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Fig. 5. NO nitrosylates dynamin. (A) Purified recombinant GST-dynamin, specific subdomains or immunoprecipitated dynamin from cells were incubated with GSNO or vehicle. Alternatively, dynamin was immunoprecipitated from aortic lysates of caveolin wild-type or caveolin-null mice. Nitrosylation of dynamin-2 was assessed by biotin-switch method. S-nitrosylation of immunoprecipitated dynamin from cells is shown (left) [anti-biotin (top panel) and anti-dyn2 (bottom panel)]. Mock represents immunoprecipitation with non-immune serum. Full-length GST dynamin, as well as the GTPase subdomain, showed nitrosylation by DEA-NO. Nitrosylation signal was blocked by preincubation of cells with the reducing agent, ascorbic acid. GST-PRD-domain nitrosylation was not detected. (B) Dynamin nitrosylation was also increased in dynamin-2 immunoprecipitates prepared from aortic lysates of mice genetically deficient in caveolin as compared with wild-type samples (left panel shows a representative autoradiograph and the right panel shows the quantitative change from three independent pooled experiments; ^*P<0.05). (C) Purified GST-dynamin or the GTPase subdomain were incubated with the NO donor, DEA-NO, or vehicle and then prepared for spectrophotometric analysis. GST-dynamin and the GST-GTPase domain alone incubated with vehicle show a flat curve throughout 200 nm to 450 nm; however, after incubation with GSNO, a characteristic S-nitrosylation peak was detected at 320 nm.

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