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Activity And Membrane Binding Of Retinol Dehydrogenase-11 And Its Deletants
- Source :
- Biophysical Journal. 96(3):613a-614a
- Publication Year :
- 2009
- Publisher :
- Elsevier BV, 2009.
-
Abstract
- Retinol dehydrogenases (RDHs) are enzymes that catalyze the interconversion between retinol and retinal. Not much is known concerning these RDHs. Indeed, the exact physiological role of many isoforms of RDH and their membrane binding remain unknown. In this work, we have overexpressed, purified and characterized the membrane binding of one isoform, RDH-11. The cDNA of RDH-11 and of the truncated RDH-11 (N-terminal deletion, N-del RDH-11) have been cloned in the pET28a plasmid. Those proteins have been overexpressed in E. coli, purified by affinity chromatography, and then concentrated by ultrafiltration. Their activity has been measured in the presence of its substrate, all-trans retinal, and the reaction was initiated by the addition of its cofactor, NADPH. The reaction products have been analyzed by HPLC. The data showed a very high activity of RDH-11. Indeed, 1 μg of protein was enough to convert nearly 100% of all-trans retinal to all-trans retinol. Membrane binding measurements have been carried out by the injecting RDH-11, N-del RDH-11 and the synthetic N-terminal peptide (NTP) into the subphase of a phospholipid monolayer at the air-water interface. Their kinetics of monolayer binding, monitored by surface pressure measurements, increases as follows : NTP > RDH-11 > N-del RDH-11. Moreover, measurements by polarization-modulated infrared reflection absorption spectroscopy have allowed to confirm the alpha helical structure of the NTP and to determine its orientation as well as to compare the structure and orientation of RDH-11 and N-del RDH-11. For example, compared to the pure protein, N-del RDH-11 undergoes a conformational change upon monolayer binding.
- Subjects :
- chemistry.chemical_classification
0303 health sciences
Conformational change
biology
Phospholipid
Biophysics
Retinal
Peptide
Retinol dehydrogenase
Cofactor
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Enzyme
Biochemistry
Affinity chromatography
chemistry
biology.protein
030217 neurology & neurosurgery
030304 developmental biology
Subjects
Details
- ISSN :
- 00063495
- Volume :
- 96
- Issue :
- 3
- Database :
- OpenAIRE
- Journal :
- Biophysical Journal
- Accession number :
- edsair.doi.dedup.....6e71e7226ee1a0b4ce80120676ce556a
- Full Text :
- https://doi.org/10.1016/j.bpj.2008.12.3244