Studies on the kallikrein-kininogen system of the ostrich (Struthio camelus)
- Authors: Bothma, Leonard Frederick
- Date: 2001
- Subjects: Kallikrein , Kinins , Ostriches
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11067 , http://hdl.handle.net/10948/275 , Kallikrein , Kinins , Ostriches
- Description: Ostrich organs/tissue/fluids were screened for plasma kallikrein-like, tissue kallikrein-like and tonin-like activity in a continuous-fluorogenic-assay system using Pro-Phe-Arg-7-amino-4-methylcoumarine, Phe- Arg-7-amino-4-methylcoumarine and Val-Leu-Arg--7-amino-4-trifluoro-methylcoumarine as substrates. Ostrich liver and kidney showed the highest specific plasma kallikrein-like activity. Ostrich adrenal glands and kidney showed the highest specific tissue kallikrein-like and tonin-like activity. Ostrich high molecular weight kininogen was purified from plasma and low molecular weight kininogen was partially purified. The N-terminal amino acid sequences of both high- and low molecular weight kininogens from ostrich plasma were determined. Ostrich plasma high molecular weight kininogen was purified as a 118 kD protein. The purified high molecular weight kininogen inhibits the cysteine proteinase papain at a ratio of one molecule HKG to two molecules of papain. Ornitho kinin-like molecules were detected in ostrich urine using reverse phase HPLC.
- Full Text:
- Date Issued: 2001
- Authors: Bothma, Leonard Frederick
- Date: 2001
- Subjects: Kallikrein , Kinins , Ostriches
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11067 , http://hdl.handle.net/10948/275 , Kallikrein , Kinins , Ostriches
- Description: Ostrich organs/tissue/fluids were screened for plasma kallikrein-like, tissue kallikrein-like and tonin-like activity in a continuous-fluorogenic-assay system using Pro-Phe-Arg-7-amino-4-methylcoumarine, Phe- Arg-7-amino-4-methylcoumarine and Val-Leu-Arg--7-amino-4-trifluoro-methylcoumarine as substrates. Ostrich liver and kidney showed the highest specific plasma kallikrein-like activity. Ostrich adrenal glands and kidney showed the highest specific tissue kallikrein-like and tonin-like activity. Ostrich high molecular weight kininogen was purified from plasma and low molecular weight kininogen was partially purified. The N-terminal amino acid sequences of both high- and low molecular weight kininogens from ostrich plasma were determined. Ostrich plasma high molecular weight kininogen was purified as a 118 kD protein. The purified high molecular weight kininogen inhibits the cysteine proteinase papain at a ratio of one molecule HKG to two molecules of papain. Ornitho kinin-like molecules were detected in ostrich urine using reverse phase HPLC.
- Full Text:
- Date Issued: 2001
Ostrich calpastatin purification and partial characterization of the liver inhibitor
- Authors: Roman, Henry James
- Date: 2000
- Subjects: Calpastatin , Protease inhibitors , Ion exchange chromatography , Ostriches
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11090 , http://hdl.handle.net/10948/d1015522
- Description: The isolation and purification of calpastatin from ostrich liver is presented, along with its physicochemical and kinetic properties. By using extraction from liver, ion-exchange chromatography on DEAE-Toyopearl, heating to 90 °C for 10 min and rechromatography on Toyopearl Super-Q 650 S, ostrich calpastatin was isolated and purified from ostrich liver. The purified intact calpastatin showed homogeneity on SDS-PAGE (Mr of 105.6 K). Amino acid analysis showed that ostrich calpastatin resembled that of rabbit liver and human erythrocyte calpastatin. An N-terminal sequence could not be obtained because the N-terminus was found to be blocked by an as yet unknown amino acid residue. The Mr values of degradative forms of ostrich liver calpastatin were determined to be 56 K and 90 K. By using PAG-IEF the pI of the intact form was determined to be 5.1. Ostrich liver calpastatin behaved characteristically like other calpastatins during kinetic analysis. Calpastatin inhibited calpain from pH 6 to 9 and was found to be unaffected by temperatures as high as 100 °C. Calpastatin also inhibited calpain activity at Ca2+ concentrations ranging from 1 to 10 mM. The inhibitor was shown to be phosphorylated because after incubation with alkaline phosphatase there was a decrease in inhibitory activity. No inhibitory effects were detected against other proteases such as chymotrypsin and trypsin, with both proteases inactivating calpastatin completely. Ostrich liver calpain was shown to have a pH optimum of 7.5 and a temperature optimum of 30 °C. In terms of its thermodynamic properties it resembled that of other ostrich proteases; DH, DS and DG being 47.07 kJ/mol, -91.1 J/mol/K and 74.237 kJ/mol, respectively. Ostrich liver calpain showed a Km of 0.14 % (w/v). The enzyme was active at both milli- and micro-molar concentrations of Ca2+. Ostrich liver calpastatin showed many physical, chemical and kinetic properties similar to those of other known calpastatins.
- Full Text: false
- Date Issued: 2000
- Authors: Roman, Henry James
- Date: 2000
- Subjects: Calpastatin , Protease inhibitors , Ion exchange chromatography , Ostriches
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:11090 , http://hdl.handle.net/10948/d1015522
- Description: The isolation and purification of calpastatin from ostrich liver is presented, along with its physicochemical and kinetic properties. By using extraction from liver, ion-exchange chromatography on DEAE-Toyopearl, heating to 90 °C for 10 min and rechromatography on Toyopearl Super-Q 650 S, ostrich calpastatin was isolated and purified from ostrich liver. The purified intact calpastatin showed homogeneity on SDS-PAGE (Mr of 105.6 K). Amino acid analysis showed that ostrich calpastatin resembled that of rabbit liver and human erythrocyte calpastatin. An N-terminal sequence could not be obtained because the N-terminus was found to be blocked by an as yet unknown amino acid residue. The Mr values of degradative forms of ostrich liver calpastatin were determined to be 56 K and 90 K. By using PAG-IEF the pI of the intact form was determined to be 5.1. Ostrich liver calpastatin behaved characteristically like other calpastatins during kinetic analysis. Calpastatin inhibited calpain from pH 6 to 9 and was found to be unaffected by temperatures as high as 100 °C. Calpastatin also inhibited calpain activity at Ca2+ concentrations ranging from 1 to 10 mM. The inhibitor was shown to be phosphorylated because after incubation with alkaline phosphatase there was a decrease in inhibitory activity. No inhibitory effects were detected against other proteases such as chymotrypsin and trypsin, with both proteases inactivating calpastatin completely. Ostrich liver calpain was shown to have a pH optimum of 7.5 and a temperature optimum of 30 °C. In terms of its thermodynamic properties it resembled that of other ostrich proteases; DH, DS and DG being 47.07 kJ/mol, -91.1 J/mol/K and 74.237 kJ/mol, respectively. Ostrich liver calpain showed a Km of 0.14 % (w/v). The enzyme was active at both milli- and micro-molar concentrations of Ca2+. Ostrich liver calpastatin showed many physical, chemical and kinetic properties similar to those of other known calpastatins.
- Full Text: false
- Date Issued: 2000
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
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