Volume 381, Issue 1

During the search for haloperoxidases in bacteria we detected a type of enzymes that catalyzed the peroxide-dependent halogenation of organic substrates. However, in contrast to already known haloperoxidases, these enzymes do not contain a prosthetic group or metal ions nor any other cofactor. Biochemical and molecular genetic studies revealed that they contain a catalytic triad consisting of a serine, a histidine, and an aspartate. The reaction they catalyze is actually the perhydrolysis of an acetic acid serine ester leading to the formation of peracetic acid. As a strong oxidizing agent the enzymatically formed peracetic acid can oxidize halide ions, resulting in the formation of hypohalous acid which then acts as the actual halogenating agent. Since hypohalous acid is also formed by the heme- and vanadium-containing haloperoxidases, enzymatic halogenation catalyzed by haloperoxidases and perhydrolases in general lacks substrate specificity and regioselectivity. However, detailed studies on the biosynthesis of several halometabolites led to the detection of a novel type of halogenases. These enzymes consist of a two-component system and require NADH and FAD for activity. Whereas the gene for one of the components is part of the biosynthetic cluster of the halometabolite, the second component is an enzyme which is also present in bacteria from which no halometabolites have ever been isolated, like Escherichia coli . In contrast to haloperoxidases and perhydrolases the newly detected NADH/FAD-dependent halogenases are substrate-specific and regioselective and might provide ideal tools for specific halogenation reactions.

The present study focused on the investigation of the chemical stability of recombinant human interferon-β (rhIFN-β) tested in vitro by chemical treatments that simulate stress-induced conditions that may occur during handling, storage or ageing of protein samples. Mild oxidation and/or alkylation of the recombinant protein showed that the four methionines occurring in the interferon displayed different chemical susceptibility in that Met36 and Met117 were fully modified, whereas Met1 showed only little modification and Met62 was completely resistant. Moreover, incubation of rhIFN-β under alkaline conditions resulted in the formation of a covalent dimeric species stabilised by an intermolecular disulphide bridge involving the free SH group of Cys17 from each polypeptide chain. Analysis of biological activity of the different IFN-β derivatives showed that rhIFN-β fully retains its specific activity following mild oxidation treatments whereas reaction with a high concentration of alkylating agents or incubation under alkaline conditions strongly reduce its specific antiviral activity.

A synthetic peptide corresponding to the amino acid sequence Arg 1487 -Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg 1500 of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca 2+ , TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487–1500)] with a K m of 11μM and V max of 24 μmol × min −1 × mg −1 . HCV(1487–1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC 50 corresponding to the K m values measured for the substrates. N- or C-terminally deleted analogs of HCV(1487–1500) did not show inhibitory effects and were only marginally or not phosphorylatable. We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe 1499 ]HCV(1487–1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe 1499 ]HCV(1487–1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and P i allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that [Phe 1499 ]HCV(1487–1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.

The neoglycolipid (NeoGL) N -acetyl-1-deoxy-1-phosphatidylethanolamino lacto-N-tetraositol [Lc 4 Ose-PtdEtn( N Ac)] and the radioactivly labeled analog [Lc 4 Ose-PtdEtn( N [ 14 C]Ac)] were synthesized by coupling the corresponding oligosaccharide to phosphatidylethanolamine (dihexadecyl) via reductive amination and subsequent N -acetylation with unlabeled and [ 14 C]acetic acid anhydride, respectively. Lc 4 Ose-PtdEtn( N [ 14 C]Ac) was then incubated with homogenates of rat small intestine epithelial cells (IEC-6) at pH 4. The reaction products were shown to be the degradation products formed by glycosidases by fast atom bombardment mass spectrometry (FAB MS). On the other hand, incubation of Lc 4 Ose-PtdEtn( N Ac) with IEC-6 cell homogenates in sialyltransferase assays yielded the corresponding sialylated product. When Lc 4 Ose-PtdEtn( N [ 14 C]Ac) was fed to IEC-6 cells as BSA complex, up to 5% of the NeoGL administered were taken up by the cells. After extraction of the NeoGL and separation by thin layer chromatography (TLC) the catabolic products Lc 3 Ose-PtdEtn( N [ 14 C]Ac), Lac-PtdEtn( N [ 14 C]Ac), and Glc-PtdEtn( N [ 14 C]Ac), as well as the main anabolic product NeuGc-Lc 4 Ose-PtdEtn( N [ 14 C]Ac) could be identified by FAB MS. These results demonstrate that PtdEtn-derived NeoGL can be used as probes for studies on the metabolism of specific oligosaccharide structures in cell culture.

Binding of adenosine diphosphate to skeletal muscle myosin was studied using a range of concentrations from 0 to 2mM. Up to 0.2mM adenosine diphosphate two equivalent and independent nucleotide binding sites were detected, characterized by the single association constant of 5 × 10 4 M −1 . At greater adenosine diphosphate concentrations a decreasing binding capacity was noticed, bound nucleotide being essentially ~ 0.1 mol/mol at a 1–2mM adenosine diphosphate concentration. We tentatively propose that nucleotides act indirectly on myosin by promoting the perturbation of the solvent, which is supported by the fact that polyphosphates are known powerful kosmotropes.

Type I allergy, an immunodisorder affecting almost 20% of the population worldwide, is based on the production of IgE antibodies against per se harmless allergens. We report the expression of hexahistidine-tagged antibody fragments (Fabs) with specificity for Bet v1, the major birch pollen allergen, in Escherichia coli . The cDNA coding for the heavy chain fragment of a mouse monoclonal anti-Bet v1 antibody, Bip 1, was engineered by PCR to contain a hexahistidine-encoding 3′ end. The modified Bip 1 heavy chain cDNA was co-expressed in E. coli XL-1 Blue with the Bip 1 light chain cDNA using the combinatorial plasmid pComb3H. His-tagged recombinant (r) Bip1 Fabs were isolated by nickel affinity chromatography and rBip 1 Fabs without His-tag were purified via affinity to rBet v1. rBip 1 Fabs with and without His-tag bound specifically to rBet v 1 and, like Bet v1-specific human serum IgE and rabbit-anti rBet v1 antibodies, cross-reacted with Bet v1-related allergens in other plant-species (alder, oak, hazelnut). We demonstrate the usefulness of His-tagged rBip 1 Fabs (1) for the identification of pollen samples containing Bet v1 by particle blotting, (2) for the detection of Bet v1-specific IgE antibodies in human serum samples by sandwich ELISA and (3) for the quantification of Bet v1 in solution. Based on these examples we suggest to use rBip 1 Fabs for the detection of Bet v1 and Bet v1-related allergens in natural allergen sources for allergy prevention, as well as for the standardization of natural allergen extracts produced for diagnosis and immunotherapy of birch pollen allergy.

Isolated human granulocyte plasma membranes contain progelatinase B. The binding of progelatinase B to the membrane, however, is relatively weak, and a considerable part of progelatinase B can be removed by simply washing the membrane with buffer. This detachment does not depend on the ionic strength of the buffer, indicating that electrostatic forces do not play an important role in the binding of progelatinase B to the membrane. A complete removal of progelatinase B is achieved by chromatography of neutrophil membranes on gelatin-agarose. The plasma membrane of human granulocytes activates added progelatinase B. This activation is inhibited by soybean trypsin inhibitor and is thus performed by membrane bound serine proteinases. In contrast to other reports that claimed an important role of elastase in activating progelatinase B, we found that this activation is mostly inhibited by chymostatin and not by elastatinal and is thus primarily due to cathepsin G. Proteinase 3 was shown to activate progelatinase B as efficient as neutrophil elastase, i. e. much weaker than cathepsin G. Binding of cathepsin G and elastase to the neutrophil membrane does not change their ability to activate progelatinase B. However, cathepsin G , the most potent activator of the three neutrophil serine proteinases, is only a weak activator, when compared to stromelysin-1. This, as well as only a weak binding of progelatinase B, make it doubtful that activation of membrane-bound progelatinase B by membrane-bound serine proteinases is of significant physiological importance.

Three isoforms of extracellular Arg-specific proteases of P. gingivalis , W50, HRgpA, RgpA cat and mt-RgpA cat , which are all products of the same gene, show identical enzymatic properties toward small chromogenic substrates but have different subunit organisation and molecular size. In order to examine the potential inhibition of these proteases in vivo by host protease inhibitors, the interaction of HRgpA (~ 110 kDa) and RgpA cat (~ 55 kDa) with human α 2 M and their cytotoxicity toward cultured fibroblasts were investigated. Both enzymes formed complexes with α 2 M as shown by gel filtration chromatography and both cleaved the ‘bait’ region at Arg 696 -Leu 697 . However, whereas (α 2 M-RgpA cat ) complex was unable to hydrolyse large substrates such as hide powder azure, (α 2 M-HRgpA) complex hydrolysed both small and large substrates. HRgpA was able to bind to α 2 M saturated with trypsin and also to methylamine-treated α 2 M. This suggested that HRgpA is able to bind to both ‘slow’ and ‘fast’ forms of α 2 M and formation of (α 2 M:HRgpA) complex does not trap HRgpA and cause inhibition of activity toward hide powder azure. However, the (α 2 M-HRgpA) complex is not able to cleave other α 2 M molecules, which suggests that the active site of HRgpA in the complex is constrained probably due to steric reasons. The (α 2 M-HRgpA) complex was cytotoxic to 3T3 cells, causing them to round up and detach from the surface with a reduction in metabolic activity.

Adjuvant and haemolytic activities of 47 saponins purified from medicinal and food plants were examined. The compounds showed various levels of both adjuvant and haemolytic activities. Soyasaponins and lablabosides showed strong adjuvant activity but little haemolytic activity. Jujubosides showed strong adjuvant and haemolytic activities. Escins showed weaker adjuvant activity than the adjuvant-control, but strong haemolytic activity. Comparison of the functional groups of each saponin revealed that the acyl residue in saponin, the aldehyde group at carbon 4 in aglycone, and branched sugar chains attached to aglycone, were not essential for adjuvant activity. Furthermore, saponins with an acyl residue or oxide-ring moiety tended to show haemolytic activity. These results suggest that the adjuvant activity of saponins does not relate with haemolytic activity. It is considered that not only the functional groups themselves, but the overall conformation harmoniously consisting of such functional groups, affects adjuvant activity of saponins.

We have examined the insulin-stimulated IRS-2 association with PI 3-kinase and the phosphorylation of AKT/PKB, which is functionally located downstream of the PI 3-kinase, in aged (obese) rats. The IRS-2 protein levels were similar in 2 and 20 month-old rats in both tissues, liver and muscle. There were reductions in insulin-induced IRS-2 tyrosine phosphorylation in liver and muscle, accompanied by a decrease in IRS-2/PI 3-kinase association and in AKT/PKB phosphorylation only in muscle tissue of aged rats. This regulation may be important in the altered glucose metabolism observed in aged (obese) rats.

In order to investigate the effect of freezing on aqueous protease-catalyzed peptide synthesis systems, the influence of polyethylene glycols as cryoprotecting substances on α-chymotrypsin-catalyzed coupling of a N-protected acyl donor ester and various nucleophilic amino components was studied. Changes in S'-specificity of α-chymotrypsin in frozen aqueous systems were suppressed by polyethylene glycols even at concentrations below 1% (w/v). Furthermore, the influence of freeze-concentration in organic solvents on protease-catalyzed peptide synthesis was investigated for the first time. In frozen tert -butanol, α-chymotrypsin-catalyzed peptide synthesis took advantage from freeze-concentration, but in contrast to frozen aqueous systems, no changes in S'-specificity of the biocatalyst were observed. The results suggest that freeze-concentration is not the only cause of freezing-induced yield improvement in aqueous peptide synthesis systems, but interactions between enzyme and ice structures strongly contribute to the observed effects.

Equistatin is a 199-residue protein composed of three thyroglobulin type-1 domains. It strongly inhibits cysteine proteinases as well as the aspartic proteinase cathepsin D. In order to initiate structure-function studies by protein engineering, a cDNA library from sea anemone, Actinia equina , was screened. A positive clone of 888 nucleotides was shown to encode a protein of 231 amino acids, including the signal sequence. The mature protein region was amplified by PCR, cloned into the pET22b(s+)cas expression vector and expressed in Escherichia coli . Isolation of active recombinant equistatin required only one purification step, the His-tag affinity column. The protein displays physical and inhibitory properties closely similar to the native inhibitor.