Effects of Centrifugal Force and Centrifugation Time on the Sedimentation of Plant Organelles

Abstract

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,...

Evidence for a KCl-Stimulated, Mg2+-ATPase on the Golgi of Corn Coleoptiles

“…They reported that 93% of the mitochondria (Cyt c oxidase) were pelleted under these conditions, together with 35% of the PM (pH 6.5 K+-stimulated ATPase) and 28% of the ER (Antimycin A-insensitive NADH-Cyt c reductase). Under the same conditions (Table I), we pelleted 92% of the mitochondria, in agreement with Nagahashi and Hiraike (30), but substantially more PM (67% UDPG-ST), ER (37%) NADPH-Cyt c reductase), and Golgi (53% latent UDPase) were also removed. These differences could be due to several factors.…”

“…The pH 7.5 KCl, Mg2+-ATPase again had a distribution similar to the Golgi marker, latent UDPase. (30) showed that an optimal separation of mitochondria and microsomes in corn roots could be achieved by an initial centrifugation of 6000g for 20 min. They reported that 93% of the mitochondria (Cyt c oxidase) were pelleted under these conditions, together with 35% of the PM (pH 6.5 K+-stimulated ATPase) and 28% of the ER (Antimycin A-insensitive NADH-Cyt c reductase).…”

Endosome-Associated CRT1 Functions Early in Resistance Gene–Mediated Defense Signaling in Arabidopsis and Tobacco

“…Similar analyses using either wild-type or crt1-2 crh1-1 plants containing an RPM1-Myc transgene revealed that RPM1-Myc also was predominantly located in the endomembranes, regardless of mock or Pst inoculation ( Figure 7B). Two-step differential centrifugation at 13,000 and 100,000g was then performed to fractionate organelles/vesicles based on size and density (Nagahashi and Hiraike, 1982). Consistent with the heterogeneous size of vesicles carrying CRT1, Myc-CRT1 was enriched in both the 13,000g (p13) and the 100,000g (p100) pellet fractions ( Figure 7C).…”

Evidence for and Subcellular Localization of a Ca-Stimulated Phospholipase D from Maize Roots

“…Pellets from subsequent, faster centrifugation steps were difficult to resuspend if the 1,lOOg centrifugation step was omitted. The crude homogenate was subjected to the differential centrifugation scheme of Nagahashi and Hiraike (20 …”