ARCHIVES
OF BIOCHEMISTRY
AND
Proline
BIOPHYSICS
178,
Transport
387-395
(1977)
in Rat Liver Mitochondria J. MEYER
Laboratoire
de Biochimie,
Dkpartement
de Recherche Fondamentale, C.E.N.-G. et Midicale, Grenoble, France
and Universiti
Scientifique
Received June 22, 1976 Proline transport across the inner membrane of rat liver mitochondria shows the following properties: (a) It is stereospecific; the penetration of L-proline is two times faster than the penetration of nn-proline. (b) Proline is accumulated against a concentration gradient. (c) The transport of proline is enhanced in the presence of respiratory substrates such as succinate or tetramethylphenylenediamine + ascorbate; it is inhibited by uncouplers of oxidative phosphorylation. (d) Proline transport is inhibited by mersalyl andp-chloromercuribenzoate, but not by hydrophobic thiol blocking reagents; thus, proline transport involves thiol groups located in a very hydrophilic environment. The penetration of several other neutral amino acids (alanine, glycine, serine) is almost insensitive to mersalyl. These results suggest that proline does not travel across the mitochondrial membrane by free diffusion, but that its transport is mediated by a specific carrier. The rate of proline transport has been compared with the rates of the first two steps of proline oxidation: All of these rates are very similar, indicating that proline transport is not a limiting factor of proline metabolism in rat liver mitochondria.
The oxidation of proline in the liver cell is mainly mitochondrial. The first enzyme of this pathway is proline oxidase (11, which is bound to the inner side of the mitochondrial inner membrane (2); this enzyme interacts with the respiratory chain at the level of ubiquinone or cytochrome b (1). During the second step of proline oxidation, glutamate is produced by A-pyrroline-5-carboxylate dehydrogenase (EC 1.2.1.a); this enzyme is NAD dependent (3) and is found in both the mitochondria and the cytosol (2). The physiological importance of proline has been overlooked for a long time. This amino acid is one of the most active substrates of gluconeogenesis: In the perfused liver, the rate of gluconeogenesis obtained with proline as precursor is 80% of that obtained with alanine (4); similar results have been obtained with isolated rat liver cells (5). Besides, proline is one of the major free amino acids in the plasma: Its concentration is 0.15 to 0.25 mM in man (6), and 0.25 to 0.3 mM in the rat (7). It is well known that proline stimulates the synthe387 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
sis of collagen and other proteins in connective tissues. Proline may thus have a role in the development of cirrhosis. Hakkinen and Kulonen (8) have shown that administration of ethanol induced a 50% increase of proline concentration in rat livers. Recent investigations (9) suggest that proline may serve as a tool for the study of glutamate metabolism in intact liver cells. Since the plasma membrane is permeable to proline but not to glutamate, and since proline is oxidized to glutamate in the mitochondria, it is possible to load the mitochondrial compartment of hepatocytes with glutamate by incubating them in the presence of proline. For all of these reasons, the transport of proline across the mitochondrial membrane is of obvious interest. It is also worth noticing that proline is a neutral amino acid and that little is known so far about mitochondrial permeability to this kind of amino acid, although several papers have been published on this subject (lo14).
388
J. MEYER mi 0 1
GLUTAMATE AW
6CQnmoles
12
\
-4 2mm
PROLINE -
“1” /
ADP 600 rides
c
FIG. 1. Oxygen consumption by rat liver mitochondria in the presence of glutamate or proline. Mitochondria (4.1 mg) were added to 1.9 ml of the following medium: 110 mM KCl, 15 mM phosphate, 6 mM MgCl,, 10 mM glutamate or proline, pH 7.2. The temperature was 30°C. Rates of oxygen consumption are expressed in nano-atom-grams per minute per milligram.
METHODS
CHEMICALS
Rat liver mitochondria were prepared as described by Hogeboom (15). Rats were fasted for 14-16 h before sacrifice. Mitochondrial proteins were assayed by the biuret method. For swelling experiments, mitochondria (1.5 to 2 mg of protein) were added to 2 ml of a medium containing 250 mM proline (or other neutral amino acids), 20 mM Tris buffer, pH 7.5,0.5 mM EDTA, and 10 PM rotenone. The absorbance decrease was recorded at 520 nm on a Cary 15 spectrophotometer. The penetration of [14C]proline was initiated by adding mitochondria (2 to 3 mg of protein) to the incubation medium in 1.5-ml plastic centrifuge tubes and was stopped by centrifugation in an Eppendorf 3200 microcentrifuge. The radioactivity of the mitochondrial pellet was measured in an Intertechnique SL30 scintillation counter in the POPOP/ PPO’ system. For more details, see Ref. (16). The reduction of intramitochondrial NAD(P) by externally added proline was measured at 340-373 nm, using an Aminco-Chance double-beam spectrophotometer. Glutamate was assayed enzymically according to Bernt and Bergmeyer (17), and proline was assayed by the calorimetric methods of either Abeles (18) or Boctor (19). .-.1 Abbreviations used: Tris, Tris(hydroxymethyl)aminomethane; MES, morpholinoethanesulfonic acid, FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; TMPD, tetramethylphenylenediamine; p-chloromercuribenzoate; PC-m NEM, N-ethylmaleimide; PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis[2-(5-phenyloxazolyl)lbenzene.
Mersalyl, N-ethylmaleimide, p-chloromercuribenzoate, rotenone, and amino acids were purchased from Sigma (St. Louis, MO.); isatin and L-Cthiazolidine-carboxylic acid from Fluka (Buchs, Switzerland); glutamate dehydrogenase (EC 1.4.1.3) from Boehringer (Mannheim, West Germany); L-[U-‘~CI proline from CEA (Saclay, France). Avenaciolide was a generous gift from Dr. W.B. Turner, I.C.I. LM, Macclesfield, United Kingdom. RESULTS
Figure 1 shows that rat liver mitochondria consume oxygen at the same rate with proline or with glutamate as substrate; however, the P:O ratio in the presence of proline is only 2.5. This can be explained by the fact that the first step of proline oxidation involves only two phosphorylation sites, whereas the second step involves three sites. The measured P:O might thus be an average value for the two first steps of proline oxidation. Stereospecificity and Inhibitors of Proline Penetration in Rat Liver Mitochondria
In Table I we have collected the initial rates of mitochondrial swelling in the presence of various D- and L-amino acids. The permeability of mitochondria is strikingly stereospecific for all these compounds; the initial rates as well as the extents of swelling are about three times higher with the
PROLINE
TRANSPORT
389
truly specific inhibitor of proline transport; the heterocyclic analog of proline, L4-thiazolidine carboxylic acid, has no effect on mitochondrial swelling in proline, nor Amino acid Initial velocity of swelling on [ 14C]proline uptake; it competitively in(AOD/min) hibits the reduction of mitochondrial enL-Proline 0.16 dogenous NAD (Ki = 100 PM), but this DL-Proline 0.09 inhibition remains unchanged after sonication of the mitochondria. It is, therefore, L-Alanine 0.18 likely that the inhibition takes place at n-Alanine 0.06 one of the oxidative steps, and not on the translocation step. L-Serine 0.19 The inhibitory effect of hydrophilic thiol n-Serine 0.06 reagents such as mersalyl and pCMB, and L-Asparagine 0.18 the inefficiency of the lipophilic reagents n-Asparagine 0.04 avenaciolide and NEM, suggest that the -SH groups involved in proline transport D The experimental conditions are as given in the legend to Fig. 2. are located on the outer side of the mitochondrial membrane in a very hydrophilic environment. The thiols of the glutamatehydroxyl carrier, on the other hand, can only be reached by lipophilic reagents able L- than with the n-isomers. Unfortunately, we had no n-proline available, but from to penetrate in the membrane; they are thus located in a hydrophobic region, or on the difference between the mitochondrial osmotic behavior in L- and m-proline, we the matrix side of the membrane (16). The can assume reasonably that the difference phosphate-hydroxyl carrier is inhibited by between D- and L-proline would be as im- mersalyl and by NEM, the latter inhibitor portant as for the other neutral amino being less efficient (20); one can thus assume that the thiol groups involved in acids. The stereospecificity of proline transport phosphate transport are neighboring both suggests that this amino acid travels the hydrophobic membrane phase and the across the mitochondrial membrane by a hydrophilic external medium. These recarrier-mediated mechanism rather than sults are summarized on Fig. 3. by free diffusion. Another argument against free diffusion can be drawn from Fig. 2. Mitochondrial PROLINE ALANINE swelling in proline is inhibited by mersalyl (50% inhibition with 50 PM mersalyl); it is also inhibited, but less effectively, by pCMB. On the other handN-ethylmaleimide and avenaciolide, two lipophilic -SH reagents, do not inhibit proline permeation. It is noteworthy that swelling in alanine (as well as in glycine or serine, not shown in the figure) is much less sensitive FIG. 2. Effect of various thiol blocking comto mersalyl, if not insensitive. This suggests that proline does not share its trans- pounds on mitochondrial swelling in isotonic soluport system with other neutral amino tions of L-proline and L-alanine. Mitochondria (1.9 mg) were added to 2 ml of the following medium: 250 acids, except possibly with hydroxyproline mM proline (or alanine), 20 mM Tris, 0.5 mM EDTA, (mitochondria swell in hydroxyproline at 10 FM rotenone, at pH 7.5 and 25°C. The optical the same rate as in proline, and this swell- density was measured at 520 nm. The inhibitors ing is also inhibited by mersalyl). How- were present in the medium before the addition of ever, we have not been able yet to find a mitochondria. TABLE
I
STEREOSPECIFICITY OF MITOCHONDRIAL SWELLING IN ISOTONIC SOLUTIONS OF NEUTRAL AMINO ACIDS”
J. MEYER
390
PROLINE
PHOSPHATE
GLUTAMATE
MATRIX
FIG. 3. Thiols involved in the transports of glutamate, phosphate, and proline; their positions in the mitochondrial membrane as deduced from (16, 20) and Fig. 2.
“C-prd~ne
in matrix
lyov
0.75 1
(“md+q)
( v m nmoles/
min 1 mg 1
. .
0.50.
\
.
. \
0.25
U,T)x103 3.4
35
36
5. Temperature effect on [Wproline uptake. Experimental conditions as in Fig. 4, except that the incubation time was 20 s. FIG.
minutes 1
2
3
FIG. 4. Time course of [Wlproline uptake by rat liver mitochondria. Mitochondria (2.6 mg) were added to 0.9 ml of the following medium: 100 mM KCl, 10 mM Tris, 10 mM MES, 10 pM rotenone, 1 mM [Wlproline, at pH 6.5 and 15°C. For other conditions see Methods.
Quantitati‘ve Uptake
Characteristics
of Proline
Time course of [Wlproline uptake. The time course of [14Clproline uptake is shown in Fig. 4. The curve remains linear for at least 30 s (the uptake is not starting from time zero, because the reaction is not stopped by an inhibitor, but by flash centrifugation; see (16)), and equilibrium is reached after more than 3 min. The uptake
shows first-order kinetics with a time constant of 0.69 min-‘. Figure 4 also shows that mitochondria accumulate proline against a concentration gradient. After 3 min of incubation, 2.5 nmol of proline/mg of protein have been accumulated, which is equivalent to a concentration of 3 mM in the matrix space, whereas the outer concentration is 1 mM. McGivan et al. (21) have reported a similar accumulation of [14Clleucine, but the rate of uptake was too high to be followed by the usual techniques. They found an internal:external ratio of 2.5. Temperature effect. Proline uptake is less temperature dependent (Fig. 5) than other mitochondrial transport systems; we found an activation energy of 9 kcal/mol and Qlo = 1.7. Most other carrier systems have an activation energy of between 15 and 20 kcal/mol, although Bradford et al.
PROLINE
(22) reported a value of 8.8 for glutamate transport, in contradiction with our value of 17.5 kcal/mol (16). Saturation kinetics. We have not been able to demonstrate unequivocally that the initial velocity of proline uptake is a saturable function of proline concentration; in some experiments we obtained apparent K, values averaging 7 mM, but the saturation was always lost when the concentration reached about 10 mM. It must be reminded, however, that such a loss of saturation has sometimes been observed for the transports of ornithine (14) and glutamate (16). Energy-dependence of [‘4C]proline uptake. The evidence that proline is taken up
by mitochondria against a concentration gradient (Fig. 4) suggests that the transport of this amino acid is energy dependent. In order to elucidate this point, we have measured the velocity of proline uptake as a function of the mitochondrial energetic state (Table II). When mitochondria are energized by succinate, the addition of antimycin causes a dramatic decrease of proline uptake. In the presence of antimycin mitochondria can still be energized by addition of ascorbate and TMPD to the incubation medium; the suppression
TABLE
II
ENERGY DEPENDENCE OF [‘4C]P~~~~~~ UPTAKE BY RAT LIVER MITOCHONDRIA” Additions Initial rate of prolinene~ /take P minimg) Expt 1 Expt 2 None Succinate Succinate Antimycin Antimycin FCCP
+ + + + +
antimycin ascorbate TMPD ascorbate TMPD + KCN
1.5 1.8 1.1 1.5
2.0 2.0 0.9
1.1 0.3
a Mitochondria (2.3 mg) were added to 0.95 ml of the following medium: 100 mM KCl, 10 mM Tris, 10 mM MES, 10 ELM rotenone. at pH 7.0. When indicated, substrates or inhibitors were also present at the following concentrations: 5 mM succinate, 5 pg/ ml of antimycin; 5 mM ascorbate, 100 PM TMPD, 1 mM KCN, 8 PM FCCP. After 2 mm of preincubation at 2O”C, [14Clproline was added (final concentration, 1 mM), and the mitochondria were separated by centrifugation as described in Methods.
391
TRANSPORT
FIG. 6. Proton movements accompanying the transport of glutamate or proline across the mitochondrial membrane. Mitochondria (11 mg) were added to 3 ml of a nonbuffered medium (120 mM KCl, 3 pg/ml of antimycin). The substrates were added at a final concentration of 1.7 mM after the pH had been allowed to stabilize for a few minutes. Where indicated, avenaciolide was added (final concentration, 17 PM) 1 min before the addition of glutamate. The pH and the oxygen concentration of the medium were recorded simultaneously.
of this energy source by cyanide also causes a fall of the rate of proline transport. The uncoupler FCCP has an even more pronounced effect that respiratory inhibitors. These results show that proline uptake is energy dependent. The striking effect of FCCP might lead to the conclusion that proline transport in rat liver mitochondria is associated with a transfer of protons. This is actually not the case, as shown in Fig. 6. In this experiment, antimycin A was present in the incubation medium in order to prevent proton movements due to respiratory chain activity. The penetration of glutamate is coupled with an uptake of protons (23, 24); as expected, this proton penetration is inhibited by avenaciolide, an inhibitor of glutamate transport (16, 25). The uptake of proline, on the other hand, causes no proton movement. pH Effect on Proline
Transport
In agreement with the observation that proline transport is not linked to proton translocation, we found [14Clproline penetration to be only slightly pH dependent (Fig. 7). We have also measured the effect of pH on the reduction of mitochondrial NAD by proline (Fig. 7). The initial rate of this reaction is strongly pH dependent; however, it must be kept in mind that reduc-
392
J. MEYER
tion of mitochondrial NAD by exogenous proline involves not only proline permeation, but also two oxidative steps, only the second of which is NAD-linked; actually, the pH effect described in Fig. 7 looks like that reported by Strecker (3) for purified dehydrogenase. pyrroline-5-carboxylate We have also determined the K, of inIO
v (nmolcslminlmg)
tramitochondrial NAD reduction by proline. The values are 1.7 mM at pH 6.5 and 1 mM at pH 7.5, in rather good agreement with Strecker (3), who found 0.3 mM at pH 8.2 for the isolated dehydrogenase. It is therefore likely that in this type of experiment one measures the characteristics of pyrroline4Gcarboxylate dehydrogenase or eventually of proline oxidase. This led us to look for the limiting step in the process of proline oxidation by rat liver mitochondria.
.
6
/
What Is the Rate-Limiting Step in Proline Oxidation by Rat Liver Mitochondria?
.
6
/
PH 6
7
a
FIG. 7. pH effect on: (0) [W]Proline
penetration. Experimental conditions are identical to those of Fig. 4, except mitochondria (2.4 mg) and incubation time (15 s). (0) Reduction of intramitochondrial NAD by exogenous proline. Mitochondria (1.8 mgl were preincubated for 3 min in 3 ml of the following medium: 100 mM KCl, 10 mM Tris, 10 mM MES, 4 PM FCCP. Rotenone (10 PM) was then added and 2 min later, 0.67 mM proline was added. The differential absorbance at 340-373 nm is measured on an Aminco-Chance double-beam spectrophotometer.
Table III gives the rates of proline transport, proline oxidase, and pyrrolined-carboxylate dehydrogenase under physiological conditions. When the second step is corrected for a concentration of 1 mM, and the third one for a temperature of 37°C both oxidation reactions have velocities of about 5 to 12 nmol/min/mg, values that are in the same range as the rate of proline transport. Thus, it appears that proline transport can regulate proline oxidation, but that it does not slow down its metabolism to a great extent. This situation is quite different from that of glutamate, the transport of which is about 10 times less active than ita intramitochondrial oxidation (22). This point is made clear by the experiment depicted on Fig. 8. Frozen and thawed mitochondria oxidize glutamate four to five times more actively than intact mitochondria; for proline, there is almost
TABLE III KINETIC CHARACTERISTICS OF PROLINE TRANSPORT AND PROLINE OXIDATION IN RAT LIVER MITOCHONDRIA
Temperature (“C!) PH Substrate concentration b-m) u (nmol/min/mg of mitochondrial proteins) Sources
37 7.5 1 7 Calculated from the data of Figs. 5 and 6
Pyrroline-5carboxylate dehydrogenase
Proline oxydase
Transnort
37 7.2
37 7.5 15
10
0.1
40
32
1.2
Calculated from the data of Johnson and Strecker (1)
Phang et al.
(26)
30 7.5 1 5-10 Calculated from the data of Strecker (3)
PROLINE
TRANSPORT
393
tral amino acids is stereospecific, which is in disagreement with a free diffusion mechanism. We would like to point out that the uptake of glutamate and aspartate is also stereospecific (28) and that the penetration of these amino acids into the mitochondrial matrix is mediated by specific carriers. t t The effects of -SH blocking reagents inFIG. 8. Glutamate and proline oxidation by indicate that proline does not share its transtact (01 or frozen and thawed (0) rat liver mitochonport system with other neutral amino dria. Mitochondria (85 mg) were incubated in 11 ml acids. While proline uptake is inhibited by of the following medium: 100 mM KCl, 10 mM Tris, mercurials, the transport of other neutral 10 mM MES, 2.2 mM glutamate (or proline) at pH 7.4 amino acids is unaffected by such inhibiand 30°C. At the times indicated, 0.5-ml samples were withdrawn and deproteinized by addition of 0.5 tors. Furthermore, the fact that proline ml of 6% perchloric acid; the supernatant was neuuptake is inhibited exclusively by mercuritralized with 2 M K,HPO, and assayed for glutamate als and not by lipophilic SH reagents such (or proline) as indicated in Methods. as NEM makes it different from most other mitochondrial substrate carriers (Fig. 3); this originality supports the existno difTerence. This result may be related to ence of a specific carrier for L-proline. those of Childress and Sacktor (27) who Proline can be accumulated in mitoreported that the Qo, of blowfly muscle chondria against a concentration gradient mitochondria was greatly increased by (Fig. 4). We have also shown that proline sonication or freezing and thawing with transport is energy dependent (Table II) glutamate or Krebs cycle intermediates as and that the energy can be provided by substrates, but remained unchanged when respiratory chain activity. The nature of proline was used as a substrate. the high-energy intermediate remains to be established; however, as for glutamate DISCUSSION transport (29), we have found no inhibitory The results reported here do not provide effect of oligomycin, suggesting that ATP an entirely satisfactory evidence for the is not an energy transfer intermediate in existence of a specific proline carrier in the this system. mitochondrial inner membrane; two imAn alternate energy source for the acportant characteristics of carrier-mediated tive transport of proline is the membrane transport are still lacking, namely, the potential, in which case proline would enexistence of saturation kinetics and of ter the mitochond.rion accompanied by a highly specific inhibitors. The transport of cation crossing the membrane downward proline, however, shows several features from the membrane potential (positive that cannot be accounted for by free dif?u- outside). At this stage, we are not able to sion of this amino acid across the mito- determine the nature of the cation driving chondrial membrane. the active transport of proline; in S. cereoThe uptake of proline by liver mitochon- isae (30) and Halobacterium halobium dria is stereospecific. Halling et al. (13) (311,a proline proton electrogenic symport have found no stereospecificity for the up- has been evidenced. However, in rat liver take of neutral amino acids by brain or mitochondria, the proton is an unlikely liver mitochondria, but they actually com- candidate for such a role: First, the direct pared the rates of uptake only for L- and measurement of proton movements across m-alanine and L- and nL-valine. They con- the mitochondrial membrane (Fig. 6) has cluded that neutral amino acids cross the shown that the transport of proline, unlike mitochondrial membrane by free diffusion glutamate transport, is not associated in a ring configuration. Our results show with detectable proton transfers; second, markedly that the uptake of several neu- proline uptake is not pH dependent (Fig. PB
GLUTAMATE
J. MEYER
7), while other substrate uptakes are at the same time strongly pH dependent and proton-substrate cotransports (glutamate is a case in point). It must be pointed out that an active transport can be driven by a membrane potential without involving proton transfers, as proposed for lysine transport in S. aureus (32); accordingly, we suggest that proline might be cotransported with a cation such as potassium. Although we have no direct evidence for potassium movements, this hypothesis would fit our experimental results, showing that proline uptake is an active transport and is not associated with proton transfers. Whatever the energy coupling mechanism, proline uptake is energy dependent, and this makes a point in favor of carriermediated transport. The transport systems of metabolites across the mitochondrial membrane have regulatory roles in cell metabolism (33). Such a role, however, is not clear for proline transport, which has the same activity as the two first steps of proline oxidation (Table III). This might simply reflect an adaptation of the transport activity to the rates of the intramitochondrial proline-oxidizing enzymes, rather than free diffusion of proline across the mitochondrial membrane. In this respect, it is interesting to point out that some insect flight muscle mitochondria oxidize proline 50 times more rapidly than liver mitochondria do (34); it is unlikely that such a big difference is due to different rates of diffusion across the mitochondrial membrane of the two species. It is much more probable that the proline transport system of insect mitochondria is more active than that of liver mitochondria. Thus, it would be most interesting to study proline transport in flight muscle mitochondria and to determine whether this transport does or does not show more definite characteristics of a carrier system than proline transport in rat liver mitochondria. ACKNOWLEDGMENTS This investigation was supported in part by research grants from the “Centre National de la Re-
cherche Scientifique” E.R.A. No. 36, and the “Delegation a la Recherche Scientifique et Technique.”
REFERENCES 1. JOHNSON, A. B., AND STRECKER, H. J. (1962) J. Biol. Chem. 237, 1876-1882. 2. BRUNNER, G., AND NEUPERT, W. (1969) FEBS Lett. 3, 283-286. 3. STRECKER, H. J. (1960) J. Biol. Chem. 235,32183223. 4. Ross, B. D., HEMS, R., AND KREBS, H. A. (19671 Biochem. J. 102, 942-951. 5. CORNELL, N. W., LUND, P., AND KREBS, H. A. (1974) Biochem. J. 142, 327-337. 6. HOLT, L. E., JR., AND SNYDERMAN, S. E. (1964) in Mammalian Protein Metabolism (Munro, H. N., and Allison, J. B., eds.), Vol. 11, pp. 357-358, Academic Press, New York. SCHARFF, R., AND WOOL, I. G. (1966)Biochem. J. 99, 173-178. HXKKINEN, H. M., AND KUL~NEN, E. (1975) Biothem. Pharmacol 24, 199-204. HENSGENS, H. E. S. J., HENSGENS, L. H. A., MEIJER, A. J., GIMPEL, J. A., AND TAGER, J. M. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (Tager, J. M., Soling, H. D., and Williamson, J. R., eds.) pp. 331-338, North-Holland/American Elsevier, Amsterdam. 10. GARFINKEL, D. (1963) J. Biol. Chem. 238, 24402444. 11. BUCHANAN, J., POPOVITCH, J. R., AND TAPLEY, D. F. (1969) Biochim. Biophys. Actu 173, 532539. 12. JONES, M. S., AND JONES, 0. T. G. (1970) Biothem. Biophys. Res. Commun. 41, 1072-1079. 13. HALLING, P. J., BRAND, M. D., AND CHAPPELL, J. B. (1973) FEBS Lett. 34, 169-171. 14. GAMBLE, J. G., AND LEHNINGER, A. L. (1973) J. Biol. Chem. 248, 610-618. 15. HOGEBOOM, G. H. (1965) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 16-19, Academic Press, New York. 16. MEYER, J., AND VIGNAIS, P. M. (1973) Biochim. Biophys. Actu 325, 375-384. 17. BERNT, E., AND BERGMEYER, H. U. (1974) in Methods in Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 4, pp. 1704-1708, Academic Press, New York. 18. ABELES, R. H. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17, Part B, pp. 317-318, Academic Press, New York. 19. BOCTOR, F. N. (1971) Aid. Biochem. 43, 66-70. 20. GU~RIN, B., GUBRIN, M., AND KLINGENBERG, M. (1970) FEBS L&t. 10, 265-268.
PROLINE 21. MCGIVAN, J. D., BRADFORD, N. M., CROMPTON, M., AND CHAPPELL, J. B. (1973) Biochem. J. 134, 209-215. 22. BRADFORD, N. M., AND MCGIVAN, J. D. (1973) Biochem. J. 134, 1023-1029. 23. MEIJER, A. J., BROUWER, A., REIJNGOUD, D. J., HOEK, J. B., AND TAGER, J. M. (1972) Biochim. Biophys. Actu 283, 421-429. 24. PALMIERI, F., GENCHI, G., AND QUAGLIARIELLO, E. (1973) Bull. Sot. Ital. Biol. Sper. 49, 269276. 25. MCGIVAN, J. D., AND CHAPPELL, J. B. (1970) Biochem. J. 116, 37P-38P. 26. PHANG, J. M., DOWNING, S. J., VALLJZ, D. L., AND KOWALOFF, E. M. (1975) J. Lab. Clin. Med. 85, 312-317.
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27. CHILDRESS, C. C., AND SACKTOR, B. (1966) Science 154, 268-270. 28. KING, M. J., AND DIWAN, J. J. (1972) Arch. Biochem. Biophys. 152, 670-676. 29. MEYER, J. (1975) Ph.D. thesis, Grenoble University. 30. SEASTON, A., INKSON, C., AND EDDY, A. A. (1973) Biochem. J. 134, 1031-1043. 31. HUBBARD, J. S., RINEHART, C. A., AND BAKER, R. A. (1976) J. Bacterial. 125, 181-190. 32. NIVEN, D. F., AND HAMILTON, W. A. (1974)Eur. J. Biochem. 44, 517-522. 33. MEIJER, A. J., AND VAN DAM, K. (1974) Biochim. Biophys. Acta 346, 213-244. 34. BURSELL, E. (1975) Camp. Biochem. Physiol. 523, 235-238.
OF BIOCHEMISTRY
AND
Proline
BIOPHYSICS
178,
Transport
387-395
(1977)
in Rat Liver Mitochondria J. MEYER
Laboratoire
de Biochimie,
Dkpartement
de Recherche Fondamentale, C.E.N.-G. et Midicale, Grenoble, France
and Universiti
Scientifique
Received June 22, 1976 Proline transport across the inner membrane of rat liver mitochondria shows the following properties: (a) It is stereospecific; the penetration of L-proline is two times faster than the penetration of nn-proline. (b) Proline is accumulated against a concentration gradient. (c) The transport of proline is enhanced in the presence of respiratory substrates such as succinate or tetramethylphenylenediamine + ascorbate; it is inhibited by uncouplers of oxidative phosphorylation. (d) Proline transport is inhibited by mersalyl andp-chloromercuribenzoate, but not by hydrophobic thiol blocking reagents; thus, proline transport involves thiol groups located in a very hydrophilic environment. The penetration of several other neutral amino acids (alanine, glycine, serine) is almost insensitive to mersalyl. These results suggest that proline does not travel across the mitochondrial membrane by free diffusion, but that its transport is mediated by a specific carrier. The rate of proline transport has been compared with the rates of the first two steps of proline oxidation: All of these rates are very similar, indicating that proline transport is not a limiting factor of proline metabolism in rat liver mitochondria.
The oxidation of proline in the liver cell is mainly mitochondrial. The first enzyme of this pathway is proline oxidase (11, which is bound to the inner side of the mitochondrial inner membrane (2); this enzyme interacts with the respiratory chain at the level of ubiquinone or cytochrome b (1). During the second step of proline oxidation, glutamate is produced by A-pyrroline-5-carboxylate dehydrogenase (EC 1.2.1.a); this enzyme is NAD dependent (3) and is found in both the mitochondria and the cytosol (2). The physiological importance of proline has been overlooked for a long time. This amino acid is one of the most active substrates of gluconeogenesis: In the perfused liver, the rate of gluconeogenesis obtained with proline as precursor is 80% of that obtained with alanine (4); similar results have been obtained with isolated rat liver cells (5). Besides, proline is one of the major free amino acids in the plasma: Its concentration is 0.15 to 0.25 mM in man (6), and 0.25 to 0.3 mM in the rat (7). It is well known that proline stimulates the synthe387 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
sis of collagen and other proteins in connective tissues. Proline may thus have a role in the development of cirrhosis. Hakkinen and Kulonen (8) have shown that administration of ethanol induced a 50% increase of proline concentration in rat livers. Recent investigations (9) suggest that proline may serve as a tool for the study of glutamate metabolism in intact liver cells. Since the plasma membrane is permeable to proline but not to glutamate, and since proline is oxidized to glutamate in the mitochondria, it is possible to load the mitochondrial compartment of hepatocytes with glutamate by incubating them in the presence of proline. For all of these reasons, the transport of proline across the mitochondrial membrane is of obvious interest. It is also worth noticing that proline is a neutral amino acid and that little is known so far about mitochondrial permeability to this kind of amino acid, although several papers have been published on this subject (lo14).
388
J. MEYER mi 0 1
GLUTAMATE AW
6CQnmoles
12
\
-4 2mm
PROLINE -
“1” /
ADP 600 rides
c
FIG. 1. Oxygen consumption by rat liver mitochondria in the presence of glutamate or proline. Mitochondria (4.1 mg) were added to 1.9 ml of the following medium: 110 mM KCl, 15 mM phosphate, 6 mM MgCl,, 10 mM glutamate or proline, pH 7.2. The temperature was 30°C. Rates of oxygen consumption are expressed in nano-atom-grams per minute per milligram.
METHODS
CHEMICALS
Rat liver mitochondria were prepared as described by Hogeboom (15). Rats were fasted for 14-16 h before sacrifice. Mitochondrial proteins were assayed by the biuret method. For swelling experiments, mitochondria (1.5 to 2 mg of protein) were added to 2 ml of a medium containing 250 mM proline (or other neutral amino acids), 20 mM Tris buffer, pH 7.5,0.5 mM EDTA, and 10 PM rotenone. The absorbance decrease was recorded at 520 nm on a Cary 15 spectrophotometer. The penetration of [14C]proline was initiated by adding mitochondria (2 to 3 mg of protein) to the incubation medium in 1.5-ml plastic centrifuge tubes and was stopped by centrifugation in an Eppendorf 3200 microcentrifuge. The radioactivity of the mitochondrial pellet was measured in an Intertechnique SL30 scintillation counter in the POPOP/ PPO’ system. For more details, see Ref. (16). The reduction of intramitochondrial NAD(P) by externally added proline was measured at 340-373 nm, using an Aminco-Chance double-beam spectrophotometer. Glutamate was assayed enzymically according to Bernt and Bergmeyer (17), and proline was assayed by the calorimetric methods of either Abeles (18) or Boctor (19). .-.1 Abbreviations used: Tris, Tris(hydroxymethyl)aminomethane; MES, morpholinoethanesulfonic acid, FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; TMPD, tetramethylphenylenediamine; p-chloromercuribenzoate; PC-m NEM, N-ethylmaleimide; PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis[2-(5-phenyloxazolyl)lbenzene.
Mersalyl, N-ethylmaleimide, p-chloromercuribenzoate, rotenone, and amino acids were purchased from Sigma (St. Louis, MO.); isatin and L-Cthiazolidine-carboxylic acid from Fluka (Buchs, Switzerland); glutamate dehydrogenase (EC 1.4.1.3) from Boehringer (Mannheim, West Germany); L-[U-‘~CI proline from CEA (Saclay, France). Avenaciolide was a generous gift from Dr. W.B. Turner, I.C.I. LM, Macclesfield, United Kingdom. RESULTS
Figure 1 shows that rat liver mitochondria consume oxygen at the same rate with proline or with glutamate as substrate; however, the P:O ratio in the presence of proline is only 2.5. This can be explained by the fact that the first step of proline oxidation involves only two phosphorylation sites, whereas the second step involves three sites. The measured P:O might thus be an average value for the two first steps of proline oxidation. Stereospecificity and Inhibitors of Proline Penetration in Rat Liver Mitochondria
In Table I we have collected the initial rates of mitochondrial swelling in the presence of various D- and L-amino acids. The permeability of mitochondria is strikingly stereospecific for all these compounds; the initial rates as well as the extents of swelling are about three times higher with the
PROLINE
TRANSPORT
389
truly specific inhibitor of proline transport; the heterocyclic analog of proline, L4-thiazolidine carboxylic acid, has no effect on mitochondrial swelling in proline, nor Amino acid Initial velocity of swelling on [ 14C]proline uptake; it competitively in(AOD/min) hibits the reduction of mitochondrial enL-Proline 0.16 dogenous NAD (Ki = 100 PM), but this DL-Proline 0.09 inhibition remains unchanged after sonication of the mitochondria. It is, therefore, L-Alanine 0.18 likely that the inhibition takes place at n-Alanine 0.06 one of the oxidative steps, and not on the translocation step. L-Serine 0.19 The inhibitory effect of hydrophilic thiol n-Serine 0.06 reagents such as mersalyl and pCMB, and L-Asparagine 0.18 the inefficiency of the lipophilic reagents n-Asparagine 0.04 avenaciolide and NEM, suggest that the -SH groups involved in proline transport D The experimental conditions are as given in the legend to Fig. 2. are located on the outer side of the mitochondrial membrane in a very hydrophilic environment. The thiols of the glutamatehydroxyl carrier, on the other hand, can only be reached by lipophilic reagents able L- than with the n-isomers. Unfortunately, we had no n-proline available, but from to penetrate in the membrane; they are thus located in a hydrophobic region, or on the difference between the mitochondrial osmotic behavior in L- and m-proline, we the matrix side of the membrane (16). The can assume reasonably that the difference phosphate-hydroxyl carrier is inhibited by between D- and L-proline would be as im- mersalyl and by NEM, the latter inhibitor portant as for the other neutral amino being less efficient (20); one can thus assume that the thiol groups involved in acids. The stereospecificity of proline transport phosphate transport are neighboring both suggests that this amino acid travels the hydrophobic membrane phase and the across the mitochondrial membrane by a hydrophilic external medium. These recarrier-mediated mechanism rather than sults are summarized on Fig. 3. by free diffusion. Another argument against free diffusion can be drawn from Fig. 2. Mitochondrial PROLINE ALANINE swelling in proline is inhibited by mersalyl (50% inhibition with 50 PM mersalyl); it is also inhibited, but less effectively, by pCMB. On the other handN-ethylmaleimide and avenaciolide, two lipophilic -SH reagents, do not inhibit proline permeation. It is noteworthy that swelling in alanine (as well as in glycine or serine, not shown in the figure) is much less sensitive FIG. 2. Effect of various thiol blocking comto mersalyl, if not insensitive. This suggests that proline does not share its trans- pounds on mitochondrial swelling in isotonic soluport system with other neutral amino tions of L-proline and L-alanine. Mitochondria (1.9 mg) were added to 2 ml of the following medium: 250 acids, except possibly with hydroxyproline mM proline (or alanine), 20 mM Tris, 0.5 mM EDTA, (mitochondria swell in hydroxyproline at 10 FM rotenone, at pH 7.5 and 25°C. The optical the same rate as in proline, and this swell- density was measured at 520 nm. The inhibitors ing is also inhibited by mersalyl). How- were present in the medium before the addition of ever, we have not been able yet to find a mitochondria. TABLE
I
STEREOSPECIFICITY OF MITOCHONDRIAL SWELLING IN ISOTONIC SOLUTIONS OF NEUTRAL AMINO ACIDS”
J. MEYER
390
PROLINE
PHOSPHATE
GLUTAMATE
MATRIX
FIG. 3. Thiols involved in the transports of glutamate, phosphate, and proline; their positions in the mitochondrial membrane as deduced from (16, 20) and Fig. 2.
“C-prd~ne
in matrix
lyov
0.75 1
(“md+q)
( v m nmoles/
min 1 mg 1
. .
0.50.
\
.
. \
0.25
U,T)x103 3.4
35
36
5. Temperature effect on [Wproline uptake. Experimental conditions as in Fig. 4, except that the incubation time was 20 s. FIG.
minutes 1
2
3
FIG. 4. Time course of [Wlproline uptake by rat liver mitochondria. Mitochondria (2.6 mg) were added to 0.9 ml of the following medium: 100 mM KCl, 10 mM Tris, 10 mM MES, 10 pM rotenone, 1 mM [Wlproline, at pH 6.5 and 15°C. For other conditions see Methods.
Quantitati‘ve Uptake
Characteristics
of Proline
Time course of [Wlproline uptake. The time course of [14Clproline uptake is shown in Fig. 4. The curve remains linear for at least 30 s (the uptake is not starting from time zero, because the reaction is not stopped by an inhibitor, but by flash centrifugation; see (16)), and equilibrium is reached after more than 3 min. The uptake
shows first-order kinetics with a time constant of 0.69 min-‘. Figure 4 also shows that mitochondria accumulate proline against a concentration gradient. After 3 min of incubation, 2.5 nmol of proline/mg of protein have been accumulated, which is equivalent to a concentration of 3 mM in the matrix space, whereas the outer concentration is 1 mM. McGivan et al. (21) have reported a similar accumulation of [14Clleucine, but the rate of uptake was too high to be followed by the usual techniques. They found an internal:external ratio of 2.5. Temperature effect. Proline uptake is less temperature dependent (Fig. 5) than other mitochondrial transport systems; we found an activation energy of 9 kcal/mol and Qlo = 1.7. Most other carrier systems have an activation energy of between 15 and 20 kcal/mol, although Bradford et al.
PROLINE
(22) reported a value of 8.8 for glutamate transport, in contradiction with our value of 17.5 kcal/mol (16). Saturation kinetics. We have not been able to demonstrate unequivocally that the initial velocity of proline uptake is a saturable function of proline concentration; in some experiments we obtained apparent K, values averaging 7 mM, but the saturation was always lost when the concentration reached about 10 mM. It must be reminded, however, that such a loss of saturation has sometimes been observed for the transports of ornithine (14) and glutamate (16). Energy-dependence of [‘4C]proline uptake. The evidence that proline is taken up
by mitochondria against a concentration gradient (Fig. 4) suggests that the transport of this amino acid is energy dependent. In order to elucidate this point, we have measured the velocity of proline uptake as a function of the mitochondrial energetic state (Table II). When mitochondria are energized by succinate, the addition of antimycin causes a dramatic decrease of proline uptake. In the presence of antimycin mitochondria can still be energized by addition of ascorbate and TMPD to the incubation medium; the suppression
TABLE
II
ENERGY DEPENDENCE OF [‘4C]P~~~~~~ UPTAKE BY RAT LIVER MITOCHONDRIA” Additions Initial rate of prolinene~ /take P minimg) Expt 1 Expt 2 None Succinate Succinate Antimycin Antimycin FCCP
+ + + + +
antimycin ascorbate TMPD ascorbate TMPD + KCN
1.5 1.8 1.1 1.5
2.0 2.0 0.9
1.1 0.3
a Mitochondria (2.3 mg) were added to 0.95 ml of the following medium: 100 mM KCl, 10 mM Tris, 10 mM MES, 10 ELM rotenone. at pH 7.0. When indicated, substrates or inhibitors were also present at the following concentrations: 5 mM succinate, 5 pg/ ml of antimycin; 5 mM ascorbate, 100 PM TMPD, 1 mM KCN, 8 PM FCCP. After 2 mm of preincubation at 2O”C, [14Clproline was added (final concentration, 1 mM), and the mitochondria were separated by centrifugation as described in Methods.
391
TRANSPORT
FIG. 6. Proton movements accompanying the transport of glutamate or proline across the mitochondrial membrane. Mitochondria (11 mg) were added to 3 ml of a nonbuffered medium (120 mM KCl, 3 pg/ml of antimycin). The substrates were added at a final concentration of 1.7 mM after the pH had been allowed to stabilize for a few minutes. Where indicated, avenaciolide was added (final concentration, 17 PM) 1 min before the addition of glutamate. The pH and the oxygen concentration of the medium were recorded simultaneously.
of this energy source by cyanide also causes a fall of the rate of proline transport. The uncoupler FCCP has an even more pronounced effect that respiratory inhibitors. These results show that proline uptake is energy dependent. The striking effect of FCCP might lead to the conclusion that proline transport in rat liver mitochondria is associated with a transfer of protons. This is actually not the case, as shown in Fig. 6. In this experiment, antimycin A was present in the incubation medium in order to prevent proton movements due to respiratory chain activity. The penetration of glutamate is coupled with an uptake of protons (23, 24); as expected, this proton penetration is inhibited by avenaciolide, an inhibitor of glutamate transport (16, 25). The uptake of proline, on the other hand, causes no proton movement. pH Effect on Proline
Transport
In agreement with the observation that proline transport is not linked to proton translocation, we found [14Clproline penetration to be only slightly pH dependent (Fig. 7). We have also measured the effect of pH on the reduction of mitochondrial NAD by proline (Fig. 7). The initial rate of this reaction is strongly pH dependent; however, it must be kept in mind that reduc-
392
J. MEYER
tion of mitochondrial NAD by exogenous proline involves not only proline permeation, but also two oxidative steps, only the second of which is NAD-linked; actually, the pH effect described in Fig. 7 looks like that reported by Strecker (3) for purified dehydrogenase. pyrroline-5-carboxylate We have also determined the K, of inIO
v (nmolcslminlmg)
tramitochondrial NAD reduction by proline. The values are 1.7 mM at pH 6.5 and 1 mM at pH 7.5, in rather good agreement with Strecker (3), who found 0.3 mM at pH 8.2 for the isolated dehydrogenase. It is therefore likely that in this type of experiment one measures the characteristics of pyrroline4Gcarboxylate dehydrogenase or eventually of proline oxidase. This led us to look for the limiting step in the process of proline oxidation by rat liver mitochondria.
.
6
/
What Is the Rate-Limiting Step in Proline Oxidation by Rat Liver Mitochondria?
.
6
/
PH 6
7
a
FIG. 7. pH effect on: (0) [W]Proline
penetration. Experimental conditions are identical to those of Fig. 4, except mitochondria (2.4 mg) and incubation time (15 s). (0) Reduction of intramitochondrial NAD by exogenous proline. Mitochondria (1.8 mgl were preincubated for 3 min in 3 ml of the following medium: 100 mM KCl, 10 mM Tris, 10 mM MES, 4 PM FCCP. Rotenone (10 PM) was then added and 2 min later, 0.67 mM proline was added. The differential absorbance at 340-373 nm is measured on an Aminco-Chance double-beam spectrophotometer.
Table III gives the rates of proline transport, proline oxidase, and pyrrolined-carboxylate dehydrogenase under physiological conditions. When the second step is corrected for a concentration of 1 mM, and the third one for a temperature of 37°C both oxidation reactions have velocities of about 5 to 12 nmol/min/mg, values that are in the same range as the rate of proline transport. Thus, it appears that proline transport can regulate proline oxidation, but that it does not slow down its metabolism to a great extent. This situation is quite different from that of glutamate, the transport of which is about 10 times less active than ita intramitochondrial oxidation (22). This point is made clear by the experiment depicted on Fig. 8. Frozen and thawed mitochondria oxidize glutamate four to five times more actively than intact mitochondria; for proline, there is almost
TABLE III KINETIC CHARACTERISTICS OF PROLINE TRANSPORT AND PROLINE OXIDATION IN RAT LIVER MITOCHONDRIA
Temperature (“C!) PH Substrate concentration b-m) u (nmol/min/mg of mitochondrial proteins) Sources
37 7.5 1 7 Calculated from the data of Figs. 5 and 6
Pyrroline-5carboxylate dehydrogenase
Proline oxydase
Transnort
37 7.2
37 7.5 15
10
0.1
40
32
1.2
Calculated from the data of Johnson and Strecker (1)
Phang et al.
(26)
30 7.5 1 5-10 Calculated from the data of Strecker (3)
PROLINE
TRANSPORT
393
tral amino acids is stereospecific, which is in disagreement with a free diffusion mechanism. We would like to point out that the uptake of glutamate and aspartate is also stereospecific (28) and that the penetration of these amino acids into the mitochondrial matrix is mediated by specific carriers. t t The effects of -SH blocking reagents inFIG. 8. Glutamate and proline oxidation by indicate that proline does not share its transtact (01 or frozen and thawed (0) rat liver mitochonport system with other neutral amino dria. Mitochondria (85 mg) were incubated in 11 ml acids. While proline uptake is inhibited by of the following medium: 100 mM KCl, 10 mM Tris, mercurials, the transport of other neutral 10 mM MES, 2.2 mM glutamate (or proline) at pH 7.4 amino acids is unaffected by such inhibiand 30°C. At the times indicated, 0.5-ml samples were withdrawn and deproteinized by addition of 0.5 tors. Furthermore, the fact that proline ml of 6% perchloric acid; the supernatant was neuuptake is inhibited exclusively by mercuritralized with 2 M K,HPO, and assayed for glutamate als and not by lipophilic SH reagents such (or proline) as indicated in Methods. as NEM makes it different from most other mitochondrial substrate carriers (Fig. 3); this originality supports the existno difTerence. This result may be related to ence of a specific carrier for L-proline. those of Childress and Sacktor (27) who Proline can be accumulated in mitoreported that the Qo, of blowfly muscle chondria against a concentration gradient mitochondria was greatly increased by (Fig. 4). We have also shown that proline sonication or freezing and thawing with transport is energy dependent (Table II) glutamate or Krebs cycle intermediates as and that the energy can be provided by substrates, but remained unchanged when respiratory chain activity. The nature of proline was used as a substrate. the high-energy intermediate remains to be established; however, as for glutamate DISCUSSION transport (29), we have found no inhibitory The results reported here do not provide effect of oligomycin, suggesting that ATP an entirely satisfactory evidence for the is not an energy transfer intermediate in existence of a specific proline carrier in the this system. mitochondrial inner membrane; two imAn alternate energy source for the acportant characteristics of carrier-mediated tive transport of proline is the membrane transport are still lacking, namely, the potential, in which case proline would enexistence of saturation kinetics and of ter the mitochond.rion accompanied by a highly specific inhibitors. The transport of cation crossing the membrane downward proline, however, shows several features from the membrane potential (positive that cannot be accounted for by free dif?u- outside). At this stage, we are not able to sion of this amino acid across the mito- determine the nature of the cation driving chondrial membrane. the active transport of proline; in S. cereoThe uptake of proline by liver mitochon- isae (30) and Halobacterium halobium dria is stereospecific. Halling et al. (13) (311,a proline proton electrogenic symport have found no stereospecificity for the up- has been evidenced. However, in rat liver take of neutral amino acids by brain or mitochondria, the proton is an unlikely liver mitochondria, but they actually com- candidate for such a role: First, the direct pared the rates of uptake only for L- and measurement of proton movements across m-alanine and L- and nL-valine. They con- the mitochondrial membrane (Fig. 6) has cluded that neutral amino acids cross the shown that the transport of proline, unlike mitochondrial membrane by free diffusion glutamate transport, is not associated in a ring configuration. Our results show with detectable proton transfers; second, markedly that the uptake of several neu- proline uptake is not pH dependent (Fig. PB
GLUTAMATE
J. MEYER
7), while other substrate uptakes are at the same time strongly pH dependent and proton-substrate cotransports (glutamate is a case in point). It must be pointed out that an active transport can be driven by a membrane potential without involving proton transfers, as proposed for lysine transport in S. aureus (32); accordingly, we suggest that proline might be cotransported with a cation such as potassium. Although we have no direct evidence for potassium movements, this hypothesis would fit our experimental results, showing that proline uptake is an active transport and is not associated with proton transfers. Whatever the energy coupling mechanism, proline uptake is energy dependent, and this makes a point in favor of carriermediated transport. The transport systems of metabolites across the mitochondrial membrane have regulatory roles in cell metabolism (33). Such a role, however, is not clear for proline transport, which has the same activity as the two first steps of proline oxidation (Table III). This might simply reflect an adaptation of the transport activity to the rates of the intramitochondrial proline-oxidizing enzymes, rather than free diffusion of proline across the mitochondrial membrane. In this respect, it is interesting to point out that some insect flight muscle mitochondria oxidize proline 50 times more rapidly than liver mitochondria do (34); it is unlikely that such a big difference is due to different rates of diffusion across the mitochondrial membrane of the two species. It is much more probable that the proline transport system of insect mitochondria is more active than that of liver mitochondria. Thus, it would be most interesting to study proline transport in flight muscle mitochondria and to determine whether this transport does or does not show more definite characteristics of a carrier system than proline transport in rat liver mitochondria. ACKNOWLEDGMENTS This investigation was supported in part by research grants from the “Centre National de la Re-
cherche Scientifique” E.R.A. No. 36, and the “Delegation a la Recherche Scientifique et Technique.”
REFERENCES 1. JOHNSON, A. B., AND STRECKER, H. J. (1962) J. Biol. Chem. 237, 1876-1882. 2. BRUNNER, G., AND NEUPERT, W. (1969) FEBS Lett. 3, 283-286. 3. STRECKER, H. J. (1960) J. Biol. Chem. 235,32183223. 4. Ross, B. D., HEMS, R., AND KREBS, H. A. (19671 Biochem. J. 102, 942-951. 5. CORNELL, N. W., LUND, P., AND KREBS, H. A. (1974) Biochem. J. 142, 327-337. 6. HOLT, L. E., JR., AND SNYDERMAN, S. E. (1964) in Mammalian Protein Metabolism (Munro, H. N., and Allison, J. B., eds.), Vol. 11, pp. 357-358, Academic Press, New York. SCHARFF, R., AND WOOL, I. G. (1966)Biochem. J. 99, 173-178. HXKKINEN, H. M., AND KUL~NEN, E. (1975) Biothem. Pharmacol 24, 199-204. HENSGENS, H. E. S. J., HENSGENS, L. H. A., MEIJER, A. J., GIMPEL, J. A., AND TAGER, J. M. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (Tager, J. M., Soling, H. D., and Williamson, J. R., eds.) pp. 331-338, North-Holland/American Elsevier, Amsterdam. 10. GARFINKEL, D. (1963) J. Biol. Chem. 238, 24402444. 11. BUCHANAN, J., POPOVITCH, J. R., AND TAPLEY, D. F. (1969) Biochim. Biophys. Actu 173, 532539. 12. JONES, M. S., AND JONES, 0. T. G. (1970) Biothem. Biophys. Res. Commun. 41, 1072-1079. 13. HALLING, P. J., BRAND, M. D., AND CHAPPELL, J. B. (1973) FEBS Lett. 34, 169-171. 14. GAMBLE, J. G., AND LEHNINGER, A. L. (1973) J. Biol. Chem. 248, 610-618. 15. HOGEBOOM, G. H. (1965) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 16-19, Academic Press, New York. 16. MEYER, J., AND VIGNAIS, P. M. (1973) Biochim. Biophys. Actu 325, 375-384. 17. BERNT, E., AND BERGMEYER, H. U. (1974) in Methods in Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 4, pp. 1704-1708, Academic Press, New York. 18. ABELES, R. H. (1971) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17, Part B, pp. 317-318, Academic Press, New York. 19. BOCTOR, F. N. (1971) Aid. Biochem. 43, 66-70. 20. GU~RIN, B., GUBRIN, M., AND KLINGENBERG, M. (1970) FEBS L&t. 10, 265-268.
PROLINE 21. MCGIVAN, J. D., BRADFORD, N. M., CROMPTON, M., AND CHAPPELL, J. B. (1973) Biochem. J. 134, 209-215. 22. BRADFORD, N. M., AND MCGIVAN, J. D. (1973) Biochem. J. 134, 1023-1029. 23. MEIJER, A. J., BROUWER, A., REIJNGOUD, D. J., HOEK, J. B., AND TAGER, J. M. (1972) Biochim. Biophys. Actu 283, 421-429. 24. PALMIERI, F., GENCHI, G., AND QUAGLIARIELLO, E. (1973) Bull. Sot. Ital. Biol. Sper. 49, 269276. 25. MCGIVAN, J. D., AND CHAPPELL, J. B. (1970) Biochem. J. 116, 37P-38P. 26. PHANG, J. M., DOWNING, S. J., VALLJZ, D. L., AND KOWALOFF, E. M. (1975) J. Lab. Clin. Med. 85, 312-317.
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27. CHILDRESS, C. C., AND SACKTOR, B. (1966) Science 154, 268-270. 28. KING, M. J., AND DIWAN, J. J. (1972) Arch. Biochem. Biophys. 152, 670-676. 29. MEYER, J. (1975) Ph.D. thesis, Grenoble University. 30. SEASTON, A., INKSON, C., AND EDDY, A. A. (1973) Biochem. J. 134, 1031-1043. 31. HUBBARD, J. S., RINEHART, C. A., AND BAKER, R. A. (1976) J. Bacterial. 125, 181-190. 32. NIVEN, D. F., AND HAMILTON, W. A. (1974)Eur. J. Biochem. 44, 517-522. 33. MEIJER, A. J., AND VAN DAM, K. (1974) Biochim. Biophys. Acta 346, 213-244. 34. BURSELL, E. (1975) Camp. Biochem. Physiol. 523, 235-238.
