title: Preparation of liposome-coupled NADH and evaluation of its affinity toward formate dehydrogenase based on deactivation kinetics of the enzyme.
abstract: β-Reduced nicotinamide adenine dinucleotide (NADH) has been immobilized onto the surface of liposome containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE). Amino groups of NADH were coupled to POPE via glutaraldehyde (GA) or poly(ethylene glycol) dialdehyde (PEG-ALD2). Formate dehydrogenase from Candida boidinii (CbFDH) was anchored on NADH through bioaffinity, where 5 NADH molecules on the liposome were associated with one CbFDH molecule. We evaluated the affinity between CbFDH and NADH present in various conditions based on of the first-order deactivation constant k(d) of the enzyme at 60°C. The kd value observed with the liposome-coupled NADH was apparently smaller than that with liposome alone, indicating the thermostability of the NADH-CbFDH complex on the liposome surface. On the other hand, free NADH showed the strongest affinity toward CbFDH. This can be recognized by considering that the affinity between CbFDH and liposome-coupled NADH is relatively weakened by the formation of chemical linkage between them. PEG-ALD2 provided a smaller k(d) value than GA. This bulkier PEG-ALD2 may cause a similar situation to NADH alone by shielding the effect of liposomes.
authors.affiliation: Department of Applied Molecular Bioscience, Yamaguchi University, 2-16-1 Tokiwadai, Ube, Japan. yosimoto@yamaguchi-u.ac.jp
authors.forename: Makoto
authors.initials: M
authors.lastname: Yoshimoto
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Kunihiro
authors.forename: Naoki
authors.initials: N
authors.lastname: Tsubomura
authors.forename: Masaharu
authors.initials: M
authors.lastname: Nakayama
mesh_terms: D002175:Candida / Q000201:enzymology; D005560:Formate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D009243:NAD / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008081:Liposomes; D009243:NAD; D005560:Formate Dehydrogenases