ARCHIVES
OF
BIOCHEMISTRY
Oxidative
AND
BIOPHYSICS
Properties M. ABDUL
Pre-Clinical
174, 705-712 (1976)
of Swollen MATLIB
AND
Rat Liver Mitochondria’ PAUL
A. SRERE
Science Unit, Veterans Administration Hospital, and Department of Biochemistry, The University of Texas Health Science Center, Dallas, Texas 75216 Received
November
17, 1975
Rat liver mitochondria undergo extensive swelling when they are incubated in hypotonic sucrose medium containing 5 mM P,. After 30 min of swelling at 25’C, a three- to fourfold increase in volume has occurred, accompanied by gross disorganization of the matrix as observed by electron microscopy. Succinate-supported respiration was unchanged, but the respiration of NAD-linked substrates was reduced and there was a complete and irreversible loss of phosphorylation in both cases. P-Hydroxybutyratesupported respiration was regained completely on addition of NAD to the swollen mitochondria. a-Ketoglutarateand malate + pyruvate-supported respiration was only partially restored by the addition of NAD. This inhibition of respiration in swollen mitochondria may be due to a disorganization of a putative complex of Krebs cycle enzymes on the inner surface of the inner membrane.
When the inner membrane of mitochondria is broken, a number of enzymes of the Krebs cycle is released as soluble proteins. These enzymes are generally thought to exist free throughout the entire mitochondrial matrix. A number of years ago, Srere (1) proposed that the “soluble” Krebs cycle enzymes exist as multienzyme complex near or on the inner surface (matrix side) of the inner membrane. Recently Matlib and O’Brien (2) have shown some of the matrix enzymes are actually located close to the inner membrane of rat liver mitochondria. Srere et al. (3) later showed that an immobilized multienzyme system of malate dehydrogenase, citrate synthase, and lactate dehydrogenase operated at a faster rate than the equivalent amount of free enzymes. This model of microenvironmental compartmentation demonstrated the kinetic advantage that could be attained by the postulated organization of Krebs cycle enzymes. In an effort to test this postulate, the rates of oxidation of various substrat.es were measured in intact and swollen mito’ Supported by grants from the National Foundation and Veterans Administration.
chondria. In swollen mitochondria one would expect a drastic reduction in the rate of oxidation of a substrate since the mean free path between an intermediate and the next enzyme is increased. For an eight-step reaction, such as the Krebs cycle, the decrease in rate might well be sizable. It was not expected that swelling of the mitochondria would have a rate effect on electron transport since this is carried out by the inner membrane which is a solid and, therefore, nondilutable multienzyme complex. On the other hand, sufficiently great disorganization of the matrix might well dissociate the proposed Krebs cycle assemblies. This paper contains the results of our studies on rates of respiration in swollen rat liver mitochondria. MATERIALS
METHODS
Materials. The following materials were obtained from the indicated sources: ADP, ATP, NAD, NADH, DTNB,’ L-malic acid, pyruvic acid, tu-ketoglutaric acid, L-glutamic acid, P-hydroxybutyric ’ Abbreviations used: DTNB, 5,5’-dithiobis(Z-nitro-benzoic acid); HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; DNP, 2,4-dinitrophenol; BSA, bovine serum albumin.
Science 705
Copyright C 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
706
MATLIB
AND
acid, and succinic acid from Sigma Chemical Co.; oxalacetic acid from Calbiochem; CoA from P-L Biochemicals. Acetyl-CoA was prepared by the method described previously (4). Chemicals for electron microscopy were obtained from Tousimis, Rockville, Md. The detergent NONIDET P40 was obtained from Gallard Schlesinger, Carle Place, N.Y. Isolation of mitochondria. Mitochondria were isolated from livers of 200-250-g male Sprague-Dawley rats fasted for 24 h. The isolation procedure used was the one described previously (5) with the exception that the excised livors were homogenized manually in 10 volumes of a medium composed of 220 rnM mannitol, 70 mM sucrose, 2 mM HEPES, O.l? BSA, pH 7.4, in a 40-ml Kontes glass homogenizer, During homogenization, four passes were made with a loosely fitting pestle (pestle A) and two passes with a tightly fitting pestle (pestle B), followed finally by four passes with pestle A. Mitochondria prepared by this method were reasonably free of contamination of other cell organelles as observed by electron microscopy. These preparations were tightly coupled, with very high respiratory control values, sometimes approaching an infinite value and an ADP/O ratio for each substrate near theoretical value. Mitochondria (0.75 mg Swelling of mitochondria. of protein/ml) were allowed to swell at 25°C in a medium containing 125 mM sucrose, 5 mM Tris-HCl (pH 7.41, and 5 mM potassium phosphate buffer (pH 7.4). These swelling conditions were maintained throughout the present studies. The kinetics of swelling were determined by following the loss of optical density of 3.3 ml of suspension at 520 nm using a Beckman DU spectrophotometer with a Gilford recording assembly. Measurement of respiration. Respiration was measured polarographically with a Clark oxygen electrode, according to a previously described method (6). The assays were carried out at 25’C in 3.3 ml containing 125 mM sucrose, 5 mM Tris-HCI tpH 7.4) and 5 mM potassium phosphate buffer \pH 7.4). Swelling of the mitochondria, carried out as described above, was stopped with 5 rnM MgCI, at appropriate times; mitochondrial respiration was measured after the addition of substrate and NAD. To obtain the rate of respiration before swelling, the mitochondrial suspension was directly added to the above medium supplemented with 5 mM MgCl?. ADP (0.4 pmol) or DNP (25-50 PM) was used in unincubated mitochondria to obtain maximum rates of respiration. Measurement of NAD released. At the end of the incubation period the mitochondrial suspension was centrifuged at 10,OOOgfor 15 min, and the supernatant solution was analyzed for NAD. NAD was measured fluorometrically in a spectrofluorometer (Farrand Optical Co., Valhalla, N.Y.) according to a method described previously (7). The total NAD con-
SRERE
tent of mitochondria was determined using a sample of mitochondrial suspension obtained after the incubation period. Known quantities of NAD were run simultaneously as standards. Assay for enqmcs released. Citrate synthase was assayed according to the method described earlier (8). The standard assay contains 0.1 mM DTNB, 0.25 mM acetyl-CoA, 0.5 rnM oxaiacetate, 100 mM TrisHCI (pH 8.1). Malate dehydrogenase was assayed according to a previously described method (9). The standard assay contains 50 mM potassium phosphate buffer (pH 7.4), 0.15 rnM NADH, and 0.5 mM oxalacetate.
OF
BIOCHEMISTRY
Oxidative
AND
BIOPHYSICS
Properties M. ABDUL
Pre-Clinical
174, 705-712 (1976)
of Swollen MATLIB
AND
Rat Liver Mitochondria’ PAUL
A. SRERE
Science Unit, Veterans Administration Hospital, and Department of Biochemistry, The University of Texas Health Science Center, Dallas, Texas 75216 Received
November
17, 1975
Rat liver mitochondria undergo extensive swelling when they are incubated in hypotonic sucrose medium containing 5 mM P,. After 30 min of swelling at 25’C, a three- to fourfold increase in volume has occurred, accompanied by gross disorganization of the matrix as observed by electron microscopy. Succinate-supported respiration was unchanged, but the respiration of NAD-linked substrates was reduced and there was a complete and irreversible loss of phosphorylation in both cases. P-Hydroxybutyratesupported respiration was regained completely on addition of NAD to the swollen mitochondria. a-Ketoglutarateand malate + pyruvate-supported respiration was only partially restored by the addition of NAD. This inhibition of respiration in swollen mitochondria may be due to a disorganization of a putative complex of Krebs cycle enzymes on the inner surface of the inner membrane.
When the inner membrane of mitochondria is broken, a number of enzymes of the Krebs cycle is released as soluble proteins. These enzymes are generally thought to exist free throughout the entire mitochondrial matrix. A number of years ago, Srere (1) proposed that the “soluble” Krebs cycle enzymes exist as multienzyme complex near or on the inner surface (matrix side) of the inner membrane. Recently Matlib and O’Brien (2) have shown some of the matrix enzymes are actually located close to the inner membrane of rat liver mitochondria. Srere et al. (3) later showed that an immobilized multienzyme system of malate dehydrogenase, citrate synthase, and lactate dehydrogenase operated at a faster rate than the equivalent amount of free enzymes. This model of microenvironmental compartmentation demonstrated the kinetic advantage that could be attained by the postulated organization of Krebs cycle enzymes. In an effort to test this postulate, the rates of oxidation of various substrat.es were measured in intact and swollen mito’ Supported by grants from the National Foundation and Veterans Administration.
chondria. In swollen mitochondria one would expect a drastic reduction in the rate of oxidation of a substrate since the mean free path between an intermediate and the next enzyme is increased. For an eight-step reaction, such as the Krebs cycle, the decrease in rate might well be sizable. It was not expected that swelling of the mitochondria would have a rate effect on electron transport since this is carried out by the inner membrane which is a solid and, therefore, nondilutable multienzyme complex. On the other hand, sufficiently great disorganization of the matrix might well dissociate the proposed Krebs cycle assemblies. This paper contains the results of our studies on rates of respiration in swollen rat liver mitochondria. MATERIALS
METHODS
Materials. The following materials were obtained from the indicated sources: ADP, ATP, NAD, NADH, DTNB,’ L-malic acid, pyruvic acid, tu-ketoglutaric acid, L-glutamic acid, P-hydroxybutyric ’ Abbreviations used: DTNB, 5,5’-dithiobis(Z-nitro-benzoic acid); HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; DNP, 2,4-dinitrophenol; BSA, bovine serum albumin.
Science 705
Copyright C 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
706
MATLIB
AND
acid, and succinic acid from Sigma Chemical Co.; oxalacetic acid from Calbiochem; CoA from P-L Biochemicals. Acetyl-CoA was prepared by the method described previously (4). Chemicals for electron microscopy were obtained from Tousimis, Rockville, Md. The detergent NONIDET P40 was obtained from Gallard Schlesinger, Carle Place, N.Y. Isolation of mitochondria. Mitochondria were isolated from livers of 200-250-g male Sprague-Dawley rats fasted for 24 h. The isolation procedure used was the one described previously (5) with the exception that the excised livors were homogenized manually in 10 volumes of a medium composed of 220 rnM mannitol, 70 mM sucrose, 2 mM HEPES, O.l? BSA, pH 7.4, in a 40-ml Kontes glass homogenizer, During homogenization, four passes were made with a loosely fitting pestle (pestle A) and two passes with a tightly fitting pestle (pestle B), followed finally by four passes with pestle A. Mitochondria prepared by this method were reasonably free of contamination of other cell organelles as observed by electron microscopy. These preparations were tightly coupled, with very high respiratory control values, sometimes approaching an infinite value and an ADP/O ratio for each substrate near theoretical value. Mitochondria (0.75 mg Swelling of mitochondria. of protein/ml) were allowed to swell at 25°C in a medium containing 125 mM sucrose, 5 mM Tris-HCl (pH 7.41, and 5 mM potassium phosphate buffer (pH 7.4). These swelling conditions were maintained throughout the present studies. The kinetics of swelling were determined by following the loss of optical density of 3.3 ml of suspension at 520 nm using a Beckman DU spectrophotometer with a Gilford recording assembly. Measurement of respiration. Respiration was measured polarographically with a Clark oxygen electrode, according to a previously described method (6). The assays were carried out at 25’C in 3.3 ml containing 125 mM sucrose, 5 mM Tris-HCI tpH 7.4) and 5 mM potassium phosphate buffer \pH 7.4). Swelling of the mitochondria, carried out as described above, was stopped with 5 rnM MgCI, at appropriate times; mitochondrial respiration was measured after the addition of substrate and NAD. To obtain the rate of respiration before swelling, the mitochondrial suspension was directly added to the above medium supplemented with 5 mM MgCl?. ADP (0.4 pmol) or DNP (25-50 PM) was used in unincubated mitochondria to obtain maximum rates of respiration. Measurement of NAD released. At the end of the incubation period the mitochondrial suspension was centrifuged at 10,OOOgfor 15 min, and the supernatant solution was analyzed for NAD. NAD was measured fluorometrically in a spectrofluorometer (Farrand Optical Co., Valhalla, N.Y.) according to a method described previously (7). The total NAD con-
SRERE
tent of mitochondria was determined using a sample of mitochondrial suspension obtained after the incubation period. Known quantities of NAD were run simultaneously as standards. Assay for enqmcs released. Citrate synthase was assayed according to the method described earlier (8). The standard assay contains 0.1 mM DTNB, 0.25 mM acetyl-CoA, 0.5 rnM oxaiacetate, 100 mM TrisHCI (pH 8.1). Malate dehydrogenase was assayed according to a previously described method (9). The standard assay contains 50 mM potassium phosphate buffer (pH 7.4), 0.15 rnM NADH, and 0.5 mM oxalacetate.
