PEP activity has been described in several locations and has mostly been linked to a variety of neurological disorders such as schizophrenia, amnaesia, depression as well as other disease states such as anorexia nervosa, bulimia nervosa and blood pressure regulation. The enzyme has also been previously isolated from a variety of archae, microorganisms and several eukaryotic species but no prolyl endopeptidases have been isolated from plants. Plants have high levels of proline and glutamine rich peptides in seeds. We therefore hypothesize plants must express PEPs during germination. Bioinformatics tools were used to identify known PEPs and putative plant PEPs. A global sequence alignment of putative plant PEPs and other known PEPs indicated that the active site amino acids Ser, His and Asp are conserved in putative plant PEP sequences. Furthermore, putative plant PEPs showed similar secondary structures to known PEPs and when a rice PEP was modelled onto porcine brain PEP structure, a high degree of similarity was found. Germination studies of wheat seed showed an increase of PEP activity over time with maximum PEP activity reached after 4 days of germination, which remained at this level until 9 days of germination, implying a function for PEP in plant seed germination. Wheat PEP was purified using ion exchange and gel filtration chromatography with a final yield of less than 1 percent and a relative purity (only 2 bands detected by SDS-PAGE). The purified wheat PEP had a molecular weight of approximately 55kDa, substrate specificity for chymotrypsin-like substrates (N-Suc-Ala-Ala-Pro-Phe-pNa, Km value of 0.58 mM, Kcat of 29.37 s–1; Kcat /Km 50813.14s–1 M–1); a pH optimum of 7.9; temperature optima of 37oC and a high sensitivity to temperature as indicated by loss of activity at temperatures above 40oC. Inhibition studies using E64, Leupeptin and PMSF confirmed that the wheat PEP is from the Serine protease family and is most likely a trypsin-like protease.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufh/vital:11269 |
| Date | January 2013 |
| Creators | Abrahams, Adriam Mark |
| Publisher | University of Fort Hare, Faculty of Science & Agriculture |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Masters, MSc (Biochemistry) |
| Format | 113 leaves; 30 cm, pdf |
| Rights | University of Fort Hare |
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