Regulation Mechanisms for Phospholipase Enzymes

Mezna, Mokdad (1993) Regulation Mechanisms for Phospholipase Enzymes. PhD thesis, University of Glasgow.

Abstract

The effect of hydrophobicity on susceptibility of phosphatidylcholine derivatives to PLA2 attack was studied by synthesising two series of compounds, the saturated symmetrically distributed derivatives and compounds with one long acyl-chain in the first position and a selected short-chain in the second position. The first series were used to investigate the effect of substrate morphology over the range from free monomer substrates through micelle to bilayer forms, the second series were used to examine the region of the micelle/bilayer transition at higher resolution. The symmetric substrates showed the expected properties with susceptibility increasing with hydrophobicity in the free monomer series and a sharp transition seen at the CMC for the shorter chain substrates. No such change was seen for longer chain substrates with any of these enzymes and the concept of a hydrophobic anchor increasing the rate of attack on condensed rather than monomeric substrates was questioned. These compounds were used to study the action of enzyme activated by treatment with oleoyl-imidazolide. The biggest activation factors were seen for asymmetric bilayer-forming substrates in propanolic solution, but the physico-chemical form of the substrates under these conditions was not established. Fatty acyl activation was found to alter the response to calcium activation and suggested a two-calcium site model. Studies of calcium activation showed very strong evidence for two kinetically important sites. Metal ion inhibition showed that barium and large cations were competitive inhibitors for calcium, but zinc and cadmium were not and appeared to inhibit a component of activation only found at high calcium activation. This lead to the proposal that zinc and barium bind to the enzyme at different calcium binding sites. Hydrolysis curves were shown to vary in shape depending on calcium concentration and the anomalous shape was associated with the presence of calcium at a single binding site. Addition of zinc removed the anomalous shape and without giving further enzyme activation whilst addition of calcium changes the curve shape and was activating. Some evidence was presented to suggest that the presence of a metal ion in the second (zinc) site was important for modulating the activation of the enzyme with surfaces. The use of the conductimetric assay was extended to the purification and characterisation of PLD enzymes acting as both transferases and hydrolases The transferase activity was used to generate phosphatidyl alcohols from DiC8PC and the methanol derivative was shown to be better substrate than the PC equivalent but to posess chartacteristics of the monomeric form. Polar phosphatidic acid compounds which should have high CMC values sould enable the relationship between structure, morphology and susceptibility to PLA2 to be examined in detail.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: A J Lawrence
• Keywords: Biochemistry
• Date of Award: 1993
• Depositing User: Enlighten Team
• Unique ID: glathesis:1993-74636
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 17:26
• Last Modified: 27 Sep 2019 17:26
• URI: https://theses.gla.ac.uk/id/eprint/74636