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 mem branes prepared from etiolated hypocotyls of soybean. The cleavage was stim ulated by auxins at physiological concentrations. The substrates utilized w ere principally 2,2'-dithiodippyrine (DTP) and 6,6'-dithiodinicotinic acid (DTNA). The DTP generated 2 moles of 2-pyridinethione whereas the 6,6'-dith iodinicotinic 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 mu M. Concentrations of 2,4-D greater than 100 mu M 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 cle avage 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 measu re of the disulfide-thiol interchange activity of the protein previously me asured only indirectly as an auxin-dependent ability of isolated plasma mem brane vesicles to restore activity to scrambled and inactive RNase.