- Email: [email protected]
[48] Peroxisomes and fatty acid degradation
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TABLE III LATENCY OF ENZYME ACTIVITIES IN GLYOXYSOMES FROM A SUCROSE GRADIENTa
Enzyme
Intact (nmol/min/ml)
Broken (nmol/min/ml)
Latency (%)
Malate synthase Hydroxyacyl-CoA dehydrogenase Malate dehydrogenase
0.48 0.23 0.08
26.60 1.52 0.28
98.2 84.9 71.4
a The activitieswere measuredin the presence of 1.9 M sucrose. The glyoxysomeswere broken by including 0.2% Triton X-100 in the sucrose-containingreaction mixture. From R. P. Donaldson,R. E. Tully,O. A. Young, and H. Beevers, Plant Physiol. 67, 21 (1981).
ever difficulties in reproducibility have been encountered with some tissues (Anderson and Butt, personal communication). The methods described here yield glyoxysomes from castor bean endosperm relatively free of contaminating organelles and membranes (ghosts) relatively free of matrix (Table II). We recommend that centrifugation of sucrose gradients in a vertical rotor be used as a rapid procedure for isolating peroxisomes and glyoxysomes from other tissues. Furthermore, we suggest using carbonate washing 1~as a method to obtain glyoxysomal/peroxisomal ghosts free of matrix protein. Acknowledgments We thank N. Charley (SEM), E. Erbe (freeze fracture), L. C. Frazier (TEM and sample preparations), G. Kaminski (TEM and sample preparations), and C. Pooley (photography). This work was funded by NSF PCM 8216051.
[48] P e r o x i s o m e s a n d F a t t y A c i d D e g r a d a t i o n
By BERNT GERHARDT Peroxisomes have been isolated from a wide range of plant tissues and are considered to be present in virtually all cells of higher plants. Plant peroxisomes have been subcategorized according to their formerly known metabolic function as glyoxysomes, leaf peroxisomes, and unspecialized METHODS IN ENZYMOLOGY,VOL. 148
Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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peroxisomes, i.e., peroxisomes of unknown function. Fatty acid/3-0xidation is now known to be a general, basic metabolic function of the higher plant peroxisome) Peroxisomal/3-0xidation was first discovered in glyoxysomes,2 which are organelles found only in fat-storing tissues of germinating seeds. Since glyoxysomes have already been treated in this series,3.3a the subject of this chapter will be procedures for studying fatty acid degradation by nonglyoxysomal peroxisomes. The peroxisome population of nonfatty tissues comprises approximately 1% of the organelle protein of those tissues: With the exception of the isolation on Percoll gradients, all methods described in this chapter have been successfully applied to peroxisomes from mung bean hypocotyls. Peroxisomes from spinach and pea leaves, maize seedling roots, and potato tubers have also been studied using these methods. 1,5 Isolation of Peroxisomes The isolation of glyoxysomes from castor bean endosperm TM and of leaf peroxisomes 6,7 has been described in this series. Therefore, isolation procedures outlined here will be restricted to the isolation of peroxisomes from mung bean hypocotyls on sucrose gradients and to the isolation of leaf and root peroxisomes on Percoll gradients. All procedures are carried out at 0-4 ° . Small pieces of plant material are gently ground with ice-cold homogenization medium in a chilled mortar. Only mortars with a glazed surface are suitable. The relative contents of sucrose (w/w) or Percoll (v/v) in the solutions used for the construction of density gradients are adjusted refractometrically. After centrifugation the density gradients are separated into 1-ml fractions and the location of the peroxisomes (and other organelles) is determined by assaying the fractions for marker enzyme activities. The peroxisomes are collected as a whole fraction when the location of the peroxisomes in the gradient is known and a peroxisomal band is visible.
t B. Gerhardt, Physiol. Veg. 24, 397 (1986). 2 T. G. Cooper and H. Beevers, J. Biol. Chem. 244, 3514 (1969). 3 H. Beevers and R. W. Breidenbach,this series, Vol. 31, p. 565. 3aA. H. C. Huang, this series, Vol. 72, p. 783. 4 B. Gerhardt, "Microbodies/Peroxisomenpflanzlicher Zellen." Springer-Verlag, Wien and New York, 1978. 5B. Gerhardt, Planta 159, 238 (1983). 6 N. E. Tolbert, this series, Vol. 23, p. 665. 7 N. E. Tolbert, this series, Vol. 31, p. 734.
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Isolation of Peroxisomes from Mung Bean Hypocotyls Mung bean seedlings are grown in the dark for 2.5-3 days. Hypocotyls (20 g) are homogenized in 20 ml of homogenization medium containing 170 mM Tricine-NaOH, pH 7.5, 1 M sucrose, 10 mM KC1, 1 mM MgC12, 1 mM EDTA, 10 mM mercaptoethanol, and bovine serum albumin (9 mg/ ml). The homogenate is squeezed through four layers of cheesecloth and centrifuged at 600 g for 10 min. From the 600 g supernatant, the peroxisomes are concentrated by pelleting a crude organelle fraction at 12,000 g (15 min). This fraction is gently resuspended in 2 ml of homogenization medium using a glass rod wrapped with cotton wool on its tip. The resuspended crude organelle preparation is layered onto a previously prepared sucrose gradient. The gradient is constructed from precooled sucrose solutions which are prepared by dissolving the sucrose in 1 mM EDTA, pH 7.5. A continuous gradient is formed from 20 ml of 30% and 15 ml of 60% sucrose, or alternatively a discontinuous gradient is constructed using, sequentially, 3 ml of 60%, 5 ml of 57%, 7 ml of 53%, 7 ml of 46%, 6 ml of 43%, and 6 ml of 35% sucrose. The discontinuous gradient has a loading capacity higher than that of the continuous gradient. The crude organelle fraction obtained from up to 50 g of hypocotyls can be applied to the discontinuous gradient. The continuous or discontinuous gradients are centrifuged at 83,000 g (average) for 3 hr. The peroxisomes are recovered in the discontinuous gradient at the interface between the 46 and 53% sucrose layer. Peroxisomes from mung bean hypocotyls can also be isolated using a modification8 of the procedure described by Schuh and Gerhardt. 9 The crude organelle fraction is prepared as described above and layered onto a discontinuous gradient composed of, in sequence, 5 ml of 60%, 5 ml of 53%, 5 ml of 43%, 7 ml of 38%, 7 ml of 35%, and 3 ml of 30% sucrose. The gradient is centrifuged for 7.5 min at an acceleration of 2700 revolutions × min -2 using a Beckman L2 65B ultracentrifuge and a SW 27 rotor (final centrifugal force 61,000 g, average). The plastids are very effectively separated from the other organelles. The organelle band (approximately 4 ml) stretching from the interface between the 30 and 35% sucrose layer into the upper part of the 35% sucrose layer is transferred onto a second discontinuous sucrose gradient. This gradient consists of 3 ml of 60%, 7 ml of 55%, 10 ml of 48%, 7 ml of 43%, and 5 ml of 35% sucrose. It is centrifuged at 83,000 g (average) for 3 hr. The peroxisomes are recovered at the interface between the 48 and 55% sucrose layer. Peroxisomal fractions isolated by this two-gradient method are uncontaminated by plastids and show, if at all, extremely low contamination by mitochondria. The s H. Gerbling and B. Gerhardt, Planta, in press (1987). 9 B. Schuh and B. Gerhardt, Z. Pflanzenphysiol. 114, 477 (1984).
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yield of peroxisomes however, amounts to only 1-3% based on catalase activity. Isolation of Nonglyoxysomal Peroxisomes on Percoll Gradients
Before use, the Percoll stock solution (100 ml) is dialyzed against charcoal (5 g of charcoal suspended in 1250 ml of H20). l° The stock solution becomes diluted due to water uptake during dialysis. It is adjusted to 80% Percoll with H20. An isosmotic Percoll solution is then prepared by adding 10.3 g of sucrose, 179 mg of Tricine, and 37 mg of EDTA to 100 ml of the dialyzed 80% PercoU solution. The isosmotic 80% Percoll solution is adjusted to pH 7.5. When required it is diluted with a solution consisting of 0.3 M sucrose, 1 mM EDTA, and 10 mM TricineNaOH, pH 7.5. LeafPeroxisomes. Peroxisomes are isolated from leaf tissue following a procedure developed by Betsche. and Mfiller. 1° Five to 10 g of young leaves are ground with 2.5 vol of homogenization medium consisting of 0.1 M Tricine-NaOH, pH 7.5, 0.25 M mannitol, 1 mM EDTA, 15 mM mercaptoethanol, and bovine serum albumin (1 mg/ml). The homogenate is squeezed through four layers of cheesecloth and centrifuged at 2400 g for 5 min. From the 2400 g supernatant, the peroxisomes are concentrated by pelleting a crude organelle fraction at 12,000 g (15 min). This fraction is resuspended in 2 ml of homogenization medium and layered onto a preformed continuous Percoll gradient. The gradient is constructed from 16 ml of isosmotic 15% and 16 ml of isosmotic 50% Percoll solution over a cushion (5 ml) of isosmotic 80% Percoll. The gradient is placed in a Sorvall SS 34 rotor and centrifuged in a Sorvall RC-5C centrifuge using the automatic rate control to avoid the formation of disturbances within the gradient during acceleration and deceleration. The speed is set at 20,000 rpm (48,000 g), the run time at 5-8 min. The exact centrifugation time within the given limits depends upon the plant species from which the peroxisomes are isolated. Root Peroxisomes. Peroxisomes isolated from roots on sucrose or Percoll gradients are commonly heavily contaminated by mitochondria. The separation is improved 11 if the density of the mitochondria is artificially increased by intramitochondrial deposition of an insoluble tetrazolium salt produced by the activity of succinic dehydrogenase.12 Roots (15 g) from 3-day-old maize seedlings are ground in 30 ml of medium A supplemented with 4 mM cysteine. Medium A contains 0.1 M Tricine10T. Betsche and G. MOiler, personal communication (1984). n B. Gerhardt, in "Structure, Function and Metabolism of Plant Lipids" (P.-A. Siegenthaler and W. Eichenberger, eds.), p. 189. Elsevier, Amsterdam, 1984. 12 G. Davis and F. Bloom, Anal. Biochem. 51, 429 (1973).
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NaOH, pH 7.5, 0.3 M mannitol, 1 mM EDTA, and bovine serum albumin (1 mg/ml). The homogenate is squeezed through four layers of cheesecloth and centrifuged at 600 g for 15 min. From the 600 g supernatant a crude organeUe fraction is pelleted at 12,000 g (15 min). This fraction is resuspended in 2 ml of medium A and pelleted again (12,000 g, 15 min). It is then resuspended and incubated in 4 ml of medium A containing additionally 80 mM succinate and 10 mM iodonitrotetrazolium. After 30 min incubation at 30°, 4 ml of medium A is added to the incubated mixture and the organelles are pelleted at 12,000 g (15 min). They are washed once with 4 ml of medium A, finally resuspended in 2 ml of medium A, and layered onto a preformed continuous Percoll gradient. The gradient is constructed from 15 ml of isosmotic 60% Percoll solution and 15 ml of 0.3 M sucrose dissolved in a I mM EDTA solution containing also 10 mM Tricine-NaOH, pH 7.5. The gradient is centrifuged in a fixed angle rotor (Sorvall SS 34 rotor) at 40,000 g for 20 min. Contamination
The peroxisomal fractions isolated on sucrose gradients are normally contaminated by mitochondria and especially by plastids, those isolated on Percoll gradients by mitochondria. With respect to the topic of this chapter, mitochondrial contamination of the peroxisomal fractions has to be considered in particular although mitochondrial B-oxidation activity has not yet unequivocally been demonstrated for higher plant cells.~ A contribution of contaminating mitochondria to the B-oxidation enzyme activities measured in a peroxisomal fraction is checked by the following method. 5,~3The activity of a given B-oxidation enzyme as well as that of a peroxisomal marker enzyme (catalase, glycolate oxidase) is assayed in the peroxisomal and mitochondrial fraction from one and the same gradient. For both fractions, the ratio of the B-oxidation enzyme activity to the peroxisomal marker enzyme activity is then calculated. If this ratio determined for a sufficient number of gradients does not significantly differ (according to a statistical analysis) between the peroxisomal and mitochondrial fraction, the B-oxidation enzyme activity in the peroxisomal (and mitochondrial) fraction is considered to be associated only with the peroxisomes.
Assay Methods Thiol reagents such as dithioerythritol (DTE), dithiothreitol (DTT), mercaptoethanol, and cysteine react with acyl-CoA's in a nonenzymatic 13 B. Gerhardt, FEBS Lett. 126, 71 (1981).
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reaction.14 Therefore, care has to be taken if thiol reagents are included in assay mixtures for measuring/3-oxidation pathway activities. On principle, thiol reagents are not included in the reaction mixtures described here.
Enzyme Assays R - - C H E - - C H 2 - - C O O H + ATP + CoASH ~ R - - C H 2 - - C H 2 - - C O S C o A + AMP + PPi ( I ) R - - C H 2 - - C H 2 - - C O S C o A + 02 ---> R--CH-~-CH--COSCoA + H202 (2) R--CH~---CH--COSCoA + H20 ~ R - - C H O H - - C H 2 - - C O S C o A (3) R - - C H O H - - C H 2 - - C O S C o A + NAD* ~ R - - C O - - C H 2 - - C O S C o A + NADH + H ÷ (4) R - - C O - - C H E - - C O S C o A + CoASH ~ R - - C O S C o A + CH3--COSCoA (5)
All five enzymes of fatty acid activation (reaction 1) and B-oxidation (reactions 2-5) can be assayed spectrophotometrically using a singlebeam recording spectrophotometer with zero offset capability, a full-scale absorbance of 0.1 or 0.2, and a recorder speed of 0.5-2.0 cm/min. The reaction mixtures of 1 ml total volume in self-masking microcuvettes (1cm light path) contain I0-100/zl of the peroxisomal fraction (approximately 5-50 /~g of protein). The reactions are started by adding the substrate to the reaction mixtures, unless otherwise stated. For measurements of unspecific absorbance changes, reactions without substrate are followed in parallel cuvettes. Reaction rates are calculated for the test reaction after subtracting the absorbance change of the blank from that of the test. Rates of NAD + reduction and NADH oxidation, respectively, are calculated using the extinction coefficient 6220 M -1 cm -~ for NADH at 340 rim. Acyl-CoA Synthetase (Reaction 1). The enzyme is routinely assayed by a coupled assay m e t h o d ) The acyl-CoA formed by the synthetase is oxidized by added acyl-CoA oxidase (reaction 2) and the H202 formation in the oxidase reaction is determined by a coupled peroxidatic reaction.15 The reaction mixture contains 175 mM Tris-HC1, pH 8.5, 10 mM ATP, 7.5 mM MgC12, 0.6 mM CoASH, 50/zM FAD, 13 mM p-hydroxybenzoic acid, 1 mM 4-aminoantipyrine, 1 mM NAN3, 0.1 U acyl-CoA oxidase (from Candida species; Sigma), 5.3 U of horseradish peroxidase (Sigma, type II), and 0.1 mM palmitic acid. The palmitic acid is given to the reaction mixture dissolved in 10/zl acetone. The reaction is started by the addition of CoASH. The absorbance increase at 500 nm is monitored. An t4 G. B. Stokes and P. K. Stumpf, Arch. Biochem. Biophys. 162, 638 (1974). 15 C. C. Allain, L. S. G. Poon, W. Richmond, and C. Fu, Clin. Chem. (Winston-Salem, N.C.) 20, 470 (1974).
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absorbance increase of 0.51 corresponds to the consumption of 0.1/xmol H202/ml in the coupled peroxidatic reaction.16 In certain cases (e.g., determination of substrate specificity) acyl-CoA synthetase cannot be assayed by coupling reaction 1 and 2 and following the formation of H202. Acyl-CoA synthetase may then by assayed by a spectrophotometric assay which either determines the AMP formation by a coupled reaction sequence 17or couples an assay for pyrophosphate TM to reaction 1. ATP hydrolase and pyrophosphatase activity, respectively, can substantially interfere with these assays. Acyl-CoA synthetase is also assayed by a direct method which determines the rate of [~4C]palmitoyl-CoA formation from [14C]palmitic acid. 19 Substrate and product are separated by thin-layer chromatography. 8 The reaction mixture contains, in a total volume of 0.5 ml, 175 mM Tris-HC1, pH 8.5, 10 mM ATP, 7.5 mM MgC12, 0.02% Triton X-100 (to inhibit acylCoA oxidaseS), 10-30/~g protein contained in -<200/.d of the peroxisomal fraction from a sucrose gradient, and 0.175 mM [1-14C]palmitic acid (0.7 mCi/mmol). The added palmitic acid is dissolved in 10/.d acetone. The reaction is started by adding CoASH to a final concentration of 1 mM. After a reaction time of 10 min an aliquot (10-50 ~1) of the reaction mixture is immediately spotted on a thin-layer cellulose plate (20 x 20 cm; 0.1 mm coating thickness) and chromatographed in a butanol : glacial acetic acid : water system (5 : 2 : 3, by volume) at room temperature. Developing the chromatogram takes 6-7 hr. After drying the chromatogram at room temperature, the chromatographic process is repeated once. Radioactive spots on the chromatogram are detected with a thin-layer chromatogram scanner. For locating palmitoyl-CoA, an aliquot of a reaction mixture is chromatographed which contains unlabeled instead of labeled palmitic acid and is mixed with labeled palmitoyl-CoA after incubation. For locating palmitic acid, an aliquot of the test reaction mixture is spotted on the thin-layer plate prior to incubation. The Rf values are approximately 0.60 and 0.95 for palmitoyl-CoA and palmitic acid, respectively. The formed palmitoyl-CoA is quantified either by integration of the palmitoyl-CoA peak of the scanner record or by counting the palmitoyl-CoA in a liquid scintillation counter. Before transferring the palmitoyl-CoA spot from the thin-layer plate into scintillation vials the cellulose is covered with a celluloid layer, z° A celluloid solution is prepared by dissolving 66 g of cellulose acetate and 16 D. J. Hryb and J. F. Hogg, Biochem. Biophys. Res. Commun. 87, 1200 (1979). 17 T. Tanaka, K. Hosaka, and S. Numa, this series, Vol. 71, p. 334. is j. Edwards, T. ap Rees, P. M. Wilson, and S. Morrell, Planta 162, 188 (1984). 19j. Bar-Tana, G. Rose, and B. Shapiro, this series, Vol. 35, p. 117. 20 p. Leusing, Doctoral Thesis, University of MOnster (1985).
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20 g of camphor in a solvent prepared from 750 ml acetone, 250 ml npropanol, and 28 ml diethylene glycol. Five to 7 ml of the celluloid solution is spread over the cellulose layer of the thin-layer plate. After drying, the area (1 x 1 cm) of the palmitoyl-CoA spot is cut out. The obtained piece of celluloid to which the cellulose firmly adheres is dissolved in 0.5 ml dimethyl sulfoxide and then 4.5 ml scintillation fluid is added. The radioactive assay and the three coupled spectrophotometric assays for measuring acyl-CoA synthetase activity give essentially identical results if the assays are performed with palmitic acid as substrate. 8 Acyl-CoA Oxidase (Reaction 2). The enzyme is assayed by following the H202 formation in a coupled assay which determines the H202 in a peroxidatic reaction.~5 The assay mixture consists of 175 mM Tris-HCl, pH 8.5, 50/zM FAD, 13 mM p-hydroxybenzoic acid, I mM 4-aminoantipyrine, 1 mM NaN3 (to inhibit the peroxisomal catalase), 5.3 U of horseradish peroxidase (Sigma, type II), and 50 mM palmitoyl-CoA. The dye formation is monitored at 500 nm. An absorbance increase of 0.51 corresponds to the consumption of 0. I/~mol H202/ml in the coupled peroxidatic reaction.~6 Triton X-100 at concentrations ---0.01% inhibits the acyl-CoA oxidase5 and is therefore not included in the reaction mixture. When acyl-CoA oxidase activities are measured by the spectrophotometric assay and a polarographic assay which determines directly the oxygen uptake by the acyl-CoA oxidase, essentially identical results are obtained, 5 demonstrating the reliability of the coupled spectrophotometric assay. Enoyl-CoA Hydratase (Crotonase; Reaction 3). The enzyme is assayed by a coupled assay method monitoring the absorbance increase at 340 nm due to the NAD ÷ reduction in the coupled 3-hydroxyacyl-CoA dehydrogenase reaction (reaction 4). 21 The reaction mixture consists of 175 mM Tris-HC1, pH 9.5, 2 mM EDTA, 2 mM KCN, 0.3 mM NAD ÷, 3 U of 3-hydroxyacyl-CoA dehydrogenase (from procine heart; Sigma type III), 22 0.05% Triton X-100, and 0.1 mM crotonyl-CoA. 3-Hydroxyacyl-CoA Dehydrogenase (Reaction 4). The enzyme is assayed in the backward direction, i.e., in the direction of reduction of 3oxoacyl-CoA to 3-hydroxyacyl-CoA.23 The reaction is followed by the absorbance decrease at 340 nm due to NADH oxidation. The reaction mixture contains 50 mM potassium phosphate, pH 6.8, 2 mM EDTA, 0.25 2t j. R. Stern and A. del CampiUo, J. Am. Chem. Soc. 75, 2277 (1953). 22 A 3-hydroxyacyl-CoA dehydrogenase preparation has to be used which does not contain the bifunctional protein exhibiting 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities. 23 F. Lynen, L. Wessely, O. Wieland, and W. Rueff, Angew. Chem. 64, 687 (1952).
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mM NADH, 2 mM KCN, bovine serum albumin (1 mg/ml), 0.05% Triton X-100, and 0.1 M acetoacetyl-CoA. 3-OxoacyI-CoA Thiolase (Reaction 5). The enzyme is assayed by trapping the formed acetyl-CoA with citrate synthase and oxaloacetate which is generated from malate in the NAD+-dependent malate dehydrogenase reaction. 2 Activity is therefore measured by following NAD + reduction at 340 nm. The reaction mixture consists of 100 mM potassium phosphate, pH 7.5, 0.25 mM MnC12, 2 mM CoASH, 0.3 mM NAD +, 3 mM L-malate, 12 U of malate dehydrogenase, 2.2 U of citrate synthase, 2 mM KCN, 0.05% Triton X-100, and 40/~M acetoacetyl-CoA.
Assay of Peroxisomal Overall t-Oxidation In contrast to the known mammalian mitochondrial t-oxidation system, the peroxisomal t-oxidation system is insensitive to KCN. Therefore, peroxisomal t-oxidation is assayed by measuring acyl-CoA- or fatty acid-dependent, KCN-insensitive acetyl-CoA production or NADH formation. The NADH formation results from reaction 4. Determination of NADH Formation. The low yield and t-oxidation activity of nonglyoxysomal peroxisomes normally requires an amplification of the formed NADH by a cycling method. 24 Step 1: The reaction mixture (total volume, I ml) for the t-oxidation is prepared in small test tubes. It contains 175 mM Tris-HCl, pH 8.5, 0.15 mM NAD +, 0.15 mM CoASH (to allow thiolytic cleavage of the formed 3oxoacyl-CoA which otherwise may inhibit reaction 4), 25 2 mM KCN, 2080/~g protein contained in -<300/zl of the peroxisomal fraction from a sucrose gradient, and 25/.~M palmitoyl-CoA (or 0.1 mM palmitic acid, 10 mM ATP, 7.5 mM MgCI2, and a CoASH concentration raised to 0.6 mM). The reaction is run at 25° for 15 min. Step 2: The/3-oxidation reaction is terminated by adding 1 ml of 0.2 N NaOH to the reaction mixture which is then boiled for 4 min to destroy the remaining NAD +. The mixture is cooled to room temperature and centrifuged at 4500 g for l0 min. An aliquot (0.5 ml) of the supernatant is neutralized by adding an equal volume of 0.2 N HC1. During neutralization the sample is vigorously shaken to avoid destruction of NADH by local acidification. Step 3: An aliquot (0.1-0.3 ml) of the neutralized sample is used in the cycling assay for NADH. The cycling assay is performed as described in t h i s s e r i e s . 26 The cycling reaction proceeds for 10 min. It is recommended u I. Papke and B. Gerhardt, unpublished results (1984). 25 T. Osumi, T. Hashimoto, and N. Ui, J. Biochem. (Tokyo) 87, 1735 (1980). 26 H. Matsumura and S. Miyachi, this series, Vol. 69, p. 465.
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to use black test tubes and an alcohol dehydrogenase purified from bound NAD+/NADH for the cycling reaction. A blank which does not contain palmitoyl-CoA (or palmitic acid) in the /]-oxidation mixture (step 1) is also carded through steps 1 to 3. If no standard curve has been prepared, 26rates of NADH formation are calculated using an internal standard prepared as follows: 50 pmol NADH dissolved in 0.1 ml H20 is included in a cycling reaction mixture (step 3) prepared with an aliquot of the neutralized blank. This standard is carried through step 3 in parallel with the sample and the blank. The rate of NADH formation is increased when a system for trapping the formed acetyl-CoA is included in the fl-oxidation mixture (step 1). Such a system consists of 0.5 mM aspartate, 0.5 mM 2-oxoglutarate, 0.1 mM pyridoxalphosphate, 0.02 U of L-aspartate:2-oxoglutarate aminotransferase, and 0.2 U of citrate synthase. Determination of Acetyl-CoA Formation. The reaction mixture (total volume, 0.6 ml) for the B-oxidation contains 175 mM Tris-HC1, pH 8.5, 0.15 mM NAD +, 2 mM KCN, 0.1 mM [U:4C]palmitic acid (2 mCi/mmol), 10 mM ATP, 7.5 mM MgC12, 1 mM CoASH, and 20-50/~g protein contained in -<200/~l of the peroxisomal fraction from a sucrose gradient. The added palmitic acid is dissolved in 10/~l acetone. After a reaction time of 15 min at 25° an aliquot (10-50/~l) of the reaction mixture is immediately spotted on a thin-layer cellulose plate. The chromatogram is developed and acetyl-CoA and palmitic acid are detected correspondingly to the procedures described in the radioactive acyl-CoA synthetase assay. The Rr values for palmitic acid and acetyl-CoA are approximately 0.95 and 0.45, respectively. The formed acetyl-CoA is quantified correspondingly to the palmitoyl-CoA in the acyl-CoA synthetase assay.
Protein Determination For specific activity measurements, protein in fractions from sucrose gradients is determined by a modification5 of the Lowry method. 27Protein in fractions from Percoll gradients is determined by following the procedure of Vincent and Nadeau. z8 Acknowledgment Studies in the author's laboratory were supported by the Deutsche Forschungsgcmeinschaft. 27 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 2~R. Vincent and D. Nadeau, Anal. Biochem. 135, 355 (1983).
[48]
PEROXISOMESAND GLYOXYSOMES
TABLE III LATENCY OF ENZYME ACTIVITIES IN GLYOXYSOMES FROM A SUCROSE GRADIENTa
Enzyme
Intact (nmol/min/ml)
Broken (nmol/min/ml)
Latency (%)
Malate synthase Hydroxyacyl-CoA dehydrogenase Malate dehydrogenase
0.48 0.23 0.08
26.60 1.52 0.28
98.2 84.9 71.4
a The activitieswere measuredin the presence of 1.9 M sucrose. The glyoxysomeswere broken by including 0.2% Triton X-100 in the sucrose-containingreaction mixture. From R. P. Donaldson,R. E. Tully,O. A. Young, and H. Beevers, Plant Physiol. 67, 21 (1981).
ever difficulties in reproducibility have been encountered with some tissues (Anderson and Butt, personal communication). The methods described here yield glyoxysomes from castor bean endosperm relatively free of contaminating organelles and membranes (ghosts) relatively free of matrix (Table II). We recommend that centrifugation of sucrose gradients in a vertical rotor be used as a rapid procedure for isolating peroxisomes and glyoxysomes from other tissues. Furthermore, we suggest using carbonate washing 1~as a method to obtain glyoxysomal/peroxisomal ghosts free of matrix protein. Acknowledgments We thank N. Charley (SEM), E. Erbe (freeze fracture), L. C. Frazier (TEM and sample preparations), G. Kaminski (TEM and sample preparations), and C. Pooley (photography). This work was funded by NSF PCM 8216051.
[48] P e r o x i s o m e s a n d F a t t y A c i d D e g r a d a t i o n
By BERNT GERHARDT Peroxisomes have been isolated from a wide range of plant tissues and are considered to be present in virtually all cells of higher plants. Plant peroxisomes have been subcategorized according to their formerly known metabolic function as glyoxysomes, leaf peroxisomes, and unspecialized METHODS IN ENZYMOLOGY,VOL. 148
Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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peroxisomes, i.e., peroxisomes of unknown function. Fatty acid/3-0xidation is now known to be a general, basic metabolic function of the higher plant peroxisome) Peroxisomal/3-0xidation was first discovered in glyoxysomes,2 which are organelles found only in fat-storing tissues of germinating seeds. Since glyoxysomes have already been treated in this series,3.3a the subject of this chapter will be procedures for studying fatty acid degradation by nonglyoxysomal peroxisomes. The peroxisome population of nonfatty tissues comprises approximately 1% of the organelle protein of those tissues: With the exception of the isolation on Percoll gradients, all methods described in this chapter have been successfully applied to peroxisomes from mung bean hypocotyls. Peroxisomes from spinach and pea leaves, maize seedling roots, and potato tubers have also been studied using these methods. 1,5 Isolation of Peroxisomes The isolation of glyoxysomes from castor bean endosperm TM and of leaf peroxisomes 6,7 has been described in this series. Therefore, isolation procedures outlined here will be restricted to the isolation of peroxisomes from mung bean hypocotyls on sucrose gradients and to the isolation of leaf and root peroxisomes on Percoll gradients. All procedures are carried out at 0-4 ° . Small pieces of plant material are gently ground with ice-cold homogenization medium in a chilled mortar. Only mortars with a glazed surface are suitable. The relative contents of sucrose (w/w) or Percoll (v/v) in the solutions used for the construction of density gradients are adjusted refractometrically. After centrifugation the density gradients are separated into 1-ml fractions and the location of the peroxisomes (and other organelles) is determined by assaying the fractions for marker enzyme activities. The peroxisomes are collected as a whole fraction when the location of the peroxisomes in the gradient is known and a peroxisomal band is visible.
t B. Gerhardt, Physiol. Veg. 24, 397 (1986). 2 T. G. Cooper and H. Beevers, J. Biol. Chem. 244, 3514 (1969). 3 H. Beevers and R. W. Breidenbach,this series, Vol. 31, p. 565. 3aA. H. C. Huang, this series, Vol. 72, p. 783. 4 B. Gerhardt, "Microbodies/Peroxisomenpflanzlicher Zellen." Springer-Verlag, Wien and New York, 1978. 5B. Gerhardt, Planta 159, 238 (1983). 6 N. E. Tolbert, this series, Vol. 23, p. 665. 7 N. E. Tolbert, this series, Vol. 31, p. 734.
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Isolation of Peroxisomes from Mung Bean Hypocotyls Mung bean seedlings are grown in the dark for 2.5-3 days. Hypocotyls (20 g) are homogenized in 20 ml of homogenization medium containing 170 mM Tricine-NaOH, pH 7.5, 1 M sucrose, 10 mM KC1, 1 mM MgC12, 1 mM EDTA, 10 mM mercaptoethanol, and bovine serum albumin (9 mg/ ml). The homogenate is squeezed through four layers of cheesecloth and centrifuged at 600 g for 10 min. From the 600 g supernatant, the peroxisomes are concentrated by pelleting a crude organelle fraction at 12,000 g (15 min). This fraction is gently resuspended in 2 ml of homogenization medium using a glass rod wrapped with cotton wool on its tip. The resuspended crude organelle preparation is layered onto a previously prepared sucrose gradient. The gradient is constructed from precooled sucrose solutions which are prepared by dissolving the sucrose in 1 mM EDTA, pH 7.5. A continuous gradient is formed from 20 ml of 30% and 15 ml of 60% sucrose, or alternatively a discontinuous gradient is constructed using, sequentially, 3 ml of 60%, 5 ml of 57%, 7 ml of 53%, 7 ml of 46%, 6 ml of 43%, and 6 ml of 35% sucrose. The discontinuous gradient has a loading capacity higher than that of the continuous gradient. The crude organelle fraction obtained from up to 50 g of hypocotyls can be applied to the discontinuous gradient. The continuous or discontinuous gradients are centrifuged at 83,000 g (average) for 3 hr. The peroxisomes are recovered in the discontinuous gradient at the interface between the 46 and 53% sucrose layer. Peroxisomes from mung bean hypocotyls can also be isolated using a modification8 of the procedure described by Schuh and Gerhardt. 9 The crude organelle fraction is prepared as described above and layered onto a discontinuous gradient composed of, in sequence, 5 ml of 60%, 5 ml of 53%, 5 ml of 43%, 7 ml of 38%, 7 ml of 35%, and 3 ml of 30% sucrose. The gradient is centrifuged for 7.5 min at an acceleration of 2700 revolutions × min -2 using a Beckman L2 65B ultracentrifuge and a SW 27 rotor (final centrifugal force 61,000 g, average). The plastids are very effectively separated from the other organelles. The organelle band (approximately 4 ml) stretching from the interface between the 30 and 35% sucrose layer into the upper part of the 35% sucrose layer is transferred onto a second discontinuous sucrose gradient. This gradient consists of 3 ml of 60%, 7 ml of 55%, 10 ml of 48%, 7 ml of 43%, and 5 ml of 35% sucrose. It is centrifuged at 83,000 g (average) for 3 hr. The peroxisomes are recovered at the interface between the 48 and 55% sucrose layer. Peroxisomal fractions isolated by this two-gradient method are uncontaminated by plastids and show, if at all, extremely low contamination by mitochondria. The s H. Gerbling and B. Gerhardt, Planta, in press (1987). 9 B. Schuh and B. Gerhardt, Z. Pflanzenphysiol. 114, 477 (1984).
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yield of peroxisomes however, amounts to only 1-3% based on catalase activity. Isolation of Nonglyoxysomal Peroxisomes on Percoll Gradients
Before use, the Percoll stock solution (100 ml) is dialyzed against charcoal (5 g of charcoal suspended in 1250 ml of H20). l° The stock solution becomes diluted due to water uptake during dialysis. It is adjusted to 80% Percoll with H20. An isosmotic Percoll solution is then prepared by adding 10.3 g of sucrose, 179 mg of Tricine, and 37 mg of EDTA to 100 ml of the dialyzed 80% PercoU solution. The isosmotic 80% Percoll solution is adjusted to pH 7.5. When required it is diluted with a solution consisting of 0.3 M sucrose, 1 mM EDTA, and 10 mM TricineNaOH, pH 7.5. LeafPeroxisomes. Peroxisomes are isolated from leaf tissue following a procedure developed by Betsche. and Mfiller. 1° Five to 10 g of young leaves are ground with 2.5 vol of homogenization medium consisting of 0.1 M Tricine-NaOH, pH 7.5, 0.25 M mannitol, 1 mM EDTA, 15 mM mercaptoethanol, and bovine serum albumin (1 mg/ml). The homogenate is squeezed through four layers of cheesecloth and centrifuged at 2400 g for 5 min. From the 2400 g supernatant, the peroxisomes are concentrated by pelleting a crude organelle fraction at 12,000 g (15 min). This fraction is resuspended in 2 ml of homogenization medium and layered onto a preformed continuous Percoll gradient. The gradient is constructed from 16 ml of isosmotic 15% and 16 ml of isosmotic 50% Percoll solution over a cushion (5 ml) of isosmotic 80% Percoll. The gradient is placed in a Sorvall SS 34 rotor and centrifuged in a Sorvall RC-5C centrifuge using the automatic rate control to avoid the formation of disturbances within the gradient during acceleration and deceleration. The speed is set at 20,000 rpm (48,000 g), the run time at 5-8 min. The exact centrifugation time within the given limits depends upon the plant species from which the peroxisomes are isolated. Root Peroxisomes. Peroxisomes isolated from roots on sucrose or Percoll gradients are commonly heavily contaminated by mitochondria. The separation is improved 11 if the density of the mitochondria is artificially increased by intramitochondrial deposition of an insoluble tetrazolium salt produced by the activity of succinic dehydrogenase.12 Roots (15 g) from 3-day-old maize seedlings are ground in 30 ml of medium A supplemented with 4 mM cysteine. Medium A contains 0.1 M Tricine10T. Betsche and G. MOiler, personal communication (1984). n B. Gerhardt, in "Structure, Function and Metabolism of Plant Lipids" (P.-A. Siegenthaler and W. Eichenberger, eds.), p. 189. Elsevier, Amsterdam, 1984. 12 G. Davis and F. Bloom, Anal. Biochem. 51, 429 (1973).
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NaOH, pH 7.5, 0.3 M mannitol, 1 mM EDTA, and bovine serum albumin (1 mg/ml). The homogenate is squeezed through four layers of cheesecloth and centrifuged at 600 g for 15 min. From the 600 g supernatant a crude organeUe fraction is pelleted at 12,000 g (15 min). This fraction is resuspended in 2 ml of medium A and pelleted again (12,000 g, 15 min). It is then resuspended and incubated in 4 ml of medium A containing additionally 80 mM succinate and 10 mM iodonitrotetrazolium. After 30 min incubation at 30°, 4 ml of medium A is added to the incubated mixture and the organelles are pelleted at 12,000 g (15 min). They are washed once with 4 ml of medium A, finally resuspended in 2 ml of medium A, and layered onto a preformed continuous Percoll gradient. The gradient is constructed from 15 ml of isosmotic 60% Percoll solution and 15 ml of 0.3 M sucrose dissolved in a I mM EDTA solution containing also 10 mM Tricine-NaOH, pH 7.5. The gradient is centrifuged in a fixed angle rotor (Sorvall SS 34 rotor) at 40,000 g for 20 min. Contamination
The peroxisomal fractions isolated on sucrose gradients are normally contaminated by mitochondria and especially by plastids, those isolated on Percoll gradients by mitochondria. With respect to the topic of this chapter, mitochondrial contamination of the peroxisomal fractions has to be considered in particular although mitochondrial B-oxidation activity has not yet unequivocally been demonstrated for higher plant cells.~ A contribution of contaminating mitochondria to the B-oxidation enzyme activities measured in a peroxisomal fraction is checked by the following method. 5,~3The activity of a given B-oxidation enzyme as well as that of a peroxisomal marker enzyme (catalase, glycolate oxidase) is assayed in the peroxisomal and mitochondrial fraction from one and the same gradient. For both fractions, the ratio of the B-oxidation enzyme activity to the peroxisomal marker enzyme activity is then calculated. If this ratio determined for a sufficient number of gradients does not significantly differ (according to a statistical analysis) between the peroxisomal and mitochondrial fraction, the B-oxidation enzyme activity in the peroxisomal (and mitochondrial) fraction is considered to be associated only with the peroxisomes.
Assay Methods Thiol reagents such as dithioerythritol (DTE), dithiothreitol (DTT), mercaptoethanol, and cysteine react with acyl-CoA's in a nonenzymatic 13 B. Gerhardt, FEBS Lett. 126, 71 (1981).
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reaction.14 Therefore, care has to be taken if thiol reagents are included in assay mixtures for measuring/3-oxidation pathway activities. On principle, thiol reagents are not included in the reaction mixtures described here.
Enzyme Assays R - - C H E - - C H 2 - - C O O H + ATP + CoASH ~ R - - C H 2 - - C H 2 - - C O S C o A + AMP + PPi ( I ) R - - C H 2 - - C H 2 - - C O S C o A + 02 ---> R--CH-~-CH--COSCoA + H202 (2) R--CH~---CH--COSCoA + H20 ~ R - - C H O H - - C H 2 - - C O S C o A (3) R - - C H O H - - C H 2 - - C O S C o A + NAD* ~ R - - C O - - C H 2 - - C O S C o A + NADH + H ÷ (4) R - - C O - - C H E - - C O S C o A + CoASH ~ R - - C O S C o A + CH3--COSCoA (5)
All five enzymes of fatty acid activation (reaction 1) and B-oxidation (reactions 2-5) can be assayed spectrophotometrically using a singlebeam recording spectrophotometer with zero offset capability, a full-scale absorbance of 0.1 or 0.2, and a recorder speed of 0.5-2.0 cm/min. The reaction mixtures of 1 ml total volume in self-masking microcuvettes (1cm light path) contain I0-100/zl of the peroxisomal fraction (approximately 5-50 /~g of protein). The reactions are started by adding the substrate to the reaction mixtures, unless otherwise stated. For measurements of unspecific absorbance changes, reactions without substrate are followed in parallel cuvettes. Reaction rates are calculated for the test reaction after subtracting the absorbance change of the blank from that of the test. Rates of NAD + reduction and NADH oxidation, respectively, are calculated using the extinction coefficient 6220 M -1 cm -~ for NADH at 340 rim. Acyl-CoA Synthetase (Reaction 1). The enzyme is routinely assayed by a coupled assay m e t h o d ) The acyl-CoA formed by the synthetase is oxidized by added acyl-CoA oxidase (reaction 2) and the H202 formation in the oxidase reaction is determined by a coupled peroxidatic reaction.15 The reaction mixture contains 175 mM Tris-HC1, pH 8.5, 10 mM ATP, 7.5 mM MgC12, 0.6 mM CoASH, 50/zM FAD, 13 mM p-hydroxybenzoic acid, 1 mM 4-aminoantipyrine, 1 mM NAN3, 0.1 U acyl-CoA oxidase (from Candida species; Sigma), 5.3 U of horseradish peroxidase (Sigma, type II), and 0.1 mM palmitic acid. The palmitic acid is given to the reaction mixture dissolved in 10/zl acetone. The reaction is started by the addition of CoASH. The absorbance increase at 500 nm is monitored. An t4 G. B. Stokes and P. K. Stumpf, Arch. Biochem. Biophys. 162, 638 (1974). 15 C. C. Allain, L. S. G. Poon, W. Richmond, and C. Fu, Clin. Chem. (Winston-Salem, N.C.) 20, 470 (1974).
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absorbance increase of 0.51 corresponds to the consumption of 0.1/xmol H202/ml in the coupled peroxidatic reaction.16 In certain cases (e.g., determination of substrate specificity) acyl-CoA synthetase cannot be assayed by coupling reaction 1 and 2 and following the formation of H202. Acyl-CoA synthetase may then by assayed by a spectrophotometric assay which either determines the AMP formation by a coupled reaction sequence 17or couples an assay for pyrophosphate TM to reaction 1. ATP hydrolase and pyrophosphatase activity, respectively, can substantially interfere with these assays. Acyl-CoA synthetase is also assayed by a direct method which determines the rate of [~4C]palmitoyl-CoA formation from [14C]palmitic acid. 19 Substrate and product are separated by thin-layer chromatography. 8 The reaction mixture contains, in a total volume of 0.5 ml, 175 mM Tris-HC1, pH 8.5, 10 mM ATP, 7.5 mM MgC12, 0.02% Triton X-100 (to inhibit acylCoA oxidaseS), 10-30/~g protein contained in -<200/.d of the peroxisomal fraction from a sucrose gradient, and 0.175 mM [1-14C]palmitic acid (0.7 mCi/mmol). The added palmitic acid is dissolved in 10/.d acetone. The reaction is started by adding CoASH to a final concentration of 1 mM. After a reaction time of 10 min an aliquot (10-50 ~1) of the reaction mixture is immediately spotted on a thin-layer cellulose plate (20 x 20 cm; 0.1 mm coating thickness) and chromatographed in a butanol : glacial acetic acid : water system (5 : 2 : 3, by volume) at room temperature. Developing the chromatogram takes 6-7 hr. After drying the chromatogram at room temperature, the chromatographic process is repeated once. Radioactive spots on the chromatogram are detected with a thin-layer chromatogram scanner. For locating palmitoyl-CoA, an aliquot of a reaction mixture is chromatographed which contains unlabeled instead of labeled palmitic acid and is mixed with labeled palmitoyl-CoA after incubation. For locating palmitic acid, an aliquot of the test reaction mixture is spotted on the thin-layer plate prior to incubation. The Rf values are approximately 0.60 and 0.95 for palmitoyl-CoA and palmitic acid, respectively. The formed palmitoyl-CoA is quantified either by integration of the palmitoyl-CoA peak of the scanner record or by counting the palmitoyl-CoA in a liquid scintillation counter. Before transferring the palmitoyl-CoA spot from the thin-layer plate into scintillation vials the cellulose is covered with a celluloid layer, z° A celluloid solution is prepared by dissolving 66 g of cellulose acetate and 16 D. J. Hryb and J. F. Hogg, Biochem. Biophys. Res. Commun. 87, 1200 (1979). 17 T. Tanaka, K. Hosaka, and S. Numa, this series, Vol. 71, p. 334. is j. Edwards, T. ap Rees, P. M. Wilson, and S. Morrell, Planta 162, 188 (1984). 19j. Bar-Tana, G. Rose, and B. Shapiro, this series, Vol. 35, p. 117. 20 p. Leusing, Doctoral Thesis, University of MOnster (1985).
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20 g of camphor in a solvent prepared from 750 ml acetone, 250 ml npropanol, and 28 ml diethylene glycol. Five to 7 ml of the celluloid solution is spread over the cellulose layer of the thin-layer plate. After drying, the area (1 x 1 cm) of the palmitoyl-CoA spot is cut out. The obtained piece of celluloid to which the cellulose firmly adheres is dissolved in 0.5 ml dimethyl sulfoxide and then 4.5 ml scintillation fluid is added. The radioactive assay and the three coupled spectrophotometric assays for measuring acyl-CoA synthetase activity give essentially identical results if the assays are performed with palmitic acid as substrate. 8 Acyl-CoA Oxidase (Reaction 2). The enzyme is assayed by following the H202 formation in a coupled assay which determines the H202 in a peroxidatic reaction.~5 The assay mixture consists of 175 mM Tris-HCl, pH 8.5, 50/zM FAD, 13 mM p-hydroxybenzoic acid, I mM 4-aminoantipyrine, 1 mM NaN3 (to inhibit the peroxisomal catalase), 5.3 U of horseradish peroxidase (Sigma, type II), and 50 mM palmitoyl-CoA. The dye formation is monitored at 500 nm. An absorbance increase of 0.51 corresponds to the consumption of 0. I/~mol H202/ml in the coupled peroxidatic reaction.~6 Triton X-100 at concentrations ---0.01% inhibits the acyl-CoA oxidase5 and is therefore not included in the reaction mixture. When acyl-CoA oxidase activities are measured by the spectrophotometric assay and a polarographic assay which determines directly the oxygen uptake by the acyl-CoA oxidase, essentially identical results are obtained, 5 demonstrating the reliability of the coupled spectrophotometric assay. Enoyl-CoA Hydratase (Crotonase; Reaction 3). The enzyme is assayed by a coupled assay method monitoring the absorbance increase at 340 nm due to the NAD ÷ reduction in the coupled 3-hydroxyacyl-CoA dehydrogenase reaction (reaction 4). 21 The reaction mixture consists of 175 mM Tris-HC1, pH 9.5, 2 mM EDTA, 2 mM KCN, 0.3 mM NAD ÷, 3 U of 3-hydroxyacyl-CoA dehydrogenase (from procine heart; Sigma type III), 22 0.05% Triton X-100, and 0.1 mM crotonyl-CoA. 3-Hydroxyacyl-CoA Dehydrogenase (Reaction 4). The enzyme is assayed in the backward direction, i.e., in the direction of reduction of 3oxoacyl-CoA to 3-hydroxyacyl-CoA.23 The reaction is followed by the absorbance decrease at 340 nm due to NADH oxidation. The reaction mixture contains 50 mM potassium phosphate, pH 6.8, 2 mM EDTA, 0.25 2t j. R. Stern and A. del CampiUo, J. Am. Chem. Soc. 75, 2277 (1953). 22 A 3-hydroxyacyl-CoA dehydrogenase preparation has to be used which does not contain the bifunctional protein exhibiting 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities. 23 F. Lynen, L. Wessely, O. Wieland, and W. Rueff, Angew. Chem. 64, 687 (1952).
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mM NADH, 2 mM KCN, bovine serum albumin (1 mg/ml), 0.05% Triton X-100, and 0.1 M acetoacetyl-CoA. 3-OxoacyI-CoA Thiolase (Reaction 5). The enzyme is assayed by trapping the formed acetyl-CoA with citrate synthase and oxaloacetate which is generated from malate in the NAD+-dependent malate dehydrogenase reaction. 2 Activity is therefore measured by following NAD + reduction at 340 nm. The reaction mixture consists of 100 mM potassium phosphate, pH 7.5, 0.25 mM MnC12, 2 mM CoASH, 0.3 mM NAD +, 3 mM L-malate, 12 U of malate dehydrogenase, 2.2 U of citrate synthase, 2 mM KCN, 0.05% Triton X-100, and 40/~M acetoacetyl-CoA.
Assay of Peroxisomal Overall t-Oxidation In contrast to the known mammalian mitochondrial t-oxidation system, the peroxisomal t-oxidation system is insensitive to KCN. Therefore, peroxisomal t-oxidation is assayed by measuring acyl-CoA- or fatty acid-dependent, KCN-insensitive acetyl-CoA production or NADH formation. The NADH formation results from reaction 4. Determination of NADH Formation. The low yield and t-oxidation activity of nonglyoxysomal peroxisomes normally requires an amplification of the formed NADH by a cycling method. 24 Step 1: The reaction mixture (total volume, I ml) for the t-oxidation is prepared in small test tubes. It contains 175 mM Tris-HCl, pH 8.5, 0.15 mM NAD +, 0.15 mM CoASH (to allow thiolytic cleavage of the formed 3oxoacyl-CoA which otherwise may inhibit reaction 4), 25 2 mM KCN, 2080/~g protein contained in -<300/zl of the peroxisomal fraction from a sucrose gradient, and 25/.~M palmitoyl-CoA (or 0.1 mM palmitic acid, 10 mM ATP, 7.5 mM MgCI2, and a CoASH concentration raised to 0.6 mM). The reaction is run at 25° for 15 min. Step 2: The/3-oxidation reaction is terminated by adding 1 ml of 0.2 N NaOH to the reaction mixture which is then boiled for 4 min to destroy the remaining NAD +. The mixture is cooled to room temperature and centrifuged at 4500 g for l0 min. An aliquot (0.5 ml) of the supernatant is neutralized by adding an equal volume of 0.2 N HC1. During neutralization the sample is vigorously shaken to avoid destruction of NADH by local acidification. Step 3: An aliquot (0.1-0.3 ml) of the neutralized sample is used in the cycling assay for NADH. The cycling assay is performed as described in t h i s s e r i e s . 26 The cycling reaction proceeds for 10 min. It is recommended u I. Papke and B. Gerhardt, unpublished results (1984). 25 T. Osumi, T. Hashimoto, and N. Ui, J. Biochem. (Tokyo) 87, 1735 (1980). 26 H. Matsumura and S. Miyachi, this series, Vol. 69, p. 465.
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to use black test tubes and an alcohol dehydrogenase purified from bound NAD+/NADH for the cycling reaction. A blank which does not contain palmitoyl-CoA (or palmitic acid) in the /]-oxidation mixture (step 1) is also carded through steps 1 to 3. If no standard curve has been prepared, 26rates of NADH formation are calculated using an internal standard prepared as follows: 50 pmol NADH dissolved in 0.1 ml H20 is included in a cycling reaction mixture (step 3) prepared with an aliquot of the neutralized blank. This standard is carried through step 3 in parallel with the sample and the blank. The rate of NADH formation is increased when a system for trapping the formed acetyl-CoA is included in the fl-oxidation mixture (step 1). Such a system consists of 0.5 mM aspartate, 0.5 mM 2-oxoglutarate, 0.1 mM pyridoxalphosphate, 0.02 U of L-aspartate:2-oxoglutarate aminotransferase, and 0.2 U of citrate synthase. Determination of Acetyl-CoA Formation. The reaction mixture (total volume, 0.6 ml) for the B-oxidation contains 175 mM Tris-HC1, pH 8.5, 0.15 mM NAD +, 2 mM KCN, 0.1 mM [U:4C]palmitic acid (2 mCi/mmol), 10 mM ATP, 7.5 mM MgC12, 1 mM CoASH, and 20-50/~g protein contained in -<200/~l of the peroxisomal fraction from a sucrose gradient. The added palmitic acid is dissolved in 10/~l acetone. After a reaction time of 15 min at 25° an aliquot (10-50/~l) of the reaction mixture is immediately spotted on a thin-layer cellulose plate. The chromatogram is developed and acetyl-CoA and palmitic acid are detected correspondingly to the procedures described in the radioactive acyl-CoA synthetase assay. The Rr values for palmitic acid and acetyl-CoA are approximately 0.95 and 0.45, respectively. The formed acetyl-CoA is quantified correspondingly to the palmitoyl-CoA in the acyl-CoA synthetase assay.
Protein Determination For specific activity measurements, protein in fractions from sucrose gradients is determined by a modification5 of the Lowry method. 27Protein in fractions from Percoll gradients is determined by following the procedure of Vincent and Nadeau. z8 Acknowledgment Studies in the author's laboratory were supported by the Deutsche Forschungsgcmeinschaft. 27 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 2~R. Vincent and D. Nadeau, Anal. Biochem. 135, 355 (1983).