Plant microbody proteins, III. Labelling of the peroxisomal membrane protein SP-63 in vitro and in vivo.

Abstract

Methods were developed to charcterize 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 labelled with diazotized [35S]sulphanilic acid. Labelling was also performed using proteins extracted from the membranes. Their main structural protein (SP-63) was solubilized with sodium dodecylsulphate and labelled with fluorescent compounds as well as 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, labelling of SP-63 in vivo was accomplished when labelled amino acids were infused into etiolated leaves while exposing them to light.