1. In situ monitoring of galactolipid digestion by infrared spectroscopy in both model micelles and spinach chloroplasts
- Author
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Sahaka, Moulay, Mateos-Diaz, Eduardo, Amara, Sawsan, Wattanakul, Jutarat, Gray, David, Lafont, Dominique, Gontero, Brigitte, Launay, Hélène, Carrière, Frédéric, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Lipolytech, University of Nottingham, UK (UON), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), The PhD thesis of Moulay Sahaka was supported by a fellowship from Aix Marseille Université, France. The PhD thesis of Miss Jutarat Wattanakul was supported by Thai Royal Government, Thailand., and ANR-09-CP2D-0006,GALACTOLIPASE,Utilisation de galactolipases pour extraire les acides gras de la principale source de lipides présente sur la terre. - Using galactolipases for extracting fatty acids from the main source of lipids on the earth.(2009)
- Subjects
lipid digestion ,chloroplast ,Enzyme ,[SDV]Life Sciences [q-bio] ,thin layer chromatography ,Organic Chemistry ,[CHIM]Chemical Sciences ,galactolipase ,pancreatic lipase related protein 2 ,Cell Biology ,Molecular Biology ,Biochemistry - Abstract
International audience; Galactolipids are the main lipids from plant photosynthetic membranes and they can be digested by pancreatic lipase related protein 2 (PLRP2), an enzyme found in the pancreatic secretion in many animal species.Here, we used transmission Fourier-transform infrared spectroscopy (FTIR) to monitor continuously the hydrolysis of galactolipids by PLRP2, in situ and in real time. The method was first developed with a model substrate, a synthetic monogalactosyl diacylglycerol with 8-carbon acyl chains (C8-MGDG), in the form of mixed micelles with a bile salt, sodium taurodeoxycholate (NaTDC). The concentrations of the residual substrate and reaction products (monogalactosylmonoglyceride, MGMG; monogalactosylglycerol, MGG; octanoic acid) were estimated from the carbonyl and carboxylate vibration bands after calibration with reference standards. The results were confirmed by thin layer chromatography analysis (TLC) and specific staining of galactosylated compounds with thymol and sulfuric acid.The method was then applied to the lipolysis of more complex substrates, a natural extract of MGDG with long acyl chains, micellized with NaTDC, and intact chloroplasts isolated from spinach leaves. After a calibration performed with alpha-linolenic acid, the main fatty acid (FA) found in plant galactolipids, FTIR allowed quantitative measurement of chloroplast lipolysis by PLRP2. A full release of FA from membrane galactolipids was observed, that was not dependent on the presence of bile salts. Nevertheless, the evolution of amide vibration band in FTIR spectra suggested the interaction of membrane proteins with NaTDC and lipolysis products.
- Published
- 2023