1081 ( 19t)l ~| 21 - 12:g ' 1991 ElsevierScience Publishers B.V. (Biomedical Dixi~ion]O()O5-276()/~1 S03 50 0005276091000676

Biochtmica et Btophrslca Aria.

121

A DONIS

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Kinetic advantage of the interaction between the fatty acid fl-oxidation enzymes and the complexes of the respiratory chain B a l a z s S u m e g i *. Z o l t a n P o r p a c z y a n d I s t v a n A l k o n y i C)lw~,r~tt~ "lfedl¢.¢zlSchool. In,/little of Bu~c-he,u~/rv. Pe¢'~ I lhm~¢r~'/

(Recei,,ed 23 September lqqO) Key ,xords: Fatty acid//-oxid;,lioJ'~:Mitochondrialen,,~.me;Respiralorychain Respiration-linked oxidation of 3-hydroxybuP,~l-CoA, crotonyI-CoA and saturated fattT,/ ac~l (C 4. C a and C I4)-CoA esters was studied in different mitochoudrial preparations. Oxidation of acyl-CoA esters was poor in intact mitoehondriag however, it was significant, as well as, NAD + and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotunyI-CoA and 3 - h ~ d r o x y l m ~ l - C o A proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratu~ ~chain) was also higher in gently disrupted mituchondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybuQn'l-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 p M in gently sonieated and 15 p M in completely sonicated mitochondria in spite of the obsen~ation that the gently sonicated mitnehondria oxidized the 3-hydroxybutyryI-CoA much faster than the completely sonicated mitochondria. The NAD +-dependence of 3-hydroxybutyryI-CoA oxidation showed that a much smaller NAD ÷concentration w~s enough to half-~turate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of ~-oxidation enzymes in situ. Respiration-linked oxidation of buQ'nl-, octanoyl- and palmitoyI-CoA w&s also studied and t h e ~ CoA intermediates were oxidized at approx. 50% of the rate of crotonyb and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria Ihe oxidation rate of these saturated acyI-CoA intermediates w&s hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory, chain had been disrupted.

Introduction Since intermediates of fatty acid fl-oxidation are present at very low concentrations in mitochondria, it has been proposed that mammalian ,8-oxidation enzymes may exist as a multienzyme complex to which intermediates are tightly bound [1,2]. However, some acyI-CoA intermediates accumulate in measurable amount [3-6] (about 10-15 n m o l / g dry weight 3-hydroxy-acyI-CoA and 2 - t r a n s - e n o y I - C o A ) [6] raising the possibility of the leakage of intermediates. Only a few studies deal with the organization of the fatty acid //-oxidation enzymes in the mitochondria [7-12]. It has been reported that enzymes of fatty acid B-oxidation bind to the inner mitochondrial membrane [7-12], and it was also demonstrated that (a) protein

Correspondence: ( * present address): B . Sume,~i,Universit) of Texas at Dallas. Department of Chemistry. Box 830688. Richardson. TX 75083-0688, U.S.A.

component(s) of inner membrane (is) are responsible for the binding [12.13]. Earlier. we isolated the membrane protein responsible for the binding of the 3-hydroxyacyl-CoA dehydrogenase to the inner mitochondrial membrane [12], and some data indicated that this binding protein is loosely bound to complex I (NADH : ubiquinone oxidoreductase) [13]. In several cases, interactions were demonstrated be*xs'een functionally related mitochondrial enzymes and coupled reactions were found to be much more active than expected from the individual kinetic parameters of the participant enzymes. Substrate channeling was detected both in vitro [1421] and in situ [22-24]. In spite of the numerous works dealing with substrate channeling in glycolysis [19-21], there has been some dispute about this phenomenon 125,26 I. Although. subsequent studies [27.28] appear to adequately reaffirm the substrate channeling in these systems, it seems to be necessary to establish the existence of this phenomenon in other metabolic pathways.

122

In this paper, we provide evidence for the positive catalytic consequence of the organization of #-oxidation enzymes in the microenvironment of the respiratory complexes in situ. In addition, evidence is provided to show the preferential transfer of N A D H between 3-hydroxyacyI-CoA dehydregenase and complex i is the main contributor to the kinetic advantage involving #-oxidation in situ. Experimental procedures

Malerials The following chemicals were obtained from the indicated sources: NAD, N A D H and 3-hydroxyacyI-CoA dehydrogenase from Boehringer Mannheim; acetoacetyl-CoA, 3-hydroxybutyryl-CoA, crotonyl-CoA and crotonase from Sigma. All other materials were of the highest purity commercially available. Method~ Rat liver mitochondria were isolated [10] and gently sonicated mitochondria were prepared as previously described [23,24]. In these experiments, the mitochondria (60-80 mg protein/ml) were subjected to sonic oscillation for 15 s in a bath sonicator (50 W) in the presence of 2 mg/ml cytochrome c. The gently disrupted mitochondria were collected by centrifugation (20 rain, 40000 X g), the sedimented gently sonicated mitochondria were resuspended to the original volume with 20 mM Tris-HCl buffer (pH 7.4), containing 150 mM KCI, 0.5 mM EDTA and 2 mg/ml cytochrome c. The gently sonicated mitochondria preparation was divided into two portions; one half was used as gently sonicated mitochondria, the other half was subjected to further sonic oscillation (250 W instrument) 10 x 20 s to achieve complete disruption of mitochondria (also refered as vigorously sonicated mitochondria). In the disrupted mitochondria, matrix enzymes were almost completely exposed, because the addition of 2% Triton X-100 (which dissolve the lipid membrane) did not increase significantly the activity (less than 10%) of the studied matrix enzymes (malate dehydrogenase and isocitrate dehydrogenase). In independent experiments we showed that Triton X-100 does not inhibit these enzyme activities when it was determined with purified enzymes. In addition, only a very small portion of matrix enzymes sedimented (less than 10%) in 20 mM Tris-HC! (pH 7.4)containing 150 mM KCI during centrifugation (20 min, 40000 × g) showing that more than 90% of these matrix enzymes were relesed from the mitochondria. Exogenously added cytochrome c was necessary during sonication of mitochondria to avoid toss of cytochrome c and to ensure a high respiration rate for both gently and completely disrupted mitochondria. After this treatment the gently sonicated mitochondria retained exposed matrix enzymes and respiration en-

zymes together in an easily sedimentable form. Also. gently sonicated mitochondria were permeable to macromolecules -blue dextran (M, 2000000) and blue albumin (M~ 67000 - that inhibit the enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase both in gently sonicated mitochondria and in completely disrupted ones (data not shown). Enzyme activities were determined in gently sonicated mitochondria (exposed activities) and in the completely disrupted mitochondria (total activities). Moreover. the exposed N A D H oxidase activities were also measured using a Clark type electrode both in gently and in completely disrupted mitochondria at 25°C.

Enzyme a.~sa)'s Enzyme activities were assayed as described for 3-hydroxyacyl-CoA dehydrogenase [29], crotonase [30] and thiolase [31] except that the osmolarity of the buffers was adjusted to 320 mOsm with KCI. The 3-hydroxyacyl-CoA dehydrogenase reaction was also measured in the physiological direction starting the reaction with 3-hydroxybutyryl-CoA and NAD. The N A D H formed was measured using the redox dyes phenazine methosulfate(PMS) and iodonitrotetrazolium violet (INT) at the same concentration as they were used for assaying the pyruvate dehydrogenase complex [32]. Coupled enzyme reactions Both gently disrupted mitochondria and completely disrupted mitochondria catalyzed the respiration-linked oxidation of acyl-CoA intermediates (crotonyl-CoA, 3hydroxybutyryl-CoA, butyryl-CoA, palmitoyI-CoA and octanoyI-CoA) in the presence of N A D and CoA, and the rate of this oxidation was determined by a Clark type oxygen electrode in a 1.6 ml reaction chamber containing 20 mM Tris-HC1 buffer (pH 7.4), 150 mM KCI, 0.5 mM EDTA and the substrates (see Tables in the text) at 25°C. The steady-state N A D H concentration in the same reaction chamber was determined as described in Ref. 33. Chromogen dye-linked oxidations of acyI-CoA intermediates were studied in the presence of PMS and INT both in gently disrupted mitochondria and completely disrupted mitochondria at the same substrate concentratior~s used in the respiration-linked oxidation measurements. The reduced coenzymes ( N A D H and F A D H , ) formed via #-oxidation were measured with PMS and IHT [32]. The progress of reaction was determined by a double beam recording spectrophotometer (Specord M40) at 500 nm (extinction coefficient for reduced I H T of 12.4 cm -I mmol -~) using 10 mM KN 3 or KCN to avoid the reoxidation of reduced coenzymes by the respiratory enzymes. The cuvette con.. tained 20 mM Tris-HCl buffer (pH 7.4), 150 mM KCI, 10 mM KN~, 0.008 mM PMS, 0.6 mM 1HT, 1 mM N A D and CoA derivatives (see in the Tables) and the reaction rate was determined at 25°C.

123 1

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3-hydroxybutyryl-CoA was oxidized in a Clark type reaction c h a m b e r by gently disrupted mitochondria. Oxygen consumption was recorded, and after oxygen consumption indicated that oxidation of 7 0 - 8 0 nmol of 3-hydroxybutyryt-CoA had formed, the reaction was stopped by 5% perchloric acid. Samples were deproteinized and neutralized and the a,:etyl-CoA content was determined as described in [33]. The a m o u n t of oxidized 3-hydroxybutyryl-CoA was estimated from the oxygen consumption and was c o m p a r e d to the formed acetyl-CoA,

Results and Discussion Gently sonicated mitochondria contain a significant portion of matrix enzymes in an exposed form and, at the same time, maintain respiratory activity [23,24]. This makes it a useful model system to study B-oxidation with externally added substrates in situ. As shown in Fig. 1, 3-hydroxybutyryl-CoA was readily oxidized in gently disrupted mitochondria but its oxidation was negligible in intact mitochondria, These data indicate that the C o A derivatives cannot enter intact mi:ochondria, but in gently disrupted mitochondria the enzymes are exposed and accessible for this substrate. Completely disrupted mitochondria could also oxidize 3-hydroxybutyryi-CoA but not as effectively as the gently disrupted mitochondria (Fig. 1C). It is i m p o r t a n t to know whether 3-hydroxybutyrylC o A oxidation stops at acetyI-CoA or is completely oxidized in the citric acid cycle. Since acetyl-CoA was not oxidized under the same condition as the 3-hyd r o x y b u t y r y | - C o A (Fig. 2) it is very likely that the

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3 - h y d r o x y b u t y r y I - C o A oxidation stops at acetyI-CoA in gently disrupted mitochondria. T h e finding that an acetyI-CoA accumulation was measurable during the oxidation of 3-hydroxybutyryl-CoA in the reaction c h a m b e r also supports this view. The formed acetyI-CoA could account for the 85 + 6% of the consumed 3-hyd r o x y b u t y r y I - C o A , indicating that the T C A cycle could not oxidize the formed acetyi-CoA under our experintental conditions. Oxidation of crotonyI-CoA was also studied under the same conditions with nearly identical results (details not shown).

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Kinetic advantage of the interaction between the fatty acid beta-oxidation enzymes and the complexes of the respiratory chain.

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C4, C8 and C14)-CoA esters was studied in different mitoc...
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