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B Gutte

Publications and source records attributed to B Gutte.

13 recordsLinked to original sources
PubMed1992

Carbohydrate masking of an antigenic epitope of influenza virus haemagglutinin independent of oligosaccharide size.

Comparison of the haemagglutinins (HA) of the pathogenic avian influenza viruses A/FPV/Dutch/27 (H7N7) and A/FPV/Rostock/34 (H7N1) revealed 94.7% nucleotide and 93.8% amino acid sequence homologies. Six of the seven N-glycosidic oligosaccharides of the Rostock HA are at the same positions as the six carbohydrates of the Dutch strain. The additional oligosaccharide side chain of the Rostock strain, which is of the complex type, is attached to asparagine149 in antigenic epitope B. The accessibility of this antigenic epitope has been analysed by using rabbit antisera raised against synthetic peptides comprising amino acids 143-162. The carbohydrates of the HA of the Rostock strain have been modified (i) to truncated cores by expression in insect cells using a baculovirus vector, (ii) to oligomannosidic side chains by growth in the presence of the trimming inhibitor methyldeoxynojirimycin and (iii) to a single N-acetylglucosamine residue by removal of the oligomannosidic sugar with endo-beta-N-acetylglucosaminidase H. Neither the authentic nor the modified oligosaccharides allowed antibody binding, as indicated by enzyme-linked immunosorbent assay (ELISA) and Western blot analyses. Reactivity was observed, however, after complete removal of the carbohydrate from HA of the Rostock strain by digestion with peptide-N-glycosidase F. HA of the Dutch strain was reactive without prior peptide-N-glycosidase F treatment. These results demonstrate that a single N-acetyl-glucosamine at asparagine149 is sufficient to prevent recognition of the peptide epitope.

Amino Acid Sequence↗
PubMed1991

Structure-function studies of designed DDT-binding polypeptides.

An artificial 24-residue DDT-binding polypeptide (Moser, R., Thomas, R.M., and Gutte, B. (1983) FEBS Lett. 157, 247-251) and several analogues of this peptide were characterized by ligand binding, spectroscopic, and immunological studies. Comparison of dissociation constants showed that Phe14 and His16 were important for DDT binding and that the designed peptide had noticeable ligand specificity. Measurement of the circular dichroism of the artificial DDT-binding peptide revealed a high proportion of beta-structure which was abolished only partly by 8 M urea. When Tyr15, Tyr17, and Phe3 whose side chains were on the same side of the proposed beta-sheet were replaced by non-aromatic amino acids, the cross-reactivity with antibodies against the original DDT-binding peptide decreased stepwise. In summary, the results of this study support essential features of our structural model of the designed 24-residue DDT-binding peptide.

Amino Acid Sequence↗
PubMed1990

Preparation and characterization of polyclonal and monoclonal antibodies against the insecticide DDT.

A synthetic DDT derivative in which the molecular structure of DDT was completely retained was coupled to bovine serum albumin. Animals were immunized with the DDT-bovine serum albumin conjugate and polyclonal and monoclonal antibodies against the insecticide were isolated. These antibodies seemed to be the first true anti-DDT antibodies and distinguished much better between DDT and DDT metabolites than previously prepared anti-DDT antisera. In competitive solid phase radioimmunoassays, DDT concentrations as low as 10 nM or 0.0035 mg/1 were detectable. The anti-DDT antibodies can be used for environmental analyses and lend themselves to the elucidation of the structure of the DDT binding site.

Animals↗
PubMed1989

Rapid partial degradation of DDT by a cytochrome P-450 model system.

Heme compounds, in combination with a reducing agent and oxygen, can express various activities of cytochrome P-450 enzymes. In the present study it was found that a mixture of hemin and excess cysteine was able to degrade the insecticide DDT partially. The major products were three water-soluble, non-toxic conjugates of DDT metabolites with cysteine which had lost two or three of the five chlorine atoms of DDT per molecule and whose structures were elucidated by gas chromatography/mass spectrometry. In 0.05 M NH4HCO3, pH 7.7/ethanol (5:6, by vol.), the degradation reaction catalyzed by the hemin-cysteine model system was at least 8 x 10(4) times faster than the uncatalyzed reaction. In the presence of a designed 24-residues polypeptide or beta-casein, two DDT-binding proteins, an additional fourfold increase in the rate of DDT degradation was observed. Although the concentrations of DDT and cysteine occurring in an organism would be expected to be lower than those in the experiments described, the formation of water-soluble conjugates of DDT metabolites with cysteine (and other amino acids) could also play a role in metabolism and excretion of DDT in vivo.

Binding Sites↗
PubMed1985

A crystalline synthetic peptide representing the epitope of a monoclonal antibody raised against synthetic interferon-alpha 1 fragment 111-166.

The antigenic determinant recognized by the monoclonal antibody that had been raised against synthetic human interferon-alpha 1 (IFN-alpha 1) fragment 111-166 [Arnheiter, H., Thomas, R.M., Leist, T., Fountoulakis, M., and Gutte, B. (1981) Nature (Lond.) 294, 278-280] and that cross-reacted with human IFN-alpha 1, IFN-alpha 2, and IFN-alpha A made in Escherichia coli, was localized to the region between residues 151 and 166 using synthetic COOH-terminal interferon fragments. In solid-phase radioimmunoassays neither the strongly hydrophilic COOH-terminal nonapeptide IFN 158-166 nor its mixtures with IFN 151-162 or IFN 149-158 showed any measurable interaction with the antigen binding site of the monoclonal antibody. For antibody binding, the full covalent structure of IFN 151-166 was required. Quantitatively very similar results were obtained with IFN 149-166 and IFN 143-166. The synthetic COOH-terminal hexadecapeptide of human IFN-alpha 1 (IFN 151-166) could be crystallized.

Antibodies, Monoclonal↗
PubMed1979

Design, synthesis and characterisation of a 34-residue polypeptide that interacts with nucleic acids.

Based on secondary structure prediction rules and model building a neutral artificial 34-residue polypeptide with potential nucleic acid-binding activity was synthesised. This peptide and its covalent dimer showed strong interaction with cytidine phosphates and single-stranded DNA. The dimer had considerable ribonuclease activity with high preference for cleavage at the 3'-end of C.

Amino Acid Sequence↗
PubMed1978

Synthesis of an open-chain asymmmetrical cystine peptide corresponding to the sequence A18-21--B19-26 of bovine insulin by solid phase fragment condensation.

An insulin fragment containing residues A 18-21 and B 19-26 linked by the disulfide bond between residues A 20 and B 19 was synthesized. The sequence B 21-26 was assembled on a solid support by the Merrifield technique. The protected fragments A 18-21 and B 19-20 were prepared by conventional methods. After forming the disulfide bridge through cleavage of the S-thiocarbonate derivative of A 18-21 by the thiol peptide B 19-20, the resulting assymmetrical cystine peptide A 18-21--B 19-20 was coupled via the carboxyl group of residue B 20 to the free NH 2-terminal amino group of the protected B 21-26 resin. The product was deprotected, cleaved from the resin, and purified to give the homogenous dodecapeptide A 18-21--B 19-26.

Amino Acid Sequence↗
PubMed1977

Study of RNase A mechanism and folding by means of synthetic 63-residue analogs.

A 63-residue RNase A analog containing residues 26 to 35 then alanine, 41 to 59 and 73 to 84 then glycine, 100 to 110 then glycine, and 117 to 124 was synthesized by the solid phase method. The deletions comprised ordered (an alpha helix, parts of the beta sheet) and less ordered structures including 27 of the 56 residues invariant in mammalian ribonucleases. The synthetic 63-residue analog was cleaved from the insoluble support with liquid HF, reduced-reoxidized, fractionated by gel filtration, and purified further on an affinity column specific for the active site fold of RNase A. It had an activity of 8 to 14 per cent in the transphosphorylation step using poly(C) and poly(U) as substrates. It also had low synthetic and hydrolytic activity (0.2 per cent) and showed RNase A-like specificity toward the substrates tested. This indicated that all residues essential for substrate binding and catalysis were present and that their relative positions in the three-dimensional structure were probably very similar to those in native RNase A. Therefore, structure-function studies with the 63-residue RNase A analog should allow conclusions about the mode of action of the natural enzyme. As a first step in this direction, lysine 41 which is believed to be important for catalysis was replaced in the 63-residue analog by tyrosine or glutamine. The resulting (Tyr-41)- and (Gln 41)-63-residue analogs were also bound by the affinity column and had the same substrate specificity as native RNase A. They differed from each other, from the (Lys 41)-63-residue analog, and the 124-residue natural enzyme only with respect to the relative rates of the catalyzed reactions. Thus, lysine 41 does not seem to be essential for the functioning of RNase A.

Amino Acid Sequence↗
PubMed1975

A synthetic 70-amino acid residue analog of ribonuclease S-protein with enzymic activity.

A 70-residue analog of RNase S-protein was synthesized by the solid phase method. It was obtained by omitting the NH2 terminus from positions 21 to 25 and the segments 36 to 40, 58 to 73, 87 to 96, and 113 to 114. Four residues were inserted to link the ends formed by the deletions. Half-cystine residues that had not been part of the deletions were replaced by alanine or leucine residues. The synthetic polypeptide was separated by gel filtration into a dimer and a monomer. Both fractions were purified further by ion exchange chromatography. The dimeric 70-residue S-protein analog had a specific activity of approximately 4% using RNA as substrate. It also cleaved other substrates of RNase A such as 5'-(3'-cytidylyl)-guanosine, 5'-(3'-uridylyl)-guanosine, and polycytidylic acid. The monomer of the 70-residue analog was less active but showed the same substrate specificity as the dimer. It was found that both fractions of the synthetic S-protein analog catalyzed only the transphosphorylation step of the RNase A mechanism and had very little if any activity in the hydrolysis step. Addition of natural S-peptide or S-protein did not increase the activity in the transphosphorylation reaction but greatly enhanced the reaction rate of the hydrolysis step. IN THE PRESENCE OF S-peptide, both monomeric and dimeric 70-residue S-protein, both monomeric and dimeric 70- residue S-protein analog had approximately 8% activity using cyclic cytidine 2':3'-monophosphate as substrate. The mixtures of monomer and dimer of the synthetic S-protein analog with natural S-protein generated even higher activities (151 and 74%, respectively) against this substrate despite the fact that the NH2-terminal portion of the natural enzyme (including His 12) was missing in both components of the two complexes. The 70-residue S-protein analog was completely inactive against DNA and (with one exception) against substrates for RNase T1. The close agreement of the substrate specificity of the synthetic analog with that of native RNase A in the transphosphorylation step suggested a remarkable conservation of the configuration of the active site despite drastic changes of the primary structure of the parent molecule. Possible implications of these results for the mechanism of action of RNase A are discussed.

Amino Acid Sequence↗