PLANT PHYSIOLOGY , Vol 102, Issue 2 595-602, Copyright © 1993 by American Society of Plant Biologists
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CELL BIOLOGY AND SIGNAL TRANSDUCTION |
NADH Oxidase Activity of Plasma Membranes of Soybean Hypocotyls Is Activated by Guanine Nucleotides
D. J. Morre, A. O. Brightman, R. Barr, M. Davidson and F. L. Crane
Department of Medicinal Chemistry (D.J.M., A.O.B., M.D.) and Department of Biological Sciences (R.B., F.L.C.), Purdue University, West Lafayette, Indiana 47907
The activity of an auxin-stimulated NADH oxidase of the plasma membrane of
hypocotyls of etiolated soybean (Glycine max Merr.) seedlings responded to
guanine and other nucleotides, but in a manner that differed from that of
enzymes coupled to the classic trimeric and low molecular weight monomeric
guanine nucleotide-binding proteins (G proteins). In the presence and
absence of either auxin or divalent ions, both GTP and GDP as well as
guanosine-5[prime]-O-(3-thiotriphosphate) (GTP-[gamma]-S) and other
nucleoside di- and triphosphates stimulated the oxidase activity over the
range 10 [mu]M to 1 mM. GTP and GTP-[gamma]-S stimulated the activity at 10
nM in the absence of added magnesium and at 1 nM in the presence of added
magnesium ions. Other nucleotides stimulated at 100 nM and above. The NADH
oxidase was stimulated by 10 [mu]M mastoparan and by 40 [mu]M aluminum
fluoride. Neither cholera nor pertussis toxins, tested at a concentration
sufficient to block mammalian G protein function, inhibited the activity.
Guanosine 5[prime]-O-(2-thiodi-phosphate) (GDP-[beta]-S) did not stimulate
activity, suggesting that the stimulation in response to GDP may be
mediated by a plasma membrane nucleoside diphosphate kinase through
conversion of GDP to GTP. Auxin stimulation of the NADH oxidase was
unaffected by nucleotides at either high or low nucleotide concentrations
in the absence of added divalent ions. However, pretreatment of plasma
membranes with auxin increased the apparent affinity for nucleotide
binding. This increased affinity, however, appeared not to be the mechanism
of auxin stimulation of the oxidase, since auxin stimulation was similar
with or without low concentrations of guanine nucleotides. The stimulation
by nucleotides was observed after incubating the membranes with 0.1% Triton
X-100 prior to assay. The results suggest a role of guanine (and other)
nucleotides in the regulation of plasma membrane NADH oxidase that differs
from the interactions with G proteins commonly described for animal models.
