Oxygen Uptake in Suspensions of Plant Leaf Mitochondria

The construction and operation ofa device for continuous measurement of CO2 release by suspensions of respiring mitochondria is described. A combination of this device with a Clark-type 02 electrode was used for simultaneous measurement of respiration and of CO2 release by spinach and pea leaf mitochondria with glycine as substrate. Both mitochondrial preparations showed high rates ofrespiration and high respiratory control ratios. The addition of oxaloacetate not only inhibited 02 uptake substantially, but also greatly stimulated glycine oxidation as monitored by CO2 release. In spinach leaf mitochondria, the maximal rates of glycine oxidation thus obtained, were two times higher than the rate of glycine oxidation required at average rates of photorespiration. It is concluded from these results that under saturating conditions the capacity of glycine oxidation by intact mitochondrin exceeds the capacity of glycine-dependent respiration. Respiration in suspensions ofisolated mitochondria is customarily determined by following 02 concentration changes with a polarographic 02 electrode. This is a most useful way of measuring the activity of the respiratory electron transport chain, but does not reflect the rates of preceding mitochondrial respiratory events, such as substrate decarboxylation, when these are coupled with other redox processes apart from electron transport to molecular oxygen. Thus, in isolated plant mitochondria, the oxidation of glycine can be coupled with the reduction of OAA4 to malate without electron transport occurring (6, 17). As this is an important partial reaction in the photorespiratory pathway, there is a need to measure the rate of glycine oxidation independently from electron transport. Since the oxidative conversion of two molecules of glycine to one serine yields one CO2 and one NH3, the radioactive assay of '4C release from [14C] glycine (14, 17) and the measurement of NH4' release by an NH4' electrode (2,3) have been employed for monitoring glycine oxidation. We present here an alternative approach based on the determination of CO2 by a CO2 electrode, a method which can also be used for other processes connected with CO2 release, e.g. ' Supported by the Deutsche Forschungsgemeinschaft. 2Present address: Department of Biology, University of Wollongong, P.O. Box 1144 Wollongong, N. S . W. 2500, Australia. 3 Supported by an Alexander von Humboldt Foundation Fellowship. 4Abbreviations: OAA, oxaloacetic acid; AA, antimycin A; RCR, respiratory control ratio. citric acid cycle turnover. The CO2 electrodes used widely for the clinical determination of blood CO2 partial pressure normally operate in the range of 8 to 400 mm Pco2. Because about 80% of dissolved carbon in neutral solutions is present as HCO3 , the concentration of CO2 in mitochondrial suspensions (typically around pH 7.4) is much lower than this. The lower limit of sensitivity (8 mm Pco2) corresponds to a dissolved CO2 concentration of 0.24 mm, and a total (CO2 + HCO3 ) concentration of 4.8 mM. Because of this comparatively low sensitivity, these electrodes have not been used with mitochondrial suspensions. For monitoring CO2 release by perfused organs, such as heart, the technique has been increased in its sensitivity by acidifying the measuring sample and thus converting all bicarbonate into CO2 (12). In the present report this method has been further refined to an extent that it enables CO2 measurements to be performed on diluted mitochondrial suspensions with sensitivity similar to 02 electrode measurements. The application of this method for studying the oxidation ofglycine by spinach and pea leaf mitochondria has revealed that the capacity of these mitochondria to oxidize glycine exceeds the capacity of glycinedependent respiration. MATERIALS AND METHODS Determination of CO2. A CO2 electrode (Radiometer A 5037) was mounted inside an aluminum jacket (Fig. IA), with the silicone membrane forming one side of a measuring flow chamber (C-volume 40 mm3) cut in a Perspex block (P). The inflow of acidified mitochondrial reaction mixture to the chamber was through a length of stainless steel tubing to the bottom of the measuring chamber, while the outflow left the chamber at the top (Fig. 1B). This ensured that any air bubbles in the inflow did not accumulate in the measuring chamber. The Perspex block was sandwiched between the jacket and a second aluminum block, both ofwhich contained internal channels, through which constant temperature water was pumped for temperature regulation. The electrode potential (mV) was measured by a Metrohm pH meter and recorded on a chart recorder. The conversion of recorded potentials to CO2 concentrations was done subsequently using a calibration graph prepared on the day of the experiment by measuring the potentials of a series of reaction mixtures of known CO2 concentration. Simultaneous Measurement of 02 Consumption and CO2 Release. The mitochondrial reaction mixture was made up in a potentiometric 02 electrode assembly (Hamsatech, King's Lynn, UK) operated and calibrated essentially as described by Delieu and Walker (4). The electrode output was recorded via a second pen on the chart recorder. The standard reaction vessel plunger 349 www.plantphysiol. org on May 1, 2019 Published by Downloaded from Copyright © 1987 American Society of Plant Biologists. All rights reserved.