Laboratory Exercises Teaching About Citric Acid Cycle Using Plant Mitochondrial Preparations SOME ASSAYS FOR USE IN LABORATORY COURSES* Received for publication, May 28, 2004, and in revised form, January 13, 2005 Joaquim A. F. Vicente‡, Carina S. S. Gomes-Santos‡, Ana Paula M. Sousa‡, and Vı ´tor M. C. Madeira¶ From the ‡Departamento de Bota  nica, ¶Departamento de Bioquı´mica, Universidade de Coimbra, 3004-517 Coimbra, Portugal Potato tubers and turnip roots were used to prepare purified mitochondria for laboratory practical work in the teaching of the citric acid cycle (TCA cycle). Plant mitochondria are particularly advantageous over the animal fractions to demonstrate the TCA cycle enzymatic steps, by using simple techniques to measure O 2 consumption and transmembrane potential (). The several TCA cycle intermediates induce specific enzyme activities, which can be identified by respiratory parameters. Such a strategy is also used to evidence properties of the TCA cycle enzymes: ADP stimulation of isocitrate dehydrogenase and -keto- glutarate dehydrogenase; activation by citrate of downstream oxidation steps, e.g. succinate dehydrogen- ase; and regulation of the activity of isocitrate dehydrogenase by citrate action on the citrate/isocitrate carrier. Furthermore, it has been demonstrated that, in the absence of exogenous Mg 2 , isocitrate-de- pendent respiration favors the alternative oxidase pathway, as judged by changes of the ADP/O elicited by the inhibitor n-propyl galate. These are some examples of assays related with TCA cycle intermediates we can use in laboratory courses. Keywords: Plant mitochondria, O 2 consumption and  determinations, TCA cycle features. Owing to the simplicity of the isolation process, most studies of bioenergetics are carried out with isolated mi- tochondrial fractions [1]. Plant mitochondrial fractions, easily obtained with high yield of purity, offer advantages over animal fractions [2, 3]. Among about 30 different plant tissues already tested [4], potato tubers and turnip roots were selected for isolation of mitochondrial fractions ow- ing to the high purity of fractions and the stable functional activities [3]. These mitochondria last longer and exhibit superior performance of respiration activities with several substrates, as compared with animal preparations [3]. Therefore, the use of plant mitochondrial fractions is ad- vantageous in biochemical studies, e.g. bioenergetics and metabolism, for practical works in undergraduated and graduated curricula. In potato tuber and turnip root mitochondria, the activ- ities dependent on the following citric acid (TCA) 1 cycle enzymes were assayed: malate dehydrogenase, NAD + - dependent malic enzyme, aconitase, isocitrate dehydro- genase, and -ketoglutarate dehydrogenase. The appro- priate substrates for these activities, yielding ubiquinol (succinate dehydrogenase) or NADH (the remaining en- zyme steps), activate Complex II and Complex I, respec- tively, triggering respiration (oxygen consumption) and the coupled transmembrane potential [5]. Respiratory indices report the bioenergetic competence of tested mitochon- dria and also characterize each intermediate activity. En- zyme properties of TCA cycle intermediate steps were also evaluated with the methodology proposed in this work. The present project is directed to teachers and instruc- tors of laboratory practicals, but is not immediately suita- ble for use by the students. For instance, after presentation of the TCA cycle scheme (Fig. 1), students are motivated to experimentally demonstrate activities of each TCA cycle intermediate by using mitocondrial preparations to access respiration parameters. Properties of the TCA cycle enzy- matic activities can be previously given to the students knowledge and then demonstrated in assays performed with plant mitochondria, as those expressed in this work. Or, alternatively, records and parameters can be produced and then considered for discussion and interpretation. EXPERIMENTAL PROCEDURES Young potato tubers (Solanum tuberosum L.) and turnip roots (Brassica napus L.) were peeled to expose a clean tissue kept in distilled water. All operations are performed at 0 – 4 °C. The tissue was homogenized in a juicer (Moulinex) at 2 g of fresh weigh per milliliter of homogenization medium (A) containing 250 mM su- crose, 2 mM EDTA, 40 mM HEPES (pH 8.1) (or 20 mM for turnip), 0.1% bovine serum albumin (BSA), and 4 mM cysteine (added just before homogenization) [6, 7]. After filtration through four to six * This work was supported by IMAR-Instituto do Mar. ‡ To whom correspondence should be addressed: Departa- mento de Zoologia, Universidade de Coimbra, 3004-517, Coim- bra, Portugal. Tel.: 351-39-834729; Fax: 351-39-826798; E-mail: jvicente@ci.uc.pt. 1 The abbreviations used are: TCA, citric acid; BSA, bovine serum albumin; TPP, tetraphenylphosphonium; RC, respiratory control. © 2005 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Printed in U.S.A. Vol. 33, No. 2, pp. 128 –132, 2005 This paper is available on line at http://www.bambed.org 128