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Previous research has indicated that oxidants, antioxidants and the intracellular redox state regulate the activities of a variety of protein tyrosine kinases, protein tyrosine phosphatases, phospholipases and transcription factors. In order to explore the redox regulation of the serine/threonine phosphatase calcineurin, we have investigated the effects of a variety of oxidants and antioxidants on calcineurin phosphatase activity in vitro. The oxidants hydrogen peroxide, superoxide and glutathione disulfide inhibited the phosphatase activity of calcineurin in a dose-dependent manner. Incubation of purified calcineurin with the antioxidants ascorbate, ascorbate 2-phosphate, a-lipoic acid, N-acetyl-l-cysteine and glutathione increased phosphatase activity relative to untreated controls. In contrast, several other commonly used antioxidants, including butylated hydroxytoluene, butylated hydroxyanisole, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) and dihydrolipoic acid decreased the activity of purified calcineurin, possibly through prooxidative mechanisms. Although the antioxidant pyrrolidine dithiocarbamate increased the activity of purified calcineurin, it significantly inhibited the activity of calcineurin present in crude fibroblast lysates. These results support and extend the hypothesis that redox factors modulate the phosphatase activity of calcineurin and suggest that further in vivo studies are warranted.

Th,~ molecular mechanism of Iilltht Sil~ttal trlln~du¢tion in plant~ mediated b~ tile photo,enter phytochromc bt nat well under,toted, TIt¢ po~t,tihJlity that phyto~:l~romc initi~t¢~ the ~ilt!nttl tran~duction clmin by mt~duhttin~ a G.prot~in.like receptor i~ examined in the prc~¢nt work. l~tiolatcd Avena ~ccdlinl!~ e~ntain G. proteins tt~ examined in tcrm~ of the bindinll oF OTP tt~ well a~ by t:t'o~.rcaction with m~tmmalian Ci.protctn ~nlibodic~. Th~ bindinlt or GTP w~ rcllulatcd in rive by rcd~far.rcd lisht. The possible involvement of G.protein~ in the ~hytocltromc.mediatcd ~i~nal tran~duction in ¢tiOhttcd Arena ~¢cdling~ ha~ I~cn implicated from the ~tudy or the li~tltt regulated cxpre~tion of the Cab lind ph,v Igen¢~.,,h,ena ~ari~r GTP.bindin$ protein: Photor~tltthttion; Sitlnttl Irunsductlon

The relative extent of chromophore exposure of the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of 124-kDa oat phytochrome and the secondary structure of the phytochrome apoprotein have been investigated by using zinc-induced modification of the phytochrome chromophore. The absence of bleaching of Pr in the presence of a 1:1 stoichiometric ratio of zinc ions, in contrast to extensive spectral bleaching of the Pfr form, confirms previous reports of differential exposure of the Pfr chromophore relative to the Pr chromophore [Hahn et al. (1984) Plant Physiol. 74, 755-758]. The emission of orange fluorescence by zinc-chelated Pfr indicates that the Pfr chromophore has been modified from its native extended/semi-extended conformation to a cyclohelical conformation. Circular dichroism (CD) analyses of native phytochrome in 20 mM Tris buffer suggests that the Pr-to-Pfr phototransformation is accompanied by a photoreversible change in the far-UV region consistent with an increase in the alpha-helical folding of the apoprotein. The secondary structure of phytochrome in Tris buffer, as determined by CD, differs slightly from that of phytochrome in phosphate buffer, suggesting that phytochrome is a conformationally flexible molecule. Upon the addition of a 1:1 molar ratio of zinc ions to phytochrome, a dramatic change in the CD of the Pfr form is observed, while the CD spectrum of the Pf form is unaffected. Analysis of the bleached Pfr CD spectrum by the method of Chang et al. (1978) reveals that chelation with zinc ions significantly alters the secondary structure of the phytochrome molecule, specifically by increasing the beta-sheet content primarily at the expense of alpha-helical folding.(ABSTRACT TRUNCATED AT 250 WORDS)

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