Bovine seminal ribonuclease (BS-RNase), a dimeric homolog of bovine pancreatic ribonuclease A (RNase A), is toxic to mammalian cells. In contrast to dimeric BSRNase, monomeric BS-RNase and RNase A are not cytotoxic and are bound tightly by cytosolic ribonuclease inhibitor. To elucidate the mechanism of ribonuclease cytotoxicity, we constructed a series of hybrid and semisynthetic enzymes and examined their properties. In five hybrid enzymes, divergent residues in BS-RNase were replaced with the analogous residues of RNase A so as to diminish an interaction with a putative cellular receptor. In a semisynthetic enzyme, the disulfide bonds that cross-link the monomeric subunits of dimeric BSRNase were replaced with thioether bonds, which can withstand the reducing environment of the cytosol. Each hybrid and semisynthetic enzyme had ribonucleolytic and cytotoxic activities comparable with those of wild-type BS-RNase. These results suggest that dimeric BS-RNase (pI ‫؍‬ 10.3) enters cells by adsorptive rather than receptor-mediated endocytosis and then evades cytosolic ribonuclease inhibitor so as to degrade cellular RNA. This mechanism accounts for the need for a cytosolic ribonuclease inhibitor and for the cytotoxicity of other homologs of RNase A.Bovine seminal ribonuclease (EC 3.1.27.5, BS-RNase) 1 is a cytotoxic protein. In various assays, this cytotoxicity can manifest itself as an immunosuppressive, antitumor, embryotoxic, and aspermatogenic activity (Dostá l and Motoušek, 1973; Souček et al., 1986;Tamburrini et al., 1990;Matoušek and D'Alessio, 1991;Laccetti et al., 1992), each of which has potential therapeutic value Deonarain and Epenetos, 1994). Among these cytotoxic activities, the immunosuppressive activity is most likely to be physiologically significant, since this activity may be required to suppress the female immune response against components of bull seminal fluid (James and Hargreave, 1984).The cytotoxicity of BS-RNase is related to its quaternary structure. BS-RNase is isolated as a dimer that is cross-linked by two disulfide bonds. RNase A and monomeric BS-RNase are not cytotoxic (Vescia et al., 1980;Tamburrini et al., 1990;Kim et al., 1995b). In contrast, artificially dimerized RNase A is cytotoxic, but to a lesser extent than is BS-RNase (Bartholeyns and Baudhuin, 1976;Bartholeyns and Zenebergh, 1979;Vescia et al., 1980;Di Donato et al., 1994). At equilibrium, dimeric BS-RNase is a mixture of two distinct quaternary forms, MϭM and MxM . The conversion of MϭM to MxM entails the exchange of N-terminal ␣-helices between subunits, as occurs during the artificial dimerization of RNase A. Recently, we and others demonstrated that the MxM form is responsible for the cytotoxicity of BS-RNase Cafaro et al., 1995;Kim et al., 1995b). We also demonstrated that the ribonucleolytic activity of BS-RNase is necessary for its cytotoxic activity (Kim et al., 1995a).The cytosol of cells contains a ribonuclease inhibitor (RI) (Roth, 1967;Blackburn and Moore, 1982;Lee and Vallee, 1993;Hofsteenge, 1994).2 This pro...

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 (...

Bovine seminal ribonuclease (BS-RNase) is a homolog of RNase A with special biological properties that include specific antitumor, aspermatogenic, and immuno-suppressive activities. Unlike RNase A, BS-RNase is a dimer cross-linked by disulfide bonds between Cys31 of one subunit and Cys32 of the other. At equilibrium, this dimer is a mixture of two distinct quaternary forms, M = M and M x M. The conversion of M = M to M x M entails the exchange of NH2-terminal alpha-helices between subunits. Here, the cytotoxic activities of purified M x M were shown to be greater than those of purified M = M, despite extensive equilibration of M = M and M x M during the time course of the assays. Replacing Cys31 or Cys32 with a serine residue did not compromise the enzymatic activity of dimeric BS-RNase, but reduced both the fraction of M x M at equilibrium and the cytotoxicity. We conclude that the M x M form is responsible for the special biological properties of BS-RNase. Since cytosolic ribonuclease inhibitor binds tightly to monomeric but not dimeric BS-RNase and only the M x M form can remain dimeric in the reducing environment of the cytosol, we propose that BS-RNase has evolved its M x M form to retain its lethal enzymatic activity in vivo.

Abstract. Dawn-dusk asymmetries are ubiquitous features of the coupled solar-wind-magnetosphere-ionosphere system. During the last decades, increasing availability of satellite and ground-based measurements has made it possible to study these phenomena in more detail. Numerous publications have documented the existence of persistent asymmetries in processes, properties and topology of plasma structures in various regions of geospace. In this paper, we present a review of our present knowledge of some of the most pronounced dawn-dusk asymmetries. We focus on four key aspects: (1) the role of external influences such as the solar wind and its interaction with the Earth's magnetosphere; (2) properties of the magnetosphere itself; (3) the role of the ionosphere and (4) feedback and coupling between regions. We have also identified potential inconsistencies and gaps in our understanding of dawn-dusk asymmetries in the Earth's magnetosphere and ionosphere.

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