tyltransferase, DL-acetylcarnitine, acetoacetate, and 3-methylbutyryl-. CoA were obtained from Sigma Chemical Co.; 4-pentenoic acid was from K & K Rare and ...
Communication
THEJOURNAL OF BI~LOGICAL CHEMISTRY Vol 255, No. 18. Issue of September 25. pp. 8394-8397,1980 Printed in U.S.A.
Mechanism of the Stimulationof Branched Chain Oxoacid Oxidation in Liver by Carnitine*
(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes),2 mM NaPi, pH 7.4, at 37°C) was added 50 pl of tissue preparation followed in 3 min by 50 p1 of deaerated 12 m~ sodium 2-oxo-[ 1-“Clpentanoate in buffer. Each assay tube contained -1 mg of protein with unfractionated homogenate or -1.5 rng of protein with purified mitochon(Received for publication, December 17, 1979, and in revised form, dria. The tube was sealed and incubated in a 37°C shaking water July IO, 1980) bath. After 14 min, 0.2 ml of Hyamine hydroxide was injected in a plastic cup suspended above the solution and 0.3 ml of 25% trichloMichael E. May, R. Paul Aftring, and Maria G. Buse roacetic acid was injected into the reaction mixture. After another 60 min at 37°C for I4COz evolution and trapping, the plastic cups were From the Departments of Medacine and Eiochernistv, removed and counted as described previously (8). Each assay was Medical University of South Carolina, Charleston, done in triplicate. Additions were made to the assay buffer, as indiSouth Carolina 29403 cated in the figure legends, and the pH was adjusted to 7.4 before addition of tissue. Proteins were determined by the method of Lowry The oxidation of 4-methyl-2-oxopentanoate(a-ketoi- et al. (9). By these methods, mitochondria exhibited respiratory socaproate) by rat liver mitochondriawas shown to be control ratios of 5 to 8 with succinate as substrate. independent ofexogenous coenzyme A andexogenous Male Wistar rats (150 to 200 g) from Charles River, Inc. were used NAD’. Carnitine stimulated the oxidation of 4-methyl- in thisstudy. Lithium coenzyme A, sodium 4-methyl-2-oxopentanoate, DL-octanoyl-carnitine,DL-carnitine, L-carnitine, carnitine ace2-oxopentanoate in rat liver homogenates and mitotyltransferase, DL-acetylcarnitine, acetoacetate, and3-methylbutyrylchondria; octanoate, DL-octanoyhunitine, 4-pentenoate, and 3-methylbutyrate (isovalerate)were inhibi- CoA were obtained from Sigma Chemical Co.; 4-pentenoic acid was K & K Rare and Fine Chemicals, silicone oil was from Aldrich tory, and 2-bromopalmitate had no effect. Addition of from Chemical Co., andother chemicals of reagent quality were from carnitine was found to increase the export from the Fisher Chemical Co. ~-[l-’~C]leucine (55 mCi/mmol) was obtained mitochondria of acylcarnitines derived from 4-methyl-from New England Nuclear Co. and DL-[methyl-’4C]carnitine(47.5 2-oxopentanoate in the presence or absence of 4-pen- mCi/mmol) and ~-[4,5-~H]leucine (40 Ci/mmol) were obtained from tenoate but not in the presence of octanoylcarnitine. It Amersham Corp. Radiolabeled oxoacids were prepared from leucine is concluded that the branched chain oxoacid dehydro-by the amino acid oxidase method of Rudiger et a f . (IO). 3-Methylgenase is localized on the inner surface of the inner butyrylcarnitine was synthesized from 3-methylbutyryl-CoA and DLis regulated in [’4C]carnitineusing carnitine acetyltransferase ( I I). mitochondrial membrane and that it In experiments designed to examine metabolites of 4-methyl-2part by the intramitochondrial levels of branched chain oxopentanoate released from the mitochondria into the medium fatty acyl-CoAesters and/or free CoA. during a 15 min incubation at 37OC, aliquots (usually 250 pl) were centrifuged through a layer of silicone oil (-200 pl, p = 1.05) into 50 p1 of 20% (w/v) HC104 in an Eppendorf microcentrifuge. The uppermost layer (medium) was then drawn off and an aliquot was chroThe presence of 4-carbon and 5-carbon branched chain matographed. acylcarnitines and branched chain acyltransferase activity in Thin layer chromatography was run on either (A) cellulose or (B) a variety of tissues suggests that carnitine might be involved silica gel (Eastman Kodak Co.). RFvalues in ethylacetate:2-propanol: in the metabolism of the branched chain oxoacids derived HzO:NH40H (9:7:3:1, v/v) on cellulose for 4-methyl-2-oxopentanoate, from valine, leucine, and isoleucine (1, 2). Although two lab- acetoacetate, 3-methylbutyrylcarnitine,acetylcarnitine, and carnitine oratories have reported no effect of carnitine on the oxidation were0.67 to 0.72,0.27 to 0.30, 0.50, 0.12 to 0.14, and 0.07 to 0.10, respectively. In 1-butanolacetic acidHz0 (95515, v/v) on silica gel, of 4-methyl-2-oxopentanoate (a-ketoisocaproate) in liver (3) the RF values for the above compounds were 0.62,0.73,0.20,0.13, and and muscle mitochondria (4),significant stimulatory effects of 0.08, respectively. Ketones were visualized with 0.4% 2.4-dinitrophencarnitine on leucine oxidation by muscle (5, 6) and 4-methyl- ylhydrazine in 2 N HCl (aqueous) and carnitine and its esters with 2-oxopentanoate oxidation by muscle (5, 6) and liver homog- Dragendorff reagent (12). Radioactivity on chromatograms was deenates ( 5 ) have been reported recently. In this paper, we show termined by liquid scintillation spectrometry after cutting chromatthat carnitine stimulates the oxidation of 4-methyl-2-oxopen- ograms into sections, adding 250 pl of 80%2-propanol and counting in 6 ml of toluene/Triton X-100 (2:1, v/v) containing 0.4% 2,5-diphentanoate in rat liver probably by the depletion of inhibitory yloxazole and 0.03% 1,4-bis-[2-(5-phenyloxazoloyl) ]benzene.
metabolites, acyl coenzyme A esters, in the inner mitochondrial compartment. MATERIALS AND METHODS
Homogenates and mitochondria were prepared from rat liver by the method of Schnaitmanand Greenawalt (7) and immediately assayed for 4-methyl-2-oxopentanoatedehydrogenase (branched chain ketoacid dehydrogenase, EC 1.1.4.4) activity as follows. To 500 pl of buffer (220 m mannitol, 70 m sucrose, 2 m~ MgC12, 2 mM 4” ”
* This work was supported in part by National Research Service AwardAM06015 (to M. E. M.) and by Grant AM02001 from the National Institute of Arthritis, Metabolism, and Digestive Diseases. Portions of this work were presented at theXXth annual meeting of the Federation of American Societies for Experimental Biology, Anaheim, CA, April 1980. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
RESULTS
The purpose of this study was totest the two current hypotheses for the mechanism of carnitine stimulation of branched chain oxoacid catabolism. The first (Fig. la) is that carnitine facilitates the transport into themitochondrial matrix of the carbon skeleton of the product, an acyl-CoA ester of branched chain oxoacid dehydrogenase, thus relieving inhibition of the enzyme by the product, and releasing CoA for that enzyme ( 5 ) . This scheme presumes that the branched chain oxoacid dehydrogenase is localized on the outersurface of the inner mitochondrial membrane (13). The second hypothesis (Fig. Ib) is that carnitine removes an inhibitory product and releases CoASH by facilitating fatty acyl group transport out of the mitochondrial matrix ( 6 ) .This mechanism presumes that the branched chain oxoacid dehydrogenase is localized on the inner surface of the mitochondrial inner
8394
Carnitine and Branched Chain Oxoacid Catabolism membrane or in the mitochondrial matrix. As shown in TableI, the activity of branched chain oxoacid dehydrogenase in purified, wellcoupled mitochondria was unaffected by addition of exogenous CoASHor NAD indicating that thebranched chain oxoacid dehydrogenase is probable localized on the inner surface of the mitochondrial inner membrane, inasmuch as the purified enzyme from kidney (14) or liver (15)has an absolute requirement for these cofactors. Next, we attempted to alterthe levels of intramitochondrial 3-methylbutyryl-CoA (isovaleryl-CoA) by agents that inhibit its oxidation, or facilitate or block transport of the 3-methylbutyryl group out of the mitochondria. 4-Pentenoic acid has been shown to inhibit oxidation (Fig. 1, Site 2) and to lead to increased levels of acyl-CoA esters (16, 17). Thus, 4-pentenoic acidshould increase intramitochondrial levels of 3-methylbutyryl-CoA. D-OCtanOylCarnitine has been shown to be a potent inhibitor of the carnitine acyltransferase 11, which is localized on the inner surface of the mitochondrial membrane (Fig. 1, Site 3) (18).Thus, DL-octanoylcarnitine should block the production of 3-methylbutyrylcarnitine resulting in the accumulation of 3-methylbutyryl-CoA in the mitochondria. Carnitine addition (Fig. 1, Site 1) should enhance the formation of 3-methylbutyrylcarnitine,which is transported out of the mitochondria and thus reduce the intramitochondrial level of 3-methylbutyryl-CoA. As shown in Table I, the rate of oxidation of 4-methyl-2~COASH
motrix
j 0J
~ C A T I I ~
0
ketone bodies,i COS.
Carnitine
FACD 8 + tRCSCoA CoASH
)
Hpd'
0.
I 5-
0 ReCornitinr
Mechanism I
g RCCornitine * ? RCCOOH
8395
oxopentanoate by mitochondria is inversely related to the predicted intramitochondrial concentration of 3-methylbutyryl-CoA. Carnitine was stimulatory in the absence of other additions and in the presence of 4-pentenoate (which acts independently of the carnitine acyltransferase system), but DL-carnitine had no effect on activity in the presence of DLoctanoylcarnitine or octanoate. This suggests that theinhibition of 4-methyl-2-oxopentanoate oxidation by octanoate may proceed in part through the intramitochondrial generation of L-octanoylcarnitinefrom the endogenousL-carnitinepool with L-octanoylcarnitine competing for carnitine acyltransferase 11. Also, octanoate addition markedly lowers the intramitochondrial NAD/NADH ratio (19). 2-Bromopalmitate has been reported to inhibit the carnitine acyltransferase I on the cytosolic side (Fig. 1, Site 5) of the inner mitochondrial membrane (18).Mechanism I (Fig. la) would predict that inhibition of that enzyme would lead to accumulation of 3-methylbutyryl-CoA in the same compartment as the branched chain oxoacid dehydrogenase, which should result in inhibition of the latter. Mechanism I1 (Fig. l b ) would predict no such relationship. The data of Table I shows no effect of 2-bromopalmitate on branched chain oxoacid dehydrogenase. The levels of intramitochondrial3-methylbutyryl-CoAand subsequent metabolites can also be increased by direct addition of 3-methylbutyric acid to purified mitochondria because the mitochondrial matrix contains the CoA-activatingenzyme for medium chain fatty acids (Fig. 1, Site 4) (20).As shown in Fig. 2, 3-methylbutyrate addition significantly inhibited the oxidation of 4-methyl-2-oxopentanoate by mitochondria. DLCarnitine produced signifkant increases in decarboxylating activity at each concentration of 3-methylbutyrate but was not able to restore activity fully at higher concentrations of 3methylbutyrate. Since Paul and Adibi (5) had conducted extensive studies on the effect of carnitine on branched chain oxoacid oxidation TABLEI Effect ofperturbants the of intramitochondrial fatty acyl-CoApool upon 4-methyl-2-oxopentanoate oxidation Homogenates and mitochondria were prepared from the livers of fed rats and assayed as described under "Materials and Methods." Activities are expressed as per cent of Conevolved relative to that of controls incubated without additions. Mean control activities were 3.79 f 0.3 nmol of COa (min-mg protein)" ( n = 14) for mitochondria and 1.50 0.1 nmol of COZ (minemg protein)" (n = 4) for homogenates. Values displayed are means f S.E. Numbers of animals are in parentheses.
*
Cornitine
c&oao1
Activity with
Addition to assay medium
Inner Mitochondrial Membrone
Mitochondria
Homogenates
mM
CoASH NAD DL-Carnitine 4-Pentenoate Octanoate
4 FACD @ ketone &dies,
Cop, Hz0 b. Mechonirm
FIG. 1. Proposed mechanisms for the effect of carnitine on
branched chain oxoacid oxidation. Explanation and discussion of numbered sites are in the text. Mechanism 1 is based on Ref. 5 and mechanism 2 on Ref. 6. Abbreviations: BCOAD, branched chain oxoacid dehydrogenase; CAT, carnitine acyltransferase;FACD, fatty acyl-CoA dehydrogenase.
0.8
2.0 4.0 0.5 0.5 1.0 1.0
DL-Octanoylcarnitine 2-Bromopalmitate 0.01 4-Pentenoate + car- 0.5, 4.0 nitine octanoate + DL-C&0.5, 4.0 tine 1.0,4.0 DL-Octanoylcarnitine + 1.0, 4.0 DL-carnitine
98 f 4 (8) 105 f 5 (8) 117 f 4 (14)" 46 f 3 (8)" 41 f 1 (4)" 17 f 2 (4)" 96 5 (4) 70 f 9 (4)'.'
*
148 f 10 (4)' 73 f 5 (4)" 73 f 5 (4)h 35 f 1 (4)" 89
*5
(4)
35 f 5 (8)" 47 f 6 (4)'." 20 f 2 (4)"
Op < 0.001 versus no addition. h p< 0.05 versus no addition. c p < 0.05 versus 4-pentenoate alone. " p < 0.05 versus octanoate alone.
Carnitine and Branched Chain Oxoacid Catabolism TABLE I1 Effect ofperturbants of fatty acid catabolism on the extramitochondrial accumulation of 4-methyl-2-oxopentanoate metabolites Mitochondria (1.2 to 2.2 mg of protein in 0.3 ml of medium) were preincubated 3 to 4 min then incubated a t 37°C for 15 min with 1mM 4-methyl-2-oxo[4,5-JH]pentanoate and separated from medium as described under "Materials and Methods." Values are expressed as percentage of the radioactivity in the medium at theend of incubation recovered in the fractionsindicated, after thin layer chromatography in system A. Number of experiments are in parentheses. All experiments were carried out in duplicate. Values are means f S.E.
k*
0
Perturbant added
1
1
Medium
Ketone bodies" Branched chain 4-Methyl-2-oxoacylcarnitines" pentanoate'
45.4 t 14.4 None 36.3 k 8.2 L-Carnitine (2 mM) 4-Pentenoate (0.5mM) 24.9 f 3.4 4-Pentenoate + car- 33.1 rt 3.8 nitine DL-OCtanOykarnitine 9.9 rt 2.6d DL-Octanoylcarnitine 7.2 1.7 + L-carnitine
*
(4) 2.3 f 0.4 (4) 41.3 rt 13.0 (4) (4) 26.3 f 6.0d (4) 29.2 f 12.6 (4) (3) 2.8 f 0.3 (3) 67.7 f 3.7 (3) (3) 16.7 f 3.4" (3) 43.7 rt 6.6' (3) (3) 4.5 f 0.4 (3) 80.8 f 2.5" (3) (3) 4.0 +. 0.3 (3) 85.9 rt 1.0 (3)
" RFrange, 0.11 to 0.33. 'RF range, 0.44 to 0.56. RFrange, 0.56 to 0.78.
0
0.5
I .o
4
[3- METHYLBUTYRATE]
5
d p < 0.05 versus same column, no addition. ' p < 0.05 uersus same column, no carnitine.
(mM1
FIG. 2. Effect of 3-methylbutyricacid on oxidation of 4methyl-2-oxopentanoate by rat liver mitochondria. Mitochondria were isolated and assayed as described under "Materials and Methods" with the addition of 3-methylbutyric acid (0)or 3-methto the assay buffer before pH ylbutyric acid + 4 mM DL-carnitine (0) adjustment to 7.4. Activity is expressed as per cent of CO2 evolved relative to that of the control incubated without additions. Mean control activity was 3.510.27 nmol of COZ (min.mg.protein)" (n = 6). The number of animals used was six at each point except a t 5 mM 3-methylbutyric acid ( n = 2). Bars represent S.E. * p < 0.05; * * p < 0.01.
by rat liver homogenates, we repeated the above series of experiments with unfractionated homogenate. As shown in by Table I, the rateof oxidation of 4-methyl-2-oxopentanoate liver homogenates is also enhanced under conditions favoring depletion of intramitochondrial3-methylbutyryl-CoAand diminished under conditions favoring increased intramitochondrial3-methylbutyryl-CoA. Theinhibitory effects seen in homogenates were generally of lower magnitude than those seen in mitochondria, possibly because of alternate reactions of the 4-pentenoate and octanoate and possibly due to the known presence inhomogenates of a distinct soluble 4-methylThe latter activity 2-oxopentanoate decarboxylase (21, 22). might reasonably be expected to be insensitive to intramitochondrial levels of acyl-CoA esters. Incubation of mitochondria with 4-methyl-2-0~0-[4,5"Hlpentanoate allowed the chromatographic testing of the fate of the substrate in the presence of various perturbants. As shownin Table I1 (using chromatographicsystem A), carnitine addition resulted in the extramitochondrial appearance of "-labeled branched chain acylcarnitines in the presence or absence of 4-pentenoate. The shift of radioactivity into branched chain acfrom 4-methy1-2-0xo-[~H]pentanoate ylcarnitines was confirmed chromatographic in systemB (data not shown). This effect of carnitine was completely blocked by DL-octanoylcarnitine. In each experiment,a p a r d e l mitoDL-[methylchondrialincubation wasperformedinwhich 14C]carnitine + 2 mM L-carnitine (0.4 mCi/mmol for the L isomer) was included and 4-methyl-2-oxopentanoatewas not
radiolabeled.Whenanalyzed as describedin Table I1 and under "Materials and Methods," the extramitochondrialmedium was enriched in branched chain a~yl-['~C]carnitines, 29 f 8 (n = 6) nmol/mg of mitochondrial protein, in remarkable agreement with the ["Hlacylcarnitines 27 f 9 (n = 6) nmol/ mg of protein measured with 4-methy1-2-0~0-[4,5-~H]pentanoate in the presence of carnitine. The intramitochondrial matrix volume, -2 pl (estimated from the protein content and the reported matrix water content in microliters/mg of protein) (23) proved too low to allow reliable measurement of intramitochondrial metabolites. DISCUSSION
Our results support thelocalization of branched chain oxoacid dehydrogenaseinside the mitochondrial inner membrane. The insensitivity to exogenous CoA and NAD of an enzyme which when purified requires CoA (14, 15) suggests that the enzyme utilizes the mitochondrialcofactor pools, i . e . that the branched chain oxoacid dehydrogenase is on the inside of the inner membrane. effect The resultsin Tables I and I1 show that the carnitine on 4-methyl-2-oxopentanoate oxidation is mostconsistent with the mechanism proposed by van Hinsbergh et al. (6), shown in Fig. lb: the reaction catalyzed by branched chain oxoacid dehydrogenase produces COn and an acyl-CoA ester and the acyl-CoA ester (and/or distal metabolites) inhibits the branched chainoxoacid dehydrogenase (and/or limits the availability of intramitochondrial CoA). Carnitine and acylCoA react through the mediation of carnitine acyltransferase I1 to produce an acylcarnitine ester, and the acylcarnitine is transported out of the inner mitochondrial space. Furthermore, the basal rate of carnitine acyltransferase I1 a t endogenous levels of carnitine in isolated mitochondria is not rapid enough to deplete fully the acyl-CoA pool and becomes even more limiting upon blockage of the further oxidation of the acyl-CoA ester. Although we have not measured the intramitochondrial concentration of branched chain acyl-CoA esters in these experiments, Williamson and co-workers (19) have suggested that the production of acyl-CoA esters is increased
Branched Carnitine Oxoacid Chain Catabolism and
8397
after urovision of branched chain oxoacids or amino acids to 3. Noda, C., and lchihara, A. (1974) J. Biochem. (Tokyo) 76. 11231130 hepatocytes. 4. Odessey, R., and Goldberg, A. L. (1979) Biochem. J . 178,475-489 The model shown in Fig. Ib does not take into account the 5. Paul, H. S., and Adibi, S. A. (1978) Am. J . Physiol. 234, E494transport of branched chain oxoacids across the mitochondrial E499 membrane as a possible site of regulation. There is much 6. van Hinsbergh, V. W., Veerkamp, J . H., Engelen, P. J. M., and evidence indicating that 4-methyl-2-oxopentanoate is transGhijsen, W. J. (1978) Biochem. Med. 20, 115-124 7. Schnaitman, C., and Greenawalt, J. W. (1968) J. Cell Biol. 38, ported by the monocarboxylate translocator (24-26)which 158-175 has a low K,,, for pyruvate (t0.4mM) and a high K,,, (10 mM) 8. Buse, M. G., Biggers, J. F., Friderici, K. H., and Buse, J. F. (1972) for 4-methyl-2-oxopentanoate(24). Recent data suggest the J . Biol. Chem. 274,8085-8096 presence of an additional, cyanocinnamate-insensitive carrier 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. system for branched chain oxoacids (26). Our data (e.g.the (1951) J . Biol. Chem. 193, 265-275 inhibition of carnitine’s effects by DL-octanoylcarnitine) does 10. Rudiger, H. W., Langenbeck, U., and Goedde, H. W. (1972) Biochem. J. 126,445-446 not suggest a primary action of carnitine on these transport 11. McGarry, J. D., and Foster, D. W. (1976) J. Lipid Res. 17, 277systems. 281 The inhibitory effects of octanoate reported here with iso12. Skipsky, V. P., and Barclay, M. (1969) Methods Enzymol. 14, lated liver mitochondria are inagreement with results in 530-598 isolated hepatocytes (19). In skeletal and heart muscle, the 13. Johnson, W. A., and Connelly, J. L. (1972)Biochemistry 11, 1967effect of octanoate is not clear, since both stimulation (8,27, 1973 28) and inhibition (8, 25) of branched chain amino acid oxi- 14. Pettit, F. H., Yeaman, S. J., and Reed, L. J. (1978) Proc. Natl. Acad. Sci. U. S. A . 75,4881-4885 dation by octanoate have been reported under different conditions. The former effect has been attributed to stimulation 15. Parker, P. J., and Randle, P. J. (1978) FEBS Lett. 90, 183-186 16. Williamson, J. R., Rostand, S. G., and Peterson, M. J. (1970) J . by octanoate of branched chain oxoacid transport into the BLol. Chem. 245, 3242-3251 mitochondria (28). 17. Holland, P. C., Senior, A. E.,andSherratt,H. S. A. (1973) Finally, it should be noted that by the mechanism proposed Biochem. J. 136, 173-184 in Fig. lb, the preparation of permeable mitochondria could 18. McGarry, J. D., and Foster, D. W. (1974) Diabetes 23,485-493 result simultaneously in the loss of carnitine stimulation and 19. Williamson, J. R., Walajtys-Rode, E., and Coll, K. E. (1979) J . Biol. Chem. 254, 11511-11520 the acquisition of cofactor dependence of branchedchain 20. Aas, M. (1971) Biochim. Biophys. Acta 231, 32-47 oxoacid dehydrogenase, as has been reported recently for 21. May, M. E., Mancusi, V. J., Aftring, R. P., and Buse, M. G. (1980) muscle mitochondria (29). While the location of the enzyme Am. J. Physiol. 239, E215-E222 appears to be the same in liver and in skeletal and heart 22. Sabourin, P. J., and Bieber, L. L. (1979) Fed. Proc. 38, 283 muscle (6, 25,26, 29), mechanisms of regulation may differ 23. Parvin, R., and Pande, S. V. (1979) J. Biol. Chem.254,5423-5429 24. Halestrap, A. P. (1978) Biochem. J . 172, 377-387 among tissues. 25. Buffington, C. K., DeBuysere, M. S., and Olson, M. S.(1979) J . REFERENCES 1. Bieber, L. L., and Choi, Y. (1977) Proc. Natl. Acad. Sci. L! S. A . 74, 2795-2798 2. Choi, Y., Fogle, P., Clarke, P., and Bieber, L. L. (1977) J. Biol. Chem. 252, 7930-7931
Biol. Chem. 254, 10453-10458 26. Patel, T. B., Waymack, P.P.,and Olson, M. S. (1980) Arch. Biochem. Biophys. 201,629-635 27. Paul, H. S., and Adibi, S.A. (1976) J. Nutr. 106, 1079-1088 28. Sydevold, 0. (1979) Eur. J . Biochem. 97, 389-394 29. Van Hinsbergh, V. W. M., Veerkamp, J. H., and Glatz, J. F. C. (1979) Biochem. J . 182, 353-360
