The Effect of Nucleotides and Inhibitors on Respiration in Isolated Wheat Mitochondria

Abstract

The effect of mono-, di-, and trinucleoside phosphates and respiratory inhibitors on respiration in winter wheat (Triticumn aestivuim L. cv. Rideau) mitochondria has been examined. When added during state 4 respiration, subsequent to addition of ADP, all of the dinucleotides stimulated oxidation and induced respiratory control with all substrates examined. Similar results were obtained with AMP, but other mononucleotides and all trinucleotides did not affect the rate of oxidation. Nucleoside diphosphates did not stimulate respiration when added prior to the addition of ADP, but subsequent addition of AMP, ADP, or ATP re-established coupled respiration in the presence of the dinucleotides.The duration of 2, 4-dinitrophenol stimulated respiration during oxidation of a-ketoglutarate was found to be dependent on the amount of AMP, ADP, or ATP added, either prior, or subsequent to, addition of the uncoupler. The addition of oligomycin during 2,4-dinitrophenol stimulated respiration reestablished coupled respiration with low ADP/O ratios, when added after addition of ATP or conditions which allow formation of ATP from added ADP. The nucleoside diphosphates, other than ADP, did not stimulate oxidation of a-ketoglutarate in the presence of 2,4-dinitrophenol until a small amount of adenine nucleotide was added to the system. The results suggest that dinucleotides other than ADP, are able to participate in the energy conversion processs of the mitochondria, probably via transphosphorvlation reactions.Studies on the respiratory properties of mitochondria isolated from different plant sources and by different isolation procedures have indicated that considerable variation may exist in their response to various substrates in the presence of uncouplers and respiratory inhibitors (3,16,27,32). Therefore, it is often difficult to extrapolate results obtained from one system to another. The question of which nucleotides are able to act as phosphate acceptors has been examined in several systems from both plant and animal sources (14,21,24 (14), but the role of other nucleotides in phosphorylation reactions in plants is not clear.The present study was undertaken in an attempt to characterize the effect of nucleotides and respiratory inhibitors on respiration in the winter wheat mitochondrial system currently being used in this laboratory to examine structural and functional changes in mitochondrial membranes in relation to growth at low temperature (20). MATERIALS AND METHODSIsolation of Mitochondria. Seedlings of winter wheat (Triticum aestivurn L. cv. Rideau) were germinated and grown in the dark at 24 C on moist filter paper for 2 days. Mitochondria were isolated from the shoots by differential centrifugation (22) and where indicated, further purified by sucrose density gradient centrifugation. Sucrose gradients were prepared by layering 3.7 ml each of 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2 M sucrose solutions containing 10 mm KCl, 10 mm tris-HCl 10 mM KH2PO4, and 0.75% (w/v) BSA at pH 7.2 in 30-ml centrifuge tubes and st...

Effects of Temperature on Electron Transport in Arum maculatum Mitochondria

“…It has been noted that in mitochondria containing both cyanide-sensitive and cyanide-insensitive respiratory pathways that the external NADH dehydrogenase and the Cyt pathway appear to be closely associated functionally (4,7,13). A similar functional association has been postulated between the succinate dehydrogenase and the alternative pathway (6, 10).…”