Preparation and Properties of Mitochondria from Cowpea Nodules

Abstract

Mitochondria were isolated from nodules of cowpea ( Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar 02 uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD+-malic enzyme which was 12-fold lower in the mitochondria from nodules.Each plant organ presents its own difficulties for the preparation of mitochondria. Other organelles may contaminate the preparation, and plant constituents may adhere to the mitochondria to inhibit or to produce artifactual activities. The major constituent of nodules are the bacteroids, which may occupy 40 to 50% of the volume of the infected cell (2). The The homogenate was filtered through four layers of cheesecloth and distributed to 50 ml centrifuge tubes. Bacteroids and cell debris were sedimented by centrifugation at 2,400g for 10 min. The supernatant was decanted and the mitochondria were pelleted at 12,000g for 10 min. Each pellet was gently resuspended into 40 ml of the above extraction buffer without the DTT, PVP, or BSA (washing buffer). After repelleting and resuspending in a total volume of 1.6 ml washing buffer the mitochondria were purified on a Percoll (Pharmacia) gradient 1092 www.plantphysiol.org on May 12, 2018 -Published by Downloaded from

Metabolism under Microaerobic Conditions of Mitochondria from Cowpea Nodules

“…We described a method for isolating active mitochondria from cowpea nodules (22 A controlled atmosphere glove box (Labconco, model 50004) was modified by incorporating a 2.5 L cold water bath. The bath was kept at 1°C by heat exchange with a refrigerant circulated from an external Lauda K-21RD cooler.…”

“…Bicarbonate reduces the activity of mitochondria from potato tubers and spinach leaves (6, 16). We therefore determined if mitochondria from the nodule could tolerate high concentrations of CO2.We described a method for isolating active mitochondria from cowpea nodules (22 A controlled atmosphere glove box (Labconco, model 50004) was modified by incorporating a 2.5 L cold water bath. The bath was kept at 1°C by heat exchange with a refrigerant circulated from an external Lauda K-21RD cooler.…”

“…We described a method for isolating active mitochondria from cowpea nodules (22). The procedure was modified to permit the preparation under completely anaerobic conditions, and subsequent study of the mitochondria under very low PO2.…”

“…Mitochondria from etiolated hypocotyls were prepared as described previously (22). Mitochondria from nodules were prepared essentially as before (22) Microaerobic 02 uptake was assayed by monitoring the deoxygenation of leghemoglobin solutions at 28°C in sealed cuvettes with no gas phase. The assay solution was medium A which contained 75 to 95 uM leghemoglobin.…”

Activation of O2 Uptake and NAD-Specific Isocitrate Dehydrogenase in Mitochondria Isolated from Cotyledons of Castor Bean by cis, trans-Abscisic Acid

“…Furthermore, the effect of oxygenase (e.g. lipoxygenase) on the 02 uptake estimation of this mitochondrial preparation also would be negligible, because lipoxygenase in cowpea hypocotyl mitochondria showed very low activities, as compared with the respiratory enzyme activities (12). The specific activity of 02 uptake was somewhat low herein.…”