Biochimica et BiophysicaActa, 756 (1983) 271-278 271 Elsevier Biomedical Press

BBA21414

INTRACELLULAR D I S T R I B U T I O N OF FUMARASE IN RAT SKELETAL MUSCLE

JULIAN SWIERCZYIqSKI, PIOTR W.D. SCISLOWSKI, ZENON ALEKSANDROW1CZ and MARIUSZ M. ZYDOWO *

Department of Biochemistry, I.B.M. School of Medicine, 80-211 Gda~isk, Dfbinki I (Poland)

(Received November 2nd, 1982)

Key words: Fumarase," Intracellular distribution; (Rat skeletal muscle)

The distribution of fumarase activity between the mitochondrial and cytoplasmic compartments of rat skeletal muscle was studied using the method of Fatania and Dalziel (Biochim. Biophys. Acta 631 (1980) 11-19), fractional extraction technique and a method based on the calculation of mitochondrial protein content in the tissue and on the determination of fumarase activity both in the tissue homogenate and in the isolated mitochondria. We found 10%, 5% and 0% of the total fumarase activity in the cytoplasm using these methods, respectively. The results suggest that no more than 10% of the total fumarase activity is present in the cytosolic fraction of rat skeletal muscle. The metabolic consequences of such distribution of fumarase in skeletal muscle are discussed.

Introduction

Aragon and Lowenstein [1] showed that the total level of citric acid cycle intermediates in rat skeletal muscle markedly rises during exercise. They suggested that the operation of purine nucleotide cycle is sufficient to account for the observed increase of citric acid cycle intermediates in skeletal muscle. Thus it is possible to conclude that the purine nucleotide cycle is involved in the anaplerotic supply of Krebs cycle intermediates in skeletal muscle [2]. This conclusion gained strong support from the recent experiments performed on isolated mitochondria [3]. Purine nucleotide cycle reactions are taking place in cytosol [2,4,5]. Thus, fumarate formed must reach intramitochondrial space. Theoretically this may be achieved by two ways: either fumarate could be taken up into the mitochondria, or the conversion of fumarate to malate catalyzed by extramitochondrial fumarase could take place outside the inner mitochondrial

* To whom correspondence should be addressed.

membrane, and then malate as permeant substrate could be taken up into the mitochondria.

Previous studies showed that rat liver fumarase is localized as much in the cytosol as in the mitochondria [6,7]. Recent studies of Fatania and Dalziel [8] showed that in the bovine heart muscle cells 10% of the fumarase activity are present in the cytoplasm. The results discussed above indi- cate that the intracellular distribution of fumarase depends on the organ and species studied. So far, there has been no information on intracellular distribution of fumarase in skeletal muscle. Since in this tissue extramitochondrial fumarase might play a special role in metabolizing fumarate de- rived from adenylsuccinate and hence linking the purine nucleotide cycle with the Krebs cycle, it seemed important to investigate the intracellular distribution of this enzyme in skeletal muscle.

Recent studies of Kobayashi et al. [9] on physicochemical, catalytic and immunochemical properties of fumarase crystallized separately from the mitochondria and cytosolic fraction of rat liver, suggest that the structure of these two fumarase is very similar, if not identical. Fumarase

0304-4165/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers

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purified from both cytoplasmic and mitochondrial fractions of pig heart appeared to be identical in terms of subunit molecular weight, electrophoretic mobility on cellulose acetate, substrate kinetics and inactivation by several inhibitors [10]. Also, L i n e t al. [11] detected no clear chromatographic differences between mitochondrial and cytosolic fumarase from pig heart. One may suppose that this is also true for rat skeletal muscle fumarase. Therefore, we decided to investigate the intracellu- lar distribution of fumarase activity in rat skeletal muscle without purifying this enzyme.

Mater ia ls and M e t h o d s

Oxaloacetate, malate, pyruvate, succinate, DL- a-glycerophosphate, acetyl-CoA, NADH, rote- none, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 2,6-dichlorophenylindophenol (DCIP), phenazine methosulphate, L-ascorbic acid (sodium salt), cyto- chrome c (horse heart type III), lubrol WX and Tris-base were from Sigma Chemicals Co (USA). Triton X-100 was from Serva (Feinbiochemica, Heidelberg) and N,N,N'N'-tetramethyl-p-phenyl- enediamine dihydrochloride (TMPD) was from BDH Chemicals Ltd (U.K.). All other chemicals were from P.O.Ch. Gliwice (Poland)

Male Wistar rats maintained on a commercial rat diet were used for experiments.

Enzyme assays. These were made by initial rates measurements at 30°C in a Specord ultraviolet Vis recording spectrophotometer with 1 ml (citrate synthase, fumarase and lactic dehydrogenase) or 2.5 ml (succinic and a-glycerophosphate dehydro- genase) assay in the cells of 1 cm light path length. The reactions were monitored at 412 nm for citrate synthase; at 250 nm for fumarase; at 340 nm for lactic dehydrogenase and at 600 nm for succinic and a-glycerophosphate dehydrogenase. The assay mixtures were: for citrate synthase as described by Srere [12] except that pH was 7.8; for fumarase the medium contained 50 mM potassium phosphate buffer pH 7.3, and 25 mM L-malate; for lactic dehydrogenase 50 mM potassium phosphate buffer pH 7.4, 0.15 mM N A D H and 1 mM pyruvate; for succinic dehydrogenase the same as described by Swierczyfiski and Davis [13]; for a-glyc- erophosphate dehydrogenase the same as for suc- cinic dehydrogenase except that succinate was re-

placed by 20 mM a-glycerophosphate. The homo- genate or mitochondria used for the determination the last two enzymes had been preincubated for 5 rain at 30°C in the assay mixture before the reac- tion was initiated by the addition of DCIP and phenazine methosulphate. Simultaneous blanks with substrates omitted were carried out for all enzymes activities studied.

Cytochrome c oxidase was assayed polaro- graphically by measuring oxygen consumption with a Clark electrode at 30°C. The reaction mixture contained: 50 mM potassium phosphate buffer (pH 7.4), 7.5 mM sodium ascorbate, 0.15 mM cytochrome c, 0.3 mM TMPD and the enzyme (homogenate or mitochondria) in a total volume of 2.4 ml. The reaction was initiated by the addition of cytochrome c, and the correction were made for the rate of oxygen consumption prior to the addi- tion of substrate.

Fractional extraction of fumarase, citrate syn- thase and lactic dehydrogenase. These were made following the procedure of Fatania and Dalziel [8] with minor modifications. 10 g of fresh minced muscle (mixed type) were suspended in 50 ml of the medium containing: 100 mM KCI, 3% glycerol and 20 mM Tris-HC1 pH 7.8, stirred for 5 min with a magnetic stirrer and centrifuged at 20 000 × g for 10 min. The supernatant was removed, the residue was extracted for 30 min again with 100 ml of the same medium and centrifuged at 20 000 × g for 20 min. This procedure was repeated twice. Then the residue was suspended in 100 ml of 0.1 M sodium phosphate buffer pH 7.8, stirred for 30 min and centrifuged at 20000 x g for 20 min. Finally, the residue was suspended in 100 ml of 0.1 M sodium phosphate buffer (pH 7.8) contain- ing 0.1% Triton X-100 and homogenized with a motor-driven teflon pestle homogenizer. The ho- mogenate was centrifuged at 20 000 x g for 20 min and the supernatant decanted. The pellet was sus- pended again in 0.1 M sodium phosphate buffer pH 7.8, containing 0.1% Triton X-100 and centri- fuged as before. The last two supernatants were combined. The supernatants from all the succesive extractions were assayed for citrate synthase, fumarase and lactate dehydrogenase. In control experiments 10 g tissue (wet weight) was homoge- nized in 0.1 M sodium phosphate buffer pH 7.8, containing 0.1% Triton X-100. The homogenate

was then centrifuged at 20 000 x g for 20 min. The supernatant was decanted and the pellet was sus- pended again in the same medium and centrifuged as before. These two supernatants were combined and assayed for enzyme activity. It should be pointed out that there were no significant dif- ferences between the total activities of all the enzymes tested in the control experiments and in the procedure described above.

Distribution of citrate synthase and fumarase ac- tivities in rat skeletal muscle homogenates. Rat skeletal muscle (both red and white) obtained from the hind legs immediately after decapitation were freed of connective and adipose tissue, minced finely with scisors and rinsed thoroughly with isotonic KC1. The muscle (about 100 g wet weight) was then suspended in 500 ml of cold isolation medium containing: 0.21 M mannitol, 0.07 M sucrose, 0.01 M EDTA and 0.01 M Tris-HC1 pH 7.8, and was mixed for 30 s on a vortex mixer. The resulting homogenate was centrifuged at 600 x g for 5 min and supernatant filtered through chee- secloth. The volume of the supernatant was mea- sured and the activity of citrate synthase and fumarase was assayed as soon as possible both in the presence of Triton X-100 and in its absence. Assuming that the assay in the absence of Triton X-100 represents the enzyme activity in cytosol only and the assay in the presence of Triton X-100 is the measure of total enzyme activity (contained both in the cytosol and in the mitochondria) the percentage of the activities present in the cytosol were calculated (Table I, method A).

The distribution of fumarase and citrate syn- thase activities in rat skeletal muscle homogenate was also measured by quantitative separation of the mitochonria (Table I, method B). The 600 x g supernatant was centrifuged at 20000 x g for 15 min. The pellet was washed by suspending it in the i sola t ion m e d i u m ( m a n n i t o l / s u c r o s e / T r i s - HC1/EDTA) and centrifuging at 20 000 x g for 15 min. The both supernatants were combined and assayed for citrate synthase and fumarase to ,give est imate of the activities in cytosol. The mitochondrial pellet was suspended in 100 ml of the medium containing 0.25 M sucrose plus 10 mM Tris-HCl pH 7.4, plus 2 mM EDTA and the activities of fumarase and citrate synthase were determined as described above in the presence of 0.1% Triton X-100.

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Preparation of the extracts for measuring total cell enzyme activity

Citrate synthase and fumarase. Fresh tissue (ap- prox. 1 g wet weight) were cut into small pieces and suspended in 10 ml of 0.1 M potassium phos- phate buffer pH 7.3, containing 0.1% Triton X-100 and extracted by homogenizing the suspension with a motor-driven teflon pestle homogenizer size A (Arthur H. Thomas Company Philadelphia, PA, USA) for 5 min at 0°C. The resulting homogenate was then centrifuged at 20 000 x g for 20 min. The obtained supernatant was used for citrate synthase and fumarase activities determination. Enzyme as- says were carried out as soon as possible after centrifugation.

Succinate and c~-glycerophosphate dehydro- genases. Fresh tissue (approx. 1.5 g wet weight) was cut into small pieces, suspended in 60 ml of 50 mM potassium phosphate buffer (pH 7.4) contain- ing 200 mM KC1 and extracted by homogenizing the suspension in the MSE homogenizer for 4 x 15 s. The resulting homogenate was used for enzyme assays. Usually 0.1 ml or 0.2 ml of homogenate was used for succinate and c~-glycerophosphate dehydrogenase determination. Before carrying out the assays for succinic dehydrogenase activity, ho- mogenates were left at 0°C for 2 h.

Cytochrome c oxidase. These were made in the conditions described for succinate and a- glycerophosphate dehydrogenases except that the medium was supplemented with lubrol WX (15 mg per 100 ml of medium). Prepara t ion of mitochondria: Mitochondria from rat skeletal muscle were prepared essentially as described in Ref. 14; from the heart as previously reported [13] and from liver and kidney cortex following John- son and Lardy procedure [15]. Mitochondria from all sources were finally suspended in 0.25 M sucrose, 10 mM Tris-HCl pH 7.4, and 1 mM EDTA. Protein was determined by the biuret method. Mitochondria from all sources used for citrate synthase and fumarase activity determina- tions were diluted with 250 mM sucrose contain- ing 10 mM Tris-HC1 pH 7.4, and 0.1% Triton X-100 before the assay.

Rat skeletal muscle mitochondria used for suc- cinate dehydrogenase and a-glycerophosphate de- hydrogenase activities assays were diluted with 200 mM KC1 containing 50 mM potassium phosphate

274

buffer (pH 7.4) (approx. 20 mg mitochondrial protein per 50 ml of medium) and 0.1 ml was used for the enzyme determination.

Approx. 20 mg mitochondrial protein were sus- pended in 4 ml of 200 mM KC1 plus 50 mM potassium phosphate buffer (pH 7.4) containing lubrol WX (15 mg per 100 ml of medium). After 2 min the suspension was diluted with 200 mM KC1 plus 50 mM potassium phosphate buffer pH 7.4 and 50 ~1 were used for cytochrome c oxidase activity assay.

Results and Discussion

During the typical procedure of the isolation of mitochondria from rat skeletal muscle a large pro- portion of fumarase activity in the post- mitochondrial fraction was found (see Table I). Since rat skeletal muscle is difficult to homogenize, the method used by us [14] may have damaged the mitochondria, so that soluble mitochondrial en- zyme appeared in the cytoplasmic fraction. There- fore it was decided to investigate intracellular dis- tribution of fumarase in skeletal muscle by using

TABLE 1

INTRACELLULAR DISTRIBUTION OF FUMARASE ESTIMATED IN 6 0 0 × g SUPERNATANTS FROM THE HOMOGENATES OF RAT SKELETAL MUSCLE

The total activities of fumarase and citrate synthase in 600 x g supernatants, and the percentages in free solution, were esti- mated in two ways: (A) from the assays of the 600x g super- natants with and without the addition of Triton X-100 and (B) by centrifugation of the 600 x g supernatant at 20000 x g and the separate assays of the supernatants and the pellet, after release of the enzymes by Triton X-100. Mean values were used to calculate the percentage of the activity of fumarase in the cytoplasm, on the assumption that all the citrate synthase in free solution originates from broken mitochondria. The values represent the mean_S.D, for three experiments each per- formed in triplicate.

Method % activity in free solution Calculated % fumarase

citrate fumarase activity in synthase cytosol

A 27.6 + 1.86 36.2 4- 4.0 B 31.1 + 1.34 37.4_+ 2.19 Mean 29.35 36,8 10.5

the indirect, but relatively milder, technique of fractional extraction. In this technique, first de- scribed by Pette [16] the tissue is gently disrupted and stirred in a near-iso-osmotic medium; the soluble enzymes of the cytoplasm are released into the medium whereas those of mitochondria are retained in the tissue. In experiments presented in Fig. 1 the percentage of the total activities of lactic dehydrogenase (as a marker enzyme for cytosol), fumarase and citrate synthase (as a marker enzyme for mitochondrial matrix) extracted after succesive stirring of minced skeletal muscle under conditions described in the Materials and Methods was mea- sured. After this treatment approx. 80% of the total lactic dehydrogenase was released from the tissue. At the same conditions less than 9% of fumarase activity and less than 4% of the total citrate synthase activity were released. Using simi- lar technique Halper and Srere [10] showed that both citrate synthase and fumarase were released from the fresh cardiac tissue to about the same extent amounting approx. 10%. The slightly greater fumarase than citrate synthase activity extracted

I00

8O

60U

~4o -6 ~- 2O

0 I I I I I I

20 30 40 50 60 70 80 90 Time of extraction (min)

I0

Fig. 1. Extraction of citrate synthase, fumarase and lactate dehydrogenase from minced rat skeletal muscle. The cumula- tive activities extracted after each step are expressed as per- centage of the total activity released after homogenization in the presence of Triton X-100. O O, citrate synthase; z~ A fumarase; [] •, lactate dehydrogenase.

from skeletal muscle at the same conditions (Fig. 1) may be due either to the presence of a small amount of fumarase in the cytoplasmic fraction or to the easier extraction of mitochondrial fumarase than citrate synthase. It is not possible at present to choose between these alternative explanations. Whichever one is valid, it is clear that, when corrected for fumarase activity presumably re- leased with citrate synthase from the broken mitochondria (approx. 4%), no more than 5% of total fumarase activity is present in the cytosol.

In the following experiments the intracellular distribution of fumarase in skeletal muscle was studied according to the method previously de- scribed by Fatania and Dalziel [8] for determina- tion of intracellular distribution of NADP-linked isocitrate dehydrogenase and fumarase in bovine heart muscle. This method is based on the de- termination of citrate synthase and fumarase total activities in homogenate in the presence of Triton X-100 and in the absence of Triton X-100 (Table I, method A) or in the supernatant and pellet ob- tained after centrifugation of homogenate at 20000 × g (Table I, method B). Then the per- centage of the enzyme activity in the so called 'free solution' (activity which may originate from the cytosol or from mitochondria broken during ho- mogenization) can be determined. Table I indi- cates that the percentage of the fumarase activity present in 'free solution' is greater than for citrate synthase. According to Fatania and Dalziel [8] the true cytosolic activity of the enzyme examined can be calculated using the following formula:

y % = x ( 1 0 0 - y ) / ( 1 0 0 - x )

where x denotes percentage of the citrate synthase activity in the ' free solution' (presumably originat- ing from the broken mitochondria) and y is the percentage of the enzyme examined found in the ' free solution' (fumarase in this case). Using this formula we calculated the percentage of tc;tal fumarase activity present in the cytosol of rat skeletal muscle. Table I indicates that approx. 10% of the total cellular activity of fumarase was pre- sent in cytosol. These results are virtually identical to those reported by Fatania and Dalziel [8] for the intracellular distribution of fumarase in bovine heart muscle.

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Intracellular distribution of fumarase in rat skeletal muscle was also studied by the method developed by us, which is based on the calculation of mitochondrial protein content in the tissue. This value and the specific activity of mitochondrial fumarase were used to calculate the contribution of the mitochondrial fumarase activity to the total cellular activity of this enzyme. The in vivo mitochondrial protein content in skeletal muscle can be estimated by the method of Rigault and Blanchaer [17], which is based on the measurement of succinic dehydrogenase (or other mitochondrial marker enzyme) in the isolated mitochondria and in the whole muscle homogenate from which the organelles are isolated. For instance mitochondrial protein content in 1 g of wet tissue can be calcu- lated by dividing the total activity of mitochondrial marker enzyme extracted from 1 g of wet tissue by the mitochondrial marker enzyme activity present in 1 mg mitochondrial protein prepared from the same tissue according to the formula: mg of mitochondrial protein per g of wet tissue = A/B, where A is the tissue marker enzyme activity ( /~mol /min per g wet tissue) and B, the mitochondrial marker enzyme activity ( /~mol/min per mg mitochondrial protein) Table II presents the mitochondrial protein content in mixed rat skeletal muscle estimated as described above. Citrate synthase, cytochrome c oxidase, succinic dehydrogenase and c~-glycerophosphate dehydro- genase were used as mitochondrial marker en- zymes. The total mitochondrial protein content calculated from these activities was between 13.1 and 15.7 mg per g wet tissue. Almost identical values for mitochondrial protein content in the mixed leg and back muscle of rats were reported by Behrens and Himms-Hagen [18].

Citrate synthase seems to be the most reliable marker enzyme of mitochondria for our purpose as it shows no latency when treated with Triton X-100, it is the mitochondrial matrix enzyme simi- lar as mitochondrial fumarase and it can be ex- tracted from the tissue under identical conditions as fumarase. O n e has to take also into considera- tion a great sensitivity of the assay method for citrate synthase, its high activity level and the fact that the mitochondrial protein content calculated from this enzyme activity gave the values virtually identical to these obtained while assaying the cyto-

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TABLE II

MITOCHONDRIAL MARKER ENZYMES ACTIVITIES AND MITOCHONDRIA CONTENT IN THE RAT LEG MIXED SKELETAL MUSCLE

For experimental conditions see text. The values represent the means_+ S.D. for the number of experiments (each measured two or three times) shown in parentheses.

Enzyme activity Mitochondria ( / tmol.min t.g i) content(mg

mitochondrial fresh tissue mitochondrial protein per g

protein fresh tissue)

Citrate synthase Cytochrome c oxidase ~ Succinic dehydrogenase b c~-Glycerophosphate dehydrogenase b

19.88__+2.04 (3) 1.509+__0.05 (3) 13.2 186.71__+5.76 (7) 14.3 _+0.53 (3) 13.1

8.25_+0.37 (7) 0.533_+0.042 (7) 15.5 2.14+__0.149 (8) 0.136-+0.023 (6) 15.7

a Expressed as/xatoms O uptake. b Expressed as tzmol DCIP reduced.

chrome c oxidase activity (Table II). Thus in the following experiments we used only citrate syn- thase for the calculation of mitochondrial protein content in skeletal muscle and other tissues. To establish intracellular distribution of fumarase ac- tivity, total activity of this enzyme in 1 g of wet tissue and in separate experiments the specific

activity of mitochondrial fumarase was determined (Table Ill). Using the formula: mitochondrial fumarase activity in 1 g of intact tissue = C x D, where C is the amount of mg mitochondrial pro- tein per g of wet tissue and D is the specific activity of mitochondrial fumarase ( # m o l / m i n per mg mitochondrial protein), one can calculate

TABLE Ili

ACTIVITIES OF CITRATE SYNTHASE AND FUMARASE IN SOME RAT TISSUES

For experimental conditions see text. The values represent the mean_+ S.D. for three experiments each measured three times. - - The assumption is made that specific activities of citrate synthase and fumarase in mitochondria from red and white muscle are the same as in mitochondria isolated from mixed type of skeletal muscle.

Tissue Enzyme activity

/ tmo l .min - l . g t tissue /~mol- rain- i. mg- t mitochondrial protein

citrate fumarase citrate synthase synthase

fumarase

Skeletal muscle (mixed type) 19.88 + 2.04 13.93 + 2.05 1.509 + 0.05 1.149 _-4- 0.03

Skeletal muscle (soleus) 30.80+ 1.6 21.29+ 1.51 - -

Skeletal muscle (white gastrocnemius) 11.15 + 1.24 7.25 + 0.29 -

Heart 124.60+ 12.5 76.22+ 11.8 1.839_+0.196 1.151 + 0~038 Liver 13.29 _+ 1.2 65.56 + 2.34 0.189 + 0.016 0.467 + 0,06 Kidney (cortex) 36.02 + 3.49 81.42 ___ 7.23 0.540 + 0.04 0,957 + 0.111

mitochondrial fumarase activity in the intact tis- sue. When the value of 13.2 mg of mitochondrial protein per g of wet skeletal muscle (Table IV) was multiplied by 1.149 / tmol /min per mg of mi tochondr ia l protein (specific activity of mitochondrial fumarase, Table III), 15.2 btmol/min per g of wet tissue fumarase appeared to be local- ized in the mitochondrial fraction of intact tissue. As can be seen in Table III the total activity of fumarase extracted from 1 g of wet skeletal muscle amounted to 13.93 /~mol/min. This suggests that skeletal muscle fumarase is located in mitochondria only. Similar calculations were performed for a typical red (soleus) and white (white gastro- cnemius) muscle. Because of difficulties to obtain enough mitochondria from soleus (this muscle is too small, so we could not employ our procedure for isolation of mitochondria from this muscle) we assumed that the specific activities of citrate syn- thase and fumarase in mitochondria isolated from red and while muscle are the same as in mitochondria isolated from mixed type of skeletal muscle. On this assumption we could calculate cellular content of mitochondrial protein and the amount of mitochondrial fumarase activity in in-

TABLE IV

C A L C U L A T E D CONTENT OF M I T O C H O N D R I A A N D M I T O C H O N D R I A L F U M A R A S E ACTIVITY IN RAT TIS- SUES

Calculations were made based on data presented in Table Ill.

Tissue Content of Fumarase % of total mitochon- activity fumarase dria (mg (/~mole. (activity mitochon- min - I. per g drial pro- g - t tissue) tissue) t e in /g wet tissue)

Skeletal muscle (mixed type) 13.2 15.2 109.1

Skeletal muscle (soleus) 20.4 23.5 110.4

Skeletal muscle (white gas trocnemius) 7.4 8.5 117.2

Heart 67.8 78.04 102.4 Liver 70.3 33.11 50.5 Kidney (cortex) 66.7 63.82 78.4

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tact tissue. The calculations shown in Table IV also suggest that skeletal muscle fumarase is located exclusively in mitochondria of both types of muscle investigated. It should be emphasised that the estimated ratio of fumarase activity per min per g of red muscle to the estimated fumarase activity per min per g of white muscle gave the value 2.8, which was essentially the same as the ratio of citrate synthase activity per min per g of red muscle to citrate synthase activity per min per g of white muscle. The ratio of the amount of mitochondrial protein per g of red muscle to the amount of the mitochondrial protein per g of white muscle gave also similar value. This provides further evidence that skeletal muscle fumarase ac- tivity is located exclusively in mitochondria.

Bearing in mind that the distribution of fumarase activity in rat liver is well established we employed our procedure for calculation of fumarase in liver mitochondrial compartment. As can be seen from Table IV about 50% of the total fumarase activity was found in mitochondria. This value agrees very well with that reported in the literature [6,7]. This provides further evidence that by multiplying the amount of mitochondrial pro- tein by specific activity of fumarase in mitochondria, the amount of mitochondrial fumarase activity in intact tissue may be calculated in a simple and reliable way. Using this method we also calculated the amount of fumarase activity present in mitochondria isolated from the heart and from the kidney cortex. The results of experi- ments presented in Table IV indicate that the heart fumarase is located in mitochondria only, whereas kidney cortex mitochondria contain ap- prox. 80% of the total tissue fumarase activity.

Comparison of the results obtained by using three different methods indicate that approx. 10%, 5% or 0% of the total fumarase activity is present in cytoplasmic fraction of rat skeletal muscle. At present no satisfactory explanation for the dis- crepancy between the results obtained by these methods can be offered. We suppose that in the study concerning intracellular distribution of enzymes such differences may be the results of the imperfection of all the methods available so far.

In conclusion, it may be stated that the results obtained tend to validate the assumption that in rat skeletal muscle no more than 10% of the total

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fumarase activity is located in cytoplasmic frac- tion. If so, the question arises how fumarate formed during the operation of the purine nucleotide cycle is further metabolized. About 1.4/.tmol/min per g of tissue fresh weight of the fumarase activity were calculated to be in cytoplasmic fraction of rat skeletal muscle (based on the assumption that 10% of total fumarase activity is present in the cytosol and that the total fumarase activity amounts ap- prox 14/~mol/min per g tissue; see Table III). On the other hand, in the rat hindleg muscle, activity of the rate-limiting enzymes of purine nucleotide cycle is approx. 0 .4 /~mol /min per g fresh weight of the tissue [19]. Comparing these figures one could speculate that even 3% of the total fumarase activity present in cytosol is adequate to deal with fumarate generated by purine nucleotide cycle. If we assume that no fumarase activity is present in the cytoplasmic fraction of rat skeletal muscle an alternative pathway is that fumarate is taken up into the mitochondria where it is converted to malate by intramitochondrial fumarase. This might be somewhat surprising because fumarate is claimed to be a metabolite non-permeant in rat liver mitochondria [20]. However some evidence that fumarate translocation in rat heart mitochon- dria is taking place has been reported recently [21 ]. It seems likely that this is also the case in rat skeletal muscle mitochondria.

Acknowledgment

This work was supported by the Polish Academy of Sciences within the framework of project ILl .2.4.

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