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A study of two proline specific peptidases from bovine serum

Cunningham, Damian F (1996) A study of two proline specific peptidases from bovine serum. PhD thesis, Dublin City University.

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Abstract

Proline holds an important position among twenty naturally occurring ammo acids, the building blocks of pepudes and proteins. It confers particular biological properties upon these physiologically important biomolecules due to its unique structural characteristics. There has evolved a specialised group of enzymes that recognise this residue and can introduce peptide bond cleavage at either its carboxyl or ammo terminus within a peptide chain. The variety of these specialised peptidases cover practically all situations where a proline residue ought occur in a substrate and their action can be of biological significance, leading to the inactivation or biotransformation of peptides and proteins. Two distinct proline specific peptidases were detected m bovine serum usmg the substrate Z-Gly-Pro-MCA, a reportedly specific fluonmetnc substrate for prolyl endopeptidase (PE) One of these activities was inhibited by Z-Pro-Prolinal, a PE specific inhibitor, and was subsequently designated PE Hie second activity resisted inhibition by Z-Pro-Prolinal, even at high concentrations and increased premcubation tunes. This activity was subsequently designated Z-Pro-Prohnal insensitive Z-Gly-Pro-MCA degrading peptidase (Z3P). Both PE and ZIP activities in bovine serum were successfully separated usmg SP-Sepharose cation exchange chromatography and were subsequently punfied independently of each other. PE activity was partially punfied, following its separation from ZIP, usmg Phenyl-Sepharose hydrophobic interaction, DEAE-Sepharose anion exchange and Sephacryl S-200 HR gel filtration chromatographies, with a final yield of 24% and a final purification factor of 30 achieved. The enzyme had a native molecular weight of 70,000 Da, as determined by gel filtration chromatography. The subunit structure of the enzyme could not be determined by SDS PAGE due to the appearance of multiple bands following visualisation of the gel by silver staining. A pH optimum of 8 0, with a preference for phosphate buffer was determined for the partially punfied PE. The enzyme was stable over a pH range of 5-9 Optimal activity was obtained from PE at a temperature of 37°C with little activity being detected above or below this temperature. The enzyme was inhibited by AEBSF indicating that the enzyme is a member of the serine protease family. The enzyme was also inhibited by PCMB and activated by DTT, indicating the possible presence of an essential cysteine residue, close to the active site. PE hydrolysed the substrates Z-Gly-Pro-MCA and pGlu-His-Pro-MCA, with Km values of 94pM and 62i*M respectively. The enzyme also cleaved a vanety of proline containing bioactive peptides including LHRH, bradykinin, substance P and angiotensin II. These peptides also competitively inhibited PE activity towards Z-GIy-Pro-MCA PE demonstrated relatively high specificities towards bradykimn and angiotensin n with Kj values of 136pM and 113[iM respectively. The enzyme was inhibited by a range of PE specific inhibitors with the highest inhibitory activity being observed for a-ketobenzothiazole (IC50 = 41 picomolar) and Z-Indohnyl Prolinal (IC50 = 45 picomolar). Z-Pro-Prolinal insensitive Z-Gly-Pro-MCA degrading peptidase (ZIP) activity was punfied, following its separation from PE, usmg Phenyl-Sepharose hydrophobic interaction , Calcium Phosphate Cellulose and Sephacryl S-200 HR gel filtration chromatographies, with a final yield of 14% and a final purification factor of 2250 achieved. The enzyme had a native molecular weight of 185,000 Da, as determined by gel filtration chromatography The subunit structure of ZIP was determined to be tetramenc, based on the identification of a major band of 50,000Da by SDS PAGE following visualisation of the gel by silver staining. The enzyme exhibited a pH optimum of 8 5, and was stable over a pH range of 4*9 5 Optimal activity was obtained from ZIP at a temperature of 37°C- 40°C with significant activities being observed at 4*C and 20aC. The enzyme was inhibited by AEBSF indicating that it is probably a member of the senne protease family. ZIP hydrolysed the substrate Z-Gly-Rro-MC A, with a Km value of 267^M. The enzyme also cleaved proline containing bioactive peptides including LHRH, bradykimn and substance P. These peptides also inhibited ZIP activity towards Z-Gly-Pro-MCA with bradykimn and LHRH demonstrating competitive Kj values of 2 5mM and 475pM respectively. The enzyme was inhibited by some of the PE specific inhibitors tested. Highest inhibitory activity was observed for a-ketobenzothiazole (IC50 =15 picomolar). Bovme serum PE activity, punfied and charactensed dunng this investigation, is similar to PE isolated from other sources with regard to its biophysical and biochemical characteristics, and its substrate specificity. Its localisation in serum, and it activity towards proline containing bioactive peptides indicates that it may play and important physiological role in the metabolism of such peptides. ZIP activity may represent a novel profane specific peptidase localised m serum which might also play a role m the degradation of proline containing bioactive peptides.

Item Type:Thesis (PhD)
Date of Award:1996
Refereed:No
Supervisor(s):O'Connor, Brendan
Uncontrolled Keywords:Peptidase; Proline; Amino acids; Enzymology
Subjects:Biological Sciences > Biotechnology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:19157
Deposited On:23 Aug 2013 14:35 by Celine Campbell. Last Modified 23 Aug 2013 14:35

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