Use of dipyridyl-dithio substrates to measure directly the protein disulfide-thiol interchange activity of the auxin stimulated NADH: protein disulfide reductase (NADH oxidase) of soybean plasma membranes.
Dipyridyl-dithio substrates were cleaved by isolated vesicles of plasma membranes prepared from etiolated hypocotyls of soybean. The cleavage was stimulated by auxins at physiological concentrations. The substrates utilized were principally 2,2'-dithiodipyridine (DTP) and 6,6'-dithiodinicotinic acid (DTNA). The DTP generated 2 moles of 2-pyridinethione whereas the 6,6'-dithiodinicotinic acid generated 2 moles of 6-nicotinylthionine. Both products absorbed at 340 nm. The auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) stimulated the activity approximately 2-fold to a maximum at about 10 microM. Concentrations of 2,4-D greater than 100 microM inhibited the activity. Indole-3-acetic acid stimulated the activity as well. The growth-inactive auxin, 2,3-dichlorophenoxyacetic acid (2,3-D), was without effect. DTNA cleavage correlated with oxidation of NADH and reduction of protein disulfide bonds reported earlier in terms of location at the external plasma membrane surface, absolute specific activity, pH dependence and auxin specificity. The dipyridyl-dithio substrates provide, for the first time, a direct measure of the disulfide-thiol interchange activity of the protein previously measured only indirectly as an auxin-dependent ability of isolated plasma membrane vesicles to restore activity to scrambled and inactive RNase.
Morré
D J
DJ
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907-1333, USA.
Gomez-Rey
M L
ML
Schramke
C
C
Em
O
O
Lawler
J
J
Hobeck
J
J
Morré
D M
DM
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives; D015084:2,4-Dichlorophenoxyacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004220:Disulfides; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006540:Herbicides / Q000494:pharmacology; D018546:Hypocotyl / Q000201:enzymology; D007210:Indoleacetic Acids / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009539:Nicotinic Acids; D013025:Glycine max / Q000201:enzymology*
D004220:Disulfides; D004791:Enzyme Inhibitors; D006540:Herbicides; D007210:Indoleacetic Acids; D009097:Multienzyme Complexes; D009539:Nicotinic Acids; C001102:6,6'-dithiodinicotinic acid; C001105:2,2'-dipyridyl disulfide; D015084:2,4-Dichlorophenoxyacetic Acid; D015082:2,2'-Dipyridyl; C030737:indoleacetic acid; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases