Hydrolytic activity of lipase on anion-exchange solid phase column after separation and electrotransfer by non-denaturing electrophoresis.
This study reports the initial separation of phospholipase C-alpha from porcine retina using non-denaturing two-dimensional gel electrophoresis (2-DE). Detection was by negative staining and then its hydrolytic activity was estimated using alpha-naphthyl acetate in a 2-DE gel. A spot of phospholipase C-alpha separated by 2-DE was excised. It was then electrophoretically transferred to an anion-exchange solid phase column after 40 mg, equal to dry weight of the solid resin from the cartridge (Accell Plus QMA, Waters Corporation), was packed into a disposable 1 ml syringe to make an anion-exchange solid phase column. Phosphatidylcholine was hydrolyzed in the anion-exchange solid phase column containing phospholipase C-alpha. The results indicated that a column with hydrolytic activity could be produced once lipases separated by non-denaturing 2-DE were transferred to the solid phase column.
Shimazaki
Youji
Y
Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Matsuyama City, 790-8577, Japan. yoji@dpc.ehime-u.ac.jp
D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D006868:Hydrolysis; D007475:Ion Exchange Resins; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010713:Phosphatidylcholines / Q000378:metabolism; D012160:Retina / Q000201:enzymology; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D053719:Tandem Mass Spectrometry; D010738:Type C Phospholipases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D007475:Ion Exchange Resins; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010738:Type C Phospholipases