Analysis of hydrolytic activity of phospholipase Calpha from porcine retina on retinyl ester and phosphatidylcholine using non-denaturing two-dimensional electrophoresis and mass spectrometry.
Hydrolysis of retinyl esters and phospholipids is important for visual functions of the animal retina. This study aimed to examine hydrolytic activity of an enzyme with native substrates such as retinyl esters and phospholipids responsible for this function in porcine retina. After cytosolic proteins were extracted from porcine retina, the proteins were separated using non-denaturing two-dimensional electrophoresis (2DE). Some major proteins and phospholipase Calpha were identified by matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) or electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). The phospholipase Calpha showed hydrolytic activities with not only alpha-naphtyl acetate but also with retinyl palmitate and phosphatidylcholine when effects of different substrates were investigated using enzyme activity staining on 2DE or MALDI-TOF-MS. Results indicated that hydrolytic activity of the enzyme with non-native and native substrates could be examined using a combination of non-denaturing 2DE and MALDI-TOF-MS.
Shimazaki
Youji
Y
Faculty of Science and Venture Business Laboratory, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp <yoji@dpc.ehime-u.ac.jp>
Kishi
Haruka
H
Mori
Masami
M
Yasuda
Chiyo
C
Manabe
Takashi
T
D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004952:Esters; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism*; D012160:Retina / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013552:Swine; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism*
D004952:Esters; D010713:Phosphatidylcholines; D014801:Vitamin A; D010738:Type C Phospholipases