Subcellular membrane fractions from 21-day-old pea (Pisum satdvum) cotyledons that have associated UDP-N-acetylglucosamine N-acetylglucosaminyl transferase and GDP-mannose mannosyl transferase activities have been isolated and identified. The rough endoplasmic reticulum (RER) is the principal location of glycosyl transferases involved in the assembly of Upid-linked sugar intermediates and glycoproteins. Antimycin A-insensitive NADH-cytochrome c reductase activity was used to identify RER at a density of 1.165 g/cc in sucrose gradients. The high proportion of RER in this fraction was confirmed by electron microscopy.Other mannosyl transferases are found at a density of 1.123 g/cc and1.201 g/cc but these glycosyl transferases do not appear to be involved with the formation of lipid-linked sugar intermediates utilized in glycoprotein biosynthesis.Recent investigations have indicated that lipid-linked sugar intermediates are involved in the synthesis of some glycoproteins in plants (2,7,8,14), fungi (15), and animals (for review see 33). The formation of lipid-linked sugar derivatives and glycoproteins is catalyzed by particulate fractions and various schemes have been developed to model the glycosylation process (16,21). Subcellular localization of the required glycosyl transferases is essential to support any model.Although information has been assembled concerning the subcellular location of the glycosyl transferases involved in formation of cell walls in plants (10,32) Cell Disruption and Differential Centrifugation. Seeds were removed from pods, testa and embryos were excised, and cotyledons were weighed (10-20 g fresh wt). All further manipulations were at 0 to 4 C. Tissue was razor blade-chopped with an electrically powered, mechanically driven chopper (2) for 1 to 2 min in a grinding mix consisting of 0.5 M sucrose, 5 mM 2-mercaptoethanol, and 30 mM Tris-MES (pH 7.5). One ml of GM2 was used/g fresh wt but immediately after chopping, enough GM was added to make the ratio 4:1. The homogenate was filtered through cheesecloth and centrifuged at 250g for 5 min to remove cell wall debris, intact cells, starch, and intact chloroplasts and nuclei. For the data in Table I

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The effect of centrifugal force and length of centrifugation time on the sedimentation of plant organelles was determined for corn (Zea mays L.) root homogenates. A centrifugal force of 6000g for at least 20 minutes was necessary to pellet 90% of the mitochondrial marker (cytochrome c oxidase). This initial centrifugation step is optimal for separating mitochondria from microsomes, since cross-contamination of endoplasmic reticulum and plasma membrane vesicles with mitochondria is minimized. Centrifugal forces of 80OOg or 10,OOOg for 20 minutes and 13,000g for 15 minutes pellet 90% of the mitochondrial marker, however, these centrifugation conditions also sediment more plasma membrane and endoplasmic reticulum.Inasmuch as mitochondria, RER, and pM2 have similar densities in sucrose gradients. it is necessary initially to separate the mitochondria from microsomes by differential centrifugation. A range of centrifugal forces, from 4,600g to 20,000g (1)(2)(3)(4)(5)14), has been used to achieve this separation; however, these crude mitochondrial fractions also contain substantial amounts of Golgi membrane, ER, and PM (6,9,12,15). These results can be viewed either as a loss of microsomal vesicles, if one is attempting to purify microsomes, or as a contaminated mitochondrial fraction.A recent investigator (3) has used a crude mitochondrial fraction (l0,OOOg pellet) as a direct source of plasma membranes; however, further separation of these two organelles proved to be difficult. A Ficoll density gradient was used, and it provided limited success, inasmuch as the plasma membrane markers were only slightly enriched after centrifugation and there was still considerable overlap between mitochondria and PM markers (3). These results exemplify the need for initial separation of the mitochondria from microsomes prior to density-gradient separation.This report is concerned with the determination of the optimal differential centrifugation conditions (centrifugal force and length of time) that produce the least amount of cross-contamination between mitochondria and microsomes. Only a few plant investigators (2,5,9,12) Enzyme Assays. The K+-stimulated ATPase activity was determined as described previously (9). To retain the K+-stimulated ATPase activity, corn roots were homogenized in DTT or dithioerythritol. When ,B-mercaptoethanol was used in the grinding medium, Cyt c oxidoreductase activities were maintained, but the K+-stimulated ATPase activity was very poor. Cyt c oxidase activity and NADH Cyt c reductase activity were determined as described by Hodges and Leonard (4). NADH Cyt c reductase activity was assayed in the presence and absence of antimycin A (10). Proteins were estimated according to Lowry et al. (I 1), and all enzyme assays were performed with 20 to 40 ,ug of membrane protein. RESULTSDifferential Centrifugation between 1,000g and 80,000g. Centrifugation at 3,000g for 45 min only sedimented 75% of the Cyt c oxidase activity (Fig. 2). Although we did not centrifuge for longer periods of time at 3,000g,...