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Partial purification of a plasma membrane flavoprotein and NAD(P)H‐oxidoreductase activity
Author(s) -
Van Gestelen Peter,
Asard Han,
Caubergs Roland J.
Publication year - 1996
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.1996.980222.x
Subject(s) - flavin group , flavoprotein , chemistry , flavin mononucleotide , pterin , nad+ kinase , cofactor , membrane , flavin adenine dinucleotide , oxidoreductase , biochemistry , chromatography , enzyme
Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H‐dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings ( Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two‐phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg −1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non‐covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H‐oxidoreductase activity and suggests the presence of plasma membrane bound pterins.