Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine

Hugues, Michel; Crane, M.; Thomas, Billy R.; Robertson, D C; Gazzano, Helene; O’Hanley, P; Waldman, Scott A.

doi:10.1021/bi00116a003

Cited by 32 publications

(26 citation statements)

References 22 publications

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“…Recently, a 74 kDa ST, binding protein was affinity purified from rat intestine by Hugues et al (1992). By analogy to our current studies, this protein, which could not be tested for guanylate cyclase activity, could represent either a novel ST, receptor or a proteolytically cleaved portion of the extracellular domain of the larger (121 kDa) guanylate cyclase receptor previously cloned by Schulz et al (1990).…”

Section: Discussionmentioning

confidence: 63%

Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco‐2)

Cohen

Jensen

Hawkins

et al. 1993

Journal Cellular Physiology

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Escherichia coli heat stable enterotoxin (STa) and the newly identified endogenous ligand guanylin bind to an intestinal receptor and activate membrane bound guanylate cyclase. We compared STa binding and affinity crosslinking of STa receptors in human small intestine to those in the Caco-2 human colon carcinoma cell line. STa had similar kinetics of binding in human intestinal and Caco-2 brush border membranes. In both human intestine and Caco-2 brush border membranes, multiple specifically radiolabeled bands, including a 140-165 kDa band, were identified by affinity crosslinking. However, in human intestine the most prominent autoradiographic species was a 60 kDa band. A 60 kDa protein was also specifically immunoprecipitated from solubilized human brush border membranes using antisera raised against a cloned STa receptor fusion protein. Our observations of multiple crosslinked proteins in human intestine and Caco-2 cells could be explained by the existence of several members of a family of STa receptors and/or the existence of smaller STa binding proteins generated by the protease cleavage of a larger complete STa receptor.

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Section: Discussionmentioning

confidence: 63%

Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco‐2)

Cohen

Jensen

Hawkins

et al. 1993

Journal Cellular Physiology

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“…In vitro guanylyl cyclase assays were performed essentially according to Hugues et al [15]. T84 membrane fractions, or solubilised or purified preparations of the ST receptor, were suspended in 60 mM Tris/HCl, pH.…”

Section: In Vitro Activation Of Guanylyl Cyclasementioning

confidence: 99%

“…How- ever, there is no biochemical evidence using purified receptor preparations to confirm that the ST receptor does in fact exist in the cell as suggested for the recombinant protein. Hugues et al reported the purification of the rat ST receptor from intestinal membranes using ST-ligand affinity chromatography and suggested that a protein of M, 70000 was the receptor [15]. However, the purified protein did not possess guanylyl cyclase activity, and this was attributed to the instability of the guanylyl cyclase activity from rat intestinal membranes during purification procedures.…”

mentioning

confidence: 99%

Characterization and partial purification of the human receptor for the heat‐stable enterotoxin

Visweswariah¹,

Ramachandran²,

Ramamohan³

et al. 1994

European Journal of Biochemistry

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The receptor for the Escherichia coli heat-stable enterotoxin has been characterized and partially purified from the T84 human colonic cell line. Using a novel mutant heat-stable enterotoxin peptide as a radioligand (the C-terminal tyrosine residue is replaced by phenylalanine in the mutant), a single class of high-affinity receptor sites was detected in T84 cells, with a Kd of 0.1 nM, similar in affinity to the receptor described in human intestinal tissue. The receptor was solubilised from T84 cell membranes and affinity cross-linking of the solubilised preparation indicated that a single species of M, 160000 served as the receptor. Freshly solubilised preparations of the receptor retained heat-stable enterotoxin-activable guanylyl cyclase activity. Purification of the receptor was achieved through sequential affinity chromatography on GTP-epoxy-Sepharose and wheat-germ-agglutinin columns resulting in purification of the receptor by 3000 fold. The heat-stable enterotoxin-binding characteristics of the receptor were unchanged during the purification and silver staining of the purified receptor preparation indicated a band of M, 160000, which was specifically cross-linked to the lZ51-labeled mutant peptide. The purified receptor retained guanylyl cyclase activity, but the activity was not stimulated on addition of human heat-stable enterotoxin, suggesting that accessory structural factors may be involved in the activation of the guanylyl cyclase/receptor.The heat-stable enterotoxins (ST) are a family of lowmolecular-mass peptide toxins, and are one of the major causes of watery secretory diarrhoea all over the world [1, 21. Various forms of the toxins, differing in amino acid sequence, are produced by a number of pathogenic bacteria [3, 41, and all these peptides contain a cysteine-rich core essential for full biological activity [5, 61. ST peptides bind to a receptor on intestinal cells and activate membrane-bound guanylyl cyclase [7, 81. Increased levels of cyclic GMP (cGMP) within the cell are hypothesised to lead to enhanced C1-secretion from the intestinal cell by as yet undefined mechanisms, resulting in fluid loss and subsequent diarrhoea Early biochemical studies had postulated that in rat intestinal membranes the ST-binding and guanylyl cyclase activities were located on separate molecules [lo]. However, the cloning and expression of the rat and human intestinal ST receptor [ll-131 suggested that the ST receptor was in fact a high-molecular-mass protein and a member of the guanylyl cyclase family of receptors, described earlier for atrial natriuretic factor and the sea urchin egg peptides [14]. These observations implied that the ST-binding and guanylyl cyclase activities were present in the same receptor molecule. How- ever, there is no biochemical evidence using purified receptor preparations to confirm that the ST receptor does in fact exist in the cell as suggested for the recombinant protein. Hugues et al. reported the purification of the rat ST receptor from intestinal membranes using ST-ligand af...

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“…As early as 1977, Ehrenstein & Lecar proposed the ‘barrel-stave’ model, believing that the main mechanism of action of ACPs is to cause cell membrane fragmentation or apoptosis by depolarization of the cell membrane, leading to failure of tumour cells to maintain normal osmotic pressure [ 47 ]. Subsequently, Pouny et al proposed a ‘carpet’ model, which suggested that ACPs caused cell death by destroying the cell membrane of cancer cells, leading to massive leakage of cytoplasmic contents [ 48 ]. Most ACPs act directly through this mechanism ( figure 2 a ), which endows them with unique advantages over conventional chemotherapy.…”

Section: Anti-tumour Mechanism Of Acpsmentioning

confidence: 99%

Anti-cancer peptides: classification, mechanism of action, reconstruction and modification

2020

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Anti-cancer peptides (ACPs) are a series of short peptides composed of 10–60 amino acids that can inhibit tumour cell proliferation or migration, or suppress the formation of tumour blood vessels, and are less likely to cause drug resistance. The aforementioned merits make ACPs the most promising anti-cancer candidate. However, ACPs may be degraded by proteases, or result in cytotoxicity in many cases. To overcome these drawbacks, a plethora of research has focused on reconstruction or modification of ACPs to improve their anti-cancer activity, while reducing their cytotoxicity. The modification of ACPs mainly includes main chain reconstruction and side chain modification. After summarizing the classification and mechanism of action of ACPs, this paper focuses on recent development and progress about their reconstruction and modification. The information collected here may provide some ideas for further research on ACPs, in particular their modification.

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Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine

Cited by 32 publications

References 22 publications

Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco‐2)

Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco‐2)

Characterization and partial purification of the human receptor for the heat‐stable enterotoxin

Anti-cancer peptides: classification, mechanism of action, reconstruction and modification

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