The ostrich is becoming an important source of meat for humans in developed and developing countries. This study was conducted to purify and characterize myofibrilbound serine protease (MBSP) and its endogenous inhibitor (MBSPI) from skeletal muscle of the ostrich. It is well documented that MBSP is tightly bound to myofibrils and its endogenous inhibitor has been purified from the same tissue of other studied mammalian species. Literature supports an association of MBSP and its endogenous inhibitor with the degradation of myofribrillar proteins, resulting in the softening of muscle that lead to the conversion of muscle into meat with the control of the inhibitor. MBSP was successfully dissociated from washed myofibrils by 40 percent ethylene glycol at pH 8.5. Following centrifugation, MBSP was partially purified in two chromatographic steps, namely Toyopearl Super Q 650S and p-aminobenzamidine-Agarose. On the other hand, MBSPI was fractionated from the sarcoplasmic fraction using 75 percent ammonium sulfate saturation, followed by centrifugation and partially purified by three chromatographic steps, namely Toyopearl Super Q 650S, Superdex 200 and HiTrap SP HR. Ostrich MBSP was physicochemically and kinetically characterized, while MBSPI was only physicochemically characterized. Ostrich MBSP revealed an Mr of 21 kDa, cleaving synthetic fluorogenic substrates specifically at the carboxyl side of arginine residues. Optimum pH and temperature of ostrich MBSP were 8.0 and 40˚C, respectively. Kinetic parameters (Km and Vmax values) were calculated from Lineweaver-Burk plots. The characteristics of ostrich MBSP were compared to the values obtained for commercial bovine trypsin in this study, as well as that obtained for MBSP from various fish species and mouse. The results suggest that ostrich MBSP is a trypsin-like serine protease, thereby confirming the existence of MBSP in ostrich skeletal muscle. Partially purified ostrich MBSPI (Mr 17 kDa) (one form) shares 100 percent identity to myoglobin from the same species, while 2 other forms of MBSPI (Mr values of 35 and 36 kDa) exhibited high sequence identity to glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (76 percent) from human and rat.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nmmu/vital:10330 |
| Date | January 2008 |
| Creators | Tshidino, Shonisani Cathphonia |
| Publisher | Nelson Mandela Metropolitan University, Faculty of Science |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Masters, MSc |
| Format | xix, 104 leaves, pdf |
| Rights | Nelson Mandela Metropolitan University |
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