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  • Continuous monitoring of phospholipid vesicle hydrolysis by phospholipase D (PLD) reveals differences in hydrolysis by PLDs from 2 Streptomyces species.

Continuous monitoring of phospholipid vesicle hydrolysis by phospholipase D (PLD) reveals differences in hydrolysis by PLDs from 2 Streptomyces species.

Colloids and surfaces. B, Biointerfaces (2012-03-07)
Satomi Hirano, Kazuhisa Sekine, Tetsurou Handa, Minoru Nakano
ABSTRACT

Phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PC) in large unilamellar vesicles (LUVs) consisting of PC and either glycerol monooleate (GMO) or methyl oleate (MeO) were monitored in situ and in real time by using a choline oxidase-immobilized oxygen electrode. This technique revealed reaction differences between 2 bacterial PLDs. PLD from Streptomyces chromofuscus, which is closely homologous to bacterial alkaline phosphatase, hydrolyzed only 6% of surface PC owing to product inhibition. The catalytic activity of this enzyme was not sensitive to the addition of GMO. On the other hand, typical bacterial PLD from Streptomyces sp. was found to hydrolyze all the PC molecules at the outer surface of LUVs suggesting that this enzyme is free from product inhibition. Introduction of GMO or MeO into the bilayer increased exposure of the PC headgroup and facilitated PC hydrolysis mediated by PLD from Streptomyces sp. GMO and MeO have the same lipophilic tail but the latter lacks hydroxyl groups on its polar head. From kinetic analysis by using the Michaelis-Menten model extended to the reaction at the interface, these compounds were found to activate PLD from Streptomyces sp. in different ways, i.e., MeO increased the protein binding to membranes and GMO stimulated the enzyme-substrate complex formation at membrane surface.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Methyl oleate, 99%
Supelco
Methyl oleate, analytical standard
Sigma-Aldrich
Choline Oxidase from Alcaligenes sp., lyophilized powder, ≥10 units/mg solid
Sigma-Aldrich
Methyl oleate, technical grade, 70%
Sigma-Aldrich
Methyl elaidate, ≥99% (capillary GC)
Supelco
cis-9-Octadecenoic acid methyl ester, certified reference material, 10 mg/mL in heptane
Glycerol mono-oleate, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Choline Oxidase from Arthrobacter globiformis, lyophilized powder, 8-20 units/mg solid
Supelco
Methyl elaidate, analytical standard