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Assay and subcellular localization of pyrroline-5-carboxylate dehydrogenase in rat liver
Biochimica et Biophysica Acta 1675 (2004) 81 – 86 http://www.elsevier.com/locate/bba
Assay and subcellular localization of pyrroline-5-carboxylate dehydrogenase in rat liver Michael R. Haslett, Desmond Pink, Barry Walters, Margaret E. Brosnan* Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NF Canada A1B 3X9 Received 17 May 2004; received in revised form 18 August 2004; accepted 18 August 2004 Available online 8 September 2004
Abstract D1-Pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyzes the conversion of D1-pyrroline-5-carboxylate to glutamate in a reaction requiring NAD(P)+ as a cofactor. D1-Pyrroline-5-carboxylate is formed in liver from proline by proline oxidase (EC number not assigned) or from ornithine via ornithine aminotransferase. A spectrophotometric assay for P5CDh was shown to be valid if rotenone was included in the assay to prevent reoxidation of NADH. Using this new assay, liver was fractionated using differential centrifugation and the distribution of P5CDh was compared to that of appropriate marker enzymes. P5CDh is enriched only in the mitochondrial fractions, as are the mitochondrial enzymes, succinate cytochrome c reductase, proline oxidase, glutaminase, and ornithine aminotransferase. Thus, it can be concluded that P5CDh occurs only in mitochondria, not in both mitochondria and cytoplasm, as had previously been reported. D 2004 Elsevier B.V. All rights reserved. Keywords: D1-Pyrroline-5-carboxylate dehydrogenase; Spectrophotometric assay; Subcellular location; Rat liver; Glutamic semialdehyde dehydrogenase
1. Introduction Subcellular localization of an enzyme is one of its key features, as its location impacts on its function and regulation. The enzymes which metabolize pyrroline-5carboxylate (P5C) in liver are shown in Fig. 1. It should be noted that P5C is in spontaneous equilibrium with an open chain tautomer, glutamic-gamma-semialdehyde [1]. It is not definitively known which tautomer is involved in each of the reactions shown in Fig. 1, but for simplicity’s sake, we show only P5C. The enzymes which synthesize P5C in liver, ornithine aminotransferase(OAT; EC 2.6.1.13) [2] and
Abbreviations: P5CDh, D1-pyrroline-5-carboxylate dehydrogenase; P5C, D1-pyrroline-5-carboxylate; PO, proline oxidase; OAT, ornithine aminotransferase; NAD+, nicotinamide adenine dinucleotide (oxidized); NADP+, nicotinamide adenine dinucleotide phosphate (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NADPH, nicotinamide adenine dinucleotide phosphate (reduced); Fp, flavoprotein (oxidized); FpH2, flavoprotein (reduced) * Corresponding author. Tel.: +1 709 737 2511 fax: +1 709 737 2422. E-mail address: [email protected] (M.E. Brosnan). 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.08.008
proline oxidase (PO; EC number not assigned) [3], both occur in mitochondria. The OAT reaction is not reversible in liver extracts, although it is in some other tissues [4]. Electrons from PO are thought to be transferred to a flavoprotein in the mitochondrial inner membrane [5]. Since P5C synthase (EC number not assigned) does not occur in liver [6], it is not shown. P5C can be converted to proline by P5C reductase, which occurs outside the mitochondrion [7]. P5C dehydrogenase (P5CDh; EC 1.5.1.12), which converts P5C to glutamate, has been reported to occur in both mitochondria and cytosol in the only paper which provided data on its localization [8]. Several papers report on the purification of P5CDh from mitochondria [9–11], but do not provide evidence that there is no activity anywhere else in the cell. A single P5CDh gene has been cloned and sequenced from humans [11]. It codes for a protein of 563 residues with features in the N-terminal region characteristic of a mitochondrial targeting sequence. This observation does not rule out the possibility of a cytoplasmic P5CDh as well. For example, there is a single gene for fumarase [12] and mature fumarase can be purified from mitochondria, but, in fact, there is also a cytoplasmic fumarase which
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libitum. All procedures were approved by Memorial University’s Institutional Animal Care Committee and were in accordance with the guidelines of the Canadian Council on Animal Care. 2.2. Chemicals All chemicals were from Sigma-Aldrich (Oakville, Ont., Canada) except where noted in the text. 2.3. Subcellular fractionation
Fig. 1. Enzymes involved in metabolism of D1-pyrroline-5-carboxylic acid in rat liver. Enzymes: 1, ornithine aminotransferase; 2, D1-pyrroline-5carboxylic acid dehydrogenase; 3, proline oxidase; 4, D1-pyrroline-5carboxylic acid reductase. Abbreviations; a-KG, a-ketoglutarate; Glu, glutamate; Fp, flavoprotein.
recycles fumarate in the urea cycle [13]. Thus, it is essential to provide data on the localization of the total activity of an enzyme in the cell [14], not simply on predicted localization from genomics [15] or proteomics [16]. Small and Jones [17] have pointed out that spectrophotometric assays for P5CDh activity that rely on the reduction of NAD+ to assess the progress of the reaction are unacceptable due to a lack of linearity with time and protein. In the present study, we have modified Strecker’s spectrophotometric assay [9] for P5CDh by the addition of 10 AM rotenone to inhibit oxidation of NADH by the electron transport chain. With our modified assay, we have performed a systematic subcellular fractionation in the manner proposed by De Duve [14] with measurement of the appropriate markers for each fraction. The results indicate that P5CDh is located solely in mitochondria, in contrast to the report by Brunner and Neupert [8].
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (supplied from our university’s breeding colony), weighing 225–250 g, were used in all studies. Animals were housed in polycarbonate cages and allowed water and standard Purina rat chow ad
Rats were anesthetized using 6.5 mg of sodium pentobarbital (i.p.) per 100-g rat. Following a midline incision, the liver was quickly removed, weighed, and placed in ice-cold homogenization buffer, consisting of 5 mM HEPES (pH 7.4), 1 mM EGTA, and 0.33 M sucrose (5-ml buffer per gram of liver). The liver was finely minced with scissors, washed three times in icecold homogenization buffer and homogenized at approximately 500 rev/min by five to six strokes of a motordriven, loose-fitting, teflon pestle (clearance 0.13–0.18 mm). The homogenate was diluted to 10 ml per gram of liver with ice-cold homogenization buffer and filtered through two layers of cheesecloth. Following filtration, 10 ml of the homogenate was removed and labeled as total homogenate (TH) while the remaining filtrate was fractionated by differential centrifugation into a nuclear fraction (N), a heavy mitochondrial fraction (M1), a light mitochondrial fraction (M2), a lysosomal fraction (L), a microsomal fraction (P), and a cytosolic fraction (S). The method proposed by De Duve [14] was followed with modifications given below. All procedures were completed at 4 8C. The nuclear fraction was obtained by centrifugation of the homogenate at 384g for 2 min. The resulting pellet was resuspended (by manual shaking) in 20.0 ml of icecold homogenization buffer and centrifuged again at 384 g for 2 min. The final nuclear pellet was resuspended in 10.0 ml of ice-cold homogenization buffer and the supernatant pooled with the previous supernatant, to give the post-nuclear supernatant. The heavy mitochondrial fraction was isolated employing two centrifugations of the post- nuclear supernatant at 1000g for 10 min. The same procedure for resuspension of the intermediate and final pellets was employed as for the nuclear fraction. The resultant supernatants were again pooled. The light mitochondrial fraction was obtained by an initial centrifugation of the post heavy mitochondrial supernatant at 3500g for 10 min, followed by resuspension of the intermediate pellet as above and a second centrifugation at 3000g for 10 min. The lysosomal and microsomal fractions were obtained by single centrifugations of 9090g for 20 min and 100,000g for 60 min, respectively, and resuspension of the resulting pellets as before. The
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supernatant obtained following the centrifugation at 100,000g represents the cytosolic fraction. Exact volumes were noted for all fractions and they were stored in 1.0-ml aliquots at 70 8C until analysis. 2.4. Marker enzyme assays LDH activity was measured according to Morrison et al. [18], succinate cytochrome c reductase and NADPH cytochrome c reductase activities were measured according to Sottocassa et al. [19], proline oxidase according to Herzfeld et al. [20], ornithine aminotransferase according to Herzfeld and Knox [21], with the addition of 0.05 mM pyridoxal-5-phosphate, glutaminase, according to Lacey et al. [22], and h-glucuronidase according to De Duve et al. [23]. All enzyme assays were conducted at 37 8C following disruption of organelles via three cycles of freezing and thawing, and were linear with time and protein.
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3. Results and discussion According to Small and Jones [17], spectrophotometric assays for P5CDh activity that rely on the reduction of NAD+ to assess the progress of the reaction are unacceptable due to a lack of linearity with time and protein. In this study, 10 AM rotenone was used to inhibit oxidation of NADH by the electron transport chain. The spectrophotometric assay was found to be linear with time for a minimum of 15 min following the addition of P5C (Fig. 2A) and with protein up to at least 3.4 Ag in the 300-Al assay volume (Fig. 2B). Table 1 shows the distribution of P5CDh activity as compared to that of markers in the subcellular fractions isolated from a homogenate of rat liver in the manner proposed by De Duve [14]. We have added an extra mitochondrial fraction because we were consistently getting a significant amount of cross contamination within the
2.5. Assay of P5CDh P5CDh activity was measured, using either NAD+ or NADP+ (Roche Biochemicals) as a cofactor. The method of Strecker [9] was used as a basis for designing the assay conditions used in this study. The assay was modified by the presence of 10 AM rotenone in the reaction mixture to prevent reoxidation of NADH. The reaction mixture consisted of 260 Al of a solution containing 1 mM EDTA and 12 mM HEPES at pH 7.8, 10 Al of 0.300 mM rotenone (dissolved in dimethyl sulfoxide), 10 Al of protein and 10 Al of 100 mM NAD+ or NADP+. The reaction was started by the addition of 10 Al of 12 mM DL-P5C to give a final volume of 300 Al. The resultant final concentrations in the assay were: EDTA 0.87 mM, HEPES 10.4 mM, rotenone 0.01 mM, NAD(P) 3.3 mM, P5C 0.4 mM and 1 to 2 Ag of protein. The progress of the reaction at 37 8C was measured by recording the production of NADH (or NADPH) at 340 nm. 2.6. Preparation of D 1-pyrroline-5-carboxylic acid P5C was obtained as the 2,4-dinitrophenylhydrazone derivative and prepared for use in enzyme assays according to the method of Mezl and Knox [1]. Determination of DLP5C concentration was carried out according to the method of Irreverre et al. [24]. 2.7. Other assays DNA was extracted from the fractions using the method of Schneider [25], and the concentration of DNA in each fraction was determined using the method described by Burton [26] with calf thymus DNA as standard. Protein concentration in each fraction was determined by the biuret method [27] using bovine serum albumin as standard, following solubilization of membranous material for 15 min with 5% deoxycholate [28].
Fig. 2. Validation of the assay for D1-pyrroline-5-carboxylic acid dehydrogenase in rat liver. Assay conditions are given in Materials and methods. (A) P5CDh activity as a function of time of assay. Assays were conducted with 1.2 Ag of mitochondrial protein and allowed to continue for 15 min. (B) P5CDh assay as a function of mitochondrial protein in the assay. Assays were conducted for 10 min with the amounts of mitochondrial protein indicated. Values represent meanFS.D., n=3.
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Table 1 Specific activities and recovery of P5CDh, marker enzymes and DNA from subcellular fractions of rat liver Enzyme
Ornithine aminotransferase P5CDh (NAD+) P5CDh (NADP+) Glutaminase Proline oxidase Succinate cytochrome c reductase h-Glucuronidase NADPH cytochrome c reductase Lactate dehydrogenase DNA
Fraction N
M1
M2
L
P
S
Recovery (%)
1.29F0.63 0.030F0.006 0.019F0.003 5.33F3.75 0.64F0.38 5.15F1.79 0.12F0.06 7.46F2.12 0.54F0.21 612.42F168
7.51F1.43 0.229F0.037 0.149F0.024 47.54F10.17 5.94F2.23 52.28F20.72 0.22F0.15 4.30F2.37 0.33F0.10 18.16F4.59
12.90F2.64 0.299F0.037 0.188F0.037 39.58F6.05 8.42F5.00 62.71F22.39 0.43F0.27 5.31F3.05 0.20F0.13 3.47F2.61
7.07F1.19 0.079F0.019 0.052F0.015 11.57F6.8 2.74F1.51 21.12F7.84 1.21F0.42 18.69F7.58 0.13F0.11 0.00F0.00
0.40F0.31 0.006F0.001 0.005F0.003 1.05F1.28 0.50F0.37 3.27F4.85 0.29F0.30 86.71F14.75 0.52F0.73 0.00F0.00
0.81F0.57 0.020F0.004 0.016F0.001 0.09F0.22 0.10F0.21 0.00F0.00 0.04F0.03 9.54F9.19 4.98F1.14 0.00F0.00
100 92 93 96 103 89 93 87 98 102
Specific activities of enzymes are given as nanomoles of product formed per minute per milligram of protein. DNA is expressed as micrograms per milligram of protein. All values are presented as meanFS.D. (n=6). Abbreviations; N, nuclear fraction; M1, first mitochondrial fraction; M2, second mitochondrial fraction; L, lysosomal fraction; P, microsomal fraction; S, cytosolic fraction. Recoveries are based on homogenate values as 100%.
nuclear and mitochondrial fractions obtained using the classical method which yields one mitochondrial fraction. The recoveries of enzyme activity, DNA and protein ranged from 87% to 103% (Table 1). The distribution patterns of typical nuclear (DNA), mitochondrial (succinate cytochrome c reductase), lysosomal (h-glucuronidase), microsomal (NADPH cytochrome c reductase) and cytosolic
(lactate dehydrogenase) markers, shown in Fig. 3, are similar to those reported by De Duve [14]. In our study we have used several marker enzymes for mitochondria: succinate cytochrome c reductase, an intrinsic protein in the mitochondrial inner membrane [14]; glutaminase [29] and proline oxidase [3], extrinsic proteins associated with the inner mitochondrial membrane; orni-
Fig. 3. Composite distribution pattern of P5CDh, DNA and marker enzymes in fractions for rat liver. Ordinate: mean relative specific activity of markers (specific activity in fraction/specific activity in homogenate, calculated from data given in Table 1). Abscissa: relative protein content of fractions (cumulatively from left to right). N, nuclear fraction; M1, first mitochondrial fraction; M2, second mitochondrial fraction; L, lysosomal fraction; P, microsomal fraction; S, cytosolic fraction.
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thine aminotransferase, a soluble enzyme located in the mitochondrial matrix [21]. The distribution pattern of P5CDh closely follows that of these mitochondrial enzymes (Fig. 3). There is no leakage of the membrane-associated enzymes into the cytosolic fraction, but approximately 7–8% of the matrix enzyme, ornithine aminotransferase, does appear in this compartment, indicating a slight release of soluble enzymes from the mitochondria. A similar amount of P5CDh also appears in the cytosolic fraction, whether assayed with NAD+ or NADP+ as cofactor (Table 1). It has been proposed by Phang [30] that the presence of P5CDh in the cytosol may represent an isoenzyme that could show a preference for NADP+ versus NAD+. The results obtained do not support this and in fact show that the specific activity of P5CDh decreases when NADP+ is used as a cofactor in all fractions. The fact that the specific activity for P5CDh in the cytosol does not increase when NADP+ is used as a cofactor also suggests that the activity observed in the cytosol is due to leakage of the enzyme during fractionation and not to a true cytosolic enzyme or isoenzyme. It can therefore be concluded that P5CDh occurs solely in mitochondria in liver. The finding in this study that P5CDh is located solely in mitochondria is in contrast to data from Brunner and Neupert [8]. Rat liver also contains the cytosolic enzyme P5C reductase which catalyzes the conversion of P5C to proline [31]. The assay employed by Brunner and Neupert followed the disappearance of P5C and assumed that this would be due only to P5CDh. When Brosemer and Veerabhadrappa [32] originated this assay for P5CDh in insects, more P5C disappeared than could be accounted for as glutamate, although they inhibited further glutamate metabolism. The assay as conducted by Brunner and Neupert [8] would not discriminate between the loss of P5C due to glutamate synthesis by P5CDh and due to proline synthesis by P5C reductase. It is likely that the activity which was attributed to the presence of P5CDh in the cytosol [31] was in fact due to the presence of P5C reductase which occurs in this compartment. P5C synthesis in liver from ornithine (via OAT) and proline (via PO) occurs in the mitochondrial matrix. Therefore, the P5C formed from these reactions would have immediate access to P5CDh. The glutamate formed could be converted to a-ketoglutarate by glutamate dehydrogenase in the mitochondrial matrix. The a-ketoglutarate could enter the tricarboxylic acid cycle for oxidation or for conversion to oxaloacetate and ultimately to glucose [13]. Proline synthesis via P5CR occurs outside the mitochondrion [7], so P5C would have to be transported out of the mitochondrion to reach the enzyme. Regulation of both P5CDh and P5C transport could be crucial in determining the fate of P5C produced from ornithine or proline in liver. It has been reported that p53 can induce PO in several tumors [33]. The increased PO catalyzes the generation of reactive-oxygen species [34] and it induces apoptosis in
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these tumor cells [35]. The mechanism involved is not yet understood but it has been suggested by Phang [30] that proline is converted to P5C by PO which is subsequently converted to proline by P5CR with oxidation of NADPH in the cytoplasm. For this pathway to occur, mitochondrial P5CDh would have to be absent or relatively less active than the P5C transporter so the P5C would be free to exit the mitochondrion. Little information is available on the regulation of P5CDh or on the P5C transporter in mitochondria in tumor cells.
Acknowledgements MRH and DP thank the school of Graduate Studies, Memorial University of Newfoundland for a graduate fellowship. This work was supported by a grant from Canadian Institutes of Health Research to MEB.
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Assay and subcellular localization of pyrroline-5-carboxylate dehydrogenase in rat liver Michael R. Haslett, Desmond Pink, Barry Walters, Margaret E. Brosnan* Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NF Canada A1B 3X9 Received 17 May 2004; received in revised form 18 August 2004; accepted 18 August 2004 Available online 8 September 2004
Abstract D1-Pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyzes the conversion of D1-pyrroline-5-carboxylate to glutamate in a reaction requiring NAD(P)+ as a cofactor. D1-Pyrroline-5-carboxylate is formed in liver from proline by proline oxidase (EC number not assigned) or from ornithine via ornithine aminotransferase. A spectrophotometric assay for P5CDh was shown to be valid if rotenone was included in the assay to prevent reoxidation of NADH. Using this new assay, liver was fractionated using differential centrifugation and the distribution of P5CDh was compared to that of appropriate marker enzymes. P5CDh is enriched only in the mitochondrial fractions, as are the mitochondrial enzymes, succinate cytochrome c reductase, proline oxidase, glutaminase, and ornithine aminotransferase. Thus, it can be concluded that P5CDh occurs only in mitochondria, not in both mitochondria and cytoplasm, as had previously been reported. D 2004 Elsevier B.V. All rights reserved. Keywords: D1-Pyrroline-5-carboxylate dehydrogenase; Spectrophotometric assay; Subcellular location; Rat liver; Glutamic semialdehyde dehydrogenase
1. Introduction Subcellular localization of an enzyme is one of its key features, as its location impacts on its function and regulation. The enzymes which metabolize pyrroline-5carboxylate (P5C) in liver are shown in Fig. 1. It should be noted that P5C is in spontaneous equilibrium with an open chain tautomer, glutamic-gamma-semialdehyde [1]. It is not definitively known which tautomer is involved in each of the reactions shown in Fig. 1, but for simplicity’s sake, we show only P5C. The enzymes which synthesize P5C in liver, ornithine aminotransferase(OAT; EC 2.6.1.13) [2] and
Abbreviations: P5CDh, D1-pyrroline-5-carboxylate dehydrogenase; P5C, D1-pyrroline-5-carboxylate; PO, proline oxidase; OAT, ornithine aminotransferase; NAD+, nicotinamide adenine dinucleotide (oxidized); NADP+, nicotinamide adenine dinucleotide phosphate (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NADPH, nicotinamide adenine dinucleotide phosphate (reduced); Fp, flavoprotein (oxidized); FpH2, flavoprotein (reduced) * Corresponding author. Tel.: +1 709 737 2511 fax: +1 709 737 2422. E-mail address: [email protected] (M.E. Brosnan). 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.08.008
proline oxidase (PO; EC number not assigned) [3], both occur in mitochondria. The OAT reaction is not reversible in liver extracts, although it is in some other tissues [4]. Electrons from PO are thought to be transferred to a flavoprotein in the mitochondrial inner membrane [5]. Since P5C synthase (EC number not assigned) does not occur in liver [6], it is not shown. P5C can be converted to proline by P5C reductase, which occurs outside the mitochondrion [7]. P5C dehydrogenase (P5CDh; EC 1.5.1.12), which converts P5C to glutamate, has been reported to occur in both mitochondria and cytosol in the only paper which provided data on its localization [8]. Several papers report on the purification of P5CDh from mitochondria [9–11], but do not provide evidence that there is no activity anywhere else in the cell. A single P5CDh gene has been cloned and sequenced from humans [11]. It codes for a protein of 563 residues with features in the N-terminal region characteristic of a mitochondrial targeting sequence. This observation does not rule out the possibility of a cytoplasmic P5CDh as well. For example, there is a single gene for fumarase [12] and mature fumarase can be purified from mitochondria, but, in fact, there is also a cytoplasmic fumarase which
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libitum. All procedures were approved by Memorial University’s Institutional Animal Care Committee and were in accordance with the guidelines of the Canadian Council on Animal Care. 2.2. Chemicals All chemicals were from Sigma-Aldrich (Oakville, Ont., Canada) except where noted in the text. 2.3. Subcellular fractionation
Fig. 1. Enzymes involved in metabolism of D1-pyrroline-5-carboxylic acid in rat liver. Enzymes: 1, ornithine aminotransferase; 2, D1-pyrroline-5carboxylic acid dehydrogenase; 3, proline oxidase; 4, D1-pyrroline-5carboxylic acid reductase. Abbreviations; a-KG, a-ketoglutarate; Glu, glutamate; Fp, flavoprotein.
recycles fumarate in the urea cycle [13]. Thus, it is essential to provide data on the localization of the total activity of an enzyme in the cell [14], not simply on predicted localization from genomics [15] or proteomics [16]. Small and Jones [17] have pointed out that spectrophotometric assays for P5CDh activity that rely on the reduction of NAD+ to assess the progress of the reaction are unacceptable due to a lack of linearity with time and protein. In the present study, we have modified Strecker’s spectrophotometric assay [9] for P5CDh by the addition of 10 AM rotenone to inhibit oxidation of NADH by the electron transport chain. With our modified assay, we have performed a systematic subcellular fractionation in the manner proposed by De Duve [14] with measurement of the appropriate markers for each fraction. The results indicate that P5CDh is located solely in mitochondria, in contrast to the report by Brunner and Neupert [8].
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats (supplied from our university’s breeding colony), weighing 225–250 g, were used in all studies. Animals were housed in polycarbonate cages and allowed water and standard Purina rat chow ad
Rats were anesthetized using 6.5 mg of sodium pentobarbital (i.p.) per 100-g rat. Following a midline incision, the liver was quickly removed, weighed, and placed in ice-cold homogenization buffer, consisting of 5 mM HEPES (pH 7.4), 1 mM EGTA, and 0.33 M sucrose (5-ml buffer per gram of liver). The liver was finely minced with scissors, washed three times in icecold homogenization buffer and homogenized at approximately 500 rev/min by five to six strokes of a motordriven, loose-fitting, teflon pestle (clearance 0.13–0.18 mm). The homogenate was diluted to 10 ml per gram of liver with ice-cold homogenization buffer and filtered through two layers of cheesecloth. Following filtration, 10 ml of the homogenate was removed and labeled as total homogenate (TH) while the remaining filtrate was fractionated by differential centrifugation into a nuclear fraction (N), a heavy mitochondrial fraction (M1), a light mitochondrial fraction (M2), a lysosomal fraction (L), a microsomal fraction (P), and a cytosolic fraction (S). The method proposed by De Duve [14] was followed with modifications given below. All procedures were completed at 4 8C. The nuclear fraction was obtained by centrifugation of the homogenate at 384g for 2 min. The resulting pellet was resuspended (by manual shaking) in 20.0 ml of icecold homogenization buffer and centrifuged again at 384 g for 2 min. The final nuclear pellet was resuspended in 10.0 ml of ice-cold homogenization buffer and the supernatant pooled with the previous supernatant, to give the post-nuclear supernatant. The heavy mitochondrial fraction was isolated employing two centrifugations of the post- nuclear supernatant at 1000g for 10 min. The same procedure for resuspension of the intermediate and final pellets was employed as for the nuclear fraction. The resultant supernatants were again pooled. The light mitochondrial fraction was obtained by an initial centrifugation of the post heavy mitochondrial supernatant at 3500g for 10 min, followed by resuspension of the intermediate pellet as above and a second centrifugation at 3000g for 10 min. The lysosomal and microsomal fractions were obtained by single centrifugations of 9090g for 20 min and 100,000g for 60 min, respectively, and resuspension of the resulting pellets as before. The
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supernatant obtained following the centrifugation at 100,000g represents the cytosolic fraction. Exact volumes were noted for all fractions and they were stored in 1.0-ml aliquots at 70 8C until analysis. 2.4. Marker enzyme assays LDH activity was measured according to Morrison et al. [18], succinate cytochrome c reductase and NADPH cytochrome c reductase activities were measured according to Sottocassa et al. [19], proline oxidase according to Herzfeld et al. [20], ornithine aminotransferase according to Herzfeld and Knox [21], with the addition of 0.05 mM pyridoxal-5-phosphate, glutaminase, according to Lacey et al. [22], and h-glucuronidase according to De Duve et al. [23]. All enzyme assays were conducted at 37 8C following disruption of organelles via three cycles of freezing and thawing, and were linear with time and protein.
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3. Results and discussion According to Small and Jones [17], spectrophotometric assays for P5CDh activity that rely on the reduction of NAD+ to assess the progress of the reaction are unacceptable due to a lack of linearity with time and protein. In this study, 10 AM rotenone was used to inhibit oxidation of NADH by the electron transport chain. The spectrophotometric assay was found to be linear with time for a minimum of 15 min following the addition of P5C (Fig. 2A) and with protein up to at least 3.4 Ag in the 300-Al assay volume (Fig. 2B). Table 1 shows the distribution of P5CDh activity as compared to that of markers in the subcellular fractions isolated from a homogenate of rat liver in the manner proposed by De Duve [14]. We have added an extra mitochondrial fraction because we were consistently getting a significant amount of cross contamination within the
2.5. Assay of P5CDh P5CDh activity was measured, using either NAD+ or NADP+ (Roche Biochemicals) as a cofactor. The method of Strecker [9] was used as a basis for designing the assay conditions used in this study. The assay was modified by the presence of 10 AM rotenone in the reaction mixture to prevent reoxidation of NADH. The reaction mixture consisted of 260 Al of a solution containing 1 mM EDTA and 12 mM HEPES at pH 7.8, 10 Al of 0.300 mM rotenone (dissolved in dimethyl sulfoxide), 10 Al of protein and 10 Al of 100 mM NAD+ or NADP+. The reaction was started by the addition of 10 Al of 12 mM DL-P5C to give a final volume of 300 Al. The resultant final concentrations in the assay were: EDTA 0.87 mM, HEPES 10.4 mM, rotenone 0.01 mM, NAD(P) 3.3 mM, P5C 0.4 mM and 1 to 2 Ag of protein. The progress of the reaction at 37 8C was measured by recording the production of NADH (or NADPH) at 340 nm. 2.6. Preparation of D 1-pyrroline-5-carboxylic acid P5C was obtained as the 2,4-dinitrophenylhydrazone derivative and prepared for use in enzyme assays according to the method of Mezl and Knox [1]. Determination of DLP5C concentration was carried out according to the method of Irreverre et al. [24]. 2.7. Other assays DNA was extracted from the fractions using the method of Schneider [25], and the concentration of DNA in each fraction was determined using the method described by Burton [26] with calf thymus DNA as standard. Protein concentration in each fraction was determined by the biuret method [27] using bovine serum albumin as standard, following solubilization of membranous material for 15 min with 5% deoxycholate [28].
Fig. 2. Validation of the assay for D1-pyrroline-5-carboxylic acid dehydrogenase in rat liver. Assay conditions are given in Materials and methods. (A) P5CDh activity as a function of time of assay. Assays were conducted with 1.2 Ag of mitochondrial protein and allowed to continue for 15 min. (B) P5CDh assay as a function of mitochondrial protein in the assay. Assays were conducted for 10 min with the amounts of mitochondrial protein indicated. Values represent meanFS.D., n=3.
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Table 1 Specific activities and recovery of P5CDh, marker enzymes and DNA from subcellular fractions of rat liver Enzyme
Ornithine aminotransferase P5CDh (NAD+) P5CDh (NADP+) Glutaminase Proline oxidase Succinate cytochrome c reductase h-Glucuronidase NADPH cytochrome c reductase Lactate dehydrogenase DNA
Fraction N
M1
M2
L
P
S
Recovery (%)
1.29F0.63 0.030F0.006 0.019F0.003 5.33F3.75 0.64F0.38 5.15F1.79 0.12F0.06 7.46F2.12 0.54F0.21 612.42F168
7.51F1.43 0.229F0.037 0.149F0.024 47.54F10.17 5.94F2.23 52.28F20.72 0.22F0.15 4.30F2.37 0.33F0.10 18.16F4.59
12.90F2.64 0.299F0.037 0.188F0.037 39.58F6.05 8.42F5.00 62.71F22.39 0.43F0.27 5.31F3.05 0.20F0.13 3.47F2.61
7.07F1.19 0.079F0.019 0.052F0.015 11.57F6.8 2.74F1.51 21.12F7.84 1.21F0.42 18.69F7.58 0.13F0.11 0.00F0.00
0.40F0.31 0.006F0.001 0.005F0.003 1.05F1.28 0.50F0.37 3.27F4.85 0.29F0.30 86.71F14.75 0.52F0.73 0.00F0.00
0.81F0.57 0.020F0.004 0.016F0.001 0.09F0.22 0.10F0.21 0.00F0.00 0.04F0.03 9.54F9.19 4.98F1.14 0.00F0.00
100 92 93 96 103 89 93 87 98 102
Specific activities of enzymes are given as nanomoles of product formed per minute per milligram of protein. DNA is expressed as micrograms per milligram of protein. All values are presented as meanFS.D. (n=6). Abbreviations; N, nuclear fraction; M1, first mitochondrial fraction; M2, second mitochondrial fraction; L, lysosomal fraction; P, microsomal fraction; S, cytosolic fraction. Recoveries are based on homogenate values as 100%.
nuclear and mitochondrial fractions obtained using the classical method which yields one mitochondrial fraction. The recoveries of enzyme activity, DNA and protein ranged from 87% to 103% (Table 1). The distribution patterns of typical nuclear (DNA), mitochondrial (succinate cytochrome c reductase), lysosomal (h-glucuronidase), microsomal (NADPH cytochrome c reductase) and cytosolic
(lactate dehydrogenase) markers, shown in Fig. 3, are similar to those reported by De Duve [14]. In our study we have used several marker enzymes for mitochondria: succinate cytochrome c reductase, an intrinsic protein in the mitochondrial inner membrane [14]; glutaminase [29] and proline oxidase [3], extrinsic proteins associated with the inner mitochondrial membrane; orni-
Fig. 3. Composite distribution pattern of P5CDh, DNA and marker enzymes in fractions for rat liver. Ordinate: mean relative specific activity of markers (specific activity in fraction/specific activity in homogenate, calculated from data given in Table 1). Abscissa: relative protein content of fractions (cumulatively from left to right). N, nuclear fraction; M1, first mitochondrial fraction; M2, second mitochondrial fraction; L, lysosomal fraction; P, microsomal fraction; S, cytosolic fraction.
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thine aminotransferase, a soluble enzyme located in the mitochondrial matrix [21]. The distribution pattern of P5CDh closely follows that of these mitochondrial enzymes (Fig. 3). There is no leakage of the membrane-associated enzymes into the cytosolic fraction, but approximately 7–8% of the matrix enzyme, ornithine aminotransferase, does appear in this compartment, indicating a slight release of soluble enzymes from the mitochondria. A similar amount of P5CDh also appears in the cytosolic fraction, whether assayed with NAD+ or NADP+ as cofactor (Table 1). It has been proposed by Phang [30] that the presence of P5CDh in the cytosol may represent an isoenzyme that could show a preference for NADP+ versus NAD+. The results obtained do not support this and in fact show that the specific activity of P5CDh decreases when NADP+ is used as a cofactor in all fractions. The fact that the specific activity for P5CDh in the cytosol does not increase when NADP+ is used as a cofactor also suggests that the activity observed in the cytosol is due to leakage of the enzyme during fractionation and not to a true cytosolic enzyme or isoenzyme. It can therefore be concluded that P5CDh occurs solely in mitochondria in liver. The finding in this study that P5CDh is located solely in mitochondria is in contrast to data from Brunner and Neupert [8]. Rat liver also contains the cytosolic enzyme P5C reductase which catalyzes the conversion of P5C to proline [31]. The assay employed by Brunner and Neupert followed the disappearance of P5C and assumed that this would be due only to P5CDh. When Brosemer and Veerabhadrappa [32] originated this assay for P5CDh in insects, more P5C disappeared than could be accounted for as glutamate, although they inhibited further glutamate metabolism. The assay as conducted by Brunner and Neupert [8] would not discriminate between the loss of P5C due to glutamate synthesis by P5CDh and due to proline synthesis by P5C reductase. It is likely that the activity which was attributed to the presence of P5CDh in the cytosol [31] was in fact due to the presence of P5C reductase which occurs in this compartment. P5C synthesis in liver from ornithine (via OAT) and proline (via PO) occurs in the mitochondrial matrix. Therefore, the P5C formed from these reactions would have immediate access to P5CDh. The glutamate formed could be converted to a-ketoglutarate by glutamate dehydrogenase in the mitochondrial matrix. The a-ketoglutarate could enter the tricarboxylic acid cycle for oxidation or for conversion to oxaloacetate and ultimately to glucose [13]. Proline synthesis via P5CR occurs outside the mitochondrion [7], so P5C would have to be transported out of the mitochondrion to reach the enzyme. Regulation of both P5CDh and P5C transport could be crucial in determining the fate of P5C produced from ornithine or proline in liver. It has been reported that p53 can induce PO in several tumors [33]. The increased PO catalyzes the generation of reactive-oxygen species [34] and it induces apoptosis in
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these tumor cells [35]. The mechanism involved is not yet understood but it has been suggested by Phang [30] that proline is converted to P5C by PO which is subsequently converted to proline by P5CR with oxidation of NADPH in the cytoplasm. For this pathway to occur, mitochondrial P5CDh would have to be absent or relatively less active than the P5C transporter so the P5C would be free to exit the mitochondrion. Little information is available on the regulation of P5CDh or on the P5C transporter in mitochondria in tumor cells.
Acknowledgements MRH and DP thank the school of Graduate Studies, Memorial University of Newfoundland for a graduate fellowship. This work was supported by a grant from Canadian Institutes of Health Research to MEB.
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