Proteolytic fragmentation for epitope mapping.

The use of antigen fragments generated by specific proteolytic cleavage is a relatively simple "library" approach for epitope mapping in which possible overlapping fragments are screened with the antibody on Western blots. Proteolytic fragmentation with numerous proteases having different cleavage specificites can be carried out on native and denaturated proteins, generating a small and large number of fragments, respectively. To determine the antigenic site of a monoclonal antibody, we have examined the limited proteolytic digestion of the transducin alpha -subunit with four different proteases and detected antibody binding to fragments by Western blot.

Allosteric inhibition of [(125I)] ET-1 binding to ET(A) receptors by aldoxime and hydroxamic acid derivatives.

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, exerts its physiological effects by binding and activating specific G protein-coupled receptors, named ET(A) and ET(B). An unique property of ET-1 is its ability to bind almost irreversibly to its receptors. Aspirin and salicylic acid (SA) are allosteric inhibitors of ET-1 binding to ET(A) receptors.

Endothelin-1 up-regulates p115RhoGEF in embryonic rat cardiomyocytes during the hypertrophic response.

In cardiomyocytes, certain extracellular stimuli that activate heterotrimeric G protein-coupled receptors (GPCRs) can induce hypertrophy by regulating gene expression and increasing protein synthesis. We investigated if rat embryonic cardiomyocytes (H9c2) underwent variations in the expression levels and subcellular distribution of key components of GPCR-activated signaling pathways during endothelin-1 (ET-1)-induced hypertrophic response. A significant increase of p115RhoGEF protein level was evident in ET-1-treated cells.

Driving forces in the delivery of penetratin conjugated G protein fragment.

A42 is a chimera peptide consisting of Galphas(374-394)C379A--the 21-mer C terminus of the Galphas protein, able of adenosine inhibitory activity--and penetratin--the 16 residue fragment, derived from the homeodomain of the Drosophila transcription factor Antennapedia. A42 is able to cross cell membranes and to inhibit A2A and A2B adenosine and beta-adrenergic receptor stimulated camps (D'Ursi et al. Mol.

A membrane-permeable peptide containing the last 21 residues of the G alpha(s) carboxyl terminus inhibits G(s)-coupled receptor signaling in intact cells: correlations between peptide structure and biological activity.

Cell-penetrating peptides are able to transport covalently attached cargoes such as peptide or polypeptide fragments of endogenous proteins across cell membranes. Taking advantage of the cell-penetrating properties of the 16-residue fragment penetratin, we synthesized a chimeric peptide that possesses an N-terminal sequence with membrane-penetrating activity and a C-terminal sequence corresponding to the last 21 residues of G alpha(s). This G alpha(s) peptide was an effective inhibitor of 5'-N-ethylcarboxamidoadenosine (NECA) and isoproterenol-stimulated production of cAMP in rat PC12 and human microvascular endothelial (HMEC-1) cells, whereas the carrier peptide had no effect.

An enzyme-linked immunosorbent assay to screen for inhibitors of the oncogenic anaplastic lymphoma kinase.

The discovery of novel anti-cancer drugs targeting anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, raises the need for in vitro assays suitable for screening compounds for ALK inhibition. To this aim we have developed and optimized an ALK-specific enzyme-linked immunosorbent assay that employs a novel ALK peptide substrate and purified ALK kinase domain.