Phospholipase D activity is regulated by product segregation and the structure formation of phosphatidic acid within model membranes
Kerstin Wagner 1* and Gerald Brezesinski 1
1 MPI of Colloids and Interfaces
* To whom correspondence should be addressed. E-mail: wagner{at}mpikg.mpg.de.
Submitted on March 14, 2007
Revised on April 18, 2007
Accepted on 5 June 2007
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Abstract |
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Phospholipase D from streptomyces chromofuscus (scPLD) hydrolyzes phosphatidylcholines (PC) to produce choline and phosphatidic acid (PA), a lipid messenger molecule within biological membranes. To scrutinize the influence of membrane structure on scPLD activity, three different substrate-containing monolayers are used as model systems: pure dipalmitoylphosphatidylcholine (DPPC) as well as equimolar mixtures of DPPC/n-hexadecanol (C16OH) and DPPC/dipalmitoylglycerol (DPG). The activity of scPLD towards these monolayers is tested by infrared reflection-absorption spectroscopy (IRRAS) and exhibits different dependencies on surface pressure. For pure DPPC, the catalytic turnover drastically drops above 20 mN/m. Upon the addition of C16OH, this strong decrease already starts at 5 mN/m. For the DPPC/DPG system, the reaction yield linearly decreases between 5 and 25 mN/m. The difference in scPLD activity is correlated to the phase state of the monolayers as examined by X-ray diffraction, Brewster angle microscopy, and atomic force microscopy. Because the additives C16OH and DPG mediate the miscibility of PC and PA, only a basal activity of scPLD is observed towards the mixed systems at higher surface pressures. At pure DPPC monolayers, scPLD is activated after the segregation of initially formed PA. Furthermore, scPLD is inhibited when the lipids in the PA-rich domains adopt an upright orientation. This phenomenon offers a self-regulating mechanism for the concentration of the second messenger PA within biological membranes.
Key Words:
Dipalmitoylphosphatidylcholine (DPPC), Infrared reflection-absorption spectroscopy (IRRAS), Lipid phase separation, Lipolytic enzyme regulation, Monolayer structure, X-ray diffraction
