Shivaprasad, H. V. and Rajaiah, R. and Frey, B. M. and Frey, F. J. and Vishwanath, B. S. (2010) 'Pergularain e I' - a plant cysteine protease with thrombin-like activity from Pergularia extensa latex. Thrombosis Research, 125 (3). E100-E105. ISSN 1879-2472
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Pergularain e I, a cysteine protease with thrombin-like activity, was purified by ion exchange chromatography from the latex of Pergularia extensa. Its homogeneity was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of pergularain e I by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was found to be 23.356 kDa and the N-terminal sequence is L-P-H-D-V-E. Pergularain e I is a glycoprotein containing similar to 20% of carbohydrate. Pergularain e I constituted 6.7% of the total protein with a specific activity of 9.5 units/mg/min with a 2.11-fold increased purity. Proteolytic activity of the pergularain e I was completely inhibited by iodoacetic acid (IAA). Pergularain e I exhibited procoagulant activity with citrated plasma and fibrinogen similar to thrombin. Pergularain e I increases the absorbance of fibrinogen solution in concentration-dependant and time-dependant manner. At 10 mu g concentration, an absorbance of 0.48 was reached within 10 min of incubation time. Similar absorbance was observed when 0.2 NIH units of thrombin were used. Thrombin-like activity of pergularain e I is because of the selective hydrolysis of A alpha and B beta chains of fibrinogen and gamma-chain was observed to be insusceptible to hydrolysis. Molecular masses of the two peptide fragments released from fibrinogen due to the hydrolysis by pergularain e I at 5-min incubation time were found to be 1537.21 and 1553.29 and were in close agreement with the molecular masses of 16 amino acid sequence of fibrinopeptide A and 14 amino acid sequence of fibrinopeptide B, respectively. Prolonged fibrinogen-pergularain e I incubation releases additional peptides and their sequence comparison of molecular masses of the released peptides suggested that pergularain e I hydrolyzes specifically after arginine residues. (C) 2009 Elsevier Ltd. All rights reserved.
| Item Type: | Article |
|---|---|
| Subjects: | C Chemical Science > Biochemistry |
| Divisions: | Department of > Biochemistry |
| Depositing User: | LA manjunath user |
| Date Deposited: | 15 Jul 2019 11:12 |
| Last Modified: | 15 Jul 2019 11:12 |
| URI: | http://eprints.uni-mysore.ac.in/id/eprint/5220 |
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