Crystallization and crystallographic investigations of ribonucleotide reductase protein R1 from Escherichia coli.

Crystals of Escherichia coli ribonucleotide reductase protein R1 have been grown in complex with a synthetic peptide corresponding to the carboxyl end of protein R2. Good quality crystals could only be obtained after improvement of the purification protocol and are of the space group R32 with hexagonal cell axes a = b = 226 A and c = 341 A. They contain 3 subunits per asymmetric unit and diffract to 2.5 A resolution in synchrotron radiation. A multiple isomorphous replacement map at 5.5A, improved by solvent flattening, shows that the dimeric molecules are elongated, about 110 A long. The dimer is thin in the middle around the molecular two-fold axis. The subunit is shaped like a bowl, probably with the active site in its center.

Structure of oxidized bacteriophage T4 glutaredoxin (thioredoxin). Refinement of native and mutant proteins.

The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 A resolution to a crystallographic R-factor of 0.209. A mutant T4 glutaredoxin gives orthorhombic crystals of better quality. The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 A to an R-value of 0.175. In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin. The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals. On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals. The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts. The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix alpha 1. The torsion angles of these residues are similar to those of Escherichia coli thioredoxin. The torsion angle around the S-S bond is smaller than that normally observed for disulfides: 58 degrees, 67 degrees and 67 degrees for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively. Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom. The active site is shielded from solvent on one side by the beta-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33. From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.

Detailed IgG and IgE antibody patterns during immunotherapy with honey bee venom.

Fourteen patients with a known honey bee venom (HBV) allergy were followed during 1-2 years of immunotherapy. HBV-specific IgG antibody levels increased in all patients but one. HBV-specific IgE antibodies decreased slightly during the first year of therapy. The ratio HBV-specific IgG-/IgE showed a marked increase during the first year for most of the patients, and a further increase during the second year in the four patients followed that long. As could be expected an increased radiostaining was found after 1 year of treatment to all important allergens in IgG CRIE, but after 2 years a sustained or increased radiostaining was obtained to phospholipase (PLA) alone. A decreased radiostaining might more easily be seen with weaker immunogens.

IgG and IgE antibody patterns after immunotherapy with monomethoxy polyethyleneglycol modified honey bee venom.

Antibody responses to honey bee venom (HBV) were studied in 13 patients during a 4-month course of immunotherapy with monomethoxy polyethyleneglycol (mPEG) modified venom. There was a rise of HBV-specific IgG antibodies as measured by IgG-RAST in all patients and a slight decrease of IgE antibody in most of them. The IgG-antibody responses during mPEG-HBV treatment as examined by crossed radioimmunoelectrophoresis were directed to phospholipase A, hyaluronidase, acid phosphatase and to another allergen, antigen 1. Thus, despite a high degree of mPEG-modification of HBV, the immunogenicity of the most important HBV allergens was retained.

Crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis analysis of "yellow jacket-common wasp" (Vespula spp.).

Crossed immunoelectrophoresis (CIE) pattern of Yellow jacket vespid venom showing 25 immunoprecipitates was used in crossed radioimmunoelectrophoresis (CRIE) analysis with sera from 35 subjects allergic to vespid venom. Phospholipase A and B, hyaluronidase, acid and alkaline phosphatases and a lytic active component were demonstrated in immunoprecipitates using zymography and direct haemolysis of sheep erythrocytes. CRIE analysis proved the allergenicity of these proteins. In addition, another antigen (Ag 17) with no detectable enzymatic or lytic activity was identified as an important allergen. This allergen is most likely identical to the previously described Antigen 5 in vespid venoms. By using CIE/CRIE the presence and complexity of multiple forms of the allergenic protein components were well demonstrated.

Bee keepers' IgG and IgE antibody responses to bee venom studied by means of crossed radioimmunoelectrophoresis.

The immune response to honey bee venom in thirty-seven bee keepers' sera was studied by several methods. Specific IgE antibody levels studied by RAST were generally low, whereas specific IgG antibody levels studied by a Sepharose protein A technique were high. Crossed radioimmunoelectrophoresis was applied for a detailed analysis of the antibody specificities towards the different components of venom in seventeen of the bee keepers' sera. Significant amounts of IgG antibodies were found towards most bee-venom components. The highest IgG response was directed towards phospholipase A. Hyaluronidase, acid phosphatase and two uncharacterized antigens also showed distinct IgG binding. The IgG binding to melittin was low. The IgE binding to the bee venom components was low and primarily directed to the phospholipase. IgE binding to hyaluronidase and acid phosphatase occurred, but was also in very small amounts. One bee-keeper serum caused heavy radiostaining to melittin but the others did not show IgE binding to this component. Thus a low IgE but a high IgG response was demonstrated in bee keepers. The major immunogen was phospholipase A, which is known to be the major allergen in bee venom. Generally, the strongest IgG responses were found to the components capable of inducing the strongest IgE responses.

Antigenic/allergenic composition of Poodle/Alsatian dandruff extract.

Poodle and Alsatian dog dandruff extracts were characterized by crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) using sera from 24 individuals clinically sensitive to dogs. By using a system with intermediate gel in immunoelectrophoresis, the content of dander-specific and serum-specific allergens was established. 29 antigens (18 dander-specific and 11 serum-specific) were identified in the mixed breed Poodle/Alsation dandruff extract. Of these, 24 antigens were radiostained in CRIE. 16 allergens were dander specific and the remaining eight were serum specific. Positive dog dander RAST (e5 and Poodle/Alsatian dandruff extract) results were observed in the tested dog hypersensitive subjects. Our results suggest that the mixture of Poodle/Alsatian dandruff extract may be a suitable preparation for the diagnosis and treatment of dog allergy.

A rapid procedure for crossed radio immunoelectrophoresis.

A procedure for performing crossed radio immunoelectrophoresis (CRIE) with significantly reduced exposure times has been developed. The rapid method has been effected by using a highly sensitive X-ray film (Du Pont Cronex-4) and X-ray intensifying screen (Du Pont Lighting Plus) and by exposing at low temperatures (-70 degrees C) in a Kodak X-Omatic cassette. Exposure at -70 degrees C appears to be critical to obtain optimal radiostaining. Furthermore, the isotope concentration has been increased from 250,000 to 500,000 cpm without any loss of specificity or reproducibility. The rapid CRIE procedure makes it possible to obtain a CRIE screening within 1 day and, furthermore, a complete CRIE classification (CRIE classes A-G) within 5 days.