Mitochondrial preparations from the leaves of tobacco (Nicotiana tabacum)

Abstract

A succinoxidase preparation was made from tobacco leaves by grinding them in a medium containing sucrose, 2-amino-2-hydroxymethylpropane-l:3-diol, citrate, phosphate and ethylenediaminetetra-acetic acid, and then centrifuging between 1000 and 10,000 g. Omission of any of the components of the medium, except phosphate, brought about a decrease in succinoxidase activity which was greatest for citrate and decreasingly less for 2-amino-2-hydroxymethylpropane-l :3-diol, sucrose and ethylenediaminetetra-acetic acid. These preparations contained the bulk of the succinoxidase of the leaf extract together with a third of the total chlorophyll and 15% of the total protein nitrogen. Only a third of this sedimented protein nitrogen, corresponding in about 5% of the protein nitrogen of the extract, was of non-chloroplast origin. The conditions necessary for measuring maximal activity of these succinoxidase preparations included the addition of cytochrome c, sucrose and Mg2+ions. The oxidation of succinate was inhibited re-versibly by malonate. It was inhibited strongly by cyanide, less strongly by fluoride and not at all by diethyldithiocarbamate. The preparations oxidized most of the acids of the tricarboxylic acid cycle; some of these oxidations were stimulated by mixtures of diphosphopyridine nucleotide, thiamine pyrophosphate and coenzyme A. A "sparked" oxidation of pyruvic acid was obtained only if the leaves were vacuum-infiltrated with dilute ammonia before being disintegrated. The products formed during the oxidation of these acids were other acids of the tricarboxylic acid cycle.