Bovine seminal ribonuclease, the only dimeric ribonuclease described thus far, is found to exist in two different quaternary structure forms. In one, the N-terminal segment (residues 1-17) of each subunit is interchanged with the remaining segment of the other subunit, whereas in the second, such interchange does not occur. Functionally, they differ in that the catalytic activity of the form with interchange can be modulated by the substrate, whereas the noninterchange form exhibits no cooperativity. Each form can convert into the other, up to an equilibrium ratio, which is that found for the isolated protein. The results of refolding experiments of unfolded protein chains suggest that also in vivo the form lacking interchange may be produced first and is then partially transformed into the other dimeric form until equilibrium is reached. Although the implications of these findings may not be immediately apparent, they are intriguing and may have an impact on the unusual noncatalytic actions of the protein, such as its selective cytotoxicity toward tumor cells, activated T cells, and differentiated male germ cells.Bovine seminal ribonuclease (BS-RNase) is a homodimeric ribonuclease-in fact, the only dimeric RNase isolated thus far. Its enzymic properties are unusual for a ribonuclease, as it cleaves effectively single-and double-stranded RNA and is allosterically regulated in the rate-determining step of the reaction. These properties are matched by its unusual biological, noncatalytic actions, including its selective toxicity toward tumor cells, activated T cells, and the male germ cell line (ref. 1 and references cited therein).In the structure of naturally dimeric BS-RNase determined by x-ray crystallography (2, 3), the two subunits interchange their N-terminal segments (see Fig. 1A) as do the monomers of artificially dimerized bovin' pancreatic RNase (RNase A) in the structure proposed by Crestfield and others (4, 5). A significant consequeoce of ths 'structural arrangement is the composite nature 9f the active sites of dimerized RNase A and of naturally dimeric BS-RNase. Composite, shared active sites have been previously found for a few oligomeric enzymes (6-9). However, in all of these cases, dissociation of the oligomers produced inactive monomers. In contrast, isolated BS-RNase monomers are active-in fact, more active than the parent dimeric enzyme, although they lack its typical allosteric properties (10).The dimeric structure of BS-RNase is maintained not only by noncovalent interactions between subunits (3) but also by two intersubunit disulfides, which link Cys-31 and Cys-32 of one subunit with Cys-32 and Cys-31, respectively, of the other subunit (11, 12). We have previously observed (12) that selective reduction of the intersubunit disulfides monomerizes only a fraction of the dimeric protein. This observation, which had not been fully understood thus far, has since been confirmed repeatedly, also in other laboratories (13), with protein preparations obtained with different purification ...

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Background Among the peach-derived allergens which are already known, the lipid transfer protein (Pru p 3) seems to be the one to exert severe allergic reactions. Objective To identify and characterize a new peach allergen causing a clinical picture similar to that of Pru p 3. Methods Patients were selected on the basis of their severe clinical reactivity and negative results to a panel of peach allergens available on the ISAC103 microarray. Several inhouse and commercial preparations were compared. Several methods were used to characterize the newly identified molecule. Specific IgE and inhibition assays were performed using the Allergen micro-Beads Array (ABA) assay. Results Negative ISAC results to Pru p 3 were confirmed by additional testing in contrast with the positive results obtained by commercial Pru p 3-enriched peach peel extracts. The analyses of one of these preparations led to the identification of Peamaclein, a new allergenic protein. It is a small, basic, cysteine-rich, heat-stable, digestion-resistant protein, homologous to a potato antimicrobial peptide. Peamaclein was able to trigger positive skin test reactions and to bind IgE in the ABA assay. It displays an electrophoretic mobility and chromatographic behaviour similar to that of Pru p 3; therefore, it can be hidden in Pru p 3 preparations. In fact, Pru p 3-enriched peach peel extracts were found to contain both Pru p 3 and Peamaclein by means of comparative in vivo testing, and by biochemical and immunochemical assays. Commercially available anti-Pru p 3 polyclonal antibodies were found to have a double specificity for the two molecules. Conclusions and Clinical Relevance A new allergen from peach belonging to a new family of allergenic proteins has been identified and characterized. This knowledge on Peamaclein will improve our understanding on the clinical aspects of the peach allergy and the quality of diagnostic reagents.

BackgroundFood allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet.ObjectiveUsing the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins.MethodsTwo new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out.ResultsAlignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients' diet.ConclusionThe biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention.

Kiwellin is a novel protein of 28 kDa isolated from kiwi (Actinidia chinensis) fruit. It is one of the three most abundant proteins present in the edible part of this fruit. Kiwellin has been purified by ion exchange chromatography. Its N-terminal amino acid sequence revealed high identity with that previously reported for a 28 kDa protein described as one of the most important kiwi allergens. This observation prompted us to fully characterize this protein. The complete primary structure, elucidated by direct sequencing, indicated that kiwellin is a cysteine-rich protein. Serological tests and Western Blotting analysis showed that kiwellin is specifically recognized by IgE of patients allergic to kiwi fruit.