J. Biochem., 82, 221-230 (1977)
Glyoxylate Oxidation in Rat Liver and Kidney Dorothy A. GIBBS, Sunna HAUSCHILDT, and R.W.E. WATTS Division of Inherited Metabolic Diseases, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, England Received for publication, February 23, 1977
The enzyme-catalyzed oxidation of glyoxylate to oxalate by subcellular particles of rat liver and kidney, and in thecytosol fraction (100,000 x g supernatant) of rat kidney, has been investigated. Catalytic activity is associated with liver peroxisomes and rough endoplasmic reticulum, but not with liver lysosomes and mitochondria. The activity associated with the endoplasmic reticulum has the properties of lactate dehydrogenase [EC 1.1.1.27], whereas the peroxisomal activity is attributed to both glycollate oxidase [EC 1.1.3.1.] and to lactate dehydrogenase. Among the paniculate fractions of kidney, catalytic activity with respect to the oxidation of glyoxylate to oxalate is due to lactate dehydrogenase associated with the endoplasmic reticulum. The possibility that some of the catalytic activity in kidney tissue is associated with the lysosomes cannot be completely excluded on the basis of the present evidence. No evidence was obtained for peroxisomal or mitochondrial enzymes with catalytic activity with respect to the oxidation of glyoxylate to oxalate in renal tissue. The catalytic activity for the oxidation of glyoxylate to oxalate in the kidney cytosol fraction was NAD+ dependent and associated with lactate dehydrogenase. These results are discussed in relation to previous work and to the problem of excessive oxalate production in man. It would be theoretically desirable to treat the hyperoxaluric diseases by reducing the excessive rate of oxalate biosynthesis in vivo. This could be most effectively done by inhibiting the last step on the oxalate biosynthetic pathway. The present evidence shows that it would be necessary to inhibit lactate dehydrogenase throughout the body as well as glycollate oxidase in order to achieve this.
Calcium oxalate nephrocalcinosis and intrarenal obstructive uropathy occur in the primary hyperoxalurias, ethylene glycol poisoning and methoxyfluorane nephropathy, and are due to the excretion of an increased load of metabolically formed oxalate (/). Glyoxylate is the main immediate metabolic precursor of oxalate in mammals so that specific inhibition of this oxidation in vivo might offer a way of reducing oxalate production in man. Such an approach would be especially valuable in the Vol. 82, No. 1, 1977
primary hyperoxalurias where excessive oxalate production is due to an inherited enzyme deficiency (/), and it could be most satisfactorily explored if the enzymes which catalyze the reaction in different organs were known. Only the liver and heart cytosol (100,000 xg supernatant) fractions have been previously studied in detail from this point of view by modern methods (2), lactate dehydrogenase [EC 1.1.1.27] being the enzyme principally involved with only minor contributions from xanthine oxidase [EC 1.2.3.2] and glycollate oxidase [EC 1.1.3.1]. 221
222
D.A. GIBBS, S. HAUSCHILDT, and R.W.E. WATTS
This report describes a qualitative investigation of the subcellular location and identity of the enzymes which catalyze the oxidation of glyoxylate to oxalate in rat liver and kidney. MATERIALS AND METHODS Reagents—AnalaR grade reagents and glass distilled water were used throughout unless otherwise stated. Sodium glyoxylate was purchased from Fluka & Co., Zurich, Switzerland. Sodium [l-14C]glyoxylate (3.2 mCi/mmol) was purchased from the Radiochemical Centre, Ajnersham, Bucks. Oxidized and reduced nicotinamide adenine dinucleotide (NAD+ and NADH), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and beef liver catalase [EC 1.11.1.6] were purchased from the Boehringer Corporation (London) Ltd., London W.5. Dextran-TIO was purchased from Pharmacia (G.B) Ltd., London, W.5. Sucrose was Mineral Water Sugar purchased from Tate and Lyle Ltd. Triton-WR1339 was a gift from Winthrop Laboratories, Newcastle-uponTyne. Nicotinamide adenine dinucleotide phosphate (NADP+) and rotenone were purchased from Sigma Chemical Company. Animals—Male Sprague-Dawley rats weighing 200-300 g were used. In some experiments, they were injected intraperitoneally with Triton-WR1339 (85 mg/100 g body weight, dissolved in water) 3 1/2 days before killing by cervical dislocation. Other animals were injected with Dextran-TIO [100 mg/ lOOg body weight, dissolved in NaCl (0.154 M)], 1, 3, 5, and 8 days before they were killed. The livers or kidneys were removed and chilled immediately. Biochemical Analyses—The cytochrome oxidase [EC 1.9.3.1] activity after treatment with an equal volume of digitonin solution (2% w/v) was determined by measuring the rate of oxidation of reduced cytochrome ' c '. The substrate was prepared by the method of Smith (5). The concentration of cytochrome' c ' used was 34 [IM in potassium phosphate buffer (pH 7.4, 30 ITIM) ; the reduction was followed at 550 nm in a Unicam SP 500 spectrophotometer, and the molar extinction coefficient E6i0 was taken as 27,700 liter mol"'-cm"1. Catalase was determined by the method of Luck (4). Urate oxidase [EC 1.7.3.3] was measured by following the decrease in extinction at 292 nm after
addition of sodium urate (3 ml, 42 //M) in potassium phosphate buffer (pH 7.4, 30 min) containing EDTA (1 mM) and Triton X-100 (0.1 %, v/v) to 0.1 ml of the enzyme. The reaction was carried out at 37°C in a Unicam SP500 spectrophotometer. The molar extinction co-efficient Eln was taken as 12,200 litre mol^-cirr 1 . Acid phosphatase [EC 3.1.3.2] and glucose-6-phosphatase [EC 3.1.3.9] were measured by the methods of de Duve et al. (5), and the glucose-6-phosphatase results were corrected for interference by acid phosphatase. Glycollate oxidase [EC 1.1.3.1] was measured by the method of Lord & Merrett (6). Lactate dehydrogenase [EC 1.1.1.27] and xanthine oxidase [EC 1.2.3.2] were measured as described previously (2) and arylsulphatase [EC 3.1.6.1] as described by Dodgson et al. (7). The ability of the fractions to catalyze the oxidation of glyoxylate to oxalate was measured radiochemically in disposable flat bottomed specimen tubes (1.5 cm x 9 cm) using Na[l-MC]glyoxylate as substrate (2). The reaction was started by adding the tissue fraction to be assayed, and stopped with trichloroacetic acid. The Ca-["C] oxalate formed was isolated, purified to constant specific radioactivity (2), and the rate of calcium oxalate formation calculated. Protein was determined by the method of Lowry et al. (8) and the sucrose concentrations were measured using an AbW refractometer. The assay of lactate dehydrogenase in kidney subcellular particles using lactate substrate was complicated by the presence of an enzyme or enzymes which catalyzed the oxidation of NADH. These were probably the mitochondrial NADH dehydrogenases [EC 1.6.99.3, and EC 1.6.99.5] of the electron transport chain. In thess experiments the lactate dehydrogenase activity was measured with pyruvate as substrate and a correction was made for NADH oxidized in the absence of pyruvate. The results obtained in this way agreed with those obtained in the presence of rotenone (4x 10"7 M) to inhibit the mitochondrial electron transport chain. Subcellular Fractionation of Liver and Kidney Tissue for the Study of Lysosomes, Peroxisomes, and Rough Endoplasmic Reticulum—The technique which was found to be most useful for the initial separation of these three cell organelles is shown in Fig. 1. It was based on the methods of Vandor & Tolbert (9) and of Schneider (10). Samples C, /. Biochem.
223
GLYOXYLATE OXIDATION IN TISSUES Liver from Triton-WR1339 treated rat Mince, weigh, homogenize in 0.25 M sucrose+ 1% ethanol (Sample A) Centrifuge 700 x e 10 min Sediment (Sample B) Wash. Recentrifuge
I
Sediment Discard
Supernatant
I Combined supematants Centrifuge 17,000 xg 20 min Pellet resuspended in two tubes, centrifuge
I Pellet 1 Resuspend in medium Sample C (M.L.P.ER)
1
Pellet 2
1
I
Pellet resuspend in medium Sample D (M.L.P.)
I
"Fluffy" layer
Combined supematants I Centrifuge 100,000xg 30 min Pellet. Wash Recentrifuge Resuspend in medium Sample E (ER) Fig. 1. The preparation of lysosome, peroxisome, and rough endoplasmic reticulum rich fractions from rat liver and kidney. Samples C, D, and E were obtained by resuspending the corresponding pellet in sucrose (0.25 M). These samples were further fractionated by centrifugation through a discontinuous sucrose gradient in the case of liver, and a linear sucrose gradient in the case of kidney, as described in the text. M=mitochondria, L=lysosomes, P=peroxisomes, ER=endoplasmic reticulum.
D, and E (Fig. 1) from liver were further fractionated by isopycnic density gradient centrifugation using a discontinuous tube gradient of seven layers from 3 3 % to 55% (w/w) sucrose solution containing 5% Dextran T10. Samples C, D, and E from kidney were fractionated by rate sedimentation on a linear sucrose gradient [8.5% (w/v) to 4 0 % (w/v) on a layer of 82% (w/v) sucrose solution, total volume=45 ml]. The discontinuous sucrose gradients were centrifuged at 25,000 rpm in the SW
Vol. 82, No. 1, 1977
25.2 rotor of the Beckman L2.65B centrifuge (gAv=75,5OO) for 5 h. The linear sucrose gradients were centrifuged at 2,300 rpm (g A v =l,350) for 2 h at 4°C. Fractions (2 ml) were collected and assayed for: (a) acid phosphatase or arylsulphatase as a marker for lysosomes; (b) cytochrome oxidase as a marker for mitochondria; (c) uricase or catalase as a marker for peroxisomes; (d) glucose6-phosphatase as a marker for rough endoplasmic reticulum; (e) xanthine oxidase; (f) glycollate oxi-
224
D.A. GIBBS, S. HAUSCHILDT, and R.W.E. WATTS
dase; (g) lactate dehydrogenase; (h) catalytic activity with respect to the oxidation of glyoxylate to oxalate with and without added NAD + ; (i) protein concentration; and (j) sucrose concentration. The liver fractions which contained the highest concentrations of the individual marker enzymes and least contamination were also examined by electron microscopy. The proteins from selected fractions were extracted by treatment with sodium pyrophosphate buffer (pH 9.0; 0.1 M), concentrated to their original volume using an Amicon cell fitted with a UM10 filter, and examined by electrophoresis in polyacrylamide gel (2). In other experiments, the sodi-
um pyrophosphate buffer extracts were dialyzcd against two changes of sodium orthophosphate buffer (pH 7.4, 0.01 M) and concentrated to their original volumes as described above. The extract was loaded onto a column of DEAE (A50)Sephadex (2.5x40 cm) and eluted with an approximately linear gradient of sodium orthophosphate (pH 7.4; 0.01-0.2 M). The fractions were collected and assayed for their ability to catalyze the oxidation of glyoxylate to oxalate, lactate dehydrogenase and glycollate oxidase. The Isolation of Liver Mitochondria by Zonal Centrifugation—The method is summarised in Fig. 2. The liver was cooled, homogenized in ice cold
Liver (1 g) cooled, minced, homogenized in ice cold medium (9 ml) in a Potter Elvejhem homogenizer with loose fitting pestle. Filtered through two layers of nylon bolting cloth Centrifuged at 700 Xff for 10 min
i Sediment Washed with 2.5 ml Homogenizing medium
Supernatant
Centrifuged at 700 Xff for 10 min Supernatant! combined and centrifuged at 5,000 xg for 10 min
Sediment Discarded
I i
1
Supernatants combined
Pellet Resuspended in 8 ml homogenizing medium. Centrifuged at 5,000xff for 10 min. Decanted supernatant and removed "fluffy" layer.
I
Pellet
I Supernatant + "fluffy" layer
Resuspended and centrifuged as above
J Pellet Supernatant Resuspend in 20 ml of homogenizing medium and loaded into AXII (See text for further details). Fig. 2. The preparation of mitochondria from rat liver. J. Biochem.
225
GLYOXYLATE OXIDATION IN TISSUES homogenizing medium [sucrose (0.25 M) containing dipotassium hydrogen phosphate 0.01 M] (II) and subjected to differential centrifugation (70). The mitochondria containing fraction was resuspended in homogenizing medium (20 ml) and loaded into an AXIT zonal rotor (Measuring & Scientific Equipment Ltd.) containing a sucrose gradient with a step at density 1.16. The rotor was accelerated to 4,000 rpm in a refrigerated centrifuge (Measuring & Scientific Equipment Ltd. Model 6L) and run for 1 h. It was then decelerated to 800 rpm and unloaded by pumping in sucrose (55% w/w). Fractions (15 ml) were collected and assayed for cytochrome oxidase and catalase in order to establish if there was any contamination by peroxisomes. The fraction which contained most cytochrome oxidase and least catalase was assayed for glyoxylate-to-oxalate oxidizing activity, it was also examined by electron microscopy and the mitochondria were shown to be intact. The Kidney Cytosol Fraction—Kidney tissue was minced and homogenized in sodium orthophosphate buffer (1 g tissue to 2 ml, 0.01 M pH 7.4). The homogenate was centrifuged for 1 h at 100,000 xg, in a superspeed 50 centrifuge with 8x25 ml angle rotor catalogue number 59594 (Measuring & Scientific Equipment Ltd.). The supernatant fraction from this centrifugation was applied to a column (2.5x100 cm) of DEAE-Sephadex (A-50) in the phosphate form. The proteins were eluted with an approximately linear sodium orthophosphate buffer gradient (0.01-0.2 M; pH 7.4) at a rate of lOml/h, the eluate being collected in 130-drop (8 ml) fractions using an LKB ultrorac fraction collector. Selected fractions (those containing lactate dehydrogenase) were examined by electrophoresis in polyacrylamide gels.
300,
2 4 6 8 10
14
16
18 22 26 30 20 24 28 '
Fig. 3. The subfractionation of sample C from the initial liver fractionation shown in Fig. 1, by centrifugation through a discontinuous sucrose gradient. This sample contained mitochondria, lysosomes, peroxisomes, and rough endoplasmic reticulum.
specimen C, and specimen E contained mostly rough endoplasmic reticulum. The distribution of NAD+ dependent glyoxylate-to-oxalate oxidizing ability of the fractions from specimen C (Fig. 3) follows the peaks of glucose-6-phosphatase, lactate RESULTS dehydrogenase and of uricase activity. There is Subcellular Fractionation of Liver Tissue for the no peak of glyoxylate-to-oxalate oxidizing activity Study of Lysosomes, Peroxisomes, Mitochondria, associated with the lysosomes and mitochondria and Rough Endoplasmic Reticulum—Figures 3, 4, for which acid phosphatase and cytochrome oxidase and 5 show the results of the assays of the fractions are the respective marker enzymes. Figure 4 + obtained by sucrose density gradient centrifugation shows a reduction in the NAD dependent glyoxyof specimens C, D, and E, respectively, from the late-to-oxalate oxidizing activity, which corresponds initial fractionation shown in Fig. 1. Specimen C to the reduction in glucose-6-phosphatase activity contained all three types of particles, specimen D of the fractions, while the glyoxylate-to-oxalate contained less rough endoplasmic reticulum than oxidizing activity of the peroxisome-rich fractions. Vol. 82, No. 1, 1977
226
D.A. GIBBS, S. HAUSCHILDT, and R.W.E. WATTS
| is relatively unchanged. The alteration in lactate dehydrogenase activity of the fractions also mirrors the changes in NAD+ dependent glyoxylate-tooxalate oxidizing activity. The catalytic activity of mitochondria with respect to the oxidation of glyoxylate to oxalate was further investigated in separate experiments with highly purified mitochondria, prepared by the fractionation procedure shown in Fig. 2 with a final zonal centrifugation step, and free from lysosomal contamination. Their purity was confirmed by electron microscopy. These mitochondria completely lacked glyoxylate-to-oxalate oxidizing activity and none was stimulated by adding NAD+, i0.2
NADP\ FMN, and FAD or by the use of digitonin to reveal any possible latent enzyme activity. Figure 5 shows the parallelism between glucose-6-phosphatase, lactate dehydrogenase and NAD+ dependent catalytic activity for the oxidation of glyoxylate to oxalate, when the peroxisomes and almost all of the mitochondria and lysosomes have been removed. Omitting NAD+ from the system abolished the glyoxylate-to-oxalate oxidizing ability of all the gradient fractions except for those containing peroxisomes where small amounts of activity were still detected. • The proteins extracted from the fractions which contained most rough endoplasmic reticulum and most peroxisomes contained lactate dehydrogenase isoenzyme 5, and a non-NAD+ dependent 010 ^
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Fig. 4. The subfractionation of sample D from the initial liver fractionation shown in Fig. 1, by centrifugation through a discontinuous' sucrose gradient. This sample contained mainly mitochondria, lysosomes, and peroxisomes, • with less endoplasmic reticulum than | sample C.
1 23456789 11 U 15 17 19 21 23 25 27 29 10 12 14 16 18 20 22 24 26 28 30
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Fig. 5. The subfractionation of sample E from the initial liver fractionation shown in Fig. 1, by centrifugation through a discontinuous sucrose gradient. This sample contained mainly endoplasmic reticulum, and no peroxisomes.
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GLYOXYLATE OXIDATION IN TISSUES enzyme with the same electrophoretic mobility as the non-NAD + dependent glycollate oxidizing enzyme previously reported in liver cytosol (2). Chromatography of the extracted proteins on DEAE-Sephadex showed that the N A D + dependent oxalate formation was only associated with lactate dehydrogenase, and the non-NAD + dependent activity was associated with glycollate oxidase. Subcellular Fractionation of Kidney Tissue for the Study of Lysosomes, Peroxisomes, Mitochondria, and Endoplasmic Reticulum—Figures 6, 7, and 8 show the distribution of N A D + dependent catalytic activity with respect to the oxidation of glyoxylate to oxalate compared with that of the marker enzymes in the fractions obtained by the sucrose density gradient centrifugation of specimens C, D, and E respectively from the initial fractionation 5O
227
shown in Fig. 1. No non-NAD + dependent activity was detected. The profile of the distribution of catalytic activity with respect to the oxidation of glyoxylate to oxalate most closely parallels that of the endoplasmic marker enzyme, glucose-6-phosphatase (Fig. 8). However, the presence of a lysosomal enzyme capable of catalyzing this oxidation cannot be excluded on the basis of the present evidence, because we were unable to change the density of the kidney lysosomes by pre-treating the animals with Triton-WR1339 or Dextran T10. The distribution of catalase and cytochrome oxidase activities differs from that of the N A D + dependent catalytic activity for the oxidation of glyoxylate to oxalate (Figs. 6 & 7), showing that the latter activity is not associated with kidney mitochondria and peroxisomes. •
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Fig. 6. The subfractionation of sample C from the initial kidney fractionation shown in Fig. 1 by centrifugation through a continuous sucrose gradient. This sample contained mitochondria, lysosomes, peroxisomes, and rough endoplasmic reticulum. Gel electrophoresis confirmed the presence of lactate dehydrogenase in tubes: 2, 3, 4, 15, 16, 17, 23, 24, 25, and 26. Vol. 82, No. 1, 1977
Glucos*-6-phosphat*s«
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D.A. GIBBS, S. HAUSCHILDT, and R.W.E. WATTS
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Glyoxylate Oxidation in Rat Liver and Kidney Dorothy A. GIBBS, Sunna HAUSCHILDT, and R.W.E. WATTS Division of Inherited Metabolic Diseases, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, England Received for publication, February 23, 1977
The enzyme-catalyzed oxidation of glyoxylate to oxalate by subcellular particles of rat liver and kidney, and in thecytosol fraction (100,000 x g supernatant) of rat kidney, has been investigated. Catalytic activity is associated with liver peroxisomes and rough endoplasmic reticulum, but not with liver lysosomes and mitochondria. The activity associated with the endoplasmic reticulum has the properties of lactate dehydrogenase [EC 1.1.1.27], whereas the peroxisomal activity is attributed to both glycollate oxidase [EC 1.1.3.1.] and to lactate dehydrogenase. Among the paniculate fractions of kidney, catalytic activity with respect to the oxidation of glyoxylate to oxalate is due to lactate dehydrogenase associated with the endoplasmic reticulum. The possibility that some of the catalytic activity in kidney tissue is associated with the lysosomes cannot be completely excluded on the basis of the present evidence. No evidence was obtained for peroxisomal or mitochondrial enzymes with catalytic activity with respect to the oxidation of glyoxylate to oxalate in renal tissue. The catalytic activity for the oxidation of glyoxylate to oxalate in the kidney cytosol fraction was NAD+ dependent and associated with lactate dehydrogenase. These results are discussed in relation to previous work and to the problem of excessive oxalate production in man. It would be theoretically desirable to treat the hyperoxaluric diseases by reducing the excessive rate of oxalate biosynthesis in vivo. This could be most effectively done by inhibiting the last step on the oxalate biosynthetic pathway. The present evidence shows that it would be necessary to inhibit lactate dehydrogenase throughout the body as well as glycollate oxidase in order to achieve this.
Calcium oxalate nephrocalcinosis and intrarenal obstructive uropathy occur in the primary hyperoxalurias, ethylene glycol poisoning and methoxyfluorane nephropathy, and are due to the excretion of an increased load of metabolically formed oxalate (/). Glyoxylate is the main immediate metabolic precursor of oxalate in mammals so that specific inhibition of this oxidation in vivo might offer a way of reducing oxalate production in man. Such an approach would be especially valuable in the Vol. 82, No. 1, 1977
primary hyperoxalurias where excessive oxalate production is due to an inherited enzyme deficiency (/), and it could be most satisfactorily explored if the enzymes which catalyze the reaction in different organs were known. Only the liver and heart cytosol (100,000 xg supernatant) fractions have been previously studied in detail from this point of view by modern methods (2), lactate dehydrogenase [EC 1.1.1.27] being the enzyme principally involved with only minor contributions from xanthine oxidase [EC 1.2.3.2] and glycollate oxidase [EC 1.1.3.1]. 221
222
D.A. GIBBS, S. HAUSCHILDT, and R.W.E. WATTS
This report describes a qualitative investigation of the subcellular location and identity of the enzymes which catalyze the oxidation of glyoxylate to oxalate in rat liver and kidney. MATERIALS AND METHODS Reagents—AnalaR grade reagents and glass distilled water were used throughout unless otherwise stated. Sodium glyoxylate was purchased from Fluka & Co., Zurich, Switzerland. Sodium [l-14C]glyoxylate (3.2 mCi/mmol) was purchased from the Radiochemical Centre, Ajnersham, Bucks. Oxidized and reduced nicotinamide adenine dinucleotide (NAD+ and NADH), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and beef liver catalase [EC 1.11.1.6] were purchased from the Boehringer Corporation (London) Ltd., London W.5. Dextran-TIO was purchased from Pharmacia (G.B) Ltd., London, W.5. Sucrose was Mineral Water Sugar purchased from Tate and Lyle Ltd. Triton-WR1339 was a gift from Winthrop Laboratories, Newcastle-uponTyne. Nicotinamide adenine dinucleotide phosphate (NADP+) and rotenone were purchased from Sigma Chemical Company. Animals—Male Sprague-Dawley rats weighing 200-300 g were used. In some experiments, they were injected intraperitoneally with Triton-WR1339 (85 mg/100 g body weight, dissolved in water) 3 1/2 days before killing by cervical dislocation. Other animals were injected with Dextran-TIO [100 mg/ lOOg body weight, dissolved in NaCl (0.154 M)], 1, 3, 5, and 8 days before they were killed. The livers or kidneys were removed and chilled immediately. Biochemical Analyses—The cytochrome oxidase [EC 1.9.3.1] activity after treatment with an equal volume of digitonin solution (2% w/v) was determined by measuring the rate of oxidation of reduced cytochrome ' c '. The substrate was prepared by the method of Smith (5). The concentration of cytochrome' c ' used was 34 [IM in potassium phosphate buffer (pH 7.4, 30 ITIM) ; the reduction was followed at 550 nm in a Unicam SP 500 spectrophotometer, and the molar extinction coefficient E6i0 was taken as 27,700 liter mol"'-cm"1. Catalase was determined by the method of Luck (4). Urate oxidase [EC 1.7.3.3] was measured by following the decrease in extinction at 292 nm after
addition of sodium urate (3 ml, 42 //M) in potassium phosphate buffer (pH 7.4, 30 min) containing EDTA (1 mM) and Triton X-100 (0.1 %, v/v) to 0.1 ml of the enzyme. The reaction was carried out at 37°C in a Unicam SP500 spectrophotometer. The molar extinction co-efficient Eln was taken as 12,200 litre mol^-cirr 1 . Acid phosphatase [EC 3.1.3.2] and glucose-6-phosphatase [EC 3.1.3.9] were measured by the methods of de Duve et al. (5), and the glucose-6-phosphatase results were corrected for interference by acid phosphatase. Glycollate oxidase [EC 1.1.3.1] was measured by the method of Lord & Merrett (6). Lactate dehydrogenase [EC 1.1.1.27] and xanthine oxidase [EC 1.2.3.2] were measured as described previously (2) and arylsulphatase [EC 3.1.6.1] as described by Dodgson et al. (7). The ability of the fractions to catalyze the oxidation of glyoxylate to oxalate was measured radiochemically in disposable flat bottomed specimen tubes (1.5 cm x 9 cm) using Na[l-MC]glyoxylate as substrate (2). The reaction was started by adding the tissue fraction to be assayed, and stopped with trichloroacetic acid. The Ca-["C] oxalate formed was isolated, purified to constant specific radioactivity (2), and the rate of calcium oxalate formation calculated. Protein was determined by the method of Lowry et al. (8) and the sucrose concentrations were measured using an AbW refractometer. The assay of lactate dehydrogenase in kidney subcellular particles using lactate substrate was complicated by the presence of an enzyme or enzymes which catalyzed the oxidation of NADH. These were probably the mitochondrial NADH dehydrogenases [EC 1.6.99.3, and EC 1.6.99.5] of the electron transport chain. In thess experiments the lactate dehydrogenase activity was measured with pyruvate as substrate and a correction was made for NADH oxidized in the absence of pyruvate. The results obtained in this way agreed with those obtained in the presence of rotenone (4x 10"7 M) to inhibit the mitochondrial electron transport chain. Subcellular Fractionation of Liver and Kidney Tissue for the Study of Lysosomes, Peroxisomes, and Rough Endoplasmic Reticulum—The technique which was found to be most useful for the initial separation of these three cell organelles is shown in Fig. 1. It was based on the methods of Vandor & Tolbert (9) and of Schneider (10). Samples C, /. Biochem.
223
GLYOXYLATE OXIDATION IN TISSUES Liver from Triton-WR1339 treated rat Mince, weigh, homogenize in 0.25 M sucrose+ 1% ethanol (Sample A) Centrifuge 700 x e 10 min Sediment (Sample B) Wash. Recentrifuge
I
Sediment Discard
Supernatant
I Combined supematants Centrifuge 17,000 xg 20 min Pellet resuspended in two tubes, centrifuge
I Pellet 1 Resuspend in medium Sample C (M.L.P.ER)
1
Pellet 2
1
I
Pellet resuspend in medium Sample D (M.L.P.)
I
"Fluffy" layer
Combined supematants I Centrifuge 100,000xg 30 min Pellet. Wash Recentrifuge Resuspend in medium Sample E (ER) Fig. 1. The preparation of lysosome, peroxisome, and rough endoplasmic reticulum rich fractions from rat liver and kidney. Samples C, D, and E were obtained by resuspending the corresponding pellet in sucrose (0.25 M). These samples were further fractionated by centrifugation through a discontinuous sucrose gradient in the case of liver, and a linear sucrose gradient in the case of kidney, as described in the text. M=mitochondria, L=lysosomes, P=peroxisomes, ER=endoplasmic reticulum.
D, and E (Fig. 1) from liver were further fractionated by isopycnic density gradient centrifugation using a discontinuous tube gradient of seven layers from 3 3 % to 55% (w/w) sucrose solution containing 5% Dextran T10. Samples C, D, and E from kidney were fractionated by rate sedimentation on a linear sucrose gradient [8.5% (w/v) to 4 0 % (w/v) on a layer of 82% (w/v) sucrose solution, total volume=45 ml]. The discontinuous sucrose gradients were centrifuged at 25,000 rpm in the SW
Vol. 82, No. 1, 1977
25.2 rotor of the Beckman L2.65B centrifuge (gAv=75,5OO) for 5 h. The linear sucrose gradients were centrifuged at 2,300 rpm (g A v =l,350) for 2 h at 4°C. Fractions (2 ml) were collected and assayed for: (a) acid phosphatase or arylsulphatase as a marker for lysosomes; (b) cytochrome oxidase as a marker for mitochondria; (c) uricase or catalase as a marker for peroxisomes; (d) glucose6-phosphatase as a marker for rough endoplasmic reticulum; (e) xanthine oxidase; (f) glycollate oxi-
224
D.A. GIBBS, S. HAUSCHILDT, and R.W.E. WATTS
dase; (g) lactate dehydrogenase; (h) catalytic activity with respect to the oxidation of glyoxylate to oxalate with and without added NAD + ; (i) protein concentration; and (j) sucrose concentration. The liver fractions which contained the highest concentrations of the individual marker enzymes and least contamination were also examined by electron microscopy. The proteins from selected fractions were extracted by treatment with sodium pyrophosphate buffer (pH 9.0; 0.1 M), concentrated to their original volume using an Amicon cell fitted with a UM10 filter, and examined by electrophoresis in polyacrylamide gel (2). In other experiments, the sodi-
um pyrophosphate buffer extracts were dialyzcd against two changes of sodium orthophosphate buffer (pH 7.4, 0.01 M) and concentrated to their original volumes as described above. The extract was loaded onto a column of DEAE (A50)Sephadex (2.5x40 cm) and eluted with an approximately linear gradient of sodium orthophosphate (pH 7.4; 0.01-0.2 M). The fractions were collected and assayed for their ability to catalyze the oxidation of glyoxylate to oxalate, lactate dehydrogenase and glycollate oxidase. The Isolation of Liver Mitochondria by Zonal Centrifugation—The method is summarised in Fig. 2. The liver was cooled, homogenized in ice cold
Liver (1 g) cooled, minced, homogenized in ice cold medium (9 ml) in a Potter Elvejhem homogenizer with loose fitting pestle. Filtered through two layers of nylon bolting cloth Centrifuged at 700 Xff for 10 min
i Sediment Washed with 2.5 ml Homogenizing medium
Supernatant
Centrifuged at 700 Xff for 10 min Supernatant! combined and centrifuged at 5,000 xg for 10 min
Sediment Discarded
I i
1
Supernatants combined
Pellet Resuspended in 8 ml homogenizing medium. Centrifuged at 5,000xff for 10 min. Decanted supernatant and removed "fluffy" layer.
I
Pellet
I Supernatant + "fluffy" layer
Resuspended and centrifuged as above
J Pellet Supernatant Resuspend in 20 ml of homogenizing medium and loaded into AXII (See text for further details). Fig. 2. The preparation of mitochondria from rat liver. J. Biochem.
225
GLYOXYLATE OXIDATION IN TISSUES homogenizing medium [sucrose (0.25 M) containing dipotassium hydrogen phosphate 0.01 M] (II) and subjected to differential centrifugation (70). The mitochondria containing fraction was resuspended in homogenizing medium (20 ml) and loaded into an AXIT zonal rotor (Measuring & Scientific Equipment Ltd.) containing a sucrose gradient with a step at density 1.16. The rotor was accelerated to 4,000 rpm in a refrigerated centrifuge (Measuring & Scientific Equipment Ltd. Model 6L) and run for 1 h. It was then decelerated to 800 rpm and unloaded by pumping in sucrose (55% w/w). Fractions (15 ml) were collected and assayed for cytochrome oxidase and catalase in order to establish if there was any contamination by peroxisomes. The fraction which contained most cytochrome oxidase and least catalase was assayed for glyoxylate-to-oxalate oxidizing activity, it was also examined by electron microscopy and the mitochondria were shown to be intact. The Kidney Cytosol Fraction—Kidney tissue was minced and homogenized in sodium orthophosphate buffer (1 g tissue to 2 ml, 0.01 M pH 7.4). The homogenate was centrifuged for 1 h at 100,000 xg, in a superspeed 50 centrifuge with 8x25 ml angle rotor catalogue number 59594 (Measuring & Scientific Equipment Ltd.). The supernatant fraction from this centrifugation was applied to a column (2.5x100 cm) of DEAE-Sephadex (A-50) in the phosphate form. The proteins were eluted with an approximately linear sodium orthophosphate buffer gradient (0.01-0.2 M; pH 7.4) at a rate of lOml/h, the eluate being collected in 130-drop (8 ml) fractions using an LKB ultrorac fraction collector. Selected fractions (those containing lactate dehydrogenase) were examined by electrophoresis in polyacrylamide gels.
300,
2 4 6 8 10
14
16
18 22 26 30 20 24 28 '
Fig. 3. The subfractionation of sample C from the initial liver fractionation shown in Fig. 1, by centrifugation through a discontinuous sucrose gradient. This sample contained mitochondria, lysosomes, peroxisomes, and rough endoplasmic reticulum.
specimen C, and specimen E contained mostly rough endoplasmic reticulum. The distribution of NAD+ dependent glyoxylate-to-oxalate oxidizing ability of the fractions from specimen C (Fig. 3) follows the peaks of glucose-6-phosphatase, lactate RESULTS dehydrogenase and of uricase activity. There is Subcellular Fractionation of Liver Tissue for the no peak of glyoxylate-to-oxalate oxidizing activity Study of Lysosomes, Peroxisomes, Mitochondria, associated with the lysosomes and mitochondria and Rough Endoplasmic Reticulum—Figures 3, 4, for which acid phosphatase and cytochrome oxidase and 5 show the results of the assays of the fractions are the respective marker enzymes. Figure 4 + obtained by sucrose density gradient centrifugation shows a reduction in the NAD dependent glyoxyof specimens C, D, and E, respectively, from the late-to-oxalate oxidizing activity, which corresponds initial fractionation shown in Fig. 1. Specimen C to the reduction in glucose-6-phosphatase activity contained all three types of particles, specimen D of the fractions, while the glyoxylate-to-oxalate contained less rough endoplasmic reticulum than oxidizing activity of the peroxisome-rich fractions. Vol. 82, No. 1, 1977
226
D.A. GIBBS, S. HAUSCHILDT, and R.W.E. WATTS
| is relatively unchanged. The alteration in lactate dehydrogenase activity of the fractions also mirrors the changes in NAD+ dependent glyoxylate-tooxalate oxidizing activity. The catalytic activity of mitochondria with respect to the oxidation of glyoxylate to oxalate was further investigated in separate experiments with highly purified mitochondria, prepared by the fractionation procedure shown in Fig. 2 with a final zonal centrifugation step, and free from lysosomal contamination. Their purity was confirmed by electron microscopy. These mitochondria completely lacked glyoxylate-to-oxalate oxidizing activity and none was stimulated by adding NAD+, i0.2
NADP\ FMN, and FAD or by the use of digitonin to reveal any possible latent enzyme activity. Figure 5 shows the parallelism between glucose-6-phosphatase, lactate dehydrogenase and NAD+ dependent catalytic activity for the oxidation of glyoxylate to oxalate, when the peroxisomes and almost all of the mitochondria and lysosomes have been removed. Omitting NAD+ from the system abolished the glyoxylate-to-oxalate oxidizing ability of all the gradient fractions except for those containing peroxisomes where small amounts of activity were still detected. • The proteins extracted from the fractions which contained most rough endoplasmic reticulum and most peroxisomes contained lactate dehydrogenase isoenzyme 5, and a non-NAD+ dependent 010 ^
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1 23456789 11 U 15 17 19 21 23 25 27 29 10 12 14 16 18 20 22 24 26 28 30
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/. Biochem.
GLYOXYLATE OXIDATION IN TISSUES enzyme with the same electrophoretic mobility as the non-NAD + dependent glycollate oxidizing enzyme previously reported in liver cytosol (2). Chromatography of the extracted proteins on DEAE-Sephadex showed that the N A D + dependent oxalate formation was only associated with lactate dehydrogenase, and the non-NAD + dependent activity was associated with glycollate oxidase. Subcellular Fractionation of Kidney Tissue for the Study of Lysosomes, Peroxisomes, Mitochondria, and Endoplasmic Reticulum—Figures 6, 7, and 8 show the distribution of N A D + dependent catalytic activity with respect to the oxidation of glyoxylate to oxalate compared with that of the marker enzymes in the fractions obtained by the sucrose density gradient centrifugation of specimens C, D, and E respectively from the initial fractionation 5O
227
shown in Fig. 1. No non-NAD + dependent activity was detected. The profile of the distribution of catalytic activity with respect to the oxidation of glyoxylate to oxalate most closely parallels that of the endoplasmic marker enzyme, glucose-6-phosphatase (Fig. 8). However, the presence of a lysosomal enzyme capable of catalyzing this oxidation cannot be excluded on the basis of the present evidence, because we were unable to change the density of the kidney lysosomes by pre-treating the animals with Triton-WR1339 or Dextran T10. The distribution of catalase and cytochrome oxidase activities differs from that of the N A D + dependent catalytic activity for the oxidation of glyoxylate to oxalate (Figs. 6 & 7), showing that the latter activity is not associated with kidney mitochondria and peroxisomes. •
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