Acyl group specificity of mitochondrial pools of carnitine acyltransferases

101

~iockj~ica et 3~oFhysic~ Acta, 360 (1974) 101-112 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 56474

ACYL GROUP SPECIFICITY OF MITOCHONDRIAL CARNITINE ACYLTRANSFERASES

POOLS OF

HELGE ERIK SOLBERG institute of Cli~icat ~~oche~~t~,

Un~uersjty of Oslo, ~ikshospitalet,

Oslo 1 {~or~ay~

(Received January 21st, 1974)

Summary 1. Two different pools of camitine acyltransferases are present in the inner mitochondrial membrane. 2. A new method for the selective assay of the “inner” and the “outer” pools of carnitine acyltransferases has been developed. The acyltransferases are assayed by an isotope exchange method in intact mitochond~a in the absence and the presence of external CoASH. Any endogenous external CoASH is removed by oxidation with the disulfide tetrathionate which does not penetrate the inner mitochondrial membrane. 3. This method has been used for the study of the acyl group specificity patterns of the two mitochondrial pools of carnitine acyltransferases in rat, mouse and calf liver. 4. In all three species the greatest activity in the “inner” transferase pool was found with medium-chain acylcarnitines (optimal substrate: C7 ). 5. In rat and mouse liver mitochondria the “outer” transferase pool had a specificity pattern different from that of the “inner” pool. These mitochondria had little or no activity with short-chain substrates in the “outer” pool. The optimum medium~h~ substrates were C9 or C1 0. 6. The specificity pattern of the “outer” transferase pool was verified by extracting the “outer” transferase by digitonin. 7. In calf liver mitochondria the specificity pattern of the “outer” transferase pool resembled closely that of the “inner” pool. 8. Clofibrate feeding caused increased activities only in the “inner” transferase pool of rat liver mitochondria. The short-chain activity increased 13 times, whereas the medium- and long-chain activities increased only moderately*

Present

knowledge

of camitine

acyltransferases,

relevant

to this study,

102

may be summarized as follows: (1) They catalyze the reversible acylation of (-)-carnitine by acyl-CoA derivatives [l--4] . (2) Their function is to carry acyl groups through the inner mitochondrial membrane [5-7], which represents a permeability barrier to CoASH and acyl-CoAs [S] . (3) They have a dual localization in an “outer” and an “inner” enzyme pool in that membrane [7,9-111. (4) Each of the two mitochondrial pools of CoASH/acyl-CoAs, one on each side of the inner membrane, are available only to the corresponding transferase pool [ 111, while both enzyme pools have access to the only externally located camitine and acylcamitines [ 5,8]. (5) At least three camitine acyltransferases may be distinguished on the basis of their acyl group specificities: the carnitine short-chain acyltransferase (acetyl-CoA:carnitine 0-acetyltransferase, EC 2.3.1.7) [1,2], the carnitine long-chain acyltransferase (hexadec~oyl-CoA: carnitine O-hexadec~oyltransfer~e, EC 2.3.1.X) [6,7,12], and the recently discovered [ 41 carnitine medium-chain acyltransferase [13-l 5) . The present study was undertaken to investigate the relationships between the different acyltransferases according to both their localization in two pools and their chain length specificity. Earlier studies on the specificity of carnitine acyltransferases [2,3,13,15-171, performed on purified enzymes, suffer from (1) the theoretical objection that the properties of soluble lipid-free acyltransferases may be different from those characterizing the native transferases in the phospholipid-containing inner membrane, and (2) the uncertainty from which of the two pools the purified transferases originate. Methods were therefore developed for assay of the two pools of camitine acyltr~sferases in intact mitochondria. Materials Female mice of NMRI/Bom strain (18-22 g) and male Wistar/Moll rats (150-250 g) were used. calf liver was obtained immediately after slaughter. Acyl-(-)-camitines [ 13,181 and (-)-[Ne-3 H] carnitine [ 191 were synthesized. (-)-Carnitine hydrochloride (Otsuka Pharmaceutical Co., Osaka, Japan) and clofibrate (ICI Ltd, Cheshire, Great Britain) were kind gifts from the manufacturers. Fatty acids, acyl chlorides and sodium tetrathionate were products of Fluka AG, Buchs, Switzerland. CoASH was obtained from Boehringer and Soehne GmbH, ~~nheim, Germany. Other coenzymes, nucleotides, auxiliary enzymes, ~-t~s(hydroxymethyl)methyl-2-~inoeth~e sulfonic acid, fatty acid-free bovine serum albumin and dithiothreitol were purchased from Sigma Chemical Co., St. Louis, U.S.A. The digitonin of Merck, Darmstadt, Germany, was used. Other chemicals were commercial products of high purity. Water was doubly distilled in all-quartz apparatus. Methods Mitochondria were isolated by the procedure of Myers and Slater [20]. The particles were washed once in 0.25 M sucrose and once in 0.15 M KC1 before being suspended in 0.15 M KCI. Protein was determined by the method of Lowry et aI. [21]. Radioactivity was measured as previously stated [18]. The results presented are always means of duplicates. Final concentrations in the mixtures are given.

103

Assay of carnitine acyltransferases was based upon the isotope exchange method [3,6] . The temperature was always 30°C. Acyl-(-)-carnitines with saturated, straight-chain acyl groups from CZ to C, 6 were used as substrates. The reaction mixture for assay of the “inner” pool of carnitine acyltransferases contained in a final volume of 1 ml: N-Tris(hydroxymethyl)methyl-2aminoethane sulfonic acid-KOH buffer (pH 7.4), 10 mM; KCl, 100 mM; (-)-[Me-3 H] carnitine, 500 /.LM (approx. 9 * lo5 cpm); EDTA, 2 mM; albumin, 0.63%; and mitochondria. This mixture was preincubated for 2 min to exhaust endogenous substrates. Thereafter &oxidation was inhibited by KCN, 1 mM; any external CoASH was oxidized by sodium tetrathionate, 4 mM; and the enzyme reaction was started by the addition of acyl-(-)-carnitine, usually 200 FM. The reaction was stopped after 4 or 5 min by the addition of 2 ml ethanol when substrates with chain length C+ or shorter were used. The amount of radioactivity incorporated into acylcarnitines were determined by thin layer chromatography [18]. Otherwise 2-methyl-1-propanol was used to stop the reaction, followed by extraction of the acylcarnitine into the organic phase [ 131. The same methods were used for the assay of total carnitine acyltransferase activity in mitochondria, except that tetrathionate was omitted and CoASH, 60 PM, was included in the reaction mixture. Any oxidized CoA in the stock solution was reduced by the addition of 3 times excess of dithiothreitol prior to use. The activity of the transferases in the “outer” pool was estimated as the difference between the total activity and the activity in the “inner” pool. The activities are expressed in arbitrary units (per cent of radioactivity incorporated into acylcarnitine) because of the complex kinetics of the exchange reaction [ 221. Extraction of carnitine acyltransferases by digitonin was performed as described by Hoppel and Tomec [lo] using rat liver mitochondria isolated according to Schnaitman and Greenawalt [23]. Assay of the supernatant and the pellet fractions was done by the isotope exchange method after freezing and thawing once. It was found that this treatment revealed all latent enzyme activity in the pellet fraction. Marher enzymes were assayed at 30°C in a Hitachi-Perkin-Elmer Model 124 spectrophotometer equipped with a thermostated cuvette holder and a scale expanding recorder. 3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) and glutamate dehydrogenase (EC 1.4.1.2) were assayed essentially as described by Beaufay et al. [24]. Adenylate kinase (EC 2.7.4.3) was assayed in the mixture stated by Bergmeyer [25] . Any unspecific reaction (mainly ATPase) was recorded before the adenylate kinase reaction was started by the addition of AMP. Results Assay of the “inner”poo1 of carnitine acyltransferases When the carnitine acyltransferases in intact mitochondria are assayed by the isotope exchange method using endogenous inner CoA (usually about 2 nmoles/mg of protein [26] ) any external CoA has to be removed in order to assay selectively the “inner” enzyme pool [ 271.

104 TABLE

I

OXIDATION

OF EXTERNAL

&ASH

BY TETRATHIONATE

Carnitine medium-chain acyltransferase in intact rat liver mitochondria was assayed by the isotope exchange method with the following reaction mixture (final volume 1 ml): N-t+ (hydroxymethyl) methyl2-aminoethane sulfonic acid-KOH buffer (pH 7.4). 10 mM; KCl, 100 mM; (-)-[A4e3H] -carnitine, 500 PM; ATP, 2 mM: mitochondria, 1.68 mg of protein; and additions as stated below. After a preincubation period of 3 min, KCN. 1 mM, and octanoyl-(-)-carnitine, 450 PM. was added. The reaction was run for 5 min. See Methods for further details. Additions

Radioactivity incorporated into octanoylcarnitine (%.min-l .(mg proteinfl)

None Sodium tetrathionate, 4 mM CoASH. 60 /JIM Tetrathionate, 4 mM. and CoASH,

0.65 0.63 1.17 0.62

60 PM

The disulfide tetrathionate does not penetrate the inner membrane of intact mitochondria as was shown by Skrede et al. [28]. Tetrathionate effectively oxidizes CoASH and can therefore be used for removal of any external CoASH. This method has previously been used by Skrede and Bremer [29] for studying the localization of acyl-CoA synthetases in intact mitochondria. Table I shows that the addition of tetrathionate caused a small reduction in the carnitine acyltransferase activity of intact mitochondria, indicating the presence of some external CoASH in the mitochondria used. In other experiments more CoASH obviously was present since tetrathionate caused greater decreases in activity. The same table shows that the addition of CoASH increased the carnitine acyltransferase activity, as expected when CoASH is available also to the “outer” enzyme pool. Control experiments showed that the oxidation of the small amounts of endogenous external CoASH by tetrathionate was rapid. Tetrathionate was added at the end of the preincubation period, as it was observed that its presence in the preincubation mixture caused a small, time-dependent reduction of the measured transferase activities (results not shown). This is probably related to a small leakage of inner CoA out of mitochondria [26] or to a slow penetration of tetrathionate through the inner membrane. For the same reason only short incubation periods (4 or 5 min) were used. It is essential for the tetrathionate method that mitochondria are in a good condition and that inner acyl-CoA is not lost by P-oxidation. It was found that KCN was a suitable inhibitor of oxidation and that EDTA plus albumin efficiently kept mitochondria in a contracted state. If several endogenous acyl-CoA derivatives are present in mitochondria (e.g. formed by P-oxidation) the corresponding radioactive acylcarnitines will be formed. Mitochondria were therefore always preincubated for 2-3 min in the presence of (-)-carnitine before inhibitors of respiration and acylcamitines were added, allowing endogenous substrates to be exhausted. Even then, several reaction products could be detected in variable amounts depending on the assay conditions. Usually radioactivity was found in acetylcamitine and in the

105

acylcarnitine ( Cn_? ) corresponding to one cycle of P-oxidation of the acylcarnitine (C,) used as substrate (results not shown). Stewart et al. [30] have recently reported that intermediates of the &oxidation can accumulate in mitochondria. Our observations support this view. When the reaction mixture described in Methods was used, negligible amounts of radioactivity were detected in non-substrate acylcarnitines. Only traces of radioactivity were found in addition to the carnitine peak when acylcarnitine was omitted from the mixture. Es tima tion of ex ternal transfer-use activity As the activity in both pools of camitine acyltransferases are measured together when external CoASH is present in the assay mixture, the difference between the total transferase activity (external CoASH present) and the “inner” activity (tetrathionate added) is an estimate of the activity in the “outer” acyltransferase pool. Only a small increase in total acyltransferase activity was observed when mitochondria was disrupted by sonication (results not shown). The “inner” transferase activity therefore is latent only to a small degree under the assay conditions used. However, because of this slight latency, the best estimate of the “outer” activity presumably was obtained when also the total activity was assayed on intact mitochondria. Purified camitine acyltransferases show substrate inhibition by acylcarnitines in the exchange assay [22]. This effect is more pronounced with longchain substrates. The same substrate inhibition was also observed when the

25

50

100

200

300

Fig. 1. Substrate inhibition by acylcarnitines of camitine acyltransferases in intact mouse liver mitochondria. The isotope exchange assay was used. Amount of mitochondria: 2.3 mg of protein. The concentrations of octanoyl-(-)-camitine (A). dodecanoyl-(-)-camitine (B) and hexadecanoyl-(-)-caitine (C) was “inner” activity (4 mM varied (abscissa). O-------O, total activity (60 pM CoASH present). ad, tetrathionate present). Other conditions were exactly as described in Methods. Ordinate: amount of radioactivity incorporated into acylcamitines (% . rnin-1 . (mg protein)-‘).

106

acyltransferases in intact mitochondria were assayed by the isotope exchange method (Fig. 1). At inhibiting concentrations of acylcarnitines the difference between the total and the “inner” activities was largely independent on the acylcarnitine concentration. Therefore, a slightly inhibitory concentration of acylcarnitines (200 PM) was used in most experiments. Since the optimum concentrations of short-chain acylcarnitines were much higher, some experiments were performed with very high concentrations of these substrates (up to 2 mM). The specificity patterns obtained in these experiments were nearly identical to those reported below using constant concentration (200 PM) for all substrates. Because the measured activities are plotted against the initial acylcarnitine concentrations (Fig. l), the two curves cross at low substrate concentrations. The actual equilibrium concentration of acylcarnitine obtained due to the reaction acylcarnitine + CoASH + acyl-CoA + carnitine is, however, relatively lower at high ~oASH/acylc~nitine ratios (CoASH added) than at low ratios (tetrathionate present). In the former case the exchange reaction thus actually proceeds when a lower concentration of the acylcarnitine substrates is present. Extraction of the “outer” transferase pool by digitonin Brdiczka et al. [9] and Hopped and Tomec [ 101 have previously used digitonin for selective extraction of the “outer” pool of carnitine acyltransferases from mitochondria. This method was therefore chosen for checking the specificity pattern obtained in the “outer” pool with the tetrathionate method. Table II shows the distribution of protein and some marker enzymes between the fractions obtained from rat liver mitochondria when 0.20 mg of digitonin was used per mg of mitochondrial protein. Two-thirds of the inner membrane marker (3-hydroxybutyra~ dehydrogen~e) and only 12% of the marker for the intermembrane space (adenylate kinase) were recovered in the pellet fraction. These results are in agreement with those of Hoppel and coworkers [ 10,311. In their experiments a lower digitonin to protein ratio were used. This may explain the higher amount of the matrix marker (glutamate dehydrogen~e) found in our soluble fraction. TABLE II DISTRIBUTION OF MARKER ENZYMES AND PROTEIN IN FRACTIONS EXTRACTION OF RAT LIVER MITOCHONDRIA

OBTAINED

BY DIGITONIN

An aliquot of suspended mitochondria (239 mg of protein> was treated by digitonin (0.20 mglmg protein). See Methods for details of fractionation procedure and enzymatic methods. The recoveries of adenylate kinase activity and protein were 103 and 102 %, respectively (in terms of results obtained on mitochondria before digitonin treatment). Recoveries could not be determined for 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase because insufficient amount of the mitochondrial sample was left for assay of these enzymes. The results presented are total enzyme activities (&nnoles/min) or total amount of protein (mg) in the two fractions (per cent of sum in parentheses).

Adenyiate kinase 3Hydroxybutyrate dehydrogenase Glutamate dehydrogenase Protein

Soluble fraction

Pellet fraction

495 6.13 6.90 123

58.0 12.9 6.13 120

(881 (321 (531 (511

(12) (68) (47) (49)

107

Fig. 2. Chain length specificity of fractions obtained by digitonin extraction of rat liver mitochondda (0.20 mg digitonin/mg of protein). Lower figure: soluble fraction (0.36 mg of protein in the incubation mixture). Upper figure: pellet fraction (0.89 mg of protein). The isotope exchange assay was used with the same incubation mixture as that used for assay of total transferase activity of intact mitochondria. )-eamitines: 200 @hf. Details of procedures are given in Methods. Abscissa: Concentration of acvl-(chain length of acyl group. Ordinate: amount of radioactivity incorporated into acyicarnitines (% * mm+ . (mg proteinrl ).

Fig. 2 shows that the specificity pattern obtained in the soluble fraction resembled that found in the “outer” pool by the tetrathionate method (Fig. 3, left part), i.e. relatively high activities with mediumchain and long-chain substrates (maxima at C, and C1 5’). The only difference was that the soluble digitonin fraction showed significant activities with the short-chain substrates, probably because of a partial extraction of the “inner” short-chain transferase, The pellet fraction obviously contains a mixture of “outer” and “inner” acyltr~sfer~es. This is in accordance with the obse~ation of Hoppel and Tomec IlO] that the “outer” pool of acyltransferases probably is incompletely extracted from the inner mitochondrial membrane although it is more easily solubilized by digitonin than the “inner” pool. We conclude that the extraction method cannot give pure fractions, but the experiments performed on digitonin fractions verify the “outer” specificity pattern estimated from activities found by the tetrathionate method. Further, it seems likely that the tetrathionate method gives the more true picture because it is based on the physiological barrier in the inner mitochondrial membrane, both between the two CoA-pools and between the two operationally different pools of carnitine acyltransferases. The ~tra~ionate assay was accordingly chosen for the studies reported below.

0

3

6

9

12

15

3

6

9

12

15

6

9

12

15

Fig. 3. Chain length specificity of carnitine acyltransferases of rat liver mitochondria; effect of clofibrate. The two figures to the left show results obtained on a rat fed an ordinary stock diet. The results to the right were obtained on a rat fed 0.3% clofibrate in the same diet for three weeks. The isotope exchange assay was used as described in Methods. Amounts of mitochondria used: 0.83 (left) and 0.75 (right) mg of protein. Concentration of acylcarnitines: 200 PM. The upper figures show the activities in the “inner” transferase pool (tetrathionate present in assay mixture). The lower figures show estimates of the activities in the “outer” pool (total minus “inner” activities). Abscissa: chain length of acyl group. Ordinate: amount of radioactivity incorporated into acylcarnitines (% . minWi a (mg protein)-I).

Carnitine acyltraasferases in rat and mouse liver mitochondria The chain-length specificity patterns of the “inner” and the “outer” pools of the acyltransferases in rat and mouse liver mitochondria were determined in several experiments, all showing the same pattern (Fig. 3, left part). The “inner” transferase pool had the greatest activity in the medium-chin region with a maximum at C, . The camitine short-chain and long-chain acyltransferases were, however, both present in the “inner” pool (maxima at C, and C1 6, respectively ) . The specificity pattern of the “outer” transferase pool was different from that of the “inner” pool. No significant activity was found in the short-chain region while the activities with both medium- and long-chain substrates were relatively high. The activity maxima, however, did not correspond with those found in the “inner” pool. The “outer” mediumchain acyltransferase had a maximum at C9 _ 1o and the maximum of the long-chain transferase was found at C, 4 _ , 5 in several experiments. Different concentrations of acylcarnitines were used. In one of these experiments the concentration of each acylcamitine was approximately 3 times the corresponding rC, -values reported for the purified calf liver transferase [13j (results not shown). The specificity patterns were always similar, indicating that the pattern shown in Fig. 3 is not significantly influenced by the acylcarnitine concentration used. The exchange assay shows deviations from linearity if the amount of

109

radioactivity incorporated in the acylcamitine exceeds about 20% of the amount incorporated at isotope equilibrium. Since the activity of the “outer” pool was estimated as the difference between the total and “inner” activities, the “outer” activity might be underestimated if the measured incorporation of radioactivity was too high. Therefore separate experiments with very low concentrations of mitochondria, and with all measured activities well within the linear range, was performed (results not shown). In these experiments the same specificity patterns were found, showing that the difference between the “inner” and the “outer” specificity patterns could not be explained on the basis of non-linear assay conditions. Effect of clofibrate on transferases in rat liver mitochondria In a previous study [14] it was shown that the activities of carnitine acyltransferases increased in the livers of rats fed a diet containing clofibrate. In that study only total activities were measured. The right part of Fig. 3 shows that clofibrate caused only small changes in the “outer” transferase pool, while all the additional activity was observed in the “inner” pool. Here the activity of the camitine short-chain acyltransferase increased with a factor of 13. The medium- and long-chain acyltransferase activities were only approximately doubled. The “outer” pool had no short-chain transferase neither in the clofibrate fed nor the control rat (the traces of “outer” short-chain activity seen in the clofibrate animal are within the experimental error for the high total activities measured). Carnitine acyltransferases in calf liver mitochondria The specificity of the “inner” and “outer” transferase pools of calf liver mitochondria (Fig. 4) was also determined in several experiments. The patterns always differed from those obtained with rat or mouse liver mitochondria

3

6

9

12

15

3

6

9

12

15

Fig. 4. Chain length specificity of camitine acyltramsferases of calf liver mitochondria. See legend to Fig. 3 and Methods for details. Amount of mitochondria: 0.28 mg of protein. Concentration Of acylcarktines: 200 YM.

110

(Fig. 3). Calf liver mitochondria had significant “outer” activities with shortchain substrates, while rat and mouse liver mitochondria showed no short-chain activity in the “outer” transferase pool. Moreover, the activity maxima in the “outer” pool of calf liver mitochondria were identical with those of the “inner” pool (C, , C, and C, 6 ). This was not the case with rodent mitochondria. Discussion Several methods have been used for selective assays of the “outer” and “inner” pools of carnitine acyltransferases in mitochondria. Transferases in the “outer” pool can be partially inhibited by 2-bromoacyl-CoA derivatives [ 111. Some workers [32,33] have compared the activities found in intact and disrupted mitochondria using acyl-CoA derivatives and carnitine as substrates. Hoppel and Tomec [lo] and Brdiczka et al. [9] separated easily solubilized and membrane-bound transferases by digitonin extraction of mitochondria. This method has also been used in the present study. Chase and Tubbs with coworkers [17,34] and Kopec and Fritz [15,35] have obtained different purified preparations of carnitine long-chain acyltransferases which they assume to represent completely separated “inner” and “outer” pools. Recently Brosnan et al. [36] have used antibodies which inactivate long-chain transferases in one or both pools. Since it is not possible to know when the “outer” enzyme pool is completely extracted or inactivated in the above mentioned methods, they are not suitable for the investigation of the specificity patterns of native carnitine acyltransferases in mitochondria. A new assay of the mitochondrial pools of transferases was therefore developed, using the isotope exchange method [ 3,6] and tetrathionate [ 27-291. This method can be used with intact mitochondria, as it is based on the mitochondria’s own barrier between inner and outer CoA and the two enzyme pools. The unavoidable drawback of the tetrathionate method is (1) that the “inner” and total transferase activities show a low, but significant, degree of latency under the assay conditions used, and (2) that the “outer” activities are not measured directly (estimated as the difference between the total and “inner” activities). Nevertheless, the method is capable of revealing changes which takes place in one of the two pools only, as can be seen from the results obtained by clofibrate feeding (Fig. 3). Moreover, the similarities of the patterns obtained by extraction procedures (Fig. 2) and by the tetrathionate method (Fig. 3) show that the operationally defined pools of carnitine acyltransferases (assayed by the tetrathionate method) roughly correspond to those obtained by physical separation, but the tetrathionate method probably gives a “purer” separation. By this method it has been shown that the carnitine acyltransferases in intact mouse and rat liver mitochondria have similar specificities towards acyl groups. The specificity patterns of the two pools showed differences in the localization of the activity maxima and in the relative activities in different chain-length regions. This may indicate that the two pools contain different carnitine acyltransferases, although additional evidence is necessary to verify this hypothesis.

111

The finding that rat liver mitoehondria has no “outer” camitine shortchain acyltransferases is supported by the results of Barker et al. 1321. They found that little or no short-chain transferase was available to ace&l-GoA outside the rat liver mitochond~a. The transferases of calf liver mitochondria have specificity patterns which are different from those of mouse and rat liver in two respects: (1) The “outer” transferases of calf liver are rather active with short-chain substrates. (2) The specificity patterns of the “inner” and “outer” pools are much more similar than in rat and mouse. Very different specificity patterns have been found on solub~ized and purified carnitine acyltransferases [ 2,3,13,15-1’73. These patterns are therefore difficult to correlate with those observed on intact mitochondria in the present study. It is possible that delipidization, solubilization and purification alter the acyl group specificity of the enzymes, possibly by inducing a different tertiary structure of the enzyme protein. The view that the properties of the transferase may be changed during purification is supported by the results of Kopec and Fritz [35]. Their fraction GPT-II (assumed to present the “inner” long-chain acyltransferase) can catalyze only the reaction from acylcarnitine to acyl-CoA. This irreversibility is probably an artifact induced by purification as our results show that the “inner” transferase pool of intact mitochondria is capable of catalyzing isotope exchange between earnitine and acylcamitine, and since ~tramitochond~~ly produced acyl-GoA derivatives can be converted to a~ylc~itines [5,18]. The fact that the inhibitory effect of hexadec~oyl-(~)-c~itine observed on membrane-bound long-chain acyltransferase is lost when the transferases is solubilized and purified [37], also shows that the enzyme may change its properties during pu~fication procedures. The identity of the different preparations of purified carnitine acyltransferases remains obscure. Some workers [15,17,34,35] claim that they have succeeded in separating and purifying long-chain acyltransferases from each of the two pools. According to the view of Tubbs and coworkers [17,34] the “inner” mitochond~al pool contains two separate transferases (c~itine short chains and long-chain acyltransfer~es), in contrast to the “outer” pool which contains only one transferase with a very broad acyl group specificity. The specificity pattern found by us in the “outer” transferase pool of calf liver mitochondria (Fig. 4) shows some similarities with their “outer” transferase preparation, but our results may suggest the existence of at least two “outer” transferases. However, no definite conclusions about the number of transferases in the “‘outer” pool is permitted. It is probable that the “inner” transferase pool contains at least three separate transferases differing in their acyl group specificity. All mitochondria studied here had a prominent activity in the medium-chain region. This activity is not explainable on the basis of the specificities reported for the carnitine short-chain acyltransferase [ 2,161 or the camitine long-chain acyltransferase [3]. The medium-chain activity does not correlate with the short-chain activity in mitochond~a of different organs [4], during pu~fication procedures 113) or in clofibrate feeding experiments [ 141 (Fig. 3). Moreover, the medium-chain activity is found in preparations showing low or no long-chain activity [4,15]

112

and it shows variations in activity independent of that of the long-chain transferase during pu~fication [ 131 and clofibrate feeding [ 143 . Although some of these results admittedly are difficult to interprete because the transferases are localized in two pools, the combined evidence support our view that mitochondria contain a separate carnitine medium-chain acyltransferase. The results presented in this study, show that this mediumchain transferase is present in the “inner” transferase pool. Whether the “outer” pool also contains several carnitine acyltransferases or only one enzyme with a broad specificity pattern remains an open question. The physiological significance of the high medium-chain transferase activity in liver mitochondria also is obscure at present. Acknowledgement Professor Jon Bremer is acknowledged for helpful suggestions and criticism. The technical assistance of Aasa Hegre Jenssen is greatly appreciated. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

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