Intra- and Extramitochondrial Isozymes of (NADP) Malate Dehydrogenase

Transcription

1 Eur. J. Biochem. 19 (1971) Intra- and Extramitochondrial Isozymes of (NADP) Malate Dehydrogenase Dieter BRDICZKA and Dirk PETTE Fachbereich Biologie der Universitiit Konstanz (Received December 21,197) The intracellular distribution of (NADP) malate dehydrogenase (decarboxylating) was studied by means of fractional extraction of extra- and intramitochondrial enzymes in various tissues of mouse, rat, rabbit, guinea pig, pig, beef, sea gull and pigeon. Absolute activity levels of (NADP) malate dehydrogenase in the same tissues of different species vary greatly. In white adipose tissue and adrenal medulla, (NADP) malate dehydrogenase is located exclusively within the extramitochondrial compartment. In liver, 95 Ol of (NADP) malate dehydrogenase activity is extramitochondrial and only 5 Ol intramitochondrial. Mitochondria1 subfractionation reveals a location of this enzyme within the inner mitochondrial compartment. Brain, heart, adrenal cortex and kidney also contain extra- and intramitochondrial (NADP) malate dehydrogenase. In kidney and adrenal cortex, the ratio of extra- and intramitochondrial isozymes varies between different species. In heart, a relatively constant distribution is found, i. e. 3 /, of the enzyme activity is extramitochondrial and 7 Ol is intramitochondrial. Extra- and intramitochondrial (NADP) malate dehydrogenases show different electrophoretic mobility in all tissues examined and can be characterized as two distinct isoenzymes. Extra- and intramitochondrial isozymes of (NADP) malate dehydrogenase are probably related to different metabolic functions in various tissues, e. 9. extra-intramitochondrial hydrogen transfer (malate shuttle) or transhydrogenation reactions. Two functions of (NADP) malate dehydrogenase (decarboxylating) in cellular metabolism have been proposed. The first was that (NADP) malate dehydrogenase might participate in glueoneogenesis [I -31. The second was that (NADP) malate dehydrogenase participates in lipogenesis by supplying NADPH. This hypothesis has been confirmed experimentally for liver and adipose tissue [a, 51. (NADP) malate dehydrogenase was thought to be located exclusively in the cytosol fraction. Some years ago in our laboratory data were obtained, which suggested the existence also of an intramitochondrial (NADP) malate dehydrogenase in adrenal cortex, heart and kidney [6]. As shown by Henderson [7] in several tissues of mice and pig, this mitochondrial enzyme is electrophoretically different from that of the cytoplasm. In a recent communication Simpson et al. [8] described in adrenal cortex intra- and extramitochondrial (NADP) malate dehydrogenase with different chromatographic and catalytic properties. Nevertheless, the information about (NADP) malate dehydrogenase and its metabolic functions appears to be still incomplete. The present study was undertaken to obtain more basic data on absolute activity levels, intracellular distri- Enzymes. Adenylate kinase (EC ) ; citrate synthase (EC ); (NADP) isocitrate dehydrogenase (EC ); lactate dehydrogenase (EC ); (NAD) malate dehydrogenase (EC ); (NADP) malate dehydrogenase (EC ); pyruvate carboxylase (EC ). bution and existence of isozymes of (NADP) malate dehydrogenase. The results obtained suggest different metabolic functions of the enzyme in various tissues. MATERIAL AND METHODS Animals Adult mice (BALB/C x V 57), rats (Sprague Dawley), rabbits (Deutsche Widder) and guinea pigs supplied ad libitum with water and Altromin standard diet from Fa. Altrogge (Lage/Lippe, Germany) were used for experiments. Tissues from pig and beef were obtained from the slaughterhouse. Preparation of Tissue Extracts Tissue Homogenates. Fresh tissues were homogenized in a 14-fold volume of.1 M phosphate buffer, ph 7.2, with a Polytron PT I homogenizer from Fa. Kinematic (Luzern, Switzerland). During disintegration, the samples were cooled by an ice-salt mixture. Cellular debris were removed by centrifugation for 2 min at 183 x g (ultracentrifuge Spinco L-5, rotor 5 Ti). Fractional Extraction. For separate determination of extra- and intramitochondrial enzyme activities, the tissues were subjected to the method reported recently [9]. Subfractionation of rat liver mitochondria was achieved by the digitonin method [

2 Vol. 19, No. 4, 1971 D. BRDICZKA and D. PETTE 547 Enzymes Activity of (NADP) malate dehydrogenase was determined by following the rate of NADP reduction spectrophotometrically at 366 nm. The test system contained: 5 mm triethanolamine, 5 mm EDTA, 8 mm MgCI,,.54 mm NADP, and 2 mm L-malate, ph 7.6. Control reactions with NAD showed that (NAD) malate dehydrogenase present in the investigated extracts did not interfere under the conditions of the test reaction. This was also demonstrated by the finding that pyruvate was formed stoichiometrically with NADPH. Lactate dehydrogenase is able to react with NADPH and pyruvate [13]. However, no measurable lactate dehydrogenase reaction was observed with levels of NADPH and pyruvate equivalent to those formed in the standard (NADP) malate dehydrogenase assays, since these levels are much lower than the K, values for lactate dehydrogenase. Thus, the (NADP) malate dehydrogenase assays were valid when less than.5 enzyme units were present in the assay. The activities of adenylate kinase and citrate synthase were determined as recently described [lo]. The activity of lactate dehydrogenase was measured according to [14]. Electrophoresis Horizontal agarose (1 gel electrophoresis was carried out in the cold for 5 h at a voltage of 5 V/cm in 5 mm sodium barbital buffer, ph 8.6. Tissue extracts were concentrated in vacuum dialysis against quartz-distilled water. Approximately 3 to 5 mu of (NADP) malate dehydrogenase were applicated to the agarose gels. For specific staining of (NADP) malate dehydrogenase activity, the agarose strips were incubated in the following staining mixture 1 5 mm triethanolamine, 5 mm EDTA, 8 mm MgCI,, 1.8 mm NADP, 5 mm L-malate,.2 mm phenazine methosulfate, 2 mm nitro blue tetrazolium, ph 7.6. RESULTS Intracellular Localization Separation of the fraction of "soluble" enzymes located in the extramitochondrial compartment of the cell from those situated within the mitochondria is achieved using the method of fractional extraction [S]. Nearly 1 /,, of the total cellular activity of glycolytic enzymes is obtained, when tissue is extracted successively in two changes of isotonic sucrose and one of phosphate buffer. The bulk of Table 1. Activity levels and intracellular distribution of (NADP) malate dehydrogenase in heart, kidney and Ziver of various mammalians and birds Extra- and intramitochondrial enzyme activities were separated by the fractional extraction technique. Results are expressed as mu/g wet weight and as percentage of total cellular activity. The data represent mean values. Number of experiments are give in brackets Animal extra- Heart intra- mu/g % mc/g % Mouse (2) Rat (8) Guinea pig (2) Rabbit (3) pig (3) Beef (4) Sea gull (1) Pigeon (2) extra- Kidney intra- mulg "1. muk % extra- Liver intra- mulg 'lo mulg % " = In ono case a low level of 25 mu/g wet wt. was found; this animal may have been of a different breed than the others. Table 2. Activity levels and intracellular distribution of (NADP) malate dehydrogenase in adrenal cortex and medulla, brain and adipose tissue of various mammalians Extra- and intramitochondrial enzyme activities were separated by the fractional extraction technique. Results are expressed as mu/g wet weight and as percentage of total cellular activity. The data represent mean values. Number of experiments are given in brackets Animal Adrenal cortex Adrenal medulla Brain Adipose tissue extra- intra- extra- intra- extra- intra- extra- intra- 37 Eur. J. Biochem., Vo1.19

3 548 Isozymes of (NADP) Malate Dehydrogenase Bur. J. Biochem. soluble enzymes of the citric acid cycle and other mitochondrial enzymes is set free, when the residue is disintegrated in phosphate buffer with sonification. In Tables 1 and 2 are presented the activities of (NADP) malate dehydrogenase which can be extracted separately from the extra- and intramitochondrial compartments. Most of the tissues investigated contain activities of (NADP) malate dehydrogenase in both compartments. With regard to the activity levels within the cytoplasmic and mitochondrial extracts, adrenal medulla and lipogenetically active tissues (liver and adipose tissue) represent a group of tissues in which the bulk of activity (approximately 95 o/o) is extractable from the extramitochondrial compartment. Mitochondria isolated from adipose tissue contain no measurable (NADP) malate dehydrogenase activity, as was recently shown also by Martin and Denton [15]. It appears that the small amount of activity found in the mitochondrial fraction of the fractional extraction procedure (cf Table 2) is not due to intramitochondrial location. In liver mitochondria, on the contrary, (NADP) malate dehydrogenase is present (see section on Intramitochondrial Localization). Another group of tissues, as represented by heart, brain, kidney and adrenal cortex, exhibits a considerable activity of (NADP) malate dehydrogenase in the mitochondrial fraction. Comparing results from different species, it appears that mainly in liver and heart the intracellular distribution of (NADP) malate dehydrogenase is organ-specific. In spite of large variations in the absolute activity levels, approximately 95/, of the total cellular activity of (NADP) malate dehydrogenase is found extramitochondrially in the livers of different species. Extremely low activities are found in the livers of guinea pig and rabbit, and no activity is measurable in beef liver [16]. In heart muscle of different species, a relatively constant percentage of the extramitochondrial (NADP) malate dehydrogenase (about 3 Oi) is found. In kidney, but also in adrenal cortex, there exist considerable variations in the ratio of extrraand intramitochondrial activities when different species are compared. Electrophoretic Behaviour Electrophoretic analysis of homogenates from various tissues of the rat results in a separation of two isozymes of (NADP) malate dehydrogenase. At alkaline ph (8.6), the two isozymes migrate toward the anode. As shown in Fig. 1, adipose tissue contains only one isozyme. This slowly migrating isozyme I is also present but in higher amounts in kidney, brain, and liver. In these tissues one observes additionally a faint band of a fast migrating isozyme 11. The latter represents the main band of the electrophoretically separated isozymes of heart muscle. Fig. 2 presents intra- (M) and extramitochondrial (C) subfractions of rat heart analyzed by gel electrophoresis. These fractions were obtained by fractional extraction. Evidently, the fast migrating isozyme I1 is concentrated exclusively in the mitochondrial fraction, and the slow isozyme I is present only in the cytoplasmic fraction. I Adipose Liver Brain Heart Kidney tissue Pig. 1. Electrophoresis of (NADP) malate dehydrogenase isozymes in crude extracts of various rat tissues. Electrophoresis was performed in 1 i agarose gel at ph 8.6, 5 h, 5 V/cm. Arrow marks origin. The isoeymes were identified by specific staining (see Methods)

4 Vol.19, N.4,1971 D. BRDICZKA and D. PETTE 549 Fig. 2. Electrophoretic resolution of (NADP) malate dehydrogenase isozymes in extra- (C) and intramitochondrial (M) fractions of rat heart. Fractions were separated by the fractional extraction technique. Conditions of electrophoresis were the same as in Fig. 1 Fig. 3. Electrophoretic analysis of (NADP) malate dehydrogenase isoenzymes in subfractions of rat liver mitochondria (cf. Table 3). Electrophoretic conditions were the same as in Fig. 1 Intramitochondrial Localization In order to determine the intramitochondrial location of isozyme 11, subfractions of liver mitochondria were prepared by the following method [1,11]. Supernatant I (S 1) obtained by incubation of mitochondria with low digitonin concentrations contains soluble enzymes adsorbed to the outer mitochondrial membrane as well as enzymes located in the outer mitochondrial compartment. After treatment with digitonin the remaining inner-membranematrix fraction is separated by centrifugation and exposed to sonification in order to release soluble enzymes of the inner mitochondrial compartment in supernatant 3 (S 3). Insoluble membrane fragments are collected in a pellet (P) by high speed centrifugation. Data in Table 3 show that 8/, of the total mitochondrial activity of (NADP) malate dehydrogenase is found in fraction S 1. This fraction contains also 99 Oi of the adenylate kinase activity and is thought to be representative of the soluble proteins of the outer mitochondrial compartment [lo-12,17,18]. Approximately 2 Ol of the mitochondrial (NAPD) malate dehydrogenase activity is detected in fractions S 3 and P, which also contain soluble and structure-bound enzymes of the citric acid cycle. This type of extractability suggests a location within the matrix space. (NADP) malate dehydrogenase derived from the outer mitochondrial compartment (fraction S 1) exhibits electrophoretic characteristics of the extramitochondrial isozyme I, whereas the enzyme from the matrix space (fraction S 3) is electrophoretically identical with isozyme I1 (Fig. 3). In these experiments, lactate dehydrogenase was chosen as a reference enzyme for extramitochondrial location. Considerable activity of this enzyme is, however, found in subfractions of liver mitochondria (Table 3). This mitochondrial activity of lactate Table 3. Distribution of different enzymes in subfractions of rat liver mitochondria obtained by the digitonin method Supernatant 1 (Sl) = soluble enzymes adsorbed to the outer mitochondrial membrane, or located in the outer mitochondrial compartment. Supernatant 3 (53) = soluble enzymes derived from the inner mitochondrial compartment. Pellet (P) = insoluble membrane fragments. Enzyme activities are given as U based on protein content of each fraction and as percentages of total mitochondrial activities Enzvme Supernatant 1 Supernatant 3 Pellet U U li OIL7 Adenylate kinase Citrate synthase (NADP) malate dehydrogenase Lactate dehydrogenase

5 55 Isozymes of (NADP) Malate Dehydrogenase Eur. J. Biochem. dehydrogenase amounts to 3 o/io of its total cellular activity. 98 o/o of the mitochondrial activity of lactate dehydrogenase is found within fraction S 1, which contains enzymes from the outer membrane and the outer mitochondrial compartment. Specific activity of lactate dehydrogenase in the fraction S 3 derived from the inner compartment is smaller by a factor of 16 compared to the first fraction S 1. On the contrary, the specific activity of (NADP) malate dehydrogenase is lower in fraction S 3 than in fraction S 1 by a factor of only It appears thus that lactate dehydrogenase in fraction S 3 is due to contamination, whereas the (NADP) malate dehydrogenase in this fraction results from a true location within the inner compartment. As far as lactate dehydrogenase in fraction S 1 is concerned, it must be considered that this activity is either adsorbed to the outer mitochondrial membrane or is truely present within the outer mitochondrial compartment, as has recently been reported [19]. The same considerations are valid also for (NADP) malate dehydrogenase present in fraction S 1. DISCUSSION The observation of extra- and intramitochondrial isozymes of (NADP) malate dehydrogenase in a variety of tissues extends earlier findings [6-8, 21. The intracellular distribution of the enzyme appears to be in some cases organ-specific (e. g. heart) and in others species-specific (e. 9. kidney). The almost exclusively extramitochondrial location of the enzyme in liver and especially in adipose tissue [i5] is consistent with the main site of lipogenesis and with the concept that (NADP) malate dehydrogenase provides NADPH for fatty acid synthesis [4,5]. Large variations in the levels of (NADP) malate dehydrogenase in livers of different species indicate probable differences in lipogenetic activity (e. g. practically no activity in beef liver and 69 U/g total activity in humming-bird liver) or suggest that this reaction is not the major source of NADPH for fatty acid synthesis. Further metabolic functions of (NADP) malate dehydrogenase may be deduced from the relatively high total activities in heart, adrenal cortex, brain and kidney and from the presence of this enzyme in mitochondria of these tissues. Based on the existence of two isozymes of (NADP) malate dehydrogenases in adrenal cortex Simpson and Estabrook have already postulated a malate shuttle [8] effecting the transfer of NADPH reducing equivalents into the mitochondria, thus providing NADPH for 1 I/% hydroxylation of steroids. The authors proposed that the extramitochondrial enzyme might operate to produce malate from pyruvate. The NADPH for this reaction could be generated by the action of the pentose phosphate cycle. Kinetic parameters of extra- and intramitochondrial enzymes in adrenal cortex suggest that the mitochondrial enzyme preferentially operates in the direction from malate to pyruvate, whereas the extramitochondrial enzyme favours the reverse reaction. It is unknown of course, whether this malate shut.tle [8) operates also in other tissues. With regard to the function of (NADP) malate dehydrogenase in heart, it is remarkable that a constant percentage distribution of extra- and intramitochondrial isozymes was observed in so many different species (Table 1). This suggests an essential role of the two isozymes in heart metabolism. One function, especially of the mitochondrial isozyme, might be replenishing the dicarboxylic acid pool in order to maintain the operation of the citric acid cycle during fatty acid oxidation. The activity level of intramitochondrial (NADP) malate dehydrogenase in heart is remarkably high and is comparable with that of the extramitochondrial isozyme in liver. NADPH for reductive carboxylation in heart mitochondria might well be provided from the NADPlinked isocitrate dehydrogenase reaction. An interesting property of mitochondrial (NADP) malate dehydrogenase in heart (but also in adrenal cortex) is that the production of pyruvate from malate is inhibited by acetyl-coa [ZO]. This might be a mechanism to hinder an efflux of malate out of the citric acid cycle at high levels of acetyl-coa. A further metabolic function of (NADP) malate dehydrogenase might be linked to transhydrogenation. This reaction involves an interplay with pyruvate carboxylase and (NAD) malate dehydrogenase C5.211: I. Pyruvate + CO, + ATP + Oxalacetate + ADP. 2. Oxalacetate + NADH + I-Ic -i- T,-malate + NAD 3. L-malate + NADP+ --f pyruvate + CO, + NADPH + H+. NADH + H+ + NADP + ATP --f NAD+ + NADPH + H+ + ADP. With regard to the activity levels of pyruvate carboxylase in rat tissues [as], this ATP-requiring transhydrogenation might be operative within the intramitochondrial compartment of kidney, brain and heart. Considering the extramitochondrial location of (NADP) malate dehydrogenase and the exclusively intramitochondrial location of pyruvate carboxylase in liver and adipose tissue [22j, an operation of this transhydrogenation reaction would imply oxaloacetate or malate transfer across the mitochondrial membrane in these tissues. This study was supported by the Deutsche Forschimgsgemeinschaft. REFERENCES 1. Krebs, H. A., Bull. Johns Hopkins I-lorp. 95 (1954) Utter, WL E., Ann. N.Y. Acad. Sci. 72 (1959) Ochoa, S., Veign Salles, J. R., and Oritz, P. J., J. Bwl. Ghem. 187 (195) 863.

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