Plant models to replace classical biochemistry practicals

154

Plant M o d e l s Practicals

to

Replace

Classical

Biochemistry

M D B R O W N L E A D E R , a M TREVAN b and P M DEY a

"Department of Biochemist~3, Royal Holloway, University of London Egham, Surrey TW20 OEX, UK b Faculty of Science, Technology, Health and Society South Bank University 103 Borough Road, London SE10AA, UK Introduction There is an ever-increasing need to reduce the cost of undergraduate praeticals and at the same time provide the student with the same learning objectives. Sometimes this requires a little abstract thought because the student must find the experiments exciting, challenging and informative. However, it is very difficult to propose an alternative technique without prior knowledge of the learning objective in mind. Does the lecturer for example, wish to develop the self-reliance and manual dexterity of the student or does he/she wish to illustrate the complex and interactive nature of mammalian biology? In addition not all life science students will pursue a career in biochemical research. Furthermore, whether we like it or not, there are always students in every facet of biochemistry education that object to using animal tissues in practical demonstrations. The question may therefore be assessed on both financial and ethical grounds. Plant models such as red beet are significantly cheaper than animal models to demonstrate certain biochemical phenomena because animals require specialized feeding and housing facilities coupled with the appropriate technical expertise. Here we exemplify some alternative protocols developed for classic biochemistry practicals. Effect of respiratory chain inhibitors and uncouplers upon mitochondrial oxidative phosphorylation The rat or guinea-pig liver has traditionally served as a source of mitochondria because these organelles occupy about one fifth of the total cell volume. There is no reason why mitochondria isolated from plant material cannot be used to study the effect of respiratory chain inhibitors such as rotenone and antimycin A or uncouplers such as 2,4-DNP upon oxidative phosphorylation. The procedure for the isolation of mitochondria from plant material is very simple and would involve the homogenization of the tissue for a defined length of time, filtration of the homogenate through muslin and the subsequent centrifugation of the filtrate at 4°C for 20 rain and at 10 000g. In order to avoid any chloroplast contamination, we suggest that beet, cauliflower or potato tuber may serve as a starting material. Certain aspects of plant mitochondrial metabolism are different from their animal counterparts, for example in the respiration of exogenous N A D H . However, components of the electron transport chain in plant mitochondria have been established by observing changes in the oxidized-reduced absorption spectra produced by inhibitors and uncouplers. In some plant species, blocking electron transfer with antim~,cin A or cyanide does not completely inhibit respiration. L This cyanide-resistant respiration can depend upon the plant species, organs and stage of development. Mitchondria from fresh potato tuber tissue are almost totally cyanide-sensitive and the ageing of dormant storage tissue can introduce a significant amount of cyanide insensitivity. The simple observation that respiratory chain inhibitors and uncouplers can modify plant mitochondrial activity can be established very easily without the need to purify the mitochondria extensively. The following section gives a recipe for the isolation of mitochondria. 2 BIOCHEMICAL

E D U C A T I O N 22(3) 1994

Preparation of Mitochondria from Red Beet Tissue Homogenization Medium: 10 mM D,L-glycerophosphate, 0.65 M ethanolamine (adjusted to pH 8.0 with concentrated H2S04), 0.28 M choline chloride, 2 mM salicylhydroxamic acid, 0.2% w/v BSA (fraction V, eseentially fatty acid-free), 0.5 mM butylated hydroxytoluene, 200 mM Tris-EDTA, pH to 8.0 with saturated Tris/MES solution (stored at -20°C). Immediately before use, the following should be added to 330 ml homogenization medium: 26 mM potassium metabisulphite (1.9 g), 115 jxl 2-mercaptoethanol, pH adjusted to 8.0 with saturated Tris solution, 0.4 w/v soluble polyvinylpyrrolidone (1.32 g). The medium for resuspension contains a much less elaborate cocktail of protectants. Glycerol and butylated hydroxytoluene are commonly used in the suspension medium. Dithiothreitol is added to the suspension medium in order to prevent oxidation of essential sulphydryl groups.

Suspension Medium: 1.1 M glycerol, 1 mM Tris-EDTA, 0.5 mM butylated hydroxytoluene, pH adjusted to 8.0 with saturated Tris solution, medium sterilized by Buchner filtration, store at -20°C. A d d 5 mM DTT (freshly prepared) before use and adjust pH to 8.0 with saturated Tris/MES solution.

Preparation After both the homogenization and suspension medium have been prepared, the following protocol may be used to prepare mitochondria from red beet. (1) Homogenize 330 g chopped red beet storage tissue for 2 x 10 s pulses with 330 ml homogenization medium in a Waring blender. (3) Filter homogenate through six layers of cheesecloth. (3) Repeat the homogenization and subsequent filtration twice with 2 x 330 g chopped red beet. (4) Centrifuge the total filtrate for 20 rain at 10 000g(av) in order to pellet heavy nuclei, cell debris and mitochondria. (5) Resuspend mitochondrial pellet and wash in suspension medium and centrifuge for 35 min at 10 000g(av) at 4°C. Subceilular localization of membrane fractions using enzyme marker analysis Glycolysis occurs in the cytosol and the tricarboxylic acid cycle occurs in the mitochondria. Succinate dehydrogenase and glucose-6-phosphatase may be used as markers for the mitochondria and (soluble) cytosol, respectively. Sodium azide and oligomycin, inhibitors of the F J F t-ATpase, have also been used as markers for the mitochondria. Subcellular fractions may be isolated from plant material. The procedure for isolation of these fractions is very similar and utilizes differential centrifugation. The isolation of mitochondria from red beet (Beta vulgaris L) for example, involves homogenization of the tissue for two 10 second bursts in homogenization medium, filtration of the homogenate through muslin and subsequent centrifugation of the filtrate at +4°C for 20 min and at 10 000g(av) as described above. The supernatant is then centrifuged at +4°C for 35 rain and at 80 000g(av) in order to pellet the microsomal fraction. Once each pellet has been resuspended and washed in suspension medium, the fractions are ready for enzyme marker analysis. All the learning objectives are fulfilled with the use of plant material. We have routinely performed enzyme marker analysis upon red beet plasma membrane and tonoplast vesicles and have isolated mitochondria and monitored ATP synthase activity. Subsequent enzyme marker analysis can be performed upon the mitochondria and the soluble (cytosol) fraction. All stages of cell fractionation must be performed at 4°C in order to minimize membrane protease activity and denaturation of proteins by excessive heat. All media and apparatus should be pre-cooled and maintained at this low temperature throughout the procedure. All the ingredients of the homogenization and suspension medium are either commonly found in teaching stores or

155 can be purchased very cheaply. Each individual homogenization medium may contain specific inhibitors, such as glycerophosphate, ethanolamine, E D T A and choline chloride, of phospholipases and phosphatases. Polyvinylpyrrolidone in either a watersoluble form or as the highly cross-linked insoluble 'Polyclar' may be added to the isolation medium in order to remove phenols. Polyvinylpyrrolidone adsorbs phenolic compounds found in high amounts in some plant tissues in order to prevent oxidation and polymerization of quinones by phenol peroxidase to the plant pigment melanin which inhibits many enzymes. 2-Mercaptoethanol which inhibits phenol oxidase and prevents oxidation of essential sulphydryl groups should also be incorporated within the homogenization medium. Defatted BSA will adsorb free fatty acids that would otherwise have stimulated acyl hydrolase activities. Antioxidants such as butylated hydroxytoluene and potassium metabisulphite will prevent unnecessary tissue damage during homogenization. The chelating agent E D T A will remove divalent cations such as Mg 2+ and C a 2÷ ions which are required by many membrane proteases. A variety of protease inhibitors such as diisopropylfluorophosphate and phenylmethylsulphonylfluoride which inhibit serine proteases may be added to the isolation medium. The procedure described above is not intended to purify the isolated mitochondria. Further continuous or discontinuous sucrose centrifugation of mitochondria which band at about 1.18 g/cm 3 is typically used to purify these organelles and is probably outside the scope of an undergraduate practical. However, biochemical research is invariably pre-occupied with establishing the purity of the material before further analysis is performed. Contamination is always a problem and establishing that enzymatic activities associated with isolated mitochondria really do represent mitochondrial enzymes needs to be determined. This contamination can, however, be reduced by repeated cycles of washing by centrifugation to reduce the levels of adhering soluble enzymes. We believe that many mitochondria remain intact during the isolation procedure. Intact and sealed tonoplast vesicles are isolated routinely from red beet after a more elaborate procedure that includes differential and sucrose density gradient centrifugation. These vesicles are capable of H ÷ transport by the tonoplast H+-ATPase and H+-PPase. Plasma membrane and tonoplast vesicles are stable in the appropriate suspension medium for at least 1 year at -70°C. Other ways of cell disruption involve the generation of protoplasts. 3 This would subsequently mean that less harsh homogenization conditions could be employed that would protect the mitochondria from further damage. There are a whole series of enzymes that will digest the plant cell wall and subsequently generate plant protoplasts. Cellulases, pectinases, hemicellulases can be purchased cheaply. Naturally, the price of the enzyme will depend upon source and purity. Any risk of disrupting the mitochondria is excluded after digestion of the plant cell wall. However, it should not be necessary to generate protoplasts from red beet disks prior to isolation of subcellular organelles.

Phosphatase activity measurements Some experiments require that the students monitor phosphatase activity of tissues. Although the purified alkaline phosphatase can be obtained from either Escherichia coli or the human placenta* which can be readily obtained from maternity hospitals and could therefore be used as an abundant source of enzyme, the experiment can be performed with plant alkaline phosphatases or ATPases that release inorganic phosphate during their catalytic reaction cycle.

*Care should be taken in using unscreened human material which may contain HIV, hepatitis, etc

B I O C H E M I C A L E D U C A T I O N 22(3) 1994

Summary The object of this general article is simply to suggest alternative inexpensive and novel procedures for biochemistry laboratory demonstrators and still retain all of the required learning outcomes. Mitochondria may be prepared by the students using common laboratory equipment in a very short period of time. Potato, red beet or sugar beet can be bought from a nursery or grown throughout the year in heated glasshouses. Countries in the sub-continent where the stable diet comprises primarily vegetables and the cost of using animal models to demonstrate simple metabolic concepts to first year biochemistry students needs to be stringently controlled, we suggest that plant models might be employed. The ready availability of the plant material combined with the relatively low cost of the practical experiments makes the use of plant models plausible.

References i Solomos, T (1977), Cyanide-resistant respiration of higher plants, Ann Rev Plant Physiol 28, 279-297 2Rea. P A and Poole, R J (1985), Proton-translocating inorganic phosphatase in red beet (Beta vulgaris L) tonoplast vesicles, Plant Physiol 77, 46-52 3Zhu, X-Y and Negrutru, I (1991), Isolation and culture of protoplasts, In Negrutru, I and Gharti-Chhetri, G B, editors, Laboratory guide for cellular and molecular plant biology. Birkhauser Verlag, Basel

SDS-Agarose Gel Eiectrophoresis as a Simple Procedure for Determining High Molecular Weight Protein Oligomerization M D BROWNLEADER, a M TREVAN b AND P M DEY a Department o f Biochemistry Royal Holloway, University o f London Egham, Surrey TW20 OEX, UK and b Faculty o f Science, Technology, Health and Society South Bank University 103 Borough Road L o n d o n S E 1 0 A A , UK a

Introduction The study of biochemical phenomena such as plant cell growth and development, and plant defence against pathogen attack is focussing upon the deposition and insolubilization of a high molecular weight glycoprotein in the cell wall. Many technical difficulties are encountered in the electrophoresis and subsequent staining of glycoproteins. Not all proteins behave as globular proteins and this experiment is designed to refect some of the problems found in biochemical research. A simple in vitro oligomerization of proteins coupled to a rapid and inexpensive method of protein separation such as SDS-agarose gel electrophoresis will enable a biochemistry student to mimic, at least in some respects, a complex in vivo process.

Background A simple procedure to detect oligomerization of a plant cell-wall glycoprotein, extensin, in vitro has been employed. Extensins are hydroxyprotine-rich glycoproteins (HRGPs) that are ionicallybound to the cell wall and are cross-linked in situ by a peroxidase when the plant is challenged by pathogens. Benhamou et al 1 demonstrated by immunolocalization that HRGPs accumulated in the cell wall to a greater extent in resistant than in susceptible cultivars of tomato root tissues infected by Fusarium oxysporum. Brownleader et al 2 have demonstrated that tomato extensin comprises about 65% protein and 35% carbohydrate. Stuart and Varner 3 demonstrated that the soluble H R G P does not enter