Figure 4.

Spectrofluorometric detection of NO^˙ in pea leaf peroxisomes with DAF-2 DA. Peroxisomes freshly isolated from pea leaves were added to the reaction mixtures and the fluorescence measured. Values shown are means of three independent experiments. A, Different volumes of peroxisomal fractions were added to the reaction mixture and the fluorescence produced was expressed as arbitrary units (A.U.). B, Effect on the NO^˙ generation of: (1) preincubating peroxisomes with 2 mm aminoguanidine (AG); (2) removing l-Arg from the reaction mixture (l-Arg); and (3) denaturing the samples by heating (95°C/10 min). The amount of peroxisomal proteins in the reaction mixtures was 12 μg, and fluorescence was expressed as percent of the control (relative fluorescence). C, NOS activity of peroxisomal fractions determined as protein-dependent formation of l-[³H]citrulline from l-[³H]Arg. D, Influence of the incubation time on the NOS activity of peroxisomal fractions determined by measuring the formation of l-[³H]citrulline from l-[³H]Arg.