P. Vangestelen et al., PARTIAL-PURIFICATION OF A PLASMA-MEMBRANE FLAVOPROTEIN AND NAD(P)H-OXIDOREDUCTASE ACTIVITY, Physiologia Plantarum, 98(2), 1996, pp. 389-398
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.