Structure-function properties of food protein-derived calmodulin-binding peptides: in vivo studies

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Calmodulin (CaM) is a 16.7 kDa multifunctional calcium-binding protein that is a proven major translator of intracellular calcium message. CaM is necessary for calcium-dependent control of many cellular events including cell proliferation, cell division and neurotransmission. Excessive activities of CaM-dependent enzymes can lead to metabolic disorders such as diabetes, hypertension, abnormal cell growth and arthritis. Therefore, peptides that bind to CaM and inhibit activity of CaM-dependent enzymes may be used to formulate nutritional supplements for preventing onset or progression of chronic ailments. My previous NSERC-funded work led to the discovery of several food (flaxseed and yellow field pea) protein-derived peptide sequences that work as in vitro inhibitors of CaM-dependent enzymes such as phosphodiesterase 1 (CaMPDE) and the nitric oxide synthases (NOSs). Therefore, the main objective of current proposal is to determine ability of these peptides (2-7 amino acid residues) to escape digestion in the gastrointestinal tract and be absorbed intact into the blood circulatory system. The work will also determine inhibitory ability of the absorbed peptides against tissue activities of CaMPDE and NOS enzymes (endothelial, neuronal and inducible), including peptide-induced reductions in levels of endogenous toxic free radicals as well as increased level of antioxidant capacity. To determine in vivo absorption properties, Wistar rats will be fed purified peptides or protein hydrolysate fractions with known peptide compositions. Peptide composition of the blood will be determined after gavage using liquid chromatography/mass spectrometry techniques to determine if absorption occurred. Oral feeding of peptides to rats subjected to ethanol- or lipopolysaccharide-induced oxidative stress will be followed by determination of plasma and organ tissue levels of toxic free radicals, especially superoxide whose production (catalyzed by endothelial NOS) becomes greatly enhanced during oxidative stress. The proposed work could enhance development of novel peptide-based supplements that can be used as preventive agents against accumulation of toxic free radicals in the body.