Use of Silica Sol Step Gradients to Prepare Bundle Sheath and Mesophyll Chloroplasts from Panicum maximum

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Abstract
The 1st method for direct separation of mesophyll and bundle sheath chloroplasts from whole tissue homogenates of a C4 plant is described. Centrifugation of mixed chloroplast preparations from P. maximum through low viscosity silica sol gradients effectively separates large, starch-containing chloroplasts from smaller plastids. The large chloroplasts are presumably bundle sheath chloroplasts on the basis of microscopic appearance, the presence of starch grains, the protein complement displayed on sodium dodecyl sulfate acrylamide gels and the exclusive localization of ribulose bisphosphate carboxylase activity in these plastids. As a measure of intactness both the large (bundle sheath) and small (mesophyll) chloroplasts contain glyceraldehyde-3-phosphate NADP-dependent dehydrogenase activity that is greatly enhanced by plastid lysis and both chloroplast preparations are impermeable to DNase. Chloroplast enzyme activities are inhibited by silica sol due to the Mg2+ chelating activity of this reagent. Well washed chloroplasts separated on silica gradients had enzyme activities similar to reported values in which silica sol gradients were not used.