PARTIAL-PURIFICATION OF A PLASMA-MEMBRANE FLAVOPROTEIN AND NAD(P)H-OXIDOREDUCTASE ACTIVITY

Plasma membrane flavins and pterins are considered to mediate importan t physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasm a membrane of higher plants was studied together with NAD(P)H-dependen t redox activities. Plasma membranes were isolated from the apical hoo ks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vr oege) by aqueous two-phase partitioning. Fluorescence spectroscopy rev ealed the presence of two chromophores. The first showed excitation ma xima at 370 and 460 nm and an emission peak at 520 nm and was identifi ed 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 p igments are considered intrinsic to the plasma membrane since they cou ld not be removed by treatment with hypotonic media containing high sa lt and low detergent concentrations. The flavin concentration was esti mated at about 500 pmol mg(-1) protein. However difficulties were enco untered 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 oct ylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a d istinct protein fraction showing flavin and pterin fluorescence and NA DH 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 pre sence of distinct NAD(P)H oxidases at the plasma membrane was supporte d by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intr insic chromophores, that may function in NAD(P)H-oxidoreductase activi ty and suggests the presence of plasma membrane bound pterins.