Abstract
Nitric oxide (NO) is an important signaling molecule involved in various plant physiological processes. The main effect of NO arises from its reaction with proteins. S-Nitrosation is the most studied NO-mediated protein posttranslational modification in plants. Via S-nitrosation, NO derivatives react with thiol groups (SHs) of protein cysteine residues and produce nitrosothiol groups (SNOs). From the time of discovering the biological function of NO in plants, an interesting case of study has been the detection of the endogenous S-nitrosated proteins in different plants, tissues, organelles, and various conditions. Maps of S-nitrosated proteins provide hints for deeper studies on the function of this modification in specific proteins, biochemical pathways, and physiological processes. Many functions of NO have been found to be related to plant defense; on the other hand the involvement of nuclear proteins in regulation of plant defense reactions is well studied. Here, an approach is described in which the Arabidopsis cell cultures first are treated with P. syringae, afterward their bioactive nuclear proteins are extracted, then the nuclear proteins are subjected to biotin switch assay in which S-nitrosated proteins are specifically converted to S-biotinylated proteins. The biotin switch technique (BST) which was introduced by Jaffrey et al. (Nat Cell Biol 3:193-197, 2001) solves the instability issue of SNOs. Additionally, it provides detection and purification of biotinylated proteins by anti-biotin antibody and affinity chromatography, respectively.
Highlights
Nitric oxide is a radical gas which is known as an important regulatory molecule in many different physiological processes such as growth, development and defense
The most known example is cyclic guanosine monophosphate-dependent signalling; in which binding of Nitric oxide (NO) to the heme center of soluble guanylate cyclase activates its catalytic domain. This leads to the production of cGMP from guanosine triphosphate; and cGMP in turn acts as a second messenger [3]
Several studies in various pathways have shown the involvement of metal nitrosylation and tyrosine nitration in NO-dependent signalling
Summary
Nitric oxide is a radical gas which is known as an important regulatory molecule in many different physiological processes such as growth, development and defense. The most known example is cyclic guanosine monophosphate (cGMP)-dependent signalling; in which binding of NO to the heme center of soluble guanylate cyclase (sGC) activates its catalytic domain. This leads to the production of cGMP from guanosine triphosphate; and cGMP in turn acts as a second messenger [3]. The specificity of BST to S-nitrosothiols is on the base of the fact that ascorbate can convert SNOs to SHs but not SSGs and other S-oxides [7] Thermodynamic measurements support this specificity [1], some controversial reports exist about the specificity of ascorbate [5]. A method of infection of Arabidopsis cell culture with bacteria is described which is followed by nuclear protein extraction and biotin switch assay and further with purification of biotinylated proteins
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