A coleopteran triosephosphate isomerase: X-ray structure and phylogenetic impact of insect sequences.

A coleopteran triosephosphate isomerase (TIM) from Tenebrio molitor (yellow mealworm beetle) was recombinantly expressed in Escherichia coli and characterized with respect to thermal stability, kinetic parameters and oligomeric state. The enzyme was successfully crystallized and the structure determined by X-ray analysis to 2.0 A resolution. This is the first example of an invertebrate TIM. We compare structural features with known structures of TIMs from microorganisms, plants and vertebrates, and discuss the utility of the Tenebrio TIM sequence, together with several newly sequenced insect TIMs, for molecular phylogenetic analysis.

Anti-c-myc antibody 9E10: epitope key positions and variability characterized using peptide spot synthesis on cellulose.

The 9E10 antibody epitope (EQKLISEEDL) derives from a protein sequence in the human proto-oncogen p62(c-myc) and is widely used as a protein fusion tag. This myc-tag is a powerful tool in protein localization, immunochemistry, ELISA or protein purification. Here, we characterize the myc-tag epitope by substitutional analysis and length variation using peptide spot synthesis on cellulose. The key amino acids of this interaction are the core residues LISE. The shortest peptide with a strong binding signal is KLISEEDL. Dissociation constants of selected peptide variants to the antibody 9E10 were determined. scFv constructs with the shortest possible myc-tags were successfully detected by Western blot and ELISA, giving a signal comparable to that of the original myc-tag.

Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody.

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr(32)Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe(94)Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe(94)Ala up to the level of the e-pep affinity to the wild-type scFv.

Crystal structure of a phage library-derived single-chain Fv fragment complexed with turkey egg-white lysozyme at 2.0 A resolution.

The three-dimensional structure of the single-chain Fv fragment 1F9 in complex with turkey egg-white lysozyme (TEL) has been determined to a nominal resolution of 2.0 A by X-ray diffraction. The scFv fragment 1F9 was derived from phage-display libraries in two steps and binds both hen and turkey egg-white lysozyme, although the level of binding affinity is two orders of magnitude greater for the turkey lysozyme. The comparison of the crystal structure with a model of the single-chain Fv fragment 1F9 in complex with hen egg-white lysozyme (HEL) reveals that in the latter a clash between Asp101 in lysozyme and Trp98 of the complementarity determining region H3 of the heavy chain variable domain occurs. This is the only explanation apparent from the crystal structure for the better binding of TEL compared to HEL. The binding site topology on the paratope is not simply a planar surface as is usually found in antibody-protein interfaces, but includes a cleft between the light chain variable domain and heavy chain variable domain large enough to accommodate a loop from the lysozyme. The scFv fragment 1F9 recognizes an epitope on TEL that differs from the three antigenic determinants recognized in other known crystal structures of monoclonal antibodies in complex with lysozyme.

Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments.

Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.

A phage library-derived single-chain Fv fragment in complex with turkey egg-white lysozyme: characterization, crystallization and preliminary X-ray analysis.

Using phage-display, an anti-turkey egg-white lysozyme single-chain Fv fragment was selected from a naive light chain variable region repertoire in combination with a heavy chain variable region 'mini library' of anti-hen egg-white lysozyme single-domain binders (Ward et al., 1989). Whereas the selected VH domain alone binds somewhat better hen egg-white lysozyme than turkey egg-white lysozyme, but both with comparatively low affinity, the specificity of VH is converted by addition of the VL domain. Thus, the single-chain Fv fragment is more specific for turkey egg-white lysozyme, with markedly increased affinities towards both lysozymes. The complex of single-chain Fv with turkey lysozyme has been crystallized and characterized by preliminary X-ray analysis.

High fecundity rates following in-vitro fertilization and embryo transfer in antiphospholipid antibody seropositive women treated with heparin and aspirin.

This study was undertaken to explore whether intervention with heparin and aspirin (H/A) in selected patients undergoing in-vitro fertilization (IVF) and embryo transfer could improve fecundity rates. Specifically, it explored the possibility that women diagnosed with organic pelvic disease who demonstrated antiphospholipid antibodies (APA) could benefit from H/A administration in a similar manner to that used in patients with recurrent pregnancy loss. We used an enzyme-linked immunosorbent assay for six different phospholipids to identify patients who expressed APA before they underwent IVF/embryo transfer. This study was confined to the first IVF/embryo transfer cycle that followed assessment of APA status and accordingly, the number of IVF/embryo transfer cycles corresponds with the number of patients treated. APA seropositive patients were treated with aspirin, 81 mg orally q.d., and heparin 5000 IU s.c. b.i.d., beginning on day 1 of controlled ovarian stimulation. The endpoint for success was a live birth or an ultrasound confirming fetal cardiac activity (a viable pregnancy). The prevalence of APA in patients diagnosed with organic pelvic disease (53%) was much higher than in those without female pathology (14%). The administration of H/A to APA seropositive patients significantly (P < 0.05) improved the viable pregnancy rate (49%) compared to the untreated APA seropositive group (16%). The viable pregnancy rate for APA seropositive women treated with H/A was also significantly (P < 0.001) higher than for untreated APA seronegative patients (27%). We conclude that all women undergoing IVF/embryo transfer should be tested for APA prior to initiating ovarian stimulation and those with APA seropositivity should be treated with H/A.

Structural base of the interaction of a monoclonal antibody against p24 of HIV-1 with its peptide epitope.

The interaction of a murine monoclonal antibody (CB 4-1) against the core protein p24 of HIV-1 with its peptide antigen was studied in detail. The amino acid sequence of the variable regions of the heavy and light chain as derived from DNA sequencing was used to model the structure of the antigen binding region on the basis of reported Fab structures from the Brookhaven Protein Data Base. A linear peptide epitope responsible for the p24 binding to the antibody was determined by peptide scan. Subsequent N- and C-terminal truncation of the corresponding sequence region as well as amino acid substitutions were performed to recognize the epitope and the amino acid residues critical for antibody binding. These data were used to derive a structural model of the peptide-antibody interaction.

Immunoglobulin V regions and epitope mapping of a murine monoclonal antibody against p24 core protein of HIV-1.

The nucleotide sequence of a murine monoclonal antibody (CB-mab-p24/13-5) against p24 core protein of the human immunodeficiency virus (HIV-1) was determined for variable regions of the heavy and light chain, respectively. Genetic elements encoding the VDJH- and VJL-regions of the antibody were generated from RNA by the polymerase chain reaction, cloned into the vector pICEM 19R and sequenced. Synthetic peptides, 10 amino acids overlapping served for the localization of the epitope. The residues 152-156 within the p24 sequence contain the epitope.

Oxidation of a methionine residue in subtilisin-type proteinases by the hydrogen peroxide/borate system--an active site-directed reaction.

Subtilisin-type proteinases (thermitase, subtilisin Carlsberg, alkaline proteinase ZIMET 10911, proteinase K) are partially inactivated by hydrogen peroxide in the alkaline pH range only in the presence of boric acid or phenylboronic acid. A model is presented to describe the inactivation mechanism. Both boric acid and perboric acid existing in equilibrium in the presence of hydrogen peroxide bind competitively at the active site of the enzyme. The inactivation, which is known to be caused by sulfoxide formation from the methionine residue in the active site (Stauffer, C.E. and Etson, D. (1969) J. Biol. Chem. 244, 5333-5338), is due to the enzyme-bound perboric acid species. The dissociation constants for the boric acid-thermitase and perboric acid-thermitase complexes are 36 +/- 7 and 4 +/- 1 mM, respectively. The first-order rate constant of inactivation is k = 0.63 +/- 0.14 min-1. The same mechanism of inactivation holds true for phenylboronic acid in alkaline hydrogen peroxide solutions.

[Long-term stability of enzymes in solution]. / Untersuchungen zur Langzeitstabilität von Enzymen in Lösung.

Two proteases (thermitase, a thermostable serine protease from Thermoactinomyces vulgaris and subtilisin Carlsberg) and one non-proteolytic enzyme (urate oxidase from Penicillium spp.) were used for revealing the main influences leading to the inactivation of enzyme preparations during their long-time storage at low temperatures. The temperature dependences (0 degree C-60 degrees C) of inactivation resulted in a linear Arrhenius-plot for each of the three native enzymes as well as in the presence of all stabilizing substances tested. Therefore, a method is available which shortens the time considerably needed for the experiments aimed at the discovery of substances stabilizing enzymes under storage conditions (i.e. long time at low temperatures). Because of the linear Arrhenius-plots potential stabilizers can be tested experimentally at suitable higher temperatures and one can extrapolate on their influence on the enzyme at storage temperatures (0 degree C-10 degrees C). By using this method effective combinations of stabilizers for urate oxidase were found, and possible reasons of their stabilizing influence on the enzyme are discussed.

The use of progress curves for the estimation of inactivation rate constants of enzymes.

Progress curve analysis is applied to the estimation of inactivation rate constants. The heat inactivation of inorganic pyrophosphatase from baker's yeast was studied assuming a simple one-substrate mechanism containing two irreversible inactivation steps for the free enzyme and the enzyme-substrate complex, respectively. Four kinetic parameters including Km simultaneously may be detected from one progress curve. The temperature dependences derived from the estimated rate constants for several inactivating temperatures agree well with that obtained from preincubation measurements.

The influence of growth temperature on the heat stability of inorganic pyrophosphatase from crude extracts of Bacillus stearothermophilus.

Inorganic pyrophosphatase activity of crude extracts from Bacillus stearothermophilus adapts its thermostability to the growth temperature of the cells. Magnesium ions increase the stability of the enzyme in all cases. Depending on the growth temperature of the cells two pyrophosphatase species differing in their molecular weights and heat stabilities have been detected.

[Control intensity of glycolytic enzymes in ultrasonic hemolysates of erythrocytes]. / Untersuchungen zur Kontrollstärke von Glykolyseenzymen in Ultraschall-Hämolysaten von Erythrozyten

By addition of enzyme the control intensity was determined on the pacemaker enzymes hexokinase and phosphofructokinase, as well as on glyceraldehyde-3-phosphate-dehydrogenase and the pyruvate kinase with a control intensity of almost 0 in ultrasonic hemolysates from erythrocyte concentrate. This hemolysate approximately reflects the conditions existing in the intact cell with regard to glycolytic rate, ATP supply, and metabolite concentration. It is therefore suitable as a cell model, excluding the membrane, for studying inner control factors. For HK, PFK, GAPD, and PK predictions based on the linear glycolytic model about the significance of these enzymes for the regulation of the glycolytic rate could be confirmed.