Amrit K. Judd

Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors

The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to the N‐terminus of ST1b using biotin‐X‐X‐N‐hydroxysuccinimide ester. The derivatized toxin (BST) has an apparent Kd of 11.7±10 nM for rat brush border receptors. We used BST in an affinity panning cell‐capture system, to validate its ability to discriminate between receptor‐positive and receptor‐negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase‐C (GC‐C) ST receptor cDNA) were captured to streptavidin and anti‐biotin‐coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST‐binding proteins. BST was then used with streptavidin‐gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors, are concentrated in villus entercoytes, while guanylin appears to be produced at the base of the crypts. This topographical arrangement suggests that there are autocrine or paracrine pathways by which ST receptors interact with endogenous ligands.

Isolation and characterization of a new atrial peptide-degrading enzyme from bovine kidney

An endopeptidase isolated from bovine kidney displays high affinity and selectivity for the Ser-Phe bond located in the C-terminal region of atrial peptides. Enzymatic activity converts APIII and APII to the less active peptide API. This peptidase is inhibited by both metal chelators and sulfhydryl-reactive agents, suggesting both a tightly bound metal and a cysteine residue are important for enzymatic activity. This enzyme may be important for the processing and/or degradation of atrial peptides.

Peptides: Chemistry, Biology, and Pharmacology

Publisher Summary This chapter discusses the chemistry, biology, and pharmacology of peptides. Peptides are small proteins composed of fewer than 50 amino acids. Unlike most globular proteins, peptides frequently have modified amino (N) and carboxyl (C) termini, contain D-amino acids and multiple disulfide bridges, or exist in cyclic forms with linked N and C termini. These features not only contribute to the functional role of these compounds, but also confer stability in physiological environments, specifically by preventing proteolytic degradation. The number of naturally occurring peptides that have been identified and structurally characterized has grown substantially in the past decade as a result of improved protein purification and sequencing techniques and through the isolation of cDNAs encoding putative peptide like structures. The production of synthetic derivatives of these compounds, including competitive antagonists and potent agonists, the use of these agents in increasingly refined physiological studies, and the identification of their cellular receptors are gradually clarifying their functional role.