Separation of membranes from semiprotoplasts of suspension‐cultured sycamore maple (Acer pseudoplatanus) cells

Abstract

Semiprotoplasts were produced from suspension‐cultured Acer pseudoplatanus (sycamore maple) cells prior to cell disruption by passing them through a 60 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on linear sucrose density gradients. Samples were collected by gradient fractionation and subcellular fractions were assayed for membrane markers and glycosyl transferase activities. Results of standard marker assays (cytochrome c reductase for endoplas‐mic reticulum. uridine and inosine diphosphatases for Golgi. and eosin‐5′‐maleimide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of 14C‐labeled sugars from UDP‐glucose and UDP‐xylose into ethanol precipitated polysaccharides was used to detect glucan synthases I & II (glucosyl transferases) and xylosyl transferase activities in Golgi membrane fractions. All three glycosyl transferases overlapped in fractions corresponding to both Golgi and plasma membrane markers, although peak activities for all three occurred in different fractions. More than one peak of glucan synthase I activity was found. Glucan synthase II, associated with ß‐l.3 glucan (cullose) synthesis in plasma membranes, was also detected and exhibited a 10‐fold stimulation in the presence of Ca2+.