ABSTRACT
Recent advances in protein separation technology have allowed for the isolation of whey proteins and peptides of significant biological importance. In this study, we report a novel method for isolation and purification of the neuroprotective proline-rich polypeptides, also known as Colostrinin (CLN). Although CLN was first isolated from ovine colostrum and characterized as a complex of small molecular peptides, its constituents are present also in other mammal colostrums. The previous purification protocols are very tedious, time consuming, and, due to the diverse characteristics of colostrum, also very difficult to validate. Thus, the aim of this study was to develop a simple protocol with a maximum recovery rate for CLN peptides. Here we demonstrate the two-step extraction/purification method that consists of methanol extraction and ammonium sulfate precipitation as the general principles. When compared with the original material, CLN obtained by this method shows (1) similar pattern of peptides in SDS PAGE, (2) identical amino acid analysis, characterized by high content of proline (22%), a high proportion of nonpolar amino acids, a low percentage of glycine, alanine, arginine, histidine, and no tryptophan, methionine, and cysteine residues, (3) similar pattern of HPLC profiles, and (4) its ability to induce IFN gamma and TNF alpha. More importantly, the protocol for the production of high-quality CLN can be accomplished in less than a 48 h timeframe. In addition, avoidance of excessively harsh conditions preserves the structure and biological activity of the peptides.
Subject(s)
Alcohols/pharmacology , Caseins/drug effects , Peptides/isolation & purification , Ammonium Sulfate/chemistry , Animals , Chromatography, High Pressure Liquid , Colostrum , Electrophoresis, Polyacrylamide Gel , Intercellular Signaling Peptides and Proteins , Interferon-gamma/metabolism , Methanol/chemistry , Micelles , Milk/chemistry , Peptides/pharmacology , Protein Conformation/drug effects , Sheep , Tumor Necrosis Factor-alpha/metabolismABSTRACT
In previous studies we showed that colostrinin (CLN), a complex of proline-rich polypeptides derived from ovine colostrum, induces mitogenic stimulation, as well as a variety of cytokines in human peripheral blood leukocytes, and possesses antioxidant activity in pheochromocytoma (PC12) cells. In this study we investigated the effects of CLN on 4-hydroxynonenal (4HNE)-mediated adduct formation, generation of reactive oxygen species (ROS), glutathione (GSH) metabolism, and the modification of signal transduction cascade that leads to activation of c-Jun N-terminal kinase (JNK) in PC12 cells. Here we demonstrate that CLN (1) reduced the abundance of 4HNE-protein adducts, as shown by fluorescent microscopy and Western blot analysis; (2) reduced intracellular levels of ROS, as shown by a decrease in 2',7'-dichlorodihydro-fluorescein-mediated fluorescence; (3) inhibited 4HNE-mediated GSH depletion, as determined fluorimetrically; and (4) inhibited 4HNE-induced activation of JNKs. Together, these findings suggest that CLN appears to down-regulate 4HNE-mediated lipid peroxidation and its product-induced signaling that otherwise may lead to pathological changes at the cellular and organ level. These findings also suggest further that CLN could be useful in the treatment of diseases such as Alzheimer's, as well as those in which ROS are implicated in pathogenesis.
