Isolation and Characterization of NP-POL Nonapeptide for Possible Therapeutic Use in Parkinson's Disease.

Colostrum and milk are the initial mammalian nourishment and rich reservoir of essential nutrients for newborn development. Bioactive peptides isolated from natural sources, such as colostrum, serve as endogenous regulators and can be used as alternative therapeutic agents in the treatment of neurodegenerative diseases. One example is the previously unknown NP-POL nonapeptide isolated from Colostrinin. In the present study, we investigated a method of NP-POL nonapeptide isolation using Bio-Gel P2 molecular sieve chromatography. We showed the protective effect of NP-POL on 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity using rat adrenal pheochromocytoma (PC12 Tet On) cells. Treatment of PC12 cells with NP-POL nonapeptide reduced 6-OHDA-induced apoptosis and caused transient phosphorylation of extracellular signal-regulated kinases (ERK 1/2), which were shown to promote cell survival. NP-POL nonapeptide also protected neuronal cells against oxidative injury induced by 6-OHDA. These results showed a potential use of NP-POL in the therapy of Parkinson's disease.

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis.

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.

Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds.

Seven new trypsin inhibitors, CyPTI I-VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28-30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine beta-trypsin (K(a)>10(11) M(-1)) and, more weakly, bovine alpha-chymotrypsin (K(a) approximately 10(4)-10(6) M(-1)). In the presence of 3 M NaCl the association constants of CyPTIs with alpha-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25-Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications.

LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression.

A novel chymotrypsin inhibitor of the potato I protease inhibitor family from the earthworm Lumbricus terrestris was purified. The inhibitor, named LTCI, was isolated by methanol extraction, affinity chromatography on immobilized methylchymotrypsin, and ion exchange chromatography followed by RP-HPLC. The 7076 Da inhibitor consists of a single polypeptide chain of 64-amino-acid residues without disulfide bridges. LTCI is the first of the potato I protease inhibitors with Tyr in position P1 of the reactive site. cDNA analysis revealed that LTCI is produced as a 86-amino-acid precursor with a 22-amino-acid secretory signal peptide. RT-PCR analysis demonstrates that LTCI mRNA is expressed in body wall, intestine, and coelomocytes. The recombinant LTCI was produced in Escherichia coli as a fusion protein with intein and chitin binding domain using IMPACT-CN system.

The alcohol-induced conformational changes in casein micelles: a new challenge for the purification of colostrinin.

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.

Non-conventional affinity chromatography of serine proteinases and their inhibitors.

From among a wide variety of protein purification techniques affinity chromatography has proved to be particularly effective for separation of proteolytic enzymes and their inhibitors. In this article, following a general description of affinity adsorbents used for purification of proteinases, we overview a simple separation procedure for some serine proteinases and their inhibitors by way of affinity chromatography in the presence of high NaCl concentration. It has been shown that some highly specific trypsin inhibitors exhibit also antichymotrypsin activity when high concentration of Na(+) but not K(+) or Li(+) ions are present in the reaction mixture. Taking advantage of this phenomenon the virgin forms of trypsin inhibitors from squash seeds, Kazal-type inhibitor from porcine pancreas and alpha(1)-proteinase inhibitor from human and sheep plasma, as an example, were separated using immobilized chymotrypsin or its inactive derivative methylchymotrypsin in the presence of 5 M NaCl.

The use of immobilized methylchymotrypsin for the purification of human and sheep alpha-1-proteinase inhibitor (alpha(1)-PI).

alpha(1)-proteinase inhibitor was isolated from albumin fractions of human and sheep plasma using a new method of purification using affinity chromatography on immobilized methylchymotrypsin in the presence of 5 M NaCl. The inhibitor was finally polished to homogenity either by chromatography on a Mono Q or a Sephacryl S-200 HR column. The presented method makes it possible to recover alpha(1)-proteinase inhibitor which has been added to cow milk.

Kazal-type chymotrypsin inhibitor from duck pancreas.

A chymotrypsin inhibitor of the Kazal-type has been isolated from duck pancreas, by affinity chromatography on immobilized chymotrypsin, gel filtration on Bio-Gel P-10 and reverse phase (RP)-HPLC. It inhibits bovine chymotrypsin Aalpha with an association constant (K(a)) of 2.06x10(7) M(-1). The complete amino acid sequence was determined after digestion of pyridylethylated inhibitor with Staphylococcus aureus V8 protease and chemical cleavage with CNBr. Duck pancreatic chymotrypsin inhibitor (DPCI) was found to be a single polypeptide chain composed of 65 amino acid residues, corresponding to a molecular mass of 7191 Da.