Plant Microbody Proteins, III. Labelling of the Peroxisomal Membrane Protein SP-63 in vitro and in vivo

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Abstract
Methods were developed to characterize membranes and membrane components of leaf peroxisomes from Lens culinaris. While microbodies from etiolated young leaves exhibited an equilibrium density of 1.19 g/cm3, older leaves or leaves exposed to light for increasing periods of time contained microbodies banding at higher densities up to 1.235 g/cm3. Similar values were also found for the corresponding microbody membranes, which could be labeled with diazotized [35S]sulphanilic acid. Labeling was also performed using proteins extracted from the membranes. Their main structural protein (SP-63) was solubilized with sodium dodecylsulphate and labeled with fluorescent compounds and with diazotized [35S]sulphanilic acid or [3H]iodoacetic acid. These conversions greatly facilitate all analytical procedures, e.g., tracing the migration of SP-63 in gels or the movement in sucrose gradients containing sodium dodecylsulphate during zonal centrifugation. Also, labeling of SP-63 in vivo was accomplished when labeled amino acids were infused into etiolated leaves while exposing them to light.

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