A procedure was described for preparing intact mitochondria from spinach (Spinacia oleracca L.) leaves. These mitochondria oxidized succinate, malate, pyruvate, a-ketoglutarate, and NADH with good respiratory control and ADP/O ratios comparable to those observed with mitochondria from other plant tissues. Glycine was oxidized by the prepartions. This oxidation linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport and phosphorylation inhibitors.Cyanide completely inhibited the oxidation of NADH. The oxidation of succinate, malate, and glycine was only partially inhibited.The development of techniques for the isolation of tightly coupled mitochondria from etiolated and storage tissues (12,17,18) has resulted in considerable progress toward understanding the physiological properties of the plant mitochondria (11,12,17,18). In marked contrast, relatively few methods are available in the literature for the isolation of intact mitochondria from the leaves of higher plants (8,12,17,18). Therefore, little information is available about the oxidative and phosphorylation properties of mitochondria from green leaves (10,12,17,18).The object of this paper is to describe the preparation, oxidative capacities, and response to various inhibitors of spinach leaf mitochondria. MATERIALS AND METHODSPreparation of Mitochondria. Fresh spinach (Spinacia oleracea L.) leaves were obtained from a local market and used immediately. Young spinach leaves (0.8 kg after deribbing) were cut into 3 liters of chilled medium containing 0.3 M mannitol, 4 mM cysteine, 1 mM EDTA, 30 mm MOPS3 buffer (pH 7.5), 0.2% defatted BSA, and 0.6% insoluble PVP. The leaves were disrupted at low speed for 2 sec in a 1-gallon Waring Blendor. The homogenate was squeezed through six layers of cheesecloth and mitochondria were isolated as fast as possible by differential centrifugation according to the method of Bonner (4). We found that very short grinding times in a Waring Blendor were the most successful. Longer grinding times had a deleterious effect on the mitochondrial oxidative and phosphorylative capacities.Mitochondrial Assays. Mitochondrial respiration was mea- sured polarographically using a Clark 02 electrode in a closed cell at 25 C. Oxygen consumption measurements were performed in a reaction medium (3.3 ml) containing 0.3 M mannitol, 5 mM MgCl2, 10 mm KCI, 10 mm Na-phosphate buffer (pH 7.2), and 0.1% defatted BSA. The 02 concentration in airsaturated medium was taken as 240 UM (9).Rotenone (Sigma), FCCP (Boehringer), oligomycin (Sigma), and antimycin A (Sigma) were dissolved in absolute ethanol. Salicyl-hydroxamic acid dissolved in formamide was a gift of Dr. Vignais.Mitochondrial Protein Determination. Total protein was determined by the Folin-Ciocalteau phenol reagent (16). Chl was extracted from the mitochondrial pellet in 80% acetone and measured according to Arnon (1). If we assume a protein to Chl ratio of 7, in broken thylakoids (15), the amount of mito...