A combination of unnatural phosphatidyl acceptor and tandem electrospray ionization mass spectrometry for tracing phospholipase D activity.
Phospholipase D (PLD) is a biocatalyst in the synthesis of bioactive compounds and a key enzyme in a variety of biological signal transductions. A combination of unnatural phosphatidyl acceptor, N,N,N-triethyl-N-2-hydroxyethylammonium bromide 6, as a substrate for PLD, and tandem electrospray ionization mass spectrometry (ESI MS) was found to provide information as to whether a given phospholipid serves as a substrate for the PLD-catalyzed reaction. Thus 2-(13'-hydroperoxy-octadecadienoyl)-1-palmitoylglycerophosphocholine 1, and its degradation products 2-(13'-oxo-octadecadienoyl)-1-palmitoylglycerophosphocholine 9 and 2-(13'-hydroxy-octadecadienoyl)-1-palmitoylglycerophosphocholine 11, in a mixture were found to be a substrate of the PLD-catalyzed transphosphatidylation. The sensitivity of this method was exemplified by the observation that PLD activity in cabbage leaves was detected using a small amount of crude crushed leaves with little pretreatment. This simple method can be used in screening for PLD activity and searching for inhibitors of the enzyme from various natural sources.
Oda
Keiichi
K
Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.
Imura
Megumi
M
Ueda
Yoshimi
Y
Hosokawa
Miho
M
Kobayashi
Michiyo
M
Matsubara
Junko
J
Sato
Mizuho
M
Kumura
Naokazu
N
Izumi
Minoru
M
Nakajima
Shuhei
S
Sugio
Tsuyoshi
T
Baba
Naomichi
N
D055162:Biocatalysis; D001937:Brassica / Q000201:enzymology; D002794:Choline / Q000031:analogs & derivatives* / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D000644:Quaternary Ammonium Compounds; C100239:triethylcholine; D010739:Phospholipase D; D002794:Choline