Purification and characterisation of 20S proteasome from ostrich skeletal muscle and its role in meat tenderisation
- Authors: Thomas, Adele René
- Date: 2004
- Subjects: Proteolytic enzymes , Ostrich products industry
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: vital:11081 , http://hdl.handle.net/10948/320 , Proteolytic enzymes , Ostrich products industry
- Description: The proteasome is renowned for its high molecular weight, multisubunit and mulicatalytic nature. One of its many suggested roles is the degradation of myofibrillar proteins, and therefore it has been proposed to play a role in the meat tenderisation process. The aim of this study was therefore to isolate, purify and characterise the 20S proteasome from ostrich skeletal muscle, with a view to ultimately investigating its role in the tenderisation process of ostrich meat. The 20S proteasome was successfully isolated and purified from ostrich skeletal muscle using Toyopearl Super Q-650S, Sephacryl S-300, hydroxylapatite and Mono Q chromatographies. The intact molecule showed a molecular weight of 725 K and a pI of 6.67. The subunits showed a molecular weight range of 22.2-33.5 K and a pI range of 3-9. 2D-PAGE revealed at least 14 polypeptides. The amino acid composition of the intact enzyme and of each of the eight subunits separating on SDSPAGE, as well as the N-terminal sequences of five of the eight subunits, were determined. The trypsinlike (Tr-L), chymotrypsin-like (ChT-L), peptidylglutamyl peptide hydrolase (PGPH) and caseinolytic activities showed pH optima of 11, 9, 7-8 and 10.3, and temperature optima of 40, 60, 70 and 60oC, respectively. The pH stability range for all four activities was 5-12. The ChT-L and PGPH activities showed thermostabilities up to 60oC, whereas the Tr-L and caseinolytic activities were stable up to 40o C. The enzyme showed complex kinetics. It was inhibited by the peptide aldehyde Z-LLL-CHO and cysteine protease inhibitors. Cations had negligible effects on the enzyme, excepting for Ca2+ and Mg2+. Of the detergents tested, SDS had the most potent stimulatory effect, particularly on the PGPH and caseinolytic activities. The fatty acid studies showed that unsaturation enhanced the ChT-L and the caseinolytic activities, while it completely suppressed the Tr-L activity. Heating at 60oC for 1-2 min stimulated the caseinolytic and PGPH activities. The studies on the role of ostrich skeletal muscle 20S proteasome in ostrich meat tenderisation suggested a definite but minor role of this enzyme, based on the fact that it remained active throughout the 12 days of storage of ostrich M. iliofibularis meat at 4oC and that it participated in myofibril degradation of post-mortem muscle, but to a small degree. These results support the proposal that the proteasome comes into play after the calpains have initiated degradation. However, there was a lack of improvement in tenderness values and minimal myofibrillar degradation over the 12-day storage period of the ostrich M. iliofibularis meat, leading to the conclusion that the tenderisation of this meat was incomplete after 12 days.
- Full Text:
- Date Issued: 2004
- Authors: Thomas, Adele René
- Date: 2004
- Subjects: Proteolytic enzymes , Ostrich products industry
- Language: English
- Type: Thesis , Doctoral , DPhil
- Identifier: vital:11081 , http://hdl.handle.net/10948/320 , Proteolytic enzymes , Ostrich products industry
- Description: The proteasome is renowned for its high molecular weight, multisubunit and mulicatalytic nature. One of its many suggested roles is the degradation of myofibrillar proteins, and therefore it has been proposed to play a role in the meat tenderisation process. The aim of this study was therefore to isolate, purify and characterise the 20S proteasome from ostrich skeletal muscle, with a view to ultimately investigating its role in the tenderisation process of ostrich meat. The 20S proteasome was successfully isolated and purified from ostrich skeletal muscle using Toyopearl Super Q-650S, Sephacryl S-300, hydroxylapatite and Mono Q chromatographies. The intact molecule showed a molecular weight of 725 K and a pI of 6.67. The subunits showed a molecular weight range of 22.2-33.5 K and a pI range of 3-9. 2D-PAGE revealed at least 14 polypeptides. The amino acid composition of the intact enzyme and of each of the eight subunits separating on SDSPAGE, as well as the N-terminal sequences of five of the eight subunits, were determined. The trypsinlike (Tr-L), chymotrypsin-like (ChT-L), peptidylglutamyl peptide hydrolase (PGPH) and caseinolytic activities showed pH optima of 11, 9, 7-8 and 10.3, and temperature optima of 40, 60, 70 and 60oC, respectively. The pH stability range for all four activities was 5-12. The ChT-L and PGPH activities showed thermostabilities up to 60oC, whereas the Tr-L and caseinolytic activities were stable up to 40o C. The enzyme showed complex kinetics. It was inhibited by the peptide aldehyde Z-LLL-CHO and cysteine protease inhibitors. Cations had negligible effects on the enzyme, excepting for Ca2+ and Mg2+. Of the detergents tested, SDS had the most potent stimulatory effect, particularly on the PGPH and caseinolytic activities. The fatty acid studies showed that unsaturation enhanced the ChT-L and the caseinolytic activities, while it completely suppressed the Tr-L activity. Heating at 60oC for 1-2 min stimulated the caseinolytic and PGPH activities. The studies on the role of ostrich skeletal muscle 20S proteasome in ostrich meat tenderisation suggested a definite but minor role of this enzyme, based on the fact that it remained active throughout the 12 days of storage of ostrich M. iliofibularis meat at 4oC and that it participated in myofibril degradation of post-mortem muscle, but to a small degree. These results support the proposal that the proteasome comes into play after the calpains have initiated degradation. However, there was a lack of improvement in tenderness values and minimal myofibrillar degradation over the 12-day storage period of the ostrich M. iliofibularis meat, leading to the conclusion that the tenderisation of this meat was incomplete after 12 days.
- Full Text:
- Date Issued: 2004
The isolation and partial characterization of a2-antiplasmin and plasminogen from ostrich plasma
- Authors: Thomas, Adele René
- Date: 2000
- Subjects: Serpins , Ostriches , Antifibrinolytic agents , Plasminogen , Plasmin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11080 , http://hdl.handle.net/10948/274 , http://hdl.handle.net/10948/d1005751 , Serpins , Ostriches , Antifibrinolytic agents , Plasminogen , Plasmin
- Description: This study reports the isolation, purification and partial characterisation of the ostrich serpin, a2AP, as well as its target enzyme, ostrich plasmin, in its active and inactive proenzyme, viz. plasminogen, forms. Three different procedures were undertaken to isolate and purify ostrich a2AP. The first one involved L-lysine-Sepharose chromatography, ammonium sulfate fractionation, ion-exchange chromatography on Toyopearl Super-Q 650S, and ostrich plasminogen-Sepharose affinity chromatography. The second procedure replaced the latter chromatographic step with gel filtration on Sephadex G-200 and hydroxylapatite chromatography, while the third one employed instead the theoretically more efficient LBSI-Sepharose chromatographic step. The third procedure yielded purified ostrich a2AP, but the degree of purity and yield were relatively low. Ostrich plasminogen was highly purified after L-lysine-Sepharose chromatography and ostrich plasmin was obtained by the urokinase-activation of the purified ostrich plasminogen Ostrich a2AP revealed an Mr of 77-84 K and two isoelectric forms of pI 3.85 and 6.18. Nterminal sequence analysis showed ostrich a2AP to have only 2 out of 11 residues in common with both those of human and bovine a2AP. Ostrich a2AP showed the largest inhibitory effects on ostrich plasmin, followed by comm. bovine chymotrypsin, trypsin and plasmin, in that order, and it appeared to be a much less potent plasmin inhibitor than bovine aprotinin, but a much more potent one than the synthetic inhibitors, DFP and EACA. Ostrich plasminogen showed an Mr of 92 K and multiple isoelectric forms (~7) in the pI range 6.01-9.18, with a major one of pI 6.01. It showed a total of 775 amino acid residues and its N-terminal sequence showed ~53 percent identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin revealed an Mr of 78 K, two isoelectric forms of pI 4.07 and 6.01, and a total of 638 amino acid residues. N-terminal sequence analysis showed that 2-4 residues are identical to the 5 of human, cat, dog, rabbit, and ox plasmins. The pH and temperature optima of ostrich plasmin were determined as 8.0 and 40 oC, respectively. The thermodynamic and kinetic parameters of ostrich plasmin were computed, and plasmin was shown to prefer Lys to Arg residues in the S1 position. In conclusion, ostrich a2AP, plasminogen and plasmin showed definite similarities to their mammalian counterparts, but there were also significant differences.
- Full Text:
- Date Issued: 2000
- Authors: Thomas, Adele René
- Date: 2000
- Subjects: Serpins , Ostriches , Antifibrinolytic agents , Plasminogen , Plasmin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11080 , http://hdl.handle.net/10948/274 , http://hdl.handle.net/10948/d1005751 , Serpins , Ostriches , Antifibrinolytic agents , Plasminogen , Plasmin
- Description: This study reports the isolation, purification and partial characterisation of the ostrich serpin, a2AP, as well as its target enzyme, ostrich plasmin, in its active and inactive proenzyme, viz. plasminogen, forms. Three different procedures were undertaken to isolate and purify ostrich a2AP. The first one involved L-lysine-Sepharose chromatography, ammonium sulfate fractionation, ion-exchange chromatography on Toyopearl Super-Q 650S, and ostrich plasminogen-Sepharose affinity chromatography. The second procedure replaced the latter chromatographic step with gel filtration on Sephadex G-200 and hydroxylapatite chromatography, while the third one employed instead the theoretically more efficient LBSI-Sepharose chromatographic step. The third procedure yielded purified ostrich a2AP, but the degree of purity and yield were relatively low. Ostrich plasminogen was highly purified after L-lysine-Sepharose chromatography and ostrich plasmin was obtained by the urokinase-activation of the purified ostrich plasminogen Ostrich a2AP revealed an Mr of 77-84 K and two isoelectric forms of pI 3.85 and 6.18. Nterminal sequence analysis showed ostrich a2AP to have only 2 out of 11 residues in common with both those of human and bovine a2AP. Ostrich a2AP showed the largest inhibitory effects on ostrich plasmin, followed by comm. bovine chymotrypsin, trypsin and plasmin, in that order, and it appeared to be a much less potent plasmin inhibitor than bovine aprotinin, but a much more potent one than the synthetic inhibitors, DFP and EACA. Ostrich plasminogen showed an Mr of 92 K and multiple isoelectric forms (~7) in the pI range 6.01-9.18, with a major one of pI 6.01. It showed a total of 775 amino acid residues and its N-terminal sequence showed ~53 percent identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin revealed an Mr of 78 K, two isoelectric forms of pI 4.07 and 6.01, and a total of 638 amino acid residues. N-terminal sequence analysis showed that 2-4 residues are identical to the 5 of human, cat, dog, rabbit, and ox plasmins. The pH and temperature optima of ostrich plasmin were determined as 8.0 and 40 oC, respectively. The thermodynamic and kinetic parameters of ostrich plasmin were computed, and plasmin was shown to prefer Lys to Arg residues in the S1 position. In conclusion, ostrich a2AP, plasminogen and plasmin showed definite similarities to their mammalian counterparts, but there were also significant differences.
- Full Text:
- Date Issued: 2000
- «
- ‹
- 1
- ›
- »