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B Gutte

Publications and source records attributed to B Gutte.

33 records · Page 2Linked to original sources
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↗
PubMedDate not supplied

Expression of the synthetic gene of an artificial DDT-binding polypeptide in Escherichia coli.

This paper reports the expression of an artificial functional polypeptide in bacteria. The gene of a designed 24-residue DDT-binding polypeptide (DBP) was inserted between the BamHI and PstI cleavage sites of plasmid pUR291. The hybrid plasmid, pUR291-DBP, was cloned in Escherichia coli JM109. After induction by isopropyl-beta-D-thiogalactopyranoside a fusion protein was expressed in which DBP was linked to the COOH-terminus of beta-galactosidase. DBP, which is stable to trypsin, was obtained by tryptic digestion of the fusion protein and subsequent fractionation of the tryptic peptides by reversed-phase h.p.l.c. Recombinant and chemically synthesized DBP showed identical chromatographic properties, amino acid composition, and chymotryptic digestion patterns. Both the beta-galactosidase-DBP fusion and isolated recombinant DBP bound DDT. The fusion protein was 25 times as potent as the designed 24-residue DBP in activating a cytochrome P-450 model system using equimolar catalytic amounts of the two proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Design, synthesis, and characterization of HIV-1 enhancer-binding polypeptides derived from bacteriophage 434 repressor.

We have designed and synthesized HIV-1 enhancer-binding polypeptides that were derived from bacteriophage 434 repressor. These peptides were 39-54 residues long and contained either the recognition helix or the entire helix-turn-helix motif of the DNA-binding domain of 434 repressor. The dissociation constant of the complex formed between the standard peptide (R42) and a synthetic 70-bp HIV enhancer DNA was ca. 10(-8) M. The specificity of the interaction of R42 with the two HIV enhancers was demonstrated by competitive band shift assays, stepwise displacement of the p50 subunit of transcription factor NF-kappa B from its two HIV enhancer binding sites, and DNase I footprinting; R42 seemed to protect best the two TTTCC sequences of the HIV enhancers against digestion by DNase I. R42 analogues with mutated recognition helix had lower DNA binding specificity. It remains to be investigated whether our artificial HIV enhancer-binding polypeptides are active in vivo.

Amino Acid Sequence↗