Isolation and Characterization of Golgi Membranes from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

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Abstract
A simple and rapid technique was developed for the isolation of the vesicular Golgi membranes from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). The procedure involves preparation of protoplasts and differential centrifugation of disrupted protoplasts followed by the sucrose density gradient centrifugation. Starting from broken protoplasts, sedimentable at two different centrifugal forces (10,000×g and 100,000 ×g), two Golgi-enriched fractions of lower density, GF1 and GF′1, and higher density, GF2 and GF′2, were separated. Purity of the fraction was assessed by determining the marker enzyme activities as well as the electron microscopy of the specimens obtained. Inosine diphosphatase was enriched about 15- and 6-fold, respectively, in the GF2 fraction from 10,000×g and the GF′2 one from 100,000×g pellets, whereas the enrichment in GF1 and GF′1 was approximately 6–7 fold. Galactosyl-transferase in GF2 was enriched about 25-fold. GF1 and GF2 account for 3–4% of the total protein of 10,000×g pellets, and GF′1 and GF′2 for about 6–7% of the total protein of 100,000×g pellets. Electron microscopic observations show that GF2 and GF′2 consisted principally of vesicular Golgi membranes without an internal matrix although GF1 and GF′1 were contaminated with ER membranes and ribosomes.

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