From Cellular Biochemistry, Hannah Research Institute, Ayr, KA6 5HL, Scotland, United Kingdom
Received for publication, February 5, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carnitine palmitoyltransferase I (CPT I)
of rat liver mitochondria is an integral, polytopic protein of the
outer membrane that is enriched at contact sites. As CPT I kinetics are
highly dependent on its membrane environment, we have measured the
kinetic parameters of CPT I present in rat liver submitochondrial
membrane fractions enriched in either outer membrane or contact sites. The Km for palmitoyl-CoA was 2.4-fold higher for
CPT I in outer membranes than that for the enzyme in contact sites. In
addition, whereas in contact sites malonyl-CoA behaved as a competitive
inhibitor of CPT I with respect to palmitoyl-CoA, in outer membranes
malonyl-CoA inhibition was non-competitive. As a result of the
combination of these changes, the IC50 for malonyl-CoA was
severalfold higher for CPT I in contact sites than for the enzyme in
bulk outer membrane. The Ki for malonyl-CoA, the
Km for carnitine, and the catalytic constant of the
enzyme were all unaffected. It is concluded that the different membrane
environments in outer membranes and contact sites result in an altered
conformation of L-CPT I that specifically affects the long-chain
acyl-CoA binding site. The accompanying changes in the kinetics of the
enzyme provide an additional potent mechanism for the regulation of
L-CPT I activity.
The overt carnitine palmitoyltransferase of mitochondria
(CPT I)1 catalyzes the
rate-limiting step in the transfer of acyl moieties from the cytosolic
compartment into the mitochondrial matrix, where they undergo
The property that makes CPT I rate-limiting for long-chain acyl-CoA
utilization by mitochondria is its inhibition by malonyl-CoA (13). The
mechanism of this inhibition, as studied in intact isolated
mitochondria, appears to be competitive with respect to the acyl-CoA
substrate. However, it has long been appreciated that its mode of
action is unorthodox as malonyl-CoA appears to induce sigmoidicity in
the velocity-substrate concentration curves (14). CPT I kinetics are
very sensitive to changes in the physical properties of the membrane in
which it resides (3). Fluidization of membrane lipids in
vivo or in vitro results in the desensitization of CPT
I to malonyl-CoA and vice versa (3, 15, 16). We have reasoned,
therefore, that as contact sites have a distinctive lipid (17) and
protein (18) composition, it is possible that CPT I molecules in the
two membrane microenvironments may have different kinetic properties
(6) as is already well established for other proteins
(e.g. porin; see Refs. 19 and 20) that exist in
both outer membrane and contact site environments.
We have tested this hypothesis by studying the kinetics of liver CPT I
in submitochondrial fractions enriched in the two membrane populations.
We find that the kinetic parameters with respect to one of its
substrates, palmitoyl-CoA, are markedly different for the enzyme in the
two microenvironments. Moreover, the kinetics of malonyl-CoA inhibition
of CPT I activity is different for the enzyme resident within the two
membrane populations.
Preparation and Subfractionation of Mitochondria--
Liver
mitochondria were prepared from male Wistar rats (350-400-g body
weight; maintained on a laboratory chow diet) by differential centrifugation followed by Percoll gradient purification. Mitochondrial subfractionation was performed as described previously (6). However,
the sonicated mitochondria were fractionated using step, rather than
continuous, sucrose gradients. The sucrose concentrations for the step
gradient were 1.13 g, 1.15 g, and 1.19 g/ml in 10 mM potassium phosphate buffer, pH 7.0, and chosen to yield
fractions enriched in outer membrane, contact sites, and inner
membrane, respectively (6). The volume of each sucrose density step was 3 ml. A step (1.5-ml) of intermediate density was present between the
1.13-g and 1.15 g/ml steps to ensure clean separation between the outer
membrane and contact site fractions. The gradients were centrifuged at
100,000 × g for 210 min at 4 °C in a 65V13 vertical rotor (Sorvall Instruments). The fractions were recovered by puncturing the polycarbonate tubes with a syringe needle followed by aspiration of
each membrane band. They were divided into aliquots and frozen at
Marker Protein Determination--
The activities of marker
enzymes were measured to characterize the purity of the fractions.
Rotenone-insensitive NADPH-cytochrome c reductase and
cytochrome c oxidase (assayed as in Ref. 6) were used as
markers for outer and inner membrane fractions, respectively. Porin was
used as a marker for both outer membrane and contact sites (6). The
component proteins of the submitochondrial fractions were separated
using SDS polyacrylamide gel electrophoresis (15% polyacrylamide)
followed by transfer onto nitrocellulose and immunodetection of porin
with mouse anti-human porin conjugated to alkaline phosphatase (see
Ref. 6). The band intensity of porin on the Western blots was
quantified densitometrically (Molecular Dynamics).
Assay of Activity and Immunodetection of CPT I--
In Ref. 6 we
showed that CPT II is also enriched within contact sites and that some
contamination of the outer membranes with CPT II is always present.
Therefore, samples of the sucrose gradient-purified submitochondrial
fractions enriched in outer membranes or contact sites were
preincubated for 10 min on ice in the absence and presence of
tetradecylglycidyl-CoA at a concentration sufficient to inhibit all CPT
I activity. This enabled us to quantify the proportion of total CPT
activity in these fractions that was due to CPT II in these fractions.
This was then subtracted from the total CPT activity (obtained in
fractions not incubated with the inhibitor) so as to obtain that of CPT I.
The basic assay medium for CPT I contained 80 mM sucrose,
70 mM KCl, 50 mM imadazole, 1 mM
EGTA, 5 mM MgATP, 1% defatted bovine serum albumin, 2 mM glutathione, 4 µg of rotenone/ml, 2 µg of antimycin
A/ml, and the indicated concentrations of carnitine (specific
radioactivity 770 dpm/nmol) and palmitoyl-CoA. The final pH value was
7.1. For determination of the IC50 for malonyl-CoA (concentration that gives 50% inhibition of CPT I activity) the concentrations of carnitine and palmitoyl-CoA were 520 and 35 µM, respectively. When the concentrations of carnitine or
palmitoyl-CoA were varied, the fixed concentrations of the second
substrate were 135 and 520 µM for palmitoyl-CoA and
carnitine, respectively. The specific activity of
3H-carnitine was kept constant at 1077 dpm/nmol.
Malonyl-CoA concentrations were either varied up to 200 µM or maintained constant at 20 µM when
investigating the dependence of CPT I activity on increasing palmitoyl-CoA concentrations in the absence or presence of
malonyl-CoA.
Immunodetection of CPT I was performed by Western blotting as
described previously, using an anti-peptide antibody (anti-C) raised against a linear epitope from the catalytic domain of rat liver
CPT I (1, 6, 21).
Palmitoyl-CoA, malonyl-CoA, and fatty acid-free albumin, co-factors,
and substrates for enzyme assays were from Sigma; mouse anti-human porin antibody was from Calbiochem-Novabiochem.
Tetradecylglycidyl-CoA was synthesized starting with the sodium salt of
tetradecylglycidic acid (McNeil Pharmaceutical, Spring House, PA) by
the method described in Ref. 22. Radiolabeled carnitine was from
Amersham Pharmacia Biotech and was washed with water-saturated
n-butanol before being used, to reduce radioactive
contaminants. Sheep anti-rat L-CPT I anti-peptide L antibody was
obtained as described previously (see Ref. 1). All other chemicals were
from Merck.
The outer membrane- and contact site-enriched fractions
were characterized with respect to their content of marker
proteins and immunodetectable CPT I (Table
I). The sucrose step gradients used for
their preparation in the present study gave fractions that had the
characteristics of the respective peaks obtained from continuous
gradients in our previous studies (6). There was minimal
cross-contamination between the two fractions, although as
expected this was greater for the contamination of contact sites with
outer membrane (see Table I).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-oxidation. The enzyme is an integral membrane protein with two
transmembrane segments that are thought to confer on its
kinetics a marked dependence on the physical state of the membrane
(1-4). CPT I is present within the general outer membrane of
mitochondria (5) but is especially concentrated within the contact
sites that occur between the outer membrane and the peripheral inner
membrane (6). The latent form of carnitine palmitoyltransferase (CPT
II) is also concentrated at the contact sites but on the inner aspect
of the inner membrane, suggesting that long-chain acylcarnitine
formation, and utilization may be facilitated by this submitochondrial
localization of the two proteins (6). Contact sites are loci for the
extensive trafficking of proteins and phospholipids between the extra-
and intramitochondrial compartments. They are the sites of the
mitochondrial protein import machinery (7) of the binding of hexokinase
and creatine kinase on the cytosolic and intermembrane space aspects of
the outer membrane, respectively (8, 9) and of the attachment of a
specialized population of endoplasmic reticular membranes through which
phospholipid trafficking occurs (10, 11). They are also sites of
interaction of mitochondria with the cytoskeleton (12).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C until used for enzyme activity measurements, protein determination, and SDS polyacrylamide gel electrophoresis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Characterization of representative outer membrane and contact site
fractions
Km for Palmitoyl-CoA and Carnitine--
Lineweaver-Burke
plots for CPT I activity at a different palmitoyl-CoA concentration for
the enzyme in contact site and outer membrane fractions were linear
(see Fig. 1, a and
b), as were Eadie-Hofstee plots (not shown), which allowed
us to use graphical methods to determine the kinetic parameters. As
shown in Table II, there was a 2.4-fold
difference (p = 0.0002) in the Km for palmitoyl-CoA for the enzyme in the two different membrane fractions (60.6 ± 7.5 and 25.6 ± 0.9 µM for
outer membrane and contact site fractions, respectively). The maximum
palmitoyl-CoA concentration used was 200 µM (in the
presence of 1% bovine serum albumin) to avoid the detergent properties
of this molecule. There was no significant difference between
the Km for carnitine values for the enzyme in the
two membrane fractions (Table II).
|
|
Malonyl-CoA Inhibition of CPT I: Effects of Membrane Location-- The double-reciprocal plots resulting from the measurement of CPT I activity at different palmitoyl-CoA concentrations in outer membranes and contact sites (Fig. 1, a and b) show that the kinetics of malonyl-CoA inhibition of CPT I with respect to palmitoyl-CoA were different for the enzyme in the two membrane fractions. In the outer membranes, malonyl-CoA acted exclusively by lowering the Vmax of the enzyme, without affecting the Km for palmitoyl-CoA. By contrast, in contact sites, malonyl-CoA inhibited CPT I activity almost entirely by raising the Km for palmitoyl-CoA without affecting the Vmax of the enzyme (there was a very minor effect on Vmax, but this can be accounted for by unavoidable minor contamination of contact sites with outer membranes flanking the points of physical contact between the outer and inner membranes). Malonyl-CoA (20 µM) increased the Km for palmitoyl-CoA of CPT I in contact sites by 4-fold (p > 0.0001), whereas it had no effect on the Km of the enzyme in outer membranes. By contrast, 20 µM malonyl-CoA reduced the Vmax of the enzyme resident in outer membranes by 70% (p = 0.0002) without affecting that for the enzyme in contact sites. In neither fraction did malonyl-CoA induce any non-linearity in double-reciprocal plots. Note that the Vmax values refer to activity per mg of membrane protein of each individual membrane fraction and not of whole mitochondria.
IC50 and Ki for Malonyl-CoA--
The
IC50 value for malonyl-CoA (the concentration of
malonyl-CoA required to inhibit CPT I activity by 50% at a suboptimal concentration of palmitoyl-CoA) is the parameter that is most commonly
measured to assess malonyl-CoA sensitivity of CPT I in intact
mitochondria (23-25). When this type of experiment was performed, the
inhibition curves obtained in the presence of increasing concentrations of malonyl-CoA were different for the enzyme in outer membranes and
contact sites, respectively. The enzyme in contact sites was less
sensitive to malonyl-CoA inhibition (Fig.
2) i.e it had the higher IC50 value (see Table II).
|
In view of the fact that the Km for palmitoyl-CoA and the mode of inhibition by malonyl-CoA were found to be different between the two fractions, values for the Ki for malonyl-CoA were also obtained from Dixon plots (1/v versus [malonyl-CoA]). As can be seen from Table II, the Ki value for malonyl-CoA was the same for CPT I in outer membranes and contact sites suggesting that the difference in IC50 was due solely to the difference in Km for palmitoyl-CoA between the enzyme in outer membranes and contact sites.
The Catalytic Constant for CPT I in Outer Membranes and Contact
Sites--
The catalytic constant of an enzyme is defined as the ratio
of Vmax divided by the molar amount of enzyme
protein, and is a measure of the intrinsic catalytic activity of the
protein molecules. A quantitative estimate of the number of CPT I
molecules in outer membrane and contact site fractions was obtained by
immunodetection of CPT I on Western blots for paired fractions obtained
from individual rat livers. The value of the
Vmax for CPT I activity (Table II) divided by
immunoreactive CPT I band intensity (Table I) was obtained for the two
membrane fractions. For five different preparations, the ratio between
the parameters for the two membrane fractions was not significantly
different from unity (0.92 ± 0.18) indicating that the intrinsic
catalytic activity of CPT I is not different in the two membrane
microenvironments. In this respect, it is noteworthy that the effect of
20 µM malonyl-CoA on the catalytic efficiency
(Vmax/Km) of CPT I in either
of the two membrane fractions was identical (a 75% decrease) as can be
calculated from the data in Table II.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CPT I is an integral membrane protein that adopts a polytopic conformation (1). Its two hydrophobic transmembrane segments are flanked by the N- and C-terminal domains that both protrude into the cytosol. We have suggested (1, 2) that this topology endows the protein with the potential for altering the conformation of its catalytic C-domain in response to changes in the membrane environment. It was previously shown for mitochondria in vitro (15, 16) and for outer membrane preparations isolated from rats in different physiological conditions (3) that CPT I kinetics are extremely sensitive to the molecular order of the constituent lipids of the membrane environment in which it resides or into which it is incorporated experimentally (4, 26). The observation (6) that CPT I is distributed both within the bulk outer membrane and contact sites (but enriched in the latter; see Ref. 6) raised the prospect that the kinetic properties of the enzyme are different in the two microenvironments. The present data provide evidence that this is indeed the case and that the properties of L-CPT I with respect to palmitoyl-CoA are markedly different in the two membrane environments. A possible mechanism for the changes observed could be the altered interaction of CPT I with other (lipid and/or protein) membrane components within outer membranes or contact sites resulting in a conformational change in the protein that affects specifically the acyl-CoA binding site. Previous observations (4, 27, 28) have shown that the kinetics of CPT I are highly dependent on the interaction between its cytosolic N-terminal domain and the rest of the molecule, as shown originally by cell biological studies (1, 2) and more recently by domain swapping (28) and functional mutagenesis experiments (27, 30). Therefore, it is plausible that short or long term changes in the membrane environment, because of either the formation of localized membrane microdomains of distinctive lipid and/or protein composition (i.e. in contact sites) or by changes in overall phospholipid composition (e.g. induced by diet, fasting, and/or insulin-deficiency; see Ref. 3) would induce altered kinetic characteristics of CPT I.
There are precedents of other proteins that show differences in molecular properties of populations that are partitioned between mitochondrial contact sites and outer membranes. Thus, voltage-dependent anion channel (porin), an integral mitochondrial outer membrane protein that is also enriched in contact sites (19, 20), displays different transport properties depending on its submitochondrial membrane location. For example, porin-rich domains present at contact sites of brain mitochondria bind hexokinase, whereas porin within the bulk outer membrane does not (12). Moreover, contact site porin is more difficult to extract with detergents, suggesting altered protein-lipid and/or protein-protein interactions (29).
The most striking difference between CPT I in contact sites and outer
membrane was observed for the kinetics of malonyl-CoA inhibition with
respect to palmitoyl-CoA. They were competitive for the enzyme resident
within contact sites but non-competitive within the outer membrane
fraction. It is significant that this change was accompanied by a
2.4-fold increase in the Km for palmitoyl-CoA but no
change in the intrinsic catalytic activity of the enzyme, its
Ki for malonyl-CoA, or its Km for
carnitine. Therefore, the change in CPT I appears to be specific to the
acyl-CoA binding site. This difference in kinetics is potentially very
important for the function of CPT I in the two membrane environments in vivo. Thus, not only would CPT I in the outer membranes
be more susceptible to inhibition by malonyl-CoA at any given
palmitoyl-CoA concentration (lower IC50), but the inhibitor
would be able to affect the outer membrane enzyme activity even at high
palmitoyl-CoA concentrations because of the non-competitive nature of
the inhibition for the enzyme in outer membranes. Consequently, the
effect of malonyl-Co would not be able to be over-ridden by high
palmitoyl-CoA concentrations unless the CPT I molecules reside within
the contact sites.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank C. Narain for excellent assistance, Dr. S. Brocklehurst (Biomathematics and Statistics, Scotland) for performing the statistical analyses, and Dr. R. R. Ramsay (University of St. Andrews, St. Andrews, Scotland) for helpful discussions.
![]() |
FOOTNOTES |
---|
* The work was supported in part by the British Heart Foundation (to F. F.), the British Council (to R. P.), and the Scottish Executive.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Departamento de Physiologia e Biophysicas,
Instituto de Ciencias Biomedicas, Cidade Universitaria, CEP 05508-900, Sao Paolo, Brazil.
§ To whom correspondence should be addressed. Tel.: 44-1292-674058; Fax: 44-1292-674059; E-mail: zammitv@hri.sari.ac.uk.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M101078200
![]() |
ABBREVIATIONS |
---|
The abbreviation used is: CPT overt, carnitine palmitoyltransferase of mitochondria.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Fraser, F., Corstorphine, C. G., and Zammit, V. A. (1997) Biochem. J. 323, 711-718[Medline] [Order article via Infotrieve] |
2. | Zammit, V. A., Fraser, F., and Corstorphine, C. G. (1997) Adv. Enzyme Regul. 37, 295-317[CrossRef][Medline] [Order article via Infotrieve] |
3. | Zammit, V., Corstorphine, C., Kolodziej, M., and Fraser, F. (1998) Lipids 33, 371-376[Medline] [Order article via Infotrieve] |
4. | McGarry, J. D., and Brown, N. F. (2000) Biochem. J. 349, 179-187[CrossRef][Medline] [Order article via Infotrieve] |
5. | Murthy, M. S. R., and Pande, S. V. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 378-382[Abstract] |
6. | Fraser, F., and Zammit, V. A. (1998) Biochem. J. 329, 225-229[Medline] [Order article via Infotrieve] |
7. | Pfanner, N., Rassow, J., Wienhaes, U., Hergersberg, C., Sollner, T., Becker, K., and Neupert, W. (1990) Biochim. Biophys. Acta 1018, 239-242[Medline] [Order article via Infotrieve] |
8. | Brdiczka, D., Bucheler, K., Kottke, M., Adams, V., and Nalam, V. K. (1990) Biochim. Biophys. Acta 1018, 234-238[Medline] [Order article via Infotrieve] |
9. | Brdiczka, D. (1991) Biochim. Biophys. Acta 1071, 291-312[Medline] [Order article via Infotrieve] |
10. |
Vance, J. E.
(1990)
J. Biol. Chem.
265,
7248-7256 |
11. | Daum, G., and Vance, J. (1997) Prog. Lipid Res. 36, 103-130[CrossRef][Medline] [Order article via Infotrieve] |
12. | Leterrier, J. F., Rusakov, B. D., Nelson, B. D., and Linden, M. (1994) Microsc. Res. Tech. 27, 233-261[Medline] [Order article via Infotrieve] |
13. | McGarry, J., and Foster, D. W. (1980) Annu. Rev. Biochem. 49, 395-420[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Cook, G. A.
(1984)
J. Biol. Chem.
259,
12030-12033 |
15. | Mynatt, R. L., Greenshaw, J. J., and Cook, G. A. (1994) Biochem. J. 248, 727-733 |
16. | Kolodziej, M. P., and Zammit, V. A. (1990) Biochem. J. 272, 421-425[Medline] [Order article via Infotrieve] |
17. |
Ardail, D.,
Lerme, F.,
and Louisot, P.
(1991)
J. Biol. Chem.
266,
7978-7981 |
18. | Pfanner, N., Rassow, J., Klei, I. J. V. D., and Neupert, W. (1997) Cell 68, 999-1002 |
19. | Adams, V., Bosch, W., Schlegel, J., Wallimann, T., and Brdiczka, D. (1989) Biochim. Biophys. Acta 981, 213-225[Medline] [Order article via Infotrieve] |
20. | Ohlendieck, K., Riesinger, I., Adams, V., Krause, J., and Bridicka, D. (1986) Biochim. Biophys. Acta 860, 672-689[Medline] [Order article via Infotrieve] |
21. | Fraser, F., Corstorphine, C. G., Price, N. T., and Zammit, V. A. (1999) FEBS Lett. 446, 69-74[CrossRef][Medline] [Order article via Infotrieve] |
22. | Bernert, J. T., Jr., and Sprecher, H. (1979) Biochim. Biophys. Acta 573, 436-442[Medline] [Order article via Infotrieve] |
23. | Mynatt, R. L., Lappi, M. D., and Cook, G. A. (1992) Biochim. Biophys. Acta 1128, 105-112[Medline] [Order article via Infotrieve] |
24. | Saggerson, D., Ghadiminejad, I., and Awan, M. (1992) Adv. Enzyme Regul. 32, 285-306[Medline] [Order article via Infotrieve] |
25. | Swanson, S. T., Foster, D. W., McGarry, J. D., and Brown, N. F. (1998) Biochem. J. 335, 513-519[Medline] [Order article via Infotrieve] |
26. | Broadway, N. M., and Saggerson, E. D. (1997) Biochem. J. 322, 435-440[Medline] [Order article via Infotrieve] |
27. | Jackson, V. N., Zammit, V. A., and Price, N. T. (2000) J. Biol. Chem. |
28. |
Jackson, V. N.,
Cameron, J. M.,
Fraser, F.,
Zammit, V. A.,
and Price, N. T.
(2000)
J. Biol. Chem.
275,
19560-19566 |
29. | Linden, M., Nelson, B. D., Loncar, D., and Leterrier, J. F. (1989) J. Bioenerg. Biomembr. 21, 507-518[Medline] [Order article via Infotrieve] |
30. | Shi, J., Zhu, H., Arvidson, D. N., and Woldegiorgis, G. (2000) Biochemistry 39, 712-717[CrossRef][Medline] [Order article via Infotrieve] |