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The data used to support the idea that malonyl-coenzyme A (CoA)-sensitive carnitine palmitoyltransferase (CPT-I) is localized on the outer mitochondrial membrane are based on harsh techniques that disrupt mitochondrial physiology. We have turned to the use of the French press, which produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristic of phosphate swelling. Our results indicate that the mitoplasts contain just 15–19% of the outer membrane marker enzyme activity while retaining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes. These mitoplasts were shown by electron microscopy to have the configuration of mitochondria that merely have been divested of their outer membranes. Carnitine-dependent fatty acid oxidation was retained in the mitoplasts, showing that they were physiologically intact. Moreover, protein immunoblotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction. The outer membrane fraction, which consisted of membrane “ghosts,” contained most (50–60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27–29% CPT-I activity. Because CPT-I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes. The data used to support the idea that malonyl-coenzyme A (CoA)-sensitive carnitine palmitoyltransferase (CPT-I) is localized on the outer mitochondrial membrane are based on harsh techniques that disrupt mitochondrial physiology. We have turned to the use of the French press, which produces a shearing force that denudes mitochondria of their outer membrane without the physiologically disruptive effects characteristic of phosphate swelling. Our results indicate that the mitoplasts contain just 15–19% of the outer membrane marker enzyme activity while retaining 85% of the total CPT activity and 50% of both CPT-I, as well as long-chain acyl-CoA synthase activity, the latter two supposed outer membrane enzymes. These mitoplasts were shown by electron microscopy to have the configuration of mitochondria that merely have been divested of their outer membranes. Carnitine-dependent fatty acid oxidation was retained in the mitoplasts, showing that they were physiologically intact. Moreover, protein immunoblotting analysis showed that CPT-I, as well as the inner CPT-II, was localized in the mitoplast fraction. The outer membrane fraction, which consisted of membrane “ghosts,” contained most (50–60%) of marker enzyme activity, monoamine oxidase-B and porin proteins, but only about 27–29% CPT-I activity. Because CPT-I and long-chain acyl-CoA synthetase appear to be associated with both inner and outer membranes, we postulate that these enzymes reside in contact sites, which represent a melding of both limiting membranes. generic carnitine palmitoyltransferase malonyl-CoA-sensitive and -insensitive carnitine palmitoyltransferase monoamine oxidase rotenone-insensitive cytochrome c reductase keyhole limpet hemocyanin bovine serum albumin dithiothreitol respiratory control ratio 4-morpholinepropanesulfonic acid. The two carnitine palmitoyltransferase (CPT)1 enzyme activities are essential in the mitochondrial oxidation of long-chain fatty acids. The malonyl-CoA-sensitive or outer form of CPT (variously termed CPT-1, CPT-I, CPTo, overt, or outer form) is the first committed step in mitochondrial long-chain fatty acid oxidation. The intramitochondrial localization of this enzyme is controversial. Murthy and Pande (1Murthy M.S. R Pande S.V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 378-382Crossref PubMed Scopus (204) Google Scholar) reported that CPT-I is present in the outer membrane and the catalytic site is specifically localized on its inner surface, whereas Fraser et al. (2Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1977; 323: 711-718Crossref Scopus (125) Google Scholar) recently have proposed that this site is on the outer surface. In contrast, two groups of investigators have reported that the enzyme is localized to the outer surface of the mitochondrial inner membrane (3Hoppel C.L. Tomec R.J. J. Biol. Chem. 1972; 247: 832-841Abstract Full Text PDF PubMed Google Scholar, 4Kopec B. Fritz I.B. J. Biol. Chem. 1973; 248: 4069-4074Abstract Full Text PDF PubMed Google Scholar, 5Brosnan J.T. Kopec B. Fritz I.B. J. Biol. Chem. 1973; 248: 4075-4082Abstract Full Text PDF PubMed Google Scholar). Previous studies on the localization of CPT have relied upon techniques for separating inner and outer membranes that potentially could alter the membranes by producing breaks with subsequent annealing in a non-native configuration and which might not yield membranes of absolute purity. To overcome these potential pitfalls in localizing the malonyl-CoA-sensitive CPT, we have utilized a method of mitochondrial fractionation that does not rely on methods that use either organellar swelling to rupture the outer membrane (1Murthy M.S. R Pande S.V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 378-382Crossref PubMed Scopus (204) Google Scholar, 2Fraser F. Corstorphine C.G. Zammit V.A. Biochem. J. 1977; 323: 711-718Crossref Scopus (125) Google Scholar), or exposure to detergents (3Hoppel C.L. Tomec R.J. J. Biol. Chem. 1972; 247: 832-841Abstract Full Text PDF PubMed Google Scholar), or enzymatic digestion of membrane components (5Brosnan J.T. Kopec B. Fritz I.B. J. Biol. Chem. 1973; 248: 4075-4082Abstract Full Text PDF PubMed Google Scholar). Instead, we have utilized physical shearing produced by the French press to skin isolated rat liver mitochondria so that the outer membrane can be detached and separated from the residual mitoplasts without exposure to potentially harmful agents. We have carried out functional and enzyme marker studies to monitor the procedure and the separated membranes were used to determine the localization of CPT. Moreover, we used electron microscopy to monitor the purity and structural integrity of the various membrane fractions. Our data show that CPT-I and long-chain acyl-CoA synthase, supposed outer membrane enzymes, are associated with a component of the mitoplast fraction. Livers from 4–5 rats were pooled, yielding a gross weight of 50 g for each experiment. The livers were minced, rinsed with 220 mmmannitol, 70 mm sucrose, 5 mm MOPS, pH 7.4 (MSM, isolation buffer) containing 2 mm EGTA and a 10% homogenate prepared using two strokes of a Potter-Elvehjem loose-fitting Teflon pestle. The mitochondria were isolated by differential centrifugation, washed twice in isolation buffer, and resuspended in 0.2 ml of isolation buffer/g of liver, yielding a mitochondrial protein concentration of 80–100 mg/ml (6Hoppel C.L. DiMarco J.P. Tandler B. J. Biol. Chem. 1979; 254: 4164-4170Abstract Full Text PDF PubMed Google Scholar). Further purification of mitochondria was achieved using self-forming Percoll gradient centrifugation. One ml aliquots of mitochondrial suspensions were layered on top of 20 ml of 30% Percoll in MSM (a total of eight tubes were prepared in this manner for each experiment) and centrifuged for 30 min with 35,000 rpm (50.2 Ti rotor, Beckman L8-M). The tubes each contained two distinct layers separated by a clear zone. The upper layer (which electron microscopy revealed to consist of peroxisomes) and the clear zone were removed and the lower brownish layer consisting of mitochondria collected. Following a 2-fold dilution with MSM, the mitochondria were collected by centrifugation (10 min, 7000 ×g) and washed once with isolation buffer. This preparation was designated as the Percoll-purified rat liver mitochondria. The final pellet from above was resuspended in 2× MSM containing 0.5 mg of defatted BSA/ml and protease inhibitors (1.0 μg/ml each aprotinin, antipain, pepsatin A, benzethonium chloride, and benzamidine, and 0.1 mm phenylmethyl sulfonium fluoride) and after 10 min was loaded into the French press (American Instrument Co., Silver Spring, MD) fitted with a 12-ml cell according to the directions of the manufacturer and to the protocol of Decker and Greenawalt (7Decker G.L. Greenawalt J.W. J. Ultrastruct. Res. 1977; 59: 44-56Crossref PubMed Scopus (64) Google Scholar). The mitochondria were subjected to a pressure of 2000 p.s.i. while being extruded at a rate of 7–8 ml/min. This pressure was selected after a series of preliminary experiments wherein the pressure was varied from 1400 to 2600 p.s.i. (see “Results”). The material exiting the French press was collected, diluted with 12 ml of 2× MSM containing 0.5% BSA and protease inhibitors, and fractionated by differential centrifugation (10 min, 12000 × g). The pellet was washed once with 12 ml of 2× MSM by resuspension and centrifugation as above, and the pellet, designated as the mitoplast fraction, resuspended in 3.0 ml of MSM containing protease inhibitors. The combined supernatant fluids were subjected to centrifugation for 30 min with 45,000 rpm (50.2 Ti rotor, Beckman L8-M) and the pellet, outer membrane fraction, resuspended in approximately 1.0 ml final volume of MSM. During the entire isolation procedure, the volumes of different fractions were recorded and aliquots saved for subsequent morphological, enzymological and balance studies. An aliquot of each and every mitochondrial fraction was to volume of J. Biol. Scholar), and for 30 in a The were removed from the and the in for a total of 2 The were rinsed in for 2 in of and of the of the for Scholar). with the were in of J. PubMed Scopus Google Scholar). in they were in of and in B. R.J. 1977; PubMed Scopus Google Scholar). were with J. PubMed Scopus Google Scholar) and J. Biochem. PubMed Scopus Google Scholar) and in a electron CPT activity was using different (3Hoppel C.L. Tomec R.J. J. Biol. Chem. 1972; 247: 832-841Abstract Full Text PDF PubMed Google Scholar). the in the of to the using mitochondrial fractions. The contained in a final volume of mm 50 mm MOPS, 1.0 mm defatted 1.0 mm 0.2 and pH the was with 10 of with 10 mm Following a at the was with ml of 1.0 and the into 1.0 ml of The was washed with ml of and ml of the was used to determine the of J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The CPT activity was on the of the the in the of and The contained in a final volume of mm 50 mm MOPS, 1.0 mm mm dithiothreitol defatted mm and pH the was with 10 of Following a at the was with 0.2 ml of acid and the protein and removed by centrifugation. The carnitine the was by aliquots of the is for the malonyl-CoA-sensitive CPT activity and in a final volume of mm 50 1.0 mm mm mm defatted 50 pH and 10 of Following the was mm final The was after a at and the of as The in rate in the and of CPT-I activity J. Biol. Chem. Full Text PDF PubMed Google Scholar). of CPT-I is to to of the enzyme to the PubMed Scopus Google PubMed Scopus Google Scholar), the activity was using the J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). at mg/ml mm 10 mm 0.1 mm and 0.5 mm were for at with 0.5 and aliquots as above for the acyl-CoA synthetase A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), synthase Scholar), oxidase A. Scopus Google Scholar), monoamine oxidase Greenawalt J.W. J. Biol. PubMed Scopus Google Scholar), and rotenone-insensitive c reductase G.L. B. A. J. Biol. PubMed Scopus Google Scholar) were as was by the method of the with J. Biol. Chem. Full Text PDF PubMed Google Scholar). A was used to monitor in 1.0 ml of containing mm 50 mm MOPS, mm 1.0 mm mg of defatted and 1.0 mg of mitochondrial protein R.J. C.L. Biochem. PubMed Scopus Google Scholar). The final pH was and the was 30 was in the and of cytochrome c by of to the after of by of The and their are in the liver mitochondrial was to not and were using A to the acid J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. PubMed Scopus Google Scholar) of rat liver and mitochondrial CPT-I was with a to a to keyhole limpet hemocyanin The was to using the as by the manufacturer The of was with and into in were 5 and analysis of rat liver mitochondrial aliquots to 0.5 of CPT-I total CPT activity were subjected to according to PubMed Scopus Google Scholar), and the separated to membranes. The membranes were with liver mitochondrial CPT-II, liver mitochondrial monoamine oxidase-B and porin and with the were using phosphate and rats were from and to and porin for the were from and liver mitochondrial monoamine oxidase-B was by C.L. J. Scopus Google Scholar) and PubMed Scopus Google Scholar) were as was by the method as for the of long-chain acyl-CoA PubMed Scopus Google Scholar). were of the purity isolated in (6Hoppel C.L. DiMarco J.P. Tandler B. J. Biol. Chem. 1979; 254: 4164-4170Abstract Full Text PDF PubMed Google Scholar) showed and respiratory respiratory control of and the of inner membrane This was the material for French press a first we used the pressure by Decker and Greenawalt (7Decker G.L. Greenawalt J.W. J. Ultrastruct. Res. 1977; 59: 44-56Crossref PubMed Scopus (64) Google Scholar) to be for separating inner and outer membranes, p.s.i. Decker and at a pressure of p.s.i. that only of the outer membrane marker monoamine was retained by the mitoplasts, we with French press of the enzyme associated with the mitoplasts at this pressure The results and of Decker and Greenawalt might be to a in the of the pressure cell used in the In to the of of the outer membrane from mitoplasts so that these two components could be we carried out a the pressure in French press To the of a we the monoamine oxidase activity of the from to was a in the of monoamine oxidase activity associated with the mitoplast fraction from of the total mitochondrial activity to at 2000 p.s.i. is 2000 and above mitoplast In to studies of the of monoamine studies of were carried out on the mitoplasts from the different oxidation was 50% of the mitochondrial activity in mitoplasts prepared at 2000 p.s.i. while the was 0.1 and the ratio was showing of the to in the The oxidation of as well as and was of the activity. we selected 2000 p.s.i. as the pressure to use in rat liver mitochondria. only were the mitochondria used in studies but as well by electron The consisted of mitochondria with a by of The mitochondria were retained both limiting membranes, and a inner showed a of their but that were in configuration to in their in A their outer the mitoplast being and of the isolated rat liver mitochondria were and from we to the mitochondria to a self-forming Percoll gradient to The mitochondrial fraction from Percoll the that were and The Percoll-purified mitochondria were washed and resuspended in 2× MSM 10 min by the French press at 2000 p.s.i. the French press, the mitochondrial fraction to distinct The first consisted of to in the The consisted of mitoplasts that of outer retained their characteristic whereas were In both the their mitoplasts in the manner of by and Greenawalt Greenawalt J.W. J. Biol. PubMed Scopus Google Scholar). The final of mitochondria consisted of that in of and of configuration of in the but from which a of the outer membrane been The residual outer membrane either the inner membrane in its or in that to to the The French mitochondria were fractionated using differential centrifugation to a mitoplast preparation and a The supernatant was to a pellet outer membrane and a The data associated with the mitochondrial fractionation are shown in The activity of the marker enzymes is as well as the of activity in each fraction using the activity × mg of of Percoll-purified French mitochondria as The of monoamine oxidase activity that of the mitochondrial activity was by French the outer membrane fraction contained and the supernatant fraction contained of the activity. The of monoamine oxidase activity in the mitoplasts was monoamine oxidase is a outer membrane these data indicate that the mitoplasts only of the outer while the is into the An outer membrane marker rotenone-insensitive cytochrome c reductase was and the results are to that with monoamine oxidase The outer membrane isolated from this supernatant show a of both monoamine oxidase and with the mitoplasts activity and of mitochondrial outer inner membrane and marker enzymes, and carnitine palmitoyltransferase in rat liver mitochondrial activity of French press acyl-CoA 3.0 CPT CPT represent the and of activities are as for cytochrome c oxidase in a activity of mitochondrial inner and outer mitochondrial marker enzymes, and carnitine cytochrome acyl-CoA represent the and of outer in a represent the and of activities are as for cytochrome c oxidase represent the and of outer we the of inner membrane and marker enzymes in the various fractions. of the inner membrane marker cytochrome c oxidase was in the mitoplast of the marker synthase was of both enzymes into the supernatant was only The of the of these enzymes in the isolated outer membrane be to the of mitoplasts as by electron Because the mitoplasts have retained the of the we that exposure to the French press not the The activity of CPT-I was as the of CPT activity. The activity of the malonyl-CoA-sensitive CPT was in the fractions The French press of the activity into the supernatant with the mitoplast retaining of the activity. In to the above for monoamine oxidase and in the outer membrane is only a of malonyl-CoA-sensitive CPT in the the of the malonyl-CoA-sensitive CPT activity in the mitoplasts is that for monoamine oxidase activity. The of the of monoamine oxidase and CPT in these two fractions the as to the malonyl-CoA-sensitive CPT activity is localized in the mitochondrial outer Because the on the ratio of to and as and of we used the of CPT-I, CPT-I activity was as the activity in the fractions. The results using were to using Because the results that CPT-I does not reside in the site as monoamine oxidase and we used enzyme marker for the outer long-chain acyl-CoA The data for long-chain acyl-CoA synthetase for CPT-I and The inner fraction consisted of a of mitochondria in various of from outer membrane mitoplasts with outer to mitoplasts In every the were The retained its characteristic The retained outer membrane to for the of monoamine oxidase activity present in this fraction. the mitoplasts were of A mitoplast that is divested of outer that the their A functional was used as to the of the of the enzymes in with as the of and of of These were in the of of oxidation in mitoplasts were at 2-fold and were to that in mitochondria. The data in show that was of cytochrome c to was a of the rate of oxidation in the The was and the ratio was showing of the to in the oxidation with was used to by the data for in the and in the of cytochrome c for both mitochondria and the that to in the mitochondrial electron were and were used to producing and and to cytochrome c for mitochondria the of cytochrome the for In contrast, the rate of mitoplast oxidation of was by to in mm of Percoll-purified rat liver mitochondria and mitoplasts in the and of cytochrome mm mm mm represent the and of in a mm and 0.2 mm respiratory of Percoll-purified mitochondria and French mitoplasts in the and of cytochrome mm mm mm mm mm 10 mm 5 mm carnitine 5 mm carnitine represent the and from in a represent the and of represent the and from The oxidation of fatty acid in the was using as a and with oxidation of which in CPT-I activity, and to which long-chain acyl-CoA synthetase and shown in the oxidation of carnitine and are in mitoplasts and in mitochondria with cytochrome This fraction consisted of a series of membrane in with the of B. Acad. Sci. PubMed Scopus Google Scholar), can be and of these contained membrane A mitoplasts in both or were membrane An outer membrane that is of membrane To the localization of CPT enzymes, we subjected membrane fractions to a we used a of in rat liver and a of for the both are in the outer membrane fraction Because the porin was from a cell we that the with porin from rat liver mitochondria that the the by of total CPT activity fraction not only from the isolated rat liver mitochondria and Percoll-purified but that from the In contrast, is with the from the outer membrane fraction. 2 and contain and 0.5 of liver mitochondrial activity. To the of CPT-I by the we utilized a a with a to keyhole limpet This a protein in rat liver mitochondria with a of the of CPT-I this to the membranes from the mitochondrial fractionation 0.5 of CPT-I activity, as the malonyl-CoA-sensitive activity in the was loaded the for each fraction, was of the mitoplasts as with the outer membrane is is of of CPT-I in the outer membrane fraction. The results of show that CPT activity in the mitoplast membranes and to a in the outer This is of total CPT, as the or 85% of the activity in either is present in mitoplasts, whereas activity is present in the outer membrane fraction. the malonyl-CoA-sensitive activity showed a with of the activity in the mitoplasts and in the outer To determine the of the membrane we carried out various The purity and integrity of mitoplast fraction is by the that of the marker enzymes for the inner membrane c and is retained by these In the for is the respiratory control only were these mitoplasts but electron microscopy showed that most of these are a of they have In to mitoplasts, the outer membrane contained that of the total CPT activity in the or These retained of the malonyl-CoA-sensitive CPT activity. these consisted of outer membrane is shown by the in the activity and of monoamine oxidase and outer membrane a of inner enzymes was in this fraction be that electron microscopy revealed to contain a of mitoplasts, to for this in the CPT activity is to the mitoplast fraction, these be of carnitine as well as In a long-chain acyl-CoA synthetase is to the mitoplast fraction, oxidation by mitoplasts be is that these enzymes reside in and are removed with the outer the mitoplast fraction be of out In contrast, mitoplast of the to not only the but above and the malonyl-CoA-sensitive form of CPT been removed from the mitoplasts, the rate for the oxidation of carnitine have to which acyl-CoA synthetase and oxidation in a as as Fritz and I.B. J. Res. Full Text PDF PubMed Google Scholar) proposed localization of CPT activity subsequent studies could CPT activity only in the inner membrane (3Hoppel C.L. Tomec R.J. J. Biol. Chem. 1972; 247: 832-841Abstract Full Text PDF PubMed Google Scholar, 5Brosnan J.T. Kopec B. Fritz I.B. J. Biol. Chem. 1973; 248: 4075-4082Abstract Full Text PDF PubMed Google Scholar). In and Tomec (3Hoppel C.L. Tomec R.J. J. Biol. Chem. 1972; 247: 832-841Abstract Full Text PDF PubMed Google Scholar), using a reported that CPT activity in two associated with the inner termed on the outer the termed on the inner This was in a subsequent by Fritz (5Brosnan J.T. Kopec B. Fritz I.B. J. Biol. Chem. 1973; 248: 4075-4082Abstract Full Text PDF PubMed Google Scholar), a different CPT was localized on the inner membrane was Murthy and Pande (1Murthy M.S. R Pande S.V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 378-382Crossref PubMed Scopus (204) Google Scholar) based on their These that the site is on the of the outer whereas CPT is on the inner surface of the outer In to the membranes, these investigators used the phosphate swelling procedure by et al. B. Acad. Sci. PubMed Scopus Google Scholar). with the swelling procedure of Fraser et F. Corstorphine C.G. Zammit V.A. Biochem. J. 1977; 323: 711-718Crossref Scopus (125) Google Scholar) the of enzymes on the of malonyl-CoA-sensitive CPT activity and that both the and catalytic site of CPT-I are on the of the outer we the swelling we that the of malonyl-CoA-sensitive CPT in the outer membrane was only that of monoamine the marker of outer membrane C.L. in Scholar). This of be by the that phosphate at of the CPT activity from mitochondria. CPT could in outer membrane fraction by the that et al. B. Acad. Sci. PubMed Scopus Google Scholar) to be in this fraction. The of intramitochondrial localization of enzymes is with of that were based on the of only does swelling have its but most for localizing CPT have associated not only the outer membrane by with its but the mitoplasts Greenawalt J.W. J. Biol. PubMed Scopus Google Scholar, Biochem. J. PubMed Scopus Google Scholar). which been used to the outer does so by digestion of membrane The with of these techniques is that the mitoplasts to respiratory control which is the of mitochondrial A in is the use of electron microscopy to monitor the structural integrity and purity of each and every fraction. was in the of cell the of this protocol have been of electron they to be at they a of the and not be of the as a To of are in This the that suspensions are first The which we have in are without to are selected for and of are at to of the are at to of not at lower these we not have to rely on We have of the of outer membrane and the electron microscopy can be and with Moreover, a to determine have been with a of and we turned to immunoblotting to this is at on enzyme and we that these two are in the outer membrane fraction with the localization of activity. liver CPT-II, the inner form of CPT, the protein localized to the mitoplast fraction with the activity a of CPT-I, the of the protein that was is present in the mitoplast fraction. The of the various the that CPT-I is associated not only in with the inner but that a with the outer membrane as A for a that both inner and outer mitochondrial membranes is the contact sites, which are localized the inner and outer membranes are as protein B. J. Biol. PubMed Scopus Google Scholar, A. J. Biol. PubMed Scopus Google Scholar) and PubMed Scopus Google Scholar, Biochem. Scopus Google Scholar), have been to contact Fraser and Zammit F. Zammit V.A. Biochem. J. PubMed Scopus Google Scholar) reported that CPT-I and proteins, by a with in the contact site fraction from rat liver mitochondria. The localization of CPT-I in contact for the results by the use of different membrane isolation We that swelling results in the contact being removed in with the outer whereas with the French press results in the contact being retained by the
Hoppel et al. (Tue,) studied this question.
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