapping mechanism (6); the less basic dimer, formerly named minor because of its ratio of 1:4 to the more basic dimer (2,3,5), is formed by the swapping of the Nterminal ␣-helix (residues 1-15) of each monomeric subunit, and the more basic or major dimer (2, 3, 5) is formed by the swapping of the C-terminal ␤-strand (residues 116 -124) of each monomer. On this basis, the two dimers will be called N-dimer and C-dimer, respectively. The structure of the more basic or minor trimer (2, 3) has also been solved; it is formed by three monomers linked to each other by swapping their Cterminal ␤-strands, thereby forming a circular structure that looks like a propeller (7). It will be called the C-trimer in this paper. On the basis of its dissociation products (3, 7), a plausible linear model was proposed for the less basic, major trimer (its abundance is 1.5 times that of the more basic, minor trimer). In this linear model, two monomers are linked through swapping of their N termini, and a third monomer is bound to one of them by C-terminal domain swapping (5, 7). It will be called the NC-trimer. Two linear structures for the two tetramers, the less basic minor and the more basic major (ratio, 1:1.6), have also been proposed on the basis of their dissociation products (...

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The evidence that nitric oxide (NO) production is possible by a non-enzymatic pathway has already been shown under restrictive experimental conditions. Here we show that NO can non-enzymatically be formed with short-time kinetics (min), by 'bombing' with shock waves a solution containing 1 mM hydrogen peroxide and 10 mM L-arginine. This procedure is widening its medical application with surprisingly positive effects in tissue regeneration and our finding could be one of the first steps for the understanding of the biochemical responsible for these therapeutical effects. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.