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J van Rietschoten

Publications and source records attributed to J van Rietschoten.

At least 19 recordsLinked to original sources
PubMed1996

Synthesis and characterization of leiurotoxin I analogs lacking one disulfide bridge: evidence that disulfide pairing 3-21 is not required for full toxin activity.

Leiurotoxin I (Lei-NH2), a toxin isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, is a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. It is a 31-residue polypeptide cross-linked by three disulfide bridges which are presumably between Cys3-Cys21, Cys8-Cys26, and Cys12-Cys28. To investigate the role of these disulfides, analogs of Lei-NH2 lacking one disulfide bridge (i.e., [Abu3,21]Lei-NH2, [Abu8,26]Lei-NH2, and [Abu12,28]Lei-NH2) were chemically synthesized by selective replacement of each pair of half-cystines forming a bridge by two alpha-aminobutyrate (Abu) residues. The two disulfide pairings of the main folded form of the synthetic analogs were established by enzymatic proteolysis. They were as expected between Cys8-Cys26 and Cys12-Cys28 for [Abu3,21]Lei-NH2 but were unexpectedly between Cys3-Cys12 and Cys21-Cys28 for [Abu8,26]Lei-NH2 and between Cys3-Cys8 and Cys21-Cys26 for [Abu12,28]Lei-NH2. The synthetic peptides were tested in vitro for their capacity to compete with the binding of [125I]apamin to rat brain synaptosomes and in vivo for their neurotoxicity in mice. In both assays, [Abu3,21]Lei-NH2 exhibited full Lei-NH2-like activity whereas [Abu8,26]Lei-NH2 and [Abu12,28]-Lei-NH2 possessed only residual activities (< 2% native toxin activity). This suggests that disulfide bridge Cys3-Cys21 is not essential per se for high toxin activity. Circular dichroism (CD) spectroscopy of the three analogs showed that only [Abu3,21]Lei-NH2 exhibited a CD spectrum similar to that of Lei-NH2, suggesting they both adopt closely related conformations, in agreement with the pharmacological data. Structural models of the analogs were constructed on the basis of the disulfide pairing assignment and compared with that of Lei-NH2.

Amino Acid Sequence↗
PubMed1996

Characterization of PO1, a new peptide ligand of the apamin-sensitive Ca2+ activated K+ channel.

A new peptide ligand of the small conductance Ca2+ activated K+ channels has been purified from the venom (obtained by manual rather than electrical stimulation of the scorpion Androctonus mauretanicus mauretanicus), by following the inhibition of the 125I-apamin binding to its receptor on rat brain synaptosomes. Only one step on a C18 reversed-phase high-performance liquid chromatography column was necessary to obtain PO1. Its K0.5 for the apamin binding site was 100 nM. The amino acid sequence of PO1 is different from those of leiurotoxin and PO5. For the first time the same peptide was also purified from the venoms of two other species of North African scorpions, Androctonus australis and Buthus occitanus tunetanus. PO1 was chemically synthesized by the solid-phase technique and fully characterized. A model of PO1 was constructed by amino acid replacement using PO5 nuclear magnetic resonance studies as the starting model. Structure-activity relationships between these toxins and their receptor are discussed.

Animals↗
PubMed1995

Characterization of an electrogenic sodium/glucose cotransporter in a human colon epithelial cell line.

In this study, we have characterized the Na/glucose transporter in polarized monolayers formed by the clonal human colon carcinoma cell line HT-29-D4. Isotopic tracer flux measurements show that differentiated HT-29-D4 cells possess a sodium-dependent alpha-methyl-D-glucopyranoside (AMG) uptake that is competed for by increasing concentrations of D-glucose, D-galactose, and phlorizin. This transport is exclusively localized on the apical side of the epithelium. Kinetic data demonstrate the existence of a single Michaelian sodium-dependent AMG transporter with a Km of 1.2 +/- 0.12 mM and a Vmax of 3.24 +/- 0.25 nmol/mg of protein per min. Hill analysis reveals a coefficient of 1.9 +/- 0.03, consistent with at least two sodium ions involved in AMG transport. Interestingly, the cotransporter function is not modulated by glucose in the culture medium. Transepithelial electrical parameter measurements show that the transepithelial potential difference (Vt) is glucose dependent and phlorizin sensitive. Antibodies directed against a peptide of the rabbit intestinal glucose cotransporter (Ser402-Lys420) recognize, in western blot experiments, the characteristic bands of the cotransporter on a crude membrane preparation of differentiated HT-29-D4 cells and react strongly with the apical domain of the monolayer in immunofluorescence experiments. We conclude that HT-29-D4 cells express the sodium/glucose cotransporter SGLT1 at their apical membrane and that this transporter generates the basal transepithelial potential difference.

Blotting, Western↗
PubMed1994

The kaliotoxin family enlarged. Purification, characterization, and precursor nucleotide sequence of KTX2 from Androctonus australis venom.

Kaliotoxin (KTX) has been originally described as an inhibitor of the intermediate conductance Ca(2+)-activated K+ channel (Crest, M., Jacquet, G., Gola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P., and Martin-Eauclaire, M.-F. (1992) J. Biol. Chem. 267, 1640-1647). However, the radioiodinated 125I-KTX-(1-37) was also able to bind to the dendrotoxin sensitive voltage-dependent K+ channel (Romi, R., Crest, M., Gola, M., Sampieri, F., Jacquet, G., Zerrouk, H., Mansuelle, P., Sorokine, O., Van Dorsselaer, A., Rochat, H., Martin-Eauclaire, M.-F., and Van Rietschoten, J. (1993) J. Biol. Chem. 268, 26302-26309). By following the ability to compete with 125I-KTX-(1-37) for binding to its receptor on rat brain synaptosomes, a new kaliotoxin-like peptide, KTX2, was isolated from Androctonus australis scorpion venom. It is a 37-amino acid residue peptide, and its sequence shares 76% identity with KTX. The differences between the two peptides concern the NH2-terminal region and the residues 31 and 34 located in the region involved in the channel recognition. These differences may explain the 5-fold decrease of the molluscan Ca(2+)-activated K+ channel blockage by KTX2 (kd = 135 nM) as well as of its binding affinity to rat brain synaptosomes (IC50 = 50 pM), compared with KTX. Specific antibodies raised against KTX-(1-37) were not able to recognize KTX2. Using degenerate primers, a 370-base pair cDNA encoding the KTX2 precursor was amplified by polymerase chain reaction from a cDNA library of A. australis venom glands. It encoded a presumed signal peptide of 22 residues followed by the sequence of the mature peptide.

Amino Acid Sequence↗
PubMed1994

Effects of the Tat basic domain on human immunodeficiency virus type 1 transactivation, using chemically synthesized Tat protein and Tat peptides.

To study the structure relationship of different Tat domains, the full-length Tat protein Tat1-86, the gene product of the first exon Tat1-72 which retains full activity of the protein, and a panel of shorter peptides mimicking different regions of the primary structure of the Tat protein were chemically synthesized by the solid-phase method, using an efficient protocol. Synthetic Tat1-86 and Tat1-72 transactivated beta-galactosidase activity in HeLa cells containing the lacZ gene under the control of the human immunodeficiency virus type 1 long terminal repeat. Analyses of the activity of Tat1-86 and Tat1-72 with the sulfhydryl of cysteine residues free or protected by the acetamidomethyl group showed that only the Tat fragments with deprotected cysteine residues retain transactivation ability. In contrast, peptide Tat1-48 was inactive, with cysteine residues either free or protected. Similarly, other shorter synthetic peptides covering the different Tat domains were inactive. Interestingly, when peptides Tat1-48 and Tat38-60 were used simultaneously, a significant transactivation was obtained. This result suggests that both peptide domains are implicated in transactivation, probably by acting at two different sites. This permits us to propose a fundamentally new step in the understanding of the molecular mechanism of Tat transactivation.

Amino Acid Sequence↗
PubMed1994

Multibranched V3 peptides inhibit human immunodeficiency virus infection in human lymphocytes and macrophages.

Synthetic polymeric constructions (SPCs) including the consensus sequence of the human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein gp120 V3 loop (GPGRAF) blocked the fusion between HIV-1- and HIV-2-infected cells and CD4+ uninfected cells. A structure-activity relationship study using V3 SPC analogs showed that the most efficient inhibitor of cell fusion was an eight-branched SPC with the hexapeptide motif GPGRAF (i.e., [GPGRAF]8-SPC). N-terminal acetylation or incorporation of D-amino acids in the GPGRAF sequence of this SPC resulted in significant loss of activity. Analogs with fewer than six residues in the motif (i.e., GPGRA or GPGR), as well as SPCs with a nonrelevant sequence, did not inhibit cell fusion, demonstrating the high specificity of the antifusion activity. [GPGRAF]8-SPC, which was not toxic to CEM cells at concentrations of up to 50 microM, inhibited 50% of HIV-1(LAI) replication in these cells at a concentration of 0.07 microM. Moreover, [GPGRAF]8-SPC inhibited the infection of human peripheral blood mononuclear cells by several HIV-1 and HIV-2 isolates, including laboratory strains [HIV-1(LAI), HIV-1(NDK), and HIV-2(ROD)], and fresh primary isolates, including two zidovudine-resistant HIV-1 isolates and two HIV-2 isolates obtained from infected individuals. The multibranched peptide also inhibited infection of human primary macrophages by the highly cytopathic macrophage-tropic isolate HIV-1(89.6). The antiviral activity of [GPGRAF]8-SPC was not related to a virucidal effect, since preincubation of HIV-1 with the peptide did not affect its infectious titer. This result is in agreement with the concept that the multibranched peptide mimics a part of the V3 loop and thus interacts with the host cell. The therapeutic properties of synthetic multibranched peptides based on the V3 loop consensus motif should be evaluated in HIV-infected patients.

Amino Acid Sequence↗
PubMed1990

Identification of antigenic residues on apamin recognized by polyclonal antibodies.

The structural requirements for antigenic recognition of apamin--an 18-amino acid, disulfide-bridged peptide--by rabbit antibodies were defined using a set of 18 apamin analogs in a competition liquid-phase radioimmunoassay. Some residues contribute considerably to antigenic recognition, e.g. Ala10, Arg13, and others to a lesser extent, e.g. Arg14, Glu7 and Thr8. The N- and C-terminal moieties of apamin are less antigenically important. These findings suggest that a good part of antibody specificities are directed to the central tightly folded part of the molecule. They are consistent with the observation that in saturating conditions, labeled apamin can, on average, bind one specific Fab fragment.

Amino Acid Sequence↗
PubMed1989

Identification of a common domain in calmodulin-activated eukaryotic and bacterial adenylate cyclases.

Bordetella pertussis and Bacillus anthracis, two taxonomically distinct bacteria, secrete adenylate cyclase toxins that are activated by the eukaryotic protein calmodulin. The two enzymes contain a well-conserved stretch of 24 amino acid residues [Escuyer et al. (1988) Gene 71, 293-298]. Antibodies have been obtained against two synthetic heptadecapeptides, covering part of the conserved sequences. The anti-peptide antibodies specifically reacted in Western blots with the rat brain adenylate cyclase as well as with the two bacterial enzymes. Anti-rat brain adenylate cyclase serum contained antibodies that were retained by the immobilized peptides, and the affinity-purified antibodies yielded the same recognition pattern of the eukaryotic enzyme as did the unfractionated serum. These results indicate that the eukaryotic adenylate cyclase contains an epitope closely related to that specified by the conserved bacterial sequence. The synthetic peptides and the bacterial adenylate cyclases appeared to compete for ATP (KD of the ATP-peptide complex ca. 0.2 mM), suggesting that the conserved sequence may be part of the substrate binding site in these two enzymes.

Adenylyl Cyclases↗
PubMed1983

Monoiodo-[Trp11]neurotensin, a highly radioactive ligand of neurotensin receptors. Preparation, biological activity, and binding properties to rat brain synaptic membranes.

Iodination of [Trp11]neurotensin, a neurotensin analogue in which tyrosine 11 has been substituted by a tryptophan, led to the incorporation of one or two iodine atoms on the single tyrosine residue in position 3. Both mono- and diiodinated derivatives were purified by ion exchange chromatography and their biological activity in an in vitro bioassay involving rat ileum was found to be similar to that of native neurotensin. The 125I-labeled monoiodo derivative of [Trp11]neurotensin bound specifically and reversibly to rat brain synaptic membranes. The binding isotherm was biphasic and could be described by postulating the existence of two different classes of independent binding sites with dissociation constants of 0.1 and 4.7 nM. The specificity of a series of neurotensin analogues for both high and low affinity binding sites was the same as that previously observed in other neurotensin radioreceptor assays. The low affinity binding sites appeared to be similar to the single class of sites described in other binding studies. The high affinity binding sites which were not previously detected might represent either a new class of neurotensin receptors or a high affinity state for a fraction of a single population of neurotensin receptors.

Animals↗
PubMed1982

[TRP11]-neurotensin and xenopsin discriminate between rat and guinea-pig neurotensin receptors.

The binding and biological activities of neurotensin and two analogues, [TRP11]-neurotensin and xenopsin, in which a tryptophan replaces the neurotensin residue Tyr11, were compared in rat and guinea-pig. The binding activity of three peptides was measured as their ability to inhibit the binding of [3H]neurotensin to rat and guinea-pig brain synaptic membranes. Their biological activities were measured as their effects on the contractility of rat and guinea-pig ileal smooth muscle preparations. In binding as well as biological assays, it was found that [Trp11]-neurotensin and xenopsin were as potent as neurotensin in the rat. In contrast, the two analogues were about 10 times less potent than neurotensin in the guinea-pig. These findings reveal differences between rat and guinea-pig neurotensin receptors should be considered when comparing the activity of neurotensin analogues in assays using tissue preparations from various animal species.

Amino Acid Sequence↗
PubMed1982

Structure-function relationships for cardiotoxins interacting with phospholipids.

Four cardiotoxins (CTX I-IV) from Naja mossambica mossambica were compared for their ability to interact with phospholipid vesicles and their capacity to bind erythrocytes. It is concluded that the affinity of the toxins always increases in the order: I approximately equal to II less than III less than IV. The binding is specific for charged lipids even in lipid mixtures. Proteolytic attack of the free and lipid-bound cardiotoxin indicates that at least the first loop Leu1-Thr13 is at the lipid contact. Tryptic and synthetic peptides constitutive of this loop are shown to interact with lipids. Arg5 residue increases the affinity toward the bilayer. The Raman spectra of lipid-bound cardiotoxin indicate a secondary and tertiary structure mainly similar to that of the free toxin. On charged lipids cardiotoxins induce a decrease of the enthalpy and an increase of disorder without change in the transition temperature; at saturating amounts of toxin the transition is abolished. In binary mixtures of phosphatidylcholine and charged lipids the observed effects can be accounted by a phase separation induced by the toxin.

Amino Acid Sequence↗
PubMed1982

Covalent structure of the insect toxin of the North African scorpion Androctonus australis Hector.

The complete covalent structure of the insect toxin purified from the venom of the North-African scorpion Androctonus australis Hector was described. Its amino acid sequence was established by phenylisothiocyanate degradation of several protein derivatives and proteolytic fragments in a liquid protein sequencer using either a "protein" or a "peptide" program. The position of the four disulfide bridges were deduced by analysis of proteolytic peptides before and after diperformic oxidation, and by partial labeling of the half cystine residues with [14C]-iodoacetic acid and determining the specific radioactivities of the S-[14C]-carboxymethylated phenylthiohydantoin cysteines. The sequences of the insect and mammal toxins from scorpions can be aligned with homology with the positions of seven half-cystine residues as registers. The mammal and insect toxins have three disulfide bridges at homologous positions. The mammal and insect toxins have three disulfide bridges at homologous positions. The fourth bridge is different in that Cys12 in mammal toxin II is replaced by Cys38 in the insect toxin. It is likely that the position of the disulfide bridges is the same for all scorpion neurotoxins active on mammals. We believe that the shift of one half-cystine residue in the insect toxin may induce a conformational change in the structure of the protein, which, in turn, may partially account for the total specificity of this toxin for insect nervous system.

Amino Acid Sequence↗
PubMed1977

Synthesis, purification, and chemical characterization of the amino-terminal 1-34 fragment of bovine parathyroid hormone synthesized by the solid-phase procedure.

Peptides prepared by solid-phase synthesis during a systematic study of structure-activity relations in parathyroid hormone have been subjected, after sequential purifications, to rigorous analysis of purity by a variety of analytical techniques including sequence analysis by the Edman procedure. The present paper undertakes a critical appraisal of the utility of different tests of peptide purity and the use of the procedures in guiding and monitoring optimal synthesis strategies. Sequence analysis of a peptide representing the amino-terminal 34 residues of bovine parathyroid hormone (bPTH-(1-34)) revealed the presence of at least 30% of contaminating error peptides which were undetected by other analytical procedures. The major contaminant was identified as a peptid in which glutamine at position 29 was deleted. A repeat synthesis using fluorescamine rather than ninhydrin to monitor the coupling reaction resulted in a preparation that lacked the contaminant resulting from deletion. These findings demonstrate the particular value of sequence analysis in the evaluation of purity of peptides synthesized by the solid-phase technique.

Adenylyl Cyclases↗
PubMed1975

Synthesis of apamin, a neurotoxic peptide from bee venom.

The apamin sequence has been synthesized by the solid-phase procedure. The synthetic peptide showed the same physiochemical and chemical properties as natural apamin in the following tests: paper electrophoresis, amino acid analyses after acid and enzymatic hydrolyses, sequence analysis, electrophoreses after tryptic and chymotryptic digestions. Synthetic apamin was as active as natural apamin in the neurotoxic assay in mice (LD50, after subcutaneous injection, for the 20-g mouse: 58 mug).

Amino Acid Sequence↗