Studies of the interfacial and heparin binding properties of secreted phospholipases A2
Studies of the interfacial and heparin binding properties of secreted phospholipases A2
Human non-pancreatic secreted phospholipase A2 (hnps PLA2) is a small (14 kDa) secreted protein of considerable biomedical interest. The enzyme is associated with a number of inflammatory disorders in which the extracellular concentrations of this enzyme can become highly elevated.
The major part of the project involved the production and analysis of N-terminal mutants (including met-PLA2). The primary focus was on the 3-position of the enzyme and the production of two mutants, V3W and V3C, together with their analysis in terms of interfacial binding and catalytic activity. The V3W mutant was prepared because the equivalent unique trytophan in the pancreatic enzyme has been extensively used as a natural probe for interfacial binding. The human mutant had 40% of the activity of the wild-type enzyme when assayed using anionic phospholipids, however, a dramatic feature of this mutant was its enhanced activity (250 fold) when assayed with vesicles of phosphatidyl choline. A 10-fold enhancement of activity was also seen using whole cells as substrates. The results highlight the potential importance of interfacial trytophan residues in promoting the penetration of condensed zwitterionic interfaces as also seen with the surface monolayer of cell membranes. A V3C mutant has also been constructed to provide a potential site of chemical attachment of fluorescent reporter groups. Preliminary analysis of this mutant enzyme has revealed that it still has about 40% of the activity of the wild-type, however, the new cysteine at position 3 was blocked. Mass-spectral analysis indicates that this cysteine is conjugate via a disulphide linkage to free cysteine, a major constituent of the refolding medium. A final mutant, H48Q, was constructed to produce an inactive enzyme and provide an ideal interfacial binding protein for use in competitive binding studies. In fact the H48Q mutant possessed significant catalytic activity which has considerable mechanistic implications.
University of Southampton
1998
Baker, Sharon Felicity
(1998)
Studies of the interfacial and heparin binding properties of secreted phospholipases A2.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Human non-pancreatic secreted phospholipase A2 (hnps PLA2) is a small (14 kDa) secreted protein of considerable biomedical interest. The enzyme is associated with a number of inflammatory disorders in which the extracellular concentrations of this enzyme can become highly elevated.
The major part of the project involved the production and analysis of N-terminal mutants (including met-PLA2). The primary focus was on the 3-position of the enzyme and the production of two mutants, V3W and V3C, together with their analysis in terms of interfacial binding and catalytic activity. The V3W mutant was prepared because the equivalent unique trytophan in the pancreatic enzyme has been extensively used as a natural probe for interfacial binding. The human mutant had 40% of the activity of the wild-type enzyme when assayed using anionic phospholipids, however, a dramatic feature of this mutant was its enhanced activity (250 fold) when assayed with vesicles of phosphatidyl choline. A 10-fold enhancement of activity was also seen using whole cells as substrates. The results highlight the potential importance of interfacial trytophan residues in promoting the penetration of condensed zwitterionic interfaces as also seen with the surface monolayer of cell membranes. A V3C mutant has also been constructed to provide a potential site of chemical attachment of fluorescent reporter groups. Preliminary analysis of this mutant enzyme has revealed that it still has about 40% of the activity of the wild-type, however, the new cysteine at position 3 was blocked. Mass-spectral analysis indicates that this cysteine is conjugate via a disulphide linkage to free cysteine, a major constituent of the refolding medium. A final mutant, H48Q, was constructed to produce an inactive enzyme and provide an ideal interfacial binding protein for use in competitive binding studies. In fact the H48Q mutant possessed significant catalytic activity which has considerable mechanistic implications.
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Published date: 1998
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Local EPrints ID: 463429
URI: http://eprints.soton.ac.uk/id/eprint/463429
PURE UUID: 155e524a-5ba1-4a15-906a-a65e73f381ec
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Date deposited: 04 Jul 2022 20:51
Last modified: 04 Jul 2022 20:51
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Author:
Sharon Felicity Baker
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