NADH OXIDASE ACTIVITY OF PLASMA-MEMBRANES OF SOYBEAN HYPOCOTYLS IS ACTIVATED BY GUANINE-NUCLEOTIDES

The activity of an auxin-stimulated NADH oxidase of the plasma membran e of hypocotyls of etiolated soybean (Glycine max Merr.) seedlings res ponded to guanine and other nucleotides, but in a manner that differed from that of enzymes coupled to the classic trimeric and low molecula r weight monomeric guanine nucleotide-binding proteins (G proteins). I n the presence and absence of either auxin or divalent ions, both GTP and GDP as well as guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) a nd other nucleoside di-and triphosphates stimulated the oxidase activi ty over the range 10 mum to 1 mm. GTP and GTP-gamma-S stimulated the a ctivity 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 mum mastoparan and by 40 mum aluminum fluoride. Neither cholera nor pertussis toxins, te sted at a concentration sufficient to block mammalian G protein functi on, inhibited the activity. Guanosine 5'-O-(2-thiodiphosphate) (GDP-be ta-S) did not stimulate activity, suggesting that the stimulation in r esponse to GDP may be mediated by a plasma membrane nucleoside diphosp hate kinase through conversion of GDP to GTP. Auxin stimulation of the NADH oxidase was unaffected by nucleotides at either high or low nucl eotide concentrations in the absence of added divalent ions. However, pretreatment of plasma membranes with auxin increased the apparent aff inity for nucleotide binding. This increased affinity, however, appear ed not to be the mechanism of auxin stimulation of the oxidase, since auxin stimulation was similar with or without low concentrations of gu anine nucleotides. The stimulation by nucleotides was observed after i ncubating the membranes with 0.1% Triton X-100 prior to assay. The res ults suggest a role of guanine (and other) nucleotides in the regulati on of plasma membrane NADH oxidase that differs from the interactions with G proteins commonly described for animal models.