Rabbit hemorrhagic disease virus (RHDV) causes more than 90% mortality in adult rabbits. In this study, the cDNA of the VP60 coding sequence of RHDV was cloned under the control of the polyhedrin and p10 promoters of baculovirus to be expressed in insect cells. The expression of RHDV VP60 under the control of the p10 promoter was 5–10 times higher than using the polyhedrin promoter. The p10-derived VP60 was able to assemble into virus-like particles (VLPs). RHDV VLPs were successfully used to protect rabbits against the disease even at doses as low as 0.5 µg when injected intramuscularly or sub-cutaneously. The ability to elicit an immune response was independent of the adjuvant or the route of immunization. Remarkably, oral administration of RHDV VLPs efficiently induced protecting antibodies to RHD at doses as low as 3 µg. The use of binary ethylenimine for the stabilization of the VLPs was decisive for eliciting a good oral immunity. This report demonstrates the potential use of these procapsids in obtaining RHD oral vaccines and opens the door to the use of these capsids for the prevention of the disease in wild animals. Therefore, a new, and potentially important application of recombinant VLPs in the induction of protective immunity by the oral route is foreseen.

The R2 protein of ribonucleotide reductase from Escherichia coli is a homodimeric tyrosyl-radical-containing enzyme with two identical dinuclear iron centers. Two randomly generated genomic mutants, nrdB-1 and nrdB-2, that produce R2 enzymes with low enzymatic activity, have been cloned and characterized to identify functionally important residues and areas of the enzyme. The mutations were identified as Pro348 to leucine in nrdB-1 and Leu304 to phenylalanine in nrdB-2. Both mutations are the results of single amino acid replacements of non-conserved residues. The three-dimensional structures of [L348]R2 and [F304]R2 have been determined to 0.26-nm and 0.28-nm resolution, respectively. Compared with wild-type R2, [L348]R2 binds with higher affinity to R1, probably due to increased flexibility of its C-terminus. Since the three-dimensional structure, iron-center properties and radical properties of [L348]R2 are comparable to those of wild-type R2, the low catalytic activity of the holoenzyme is probably caused by a perturbed interaction between R2 and R1. The [F304]R2 enzyme has increased radical sensitivity and low catalytic activity compared with wild-type R2. In [F304]R2 the only significant change in structure is that the evolutionary conserved Ser211 forms a different hydrogen bond to a distorted helix. The results obtained with [F304]R2 indicate that structural changes in E. coli R2 in the vicinity of this helix distortion can influence the catalytic activity of the holoenzyme.

The active form of protein R2, the small subunit of ribonucleotide reductase, contains a diferric center and a free radical localized at Tyr122. Hydroxyurea scavenges this radical but leaves the iron center intact. The resulting metR2 protein is inactive. The introduction of a radical into metR2 is dependent on the reduction of the iron center. In Escherichia coli, this is achieved by an enzyme system consisting of a NAD(P)H:flavin oxidoreductase and a poorly defined protein fraction, fraction b. Assuming that the iron center is deeply buried within the protein, electron transfer is suggested to occur over long distances. Site-directed mutagenesis allowed us to identify two invariant residues, Tyr356 at the C-terminal part of the protein and Tyr122 located 0.5 nm away from the closest iron atom, as mediators of this electron transfer. We also found that deazaflavins were excellent catalysts in the photoreduction of the iron center of metR2 and generation of the tyrosyl radical, providing the simplest and most efficient model for the physiological flavin reductase/fraction b activating system. The properties of the model reaction are described.

Ribonucleotide reductase from Escherichia coli consists of two dissociable, nonidentical homodimeric proteins called R1 and R2. The role of the C-terminal region of R2 in forming the R1R2 active complex has been studied. A heterodimeric R2 form with a full-length polypeptide chain and a truncated one missing the last 30 carboxyl-terminal residues was engineered by site-directed mutagenesis. Kinetic analysis of the binding of this protein to R1, compared with full-length or truncated homodimer, revealed that the C-terminal end of R2 accounts for all of its interactions with R1. The intrinsic dissociation constant of the heterodimeric R2 form, with only one contact to R1, 13 microM, is of the same magnitude as that obtained previously [Climent, I., Sjöberg, B.-M., & Huang, C. Y. (1991) Biochemistry 30, 5164-5171] for synthetic C-terminal peptides, 15-18 microM. We have also mutagenized the only two invariant residues localized at the C-terminal region of R2, glutamic acid-350 and tyrosine-356, to alanine. The binding of these mutant proteins to R1 remains tight, but their catalytic activity is severely affected. While E350A protein exhibits a low (240 times less active than the wild-type) but definitive activity, Y356A is completely inactive. A catalytic rather than structural role for these residues is discussed.

The active complex of Escherichia coli ribonucleotide reductase comprises two dissociable, nonidentical homodimeric proteins, B1 and B2. When B2 is the varied component, the reductase activity is competitively inhibited by synthetic peptides of varying lengths corresponding to the C-terminus of protein B2. This finding provides the first evidence that the C-terminal peptides and protein B2 share the same binding domain on protein B1. Our data also show that two molecules of peptide can bind to protein B1 with equal affinity. Similar inhibition constants (18 microM) were obtained for peptides containing the C-terminal 20, 30, and 37 residues. When the invariant residue Tyr 356 was omitted, a 2-fold decrease in peptide inhibitory ability was observed. A small peptide, lacking the last 11 residues, had virtually no inhibitory potency. These results, coupled with our previous observations that truncated protein B2, in which one or both polypeptide chains are missing approximately 24 C-terminal residues, had considerably lower or no affinity for B1, suggest that the C-terminal regions are the major determinants in the B1-B2 interaction. In the Appendix, two methods for treatment of kinetic situations pertinent to the ribonucleotide reductase system are presented. One method deals with the determination of kinetic parameters for two components present at comparable levels; the other is concerned with the differentiation of linear and nonlinear competitive inhibition involving the binding of two inhibitor molecules. Both methods should find application to other similar cases.

Metal-catalyzed oxidative modification of proteins is implicated in a number of physiologic and pathologic processes. The reaction is presumed to proceed via a site-specific free radical mechanism, with the site-specificity conferred by a cation-binding site on the protein. The oxidation of bacterial glutamine synthetase has been studied in detail, providing the opportunity to examine whether the oxidation is consistent with a site-specific radical reaction. Oxidation leads to the appearance of carbonyl groups in amino acid side chains of the protein, and labeling of those carbonyl groups with fluorescein-amine facilitated purification of the oxidized peptide from a tryptic digest. The oxidized residue was arginine-344, which was converted to a gamma-glutamyl semialdehyde residue. Histidine-269 had previously been shown to be converted to asparagine during metal-catalyzed oxidation. Both arginine-344 and histidine-269 are situated at the metal-nucleotide binding pocket of the enzyme's active site, thus establishing the site-specificity of the oxidation.

This chapter discusses methods to determine carbonyl content in oxidatively modified proteins. The methods described are (1) reduction of the carbonyl group to an alcohol with tritiated borohydride; (2) reaction of the carbonyl group with 2,4-dinitrophenylhydrazine to form the 2,4-dinitrophenylhydrazone; (3) reaction of the carbonyl with fluorescein thiosemicarbazide to form the thiosemicarbazone; and (4) reaction of the carbonyl group with fluorescein amine to form a Schiff base followed by reduction to the secondary amine with cyanoborohydride. Van Poelje and Snell have also quantitated protein-bound pyruvoyl groups through formation of a Schiff base with p-aminobenzoic acid followed by reduction with cyanoborohydride. Although a systematic investigation has not appeared, this method should also be useful in detecting other protein-bound carbonyl groups. Carbonyl content of proteins is expressed as moles carbonyl/mole subunit for purified proteins of known molecular weight. For extracts, the results may be given as nanomoles carbonyl/milligram protein. For a protein having a molecular weight of 50,000, a carbonyl content of 1 mol carbonyl/mol protein corresponds to 20 nmol carbonyl/mg proteins.

Oxidative modification of proteins is implicated in a number of physiologic and pathologic processes. Metal-catalyzed oxidative modification usually causes inactivation of enzymes and the appearance of carbonyl groups in amino acid side chains of the protein. We describe use of fluoresceinamine to label certain of those carbonyl groups. Fluoresceinamine reacted with those carbonyl groups to form a Schiff base which was reduced by cyanoborohydride to yield a stable chromophore on the oxidized residue. The high molar absorbtivity of the fluorescein moiety conferred high sensitivity upon the method. Labeled peptides were readily identified after tryptic digestion of oxidized glutamine synthetase. Further, acid hydrolysis of labeled glutamine synthetase allowed isolation of the derivatized, oxidized residue. The oxidized amino acid was identified as gamma-glutamyl semialdehyde. During metal-catalyzed oxidation, the inactivation of glutamine synthetase paralleled the appearance of gamma-glutamyl semialdehyde.

When we incubated biotin carboxylase from Escherichia coli with ATP in absence of biotin we observed HCO3- -dependent ATP hydrolysis, which was activated by 10% ethanol in the same proportion as the activity of D-biotin carboxylation assayed in the presence of biotin. The two activities exhibited identical heat stability and were protected equally by glycerol; both required Mg2+ and K+ and showed similar dependency on the concentration of ATP. Biotin assay excluded potential contamination by traces of biotin as a cause of the observed ATP hydrolysis, and this was confirmed by the findings that carboxybiotin did not accumulate and that avidin was uninhibitory. Therefore we concluded that this HCO3- -dependent ATPase was genuinely a partial activity of biotin carboxylase. This partial activity supports a sequential mechanism for enzymatic carboxylation of biotin in which HCO3- is activated by ATP in a first step. It is consistent with the initial formation of the carbonic-phosphoric anhydride (HOCO2PO3(2-)), and it does not agree with models where biotin is phosphorylated by ATP prior to reaction with HCO3-. It appears that enzymes that use HCO3- for carboxylation, including biotin-dependent carboxylases, phosphoenolpyruvate carboxylase, and carbamoyl phosphate synthetase, activate HCO3- by a common mechanism involving the initial formation of the carbonic-phosphoric anhydride.