Structural determinants for activity of glucagon-like peptide-2.

Glucagon-like peptide-2 (GLP-2) is a 33 amino acid gastrointestinal hormone that regulates epithelial growth in the intestine. Dipeptidylpeptidase IV cleaves GLP-2 at the position 2 alanine, resulting in the inactivation of peptide activity. To understand the structural basis for GLP-2 action, we studied receptor binding and activation for 56 GLP-2 analogues with either position 2 substitutions or alanine replacements along the length of the peptide.

Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2.

Glucagon-like peptide 2 (GLP-2) is a 33-aa proglucagon-derived peptide produced by intestinal enteroendocrine cells. GLP-2 stimulates intestinal growth and up-regulates villus height in the small intestine, concomitant with increased crypt cell proliferation and decreased enterocyte apoptosis. Moreover, GLP-2 prevents intestinal hypoplasia resulting from total parenteral nutrition.

Circulating and tissue forms of the intestinal growth factor, glucagon-like peptide-2.

Glucagon-like peptide-2 (GLP-2) has recently been identified as a stimulator of intestinal epithelial growth, prompting the development of RIA and HPLC methodologies to study this peptide in more detail. A GLP-2-specific antiserum (UTTH-7) was developed that recognizes amino acids 25-30 of human and rat GLP-2-(1-33). UTTH-7 cross-reacts with N- and C-terminally modified forms of GLP-2, proglucagon, and the major proglucagon fragment.

Regulation of the biological activity of glucagon-like peptide 2 in vivo by dipeptidyl peptidase IV.

Species-specific differences in the enzymatic inactivation of peptides is an important consideration in the evaluation of therapeutic efficacy. We demonstrate that glucagon-like peptide 2 (GLP-2), shown to be highly intestinotrophic in mice, promotes an increase in intestinal villus height but has no trophic effect on small bowel weight in rats. The reduced intestinotrophic activity of GLP-2 in rats is attributable to inactivation by the enzyme dipeptidyl peptidase IV (DPP-IV).

Calcium binding and conformational properties of calmodulin complexed with peptides derived from myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP).

The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein kinase C(PKC) substrates that bind calmodulin (CaM) in a manner regulated by Ca2+ and phosphorylation by PKC. The CaM binding region overlaps with the PKC phosphorylation sites, suggesting a potential coupling between Ca(2+)-CaM signalling and PKC-mediated phosphorylation cascades. We have studies Ca2+ binding of CaM complexed with CaM binding peptides from MARCKS and MRP using flow dialysis, NMR and circular dichroism (CD) spectroscopy.

Characterization of novel calmodulin-binding peptides with distinct inhibitory effects on calmodulin-dependent enzymes.

We describe the isolation and interaction with calmodulin (CaM) of two 10-amino-acid peptides (termed peptides 1 and 2; AWDTVRISFG and AWPSLQAIRG respectively) derived from a phage random peptide display library. Both peptides are shorter than previously described CaM-binding peptides and lack certain features found in the sequences of CaM-binding domains present in CaM-activated enzymes. However, 1H NMR spectroscopy and fluorimetry indicate that both peptides interact with CaM in the presence of Ca2+.

Molecular and structural basis of target recognition by calmodulin.

Calmodulin (CaM) acts as an intracellular calcium sensor that translates the Ca2+ signal into a variety of cellular processes. Ca(2+)-CaM recognition of a short polypeptide segment in target proteins induces conformational changes in both CaM and the target, enabling the target protein to become functionally active. The solution and crystal structures of Ca(2+)-CaM bound to peptides derived from three CaM-dependent enzymes reveal structural features that are common in target recognition by Ca(2+)-CaM.

Use of Marfey's reagent to quantitate racemization upon anchoring of amino acids to solid supports for peptide synthesis.

A chromatographic assay has been developed to quantitate racemization occurring during attachment of protected amino acids to peptide synthesis resins. Acidolytic cleavage of deprotected amino acids from supports and subsequent derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) gave diastereomers separable by reverse-phase HPLC using aqueous acetonitrile. The assay is reliable to 0.

Optimization of radioimmunotherapy using human malignant melanoma multicell spheroids as a model.

In vitro multicell spheroids from a human melanoma cell line and the human colon cancer cell line HT29, used as control, have been established as a model of poorly vascularized micrometastases in vivo. The antimelanoma monoclonal antibody 96.5 was radiolabeled with 131I at specific radioactivities from 1.