Loss of function of cytochrome c in Jurkat cells undergoing fas-mediated apoptosis.

Mitochondrial function was examined in Jurkat cells undergoing Fas-mediated apoptosis. With succinate or ascorbate/tetramethylphenylenediamine as substrate, oxygen uptake by digitonin-permeabilized apoptotic mitochondria was greatly decreased as compared with control. Assessment of the function of the cytochrome c-cytochrome oxidase segment of the electron transport chain of apoptotic mitochondria showed that the activity of cytochrome oxidase appeared to be normal, but that of cytochrome c was greatly diminished. A death protease was found to participate in the events leading to the loss of cytochrome c activity, but the cytochrome did not seem to be extensively degraded during the course of apoptosis. Our results suggest that a rapid loss in mitochondrial function due at least in part to the inhibition or inactivation of cytochrome c is a potentially fatal component of the apoptosis program of Jurkat cells.

Mitochondrial function was examined in Jurkat cells undergoing Fas-mediated apoptosis. With succinate or ascorbate/tetramethylphenylenediamine as substrate, oxygen uptake by digitonin-permeabilized apoptotic mitochondria was greatly decreased as compared with control. Assessment of the function of the cytochrome c-cytochrome oxidase segment of the electron transport chain of apoptotic mitochondria showed that the activity of cytochrome oxidase appeared to be normal, but that of cytochrome c was greatly diminished. A death protease was found to participate in the events leading to the loss of cytochrome c activity, but the cytochrome did not seem to be extensively degraded during the course of apoptosis. Our results suggest that a rapid loss in mitochondrial function due at least in part to the inhibition or inactivation of cytochrome c is a potentially fatal component of the apoptosis program of Jurkat cells.
Most cells are equipped with a program whose activation results in a stereotyped series of events that culminate in the death and fragmentation of the cell. In aggregate, these events are referred to as apoptosis, and their effect is to destroy the cell without releasing its contents into the external environment. During apoptosis, the surface of the dying cell is altered so as to mark the cell and its fragments as targets for macrophages, which ingest and degrade the remains.
During apoptosis, much of the cell's contents, including many of its organelles, its cytoskeleton and its plasma membrane undergo far-reaching changes. In the nucleus, the chromatin is disrupted, lamin B (1) is degraded and the DNA is digested into fragments whose length is an integral multiple of Ϸ200 base pairs, the length of the DNA in a nucleosome. A number of proteins, including among others poly(ADP-ribose) polymerase (2), the 70-kDa protein of the U1 small nuclear ribonucleoprotein (3), ␣-fodrin (4), topoisomerase I, histone H1, and phospholipase A 2 (5), are cleaved into defined fragments, presumably with an alteration in their activity. Other proteins are cross-linked by transglutamination, with eventual cornification of the cell and its fragments (6). Cytoskeletal changes result in nuclear fragmentation and blebbing at the cell periphery, substance being lost when a bleb detaches from the main body of the cell, sometimes taking with it a fragment of nucleus. Alterations in cell lipids also occur, with a large increase in levels of ceramide (7), a molecule that may activate the cell death program, and the transfer of phosphatidylserine from the inner to the outer leaflet of the plasma membrane (8).
Mitochondria are also affected by the cell death program. Many studies have shown a partial depolarization of the mitochondrial membrane potential in apoptotic cells (9 -15), and most (9 -11, 16, 17) but not all (18) investigators believe that the oxidants thought to be important in apoptosis induced by tumor necrosis factor-␣ are generated by the mitochondria. Using specific substrates and tumor necrosis factor-␣-treated L929 cells, Schulze-Osthoff et al. (17) demonstrated that electron flow through all four mitochondrial electron transport complexes fell steadily, declining at a rate that was approximately equal among the complexes as the cells progressed through apoptosis. We have studied mitochondrial function in Jurkat cells sent into apoptosis with an anti-Fas IgM, and report that the defect in mitochondrial electron transport observed by ourselves and others in apoptotic cells is due at least in part to the inactivation of cytochrome c.

MATERIALS AND METHODS
Cell Culture-Jurkat cells, a lymphoblastoid T-cell line, were the kind gift of D. Green, La Jolla Institute for Allergy and Immunology. They were cultured at 10 6 cells/ml in RPMI 1640 with 5% fetal calf serum, 2 mM L-glutamine, and penicillin-streptomycin. Individual cultures were maintained for no more than 2 months. Apoptosis was induced by treatment with anti-Fas IgM (clone CH-11, Kamiya Biomedical Co., Thousand Oaks, CA) at the concentration indicated in the figure legends.
Morphological Assessment of Apoptosis-Cells were applied to glass slides (Superfrost/Plus, Fisher), fixed for 5 min in phosphate-buffered saline containing 4% formalin solution, rinsed in methanol, and airdried. A drop of acridine orange solution (4 g/ml) was placed over the cells, a coverslip was added, and the cells were viewed by fluorescence microscopy. Apoptotic cells were scored based on characteristic changes of chromatin condensation and nuclear fragmentation. On each slide, a minimum of 200 cells were evaluated.
Flow Cytometry-DNA content and mitochondrial membrane potential were analyzed on a Coulter Elite flow cytometer. For measurements of DNA content, cells were fixed in 50% ethanol and stored at 4°C until analysis, when they were stained with propidium iodide (50 g/ml), treated with RNase (10 g/ml), and washed in Dulbecco's phosphatebuffered saline. DNA content was calculated from the flow cytometry data using Multicycle C software (Phoenix Flow Systems, San Diego) (19). To determine the mitochondrial membrane potential during apoptosis, the cells were treated with anti-Fas antibody (100 ng/ml) for 2 h in serum-free RPMI. During the last 30 min of treatment, the cells were loaded with di-OC 6 (3) 1 (0.1 M) (14). At the conclusion of the incubation, the cells were isolated by centrifugation, resuspended in Hanks' balanced salt solution and analyzed immediately. The zero-time value and the value for mitochondria whose transmembrane potential was discharged with carbonyl cyanide m-chlorophenylhydrazone (CCCP) were obtained from cells that had been incubated for 30 min with di-OC 6 (3) without exposure to anti-Fas antibody.
* This work was supported in part by United States Public Health Service Grants AG-13501 and AI/CA-01345 and by a grant from Sandoz. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Oxygen Electrode Measurements-A Clarke oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH) with a thermojacketed chamber was used. To induce apoptosis, cells were treated in culture (serum-free RPMI) with anti-Fas antibody (100 ng/ml) at the times indicated; control cells were incubated in a similar manner either with an irrelevant IgM antibody (an anti-cytochrome P450 monoclonal antibody generously provided by Dr. Eric Johnson) or without antibody. The cells were then centrifuged and resuspended in respiration buffer (0.25 M sucrose, 0.1% bovine serum albumin, 10 mM MgCl 2 , 10 mM K ϩ Hepes, 5 mM KH 2 PO 4 , pH 7.2) at a final concentration of 6 ϫ 10 7 cells/ml. One-half ml of the suspension was injected into a chamber containing 2.5 ml of air-saturated respiration buffer, 1 mM ADP that had been prewarmed to 37°C. The cells were permeabilized with digitonin (final concentration 0.005%), and substrates and inhibitors were added in the following order and final concentrations: malate, 5 mM pyruvate, 5 mM; rotenone, 100 nM; succinate, 5 mM; antimycin A, 50 nM; ascorbate, 1 mM (2 mM in experiments using horse heart cytochrome c) with TMPD (tetramethyl-p-phenylenediamine) 0.4 mM; NaN 3 , 5 mM. In most of the experiments, only antimycin A, ascorbate/TMPD, and NaN 3 were used. Where noted, the uncoupling agent CCCP was added at a final concentration of 5 M. Oxygen concentration was calibrated with air-saturated buffer, assuming 390 ng-atoms of oxygen/ml of buffer (17). Transient downward deflections indicate the points at which new reagents were added to the samples in the oxygen electrodes. Rates of azide-sensitive oxygen consumption are expressed as ng-atoms of oxygen/min/3 ϫ 10 7 cells.
Preparation of Mitochondria from Jurkat Cells-Jurkat cells (10 8 ) were incubated for 2.5-3.5 h at 37°C with anti-Fas antibody (50 ng/ml) or with no antibody. Crude mitochondria were prepared as described, with minor modifications (20). Cells were washed once with buffer A (100 mM sucrose, 1 mM EGTA, 20 mM MOPS, pH 7.4, 0.1% bovine serum albumin), then resuspended in 1 ml of buffer B (buffer A plus 10 mM triethanolamine, 5% Percoll, 0.01% digitonin, and the protease inhibitors aprotinin (10 M), pepstatin A (10 M), leupeptin (10 M), and phenylmethylsulfonyl fluoride (1 mM)). After chilling for 3 min on ice, the cells were disrupted by 20 strokes of a glass homogenizer. The homogenate was centrifuged twice to remove unbroken cells and nuclei (2500 ϫ g, 5 min, 4°C). The mitochondria were then pelleted by centrifugation at 10,000 ϫ g for 15 min and resuspended in 100 l of buffer C (300 mM sucrose, 1 mM EGTA, 20 mM MOPS, pH 7.4, 0.1% bovine serum albumin, and protease inhibitors as above, except for phenylmethylsulfonyl fluoride). All steps were performed on ice or at 4°C.
Spectrophotometric Assays of Mitochondria-Mitochondria were prepared as described above from Jurkat cells that had been incubated for 3.5 h at 37°C with or without anti-Fas antibody (50 ng/ml). Mitochondrial difference spectra were obtained by subtraction of the absorption spectra of the unreduced mitochondria from that of mitochondria that had been reduced by dithionite (20) using a DW-2000 SLM/Aminco dual wavelength spectrophotometer. Cytochrome oxidase activity of the isolated mitochondria (about 50 g of protein/assay) was measured spectrophotometrically by following the azide-inhibitable oxidation of ascorbate-reduced cytochrome c (horse heart, Sigma) at 550 nm, determining initial rates from slopes obtained between the first and third minutes of observation (21). K m values were determined from an Eadie-Hofstee plot.
Nondenaturing Polyacrylamide Gel Electrophoresis-Nondenaturing gel electrophoresis was carried out using a 15% polyacrylamide gel. Cytochrome c was detected by a heme stain (22), using horse heart cytochrome c as standard.
Preparation of Cell Lysates for Detection of Antimitochondrial Activity-Jurkat cells (5 ϫ 10 7 ) were incubated for 90 min with or without anti-Fas antibody (100 ng/ml). Cells were pelleted and resuspended in 200 l of respiration buffer, disrupted by sonication on ice (10 3-s bursts, setting 2, Heat Systems Ultrasonics), and centrifuged at 100,000 ϫ g for 30 min at 4°C. The supernatant (200 l, representing 5 ϫ 10 7 cell equivalents) was added to cells (3.6 ϫ 10 7 ) permeabilized with 0.0375% digitonin and incubated at 37°C. After 10 min, measurement of oxygen consumption was initiated.
Electron Microscopy-Control Jurkat cells and cells treated with anti-Fas antibody were collected by centrifugation (2000 rpm for 3 min at room temperature in a Beckman GPR centrifuge). The cell pellet was fixed in Karnovsky's fixative, embedded in epon resin (T. Pella Eponate 812) and cut into 100-nm sections with an LKB Ultratome V. The sections were stained with Reynold's lead citrate and examined under a Hitachi HU 12A transmission electron microscope at 75 kV.
Replicates-Each oxygen electrode tracing shown in this paper is representative of at least two experiments.

Inhibition of Mitochondrial Electron
Transport during Fasmediated Apoptosis-Mitochondrial electron transport is mediated by four multisubunit complexes, designated complexes I-IV, that reside in the inner mitochondrial membrane (Fig. 1). Complexes I and II accept electrons from NADH and succinate, respectively, then pass these electrons on to Complex III via ubiquinone, a low molecular weight redox carrier. Complex III transfers the electrons to cytochrome c, which in turn donates them to Complex IV (cytochrome oxidase). From there the electrons are transferred 4 at a time to molecular oxygen, producing 2 molecules of water. The function of these complexes in mitochondria from control cells and cells treated with anti-Fas antibody was assayed by the sequential addition of substrates specific for various segments of the electron transport path after permeabilization of the mitochondrial outer membrane with digitonin (Fig. 2). The malate-pyruvate combination, which transfers electrons to oxygen via NADH 3 I 3 III 3 IV, produced little oxygen consumption in either control or apoptotic cells. Succinate, however, passing electrons to oxygen via II 3 III 3 IV, generated oxygen uptake that was readily apparent in control cells but not detectable in apoptotic cells. Most striking were the ascorbate/TMPD results, which reflect only electron transport through cytochrome oxidase. With ascorbate/TMPD as substrate, oxygen uptake by apoptotic cells was only a small fraction of that seen with the control cells. The marked inhibition of oxygen uptake in control cells by N 3 Ϫ indicated that cytochrome oxidase was responsible for almost all the oxygen taken up by these cells. These results indicate that electron transport by cytochrome oxidase was greatly inhibited in apoptotic cells, a finding that could also explain the reduction in oxygen uptake in response to succinate, because succinate electrons have to pass through cytochrome oxidase on their way to oxygen. Fig. 3 shows the time course of cytochrome oxidase inhibition. For this experiment, a suspension of Jurkat cells was incubated with anti-Fas antibody, and samples were withdrawn at various time intervals for measurement of oxygen consumption. A reduction in oxygen consumption could be detected as early as 45 min after Fas ligation. By 60 min, oxygen consumption was inhibited by 70%. Under similar conditions, oxygen consumption by control cells (incubated with an irrele-vant IgM) remained relatively constant. At each time point, aliquots of the cells were fixed and analyzed for apoptosis by nuclear morphology (acridine orange staining) and flow cytometry (analysis of ploidy by flow cytometry after propidium iodide staining). The results (Table I) indicated that inhibition of mitochondrial respiration preceded the nuclear changes of apoptosis.
Loss of Function of Cytochrome c in Apoptotic Jurkat Cells-One possible explanation for our findings would be a generalized disruption of mitochondrial structure. To investigate this possibility, we treated Jurkat cells with anti-Fas antibody for 90 min, a time point at which oxygen consumption was inhibited by more than 95%, then examined the cells by electron microscopy (Fig. 4). The nuclei of the control cells were normal in appearance, large and containing well dispersed chromatin. In contrast, the nuclei of most of the anti-Fas-treated cells showed chromatin that was condensed and of a monotonous texture, and many of these nuclei were fragmented. In cells showing these advanced nuclear abnormalities, however, morphological changes in the mitochondria were minor; inner and outer mitochondrial membranes were clearly evident, and cristae appeared intact, although the mitochondria did appear to be slightly swollen, with a more empty-appearing matrix. Thus major mitochondrial disruption at an ultrastructural level did not appear to explain the observed inhibition of electron transport.
Electron transport in mitochondria is normally coupled tightly to the biosynthesis of ATP, which is driven by the passage of protons into the mitochondrial matrix through a channel in the ATP-synthesizing enzyme (the mitochondrial H ϩ -ATPase; Complex V). If the mitochondria assayed in these experiments were tightly coupled, a failure of Complex V would arrest electron transport. Under these circumstances, the addition of an uncoupling agent such as CCCP would be expected to restore oxygen uptake to near normal levels. We found, however, that the addition of CCCP to apoptotic cells incubated with ascorbate/TMPD did not increase oxygen uptake (Fig. 2). This finding indicates that the inhibition of mitochondrial electron transport in apoptotic cells was irrelevant to the coupling of electron transport to ATP production. Taken together, our observations up to this point suggested that apoptosis had a direct effect on electron transport through cytochrome oxidase.
We therefore focused our investigations on this redox complex.
Ascorbate/TMPD does not reduce cytochrome oxidase directly, however, but instead reduces cytochrome c, which then acts as the reducing agent for cytochrome oxidase. To measure the activity of cytochrome oxidase more directly, we employed a spectrophotometric assay based on the oxidation of exogenously added cytochrome c (21). We found to our surprise that the activities of cytochrome oxidase were similar in control and anti-Fas-treated cells (Table II). Furthermore, the interaction between cytochrome oxidase and cytochrome c did not appear to be affected by apoptosis, because the apparent K m for cytochrome c was the same for control mitochondria and mitochondria isolated from cells treated with anti-Fas antibody ( Table II).
The foregoing results suggested that the cytochrome oxidase in anti-Fas-treated cells was intact, and that the failure of electron transport through Complex IV in mitochondria from apoptotic cells was the result of cytochrome c inactivation. This formulation predicts that exogenous cytochrome c would normalize oxygen consumption in apoptotic cells. To test this prediction, cytochrome c (5-45 M in sequential additions) was added along with ascorbate/TMPD to permeabilized cells that had been treated either with anti-Fas antibody or with no antibody for 90 min before permeabilization. Assays of oxygen uptake showed that in anti-Fas-treated cells, the very low rate of cytochrome oxidase-mediated oxygen consumption (3.5 ngatoms/min) was largely corrected by the addition of cytochrome c (Fig. 5). At the highest concentration of cytochrome c, oxygen consumption was 80% of normal. Furthermore, the K m for cytochrome c calculated from these oxygen uptake measurements was 20 M, in reasonable agreement with the K m values determined from measurements of cytochrome c oxidation. In contrast, exogenous cytochrome c did not enhance oxygen consumption in control cells. These findings suggest that cytochrome c is specifically inactivated during Fas-mediated apoptosis.
Cytochrome c is located in the intermembrane space of mitochondria. One possible explanation for the inactivation of cytochrome c observed in our experiments was that digitonin permeabilization of the cells would render the intermembrane space accessible to cytosolic components that are normally excluded from that space. If cytochrome c inactivation were due solely to the invasion of the intermembrane space by such cytosolic components after digitonin permeabilization, then measurements of oxygen uptake obtained immediately after digitonin treatment should be similar for anti-Fas-treated and control cells, since the cytosolic components responsible for the inactivation of the cytochrome would not have had time to work. If however, cytochrome c inactivation had occurred in anti-Fas-treated cells before digitonin permeabilization, then inactivation of cytochrome c (and the consequent decrease in electron transport through cytochrome oxidase) would be apparent immediately upon digitonin permeabilization. To examine these possibilities, we first treated cells for 90 min with anti-Fas antibody or no antibody, then introduced the cells into the oxygen electrode in the absence of digitonin and finally Original magnification for panels A and C was ϫ3000, and for panels B and D was ϫ30,000.

TABLE I Decline in mitochondrial respiration and appearance of signs of apoptosis (apoptotic nuclei (%) as a function of time in cells treated
with anti-Fas antibody The inhibition of oxygen uptake in anti-Fas-treated cells (measured as % of control) was calculated from the rates of oxygen uptake shown in Fig. 3. Apoptotic nuclei were quantified as described under "Materials and Methods."  added digitonin together with ascorbate/TMPD. We found that oxygen uptake was already inhibited when measured immediately after digitonin permeabilization, and that it could be restored by the addition of cytochrome c (Fig. 6). These findings suggest that Fas-mediated inactivation of mitochondrial electron transport occurs in intact cells and is not merely a consequence of digitonin permeabilization. These findings also suggest that cytochrome c is a specific target of inactivation during Fas-mediated apoptosis. Cytochrome c possesses a covalently linked heme group through which electrons are transferred. To determine if cytochrome c or its prosthetic heme group was degraded during Fas-mediated apoptosis, difference spectra (oxidized minus reduced) of the mitochondrial cytochromes were obtained using whole mitochondria obtained from control or anti-Fas-treated cells, and cytochrome c in the cell lysates was analyzed by nondenaturing gel electrophoresis, visualizing with a heme stain (22). Alterations of cytochrome c could be reflected by a change in A 550 /A 563 in the absorption spectrum (i.e. a change in the apparent ratio of cytochrome c to cytochrome b), a change in the electrophoretic mobility of the cytochrome, or both. The spectra of control and apoptotic mitochondria were found to be similar, however, and on gel electrophoresis the cytochrome obtained from apoptotic cells was similar in quantity and mobility to the cytochrome from control cells (Fig. 7). The lack of a detectable change in the A 550 /A 563 ratio or in the electrophoretic mobility of the cytochrome at a time when mitochondrial electron transport was Ͼ90% inhibited suggests that the inactivation of cytochrome c did not involve extensive degradation of the protein or loss of the heme group. Limited proteolysis or other minor modifications of the cytochrome, however, might not have been detected in these experiments.
Studies on the Mechanism of Inactivation of Cytochrome c-Fas-mediated apoptosis is thought to involve the activation of one or more of a large group of death proteases that participate in apoptosis by cleaving a well defined group of proteins carboxyl to an aspartate residue (23,24). To determine if Fasmediated inactivation of cytochrome c required protease activity, cells were preincubated for 90 min with Z-VAD fluoromethyl ketone (Kamiya Biomedical), a tripeptide inhibitor of ICE and related proteases (25), then treated with 100 ng/ml anti-Fas antibody as described. Measurements of cytochrome oxidase-dependent oxygen uptake after an additional 90 min FIG. 5. Effect of cytochrome c supplementation on oxygen consumption in Fas-treated cells. Control and anti-Fas-treated cells (100 ng of anti-Fas antibody/ml) were permeabilized with digitonin. TMPD/ascorbate were then added as electron donors, and oxygen consumption was measured before adding cytochrome c and after each addition of the cytochrome. The final concentrations of cytochrome c after the three successive additions were 5, 15, and 45 M. Numerical values represent azide-sensitive oxygen consumption (ng-atoms O 2 / min/3 ϫ 10 7 cells).
FIG. 6. Oxygen consumption immediately after digitonin permeabilization. Cells were treated with anti-Fas antibody (100 ng/ml) for 90 min, then washed, resuspended in respiration buffer, and injected into an oxygen electrode cuvette containing air-saturated respiration buffer. Digitonin and ascorbate/TMPD were then added simultaneously, and measurement of oxygen consumption was begun immediately. Finally, cytochrome c (100 M final concentration) was added to determine whether the observed inactivation of electron transport could be reversed with exogenous cytochrome c. Numerical values represent azide-sensitive oxygen consumption (ng-atoms O 2 /min/3 ϫ 10 7 cells). For these experiments, the values for azide-insensitive oxygen uptake that were subtracted from the rates of oxygen consumption in the first segment of the reaction (i.e. the segment recorded before the addition of cytochrome c) were measured in separate incubations carried out on the same day with the same cell preparation but without adding cytochrome c.
showed that the ICE inhibitor protected cells against Fasmediated inactivation of cytochrome c (Fig. 8). These results indicate that an antecedent proteolytic event was needed for inactivation of electron transport through cytochrome oxidase.
To ascertain whether a component in the cytosol of the apoptotic cells was responsible for the effect of apoptosis on cytochrome c, cytosol from Jurkat cells treated for 60 -120 min with anti-Fas antibody was added to digitonin-permeabilized control cells (Fig. 9). Cytosol from Fas-treated cells effected an inhibition of cytochrome oxidase-dependent oxygen consumption that was apparent after a preincubation of 5-20 min; cytosol from control cells had no effect on oxygen consumption. The inactivation of electron transport by the cytosolic factor could be reversed by the addition of cytochrome c, indicating that, as in the previous experiments, cytochrome c was inactivated and cytochrome oxidase was spared.
Effect of Fas Ligation on Mitochondrial Membrane Potential-We evaluated the mitochondrial membrane potential using the fluorescent probe di-OC 6 (3), which is retained in mitochondria with a normal membrane potential. CCCP was used to fully discharge the membrane potential before dye loading in the negative control. Untreated cells demonstrated a mean value (in arbitrary fluorescence units) of 24.6. Fas ligation led to a slow decline in fluorescence (Fig. 10) consistent with partial loss of mitochondrial membrane potential during apoptosis. DISCUSSION The decline in mitochondrial function observed in our studies and reported from other laboratories is one of several potentially fatal functional alterations that take place in apoptotic cells. An issue often raised in connection with apoptosis has to do with which one of these lethal changes is the change that is actually responsible for the death of the cell. The role of executioner is usually assigned to the earliest of the lethal changes observed to take place in the dying cell. To the extent that any of several events that take place during apoptosis has the potential to kill the cell, however, this issue is difficult to settle.
It may be that, rather than dying by a single stroke of the sword, a cell undergoing apoptosis actually suffers "the death of a thousand cuts" (26).
The findings obtained with Z-VAD fluoromethyl ketone suggest that the loss of cytochrome c activity during apoptosis involves a step dependent on a death protease. The death protease could have either of two effects: 1) the direct proteolytic inactivation of the cytochrome, or 2) the proteolytic release of a cytochrome c inhibitor. From the results of spectrophotometry, extensive degradation of the cytochrome seems unlikely. Limited proteolysis, however, could alter one of the sites through which cytochrome c binds to other cytochromes, or could change the environment of the heme, altering its redox potential (for example) so that it could no longer carry electrons between cytochrome c 1 and cytochrome oxidase. Such a proteolytic alteration could be accomplished by one of the death proteases or by another protease released through the action of a death protease. As to inhibitors, a precedent exists in the form of IF1, a small mitochondrial polypeptide that inhibits the ATPase activity of Complex V when the potential across the inner mitochondrial membrane is low (27)(28)(29)(30)(31)(32)(33)(34). This regulatory polypeptide prevents the destruction of ATP that would otherwise occur if for some reason the mitochondrial transmem-  Anti-Fas antibody (100 ng/ml) was then added and the cells incubated for an additional 90 min. The cells were then placed in an oxygen electrode cuvette, ascorbate/TMPD added and oxygen uptake measured to assess electron transport through cytochrome oxidase. Numerical values represent azide-sensitive oxygen consumption (ng-atoms O 2 / min/3 ϫ 10 7 cells). brane potential should transiently collapse (28).
Cytochrome c is located in the intermembrane space of mitochondria, raising the question as to how a cytosolic cytochrome c antagonist could reach its target. The antagonist may be generated directly in the intermembrane space during the course of apoptosis, appearing in the cytosol as a result of leakage through the outer mitochondrial membrane. Alternatively, it may be formed in the cytosol, then move to the intermembrane space to exert its effect. In either case, these results suggest that the outer mitochondrial membrane, although appearing intact by electron microscopy, allows the cytochrome c antagonist to pass through, either via a specific carrier or through an alteration in its lipid composition.
The anti-apoptosis protein Bcl-2 is located in the outer membrane of mitochondria, with a portion extending into the intermembrane space. The mechanism by which members of the Bcl-2 family affect apoptosis is not known, and it has been a particular puzzle why Bcl-2 is located in the outer mitochondrial membrane, rather than in the inner mitochondrial membrane where the electron transport complexes and the ATPsynthesizing enzyme are found. Our observation that the apoptosis program can destroy the activity of cytochrome c, a resident of the intermembrane space, indicates that during the execution of that program, events occur in that location that could in principle be influenced by the Bcl-2 associated with the outer mitochondrial membrane.
A further point of interest is our finding that the mitochondria retain much of their transmembrane potential even after oxygen uptake has been completely abolished. The mitochondrial transmembrane potential is generated by the transport of protons out of the mitochondrial matrix during electron trans-port, and would therefore be expected to collapse with the cessation of electron transport as the excess protons flow back into the matrix through the mitochondrial ATPase (Complex V). The finding that mitochondria in apoptotic cells retain a portion of their transmembrane potential raises the possibility that, like cytochrome c, the mitochondrial ATPase is also defunctionalized in some way as cells undergo apoptosis. The function of other mitochondrial elements may also be abrogated during apoptosis. Complex III, as a hypothetical example, could be inactivated through an effect on the Rieske protein, which extends into the intermembrane space (35). Electron transport abnormalities yet to be demonstrated may account for the discrepancy in time between the early fall in oxygen uptake and the somewhat later decline in cytochrome c function in cells undergoing apoptosis.  9. Effect of Fas-treated cytosol on oxygen consumption in digitonin-permeabilized control cells. Untreated whole Jurkat cells in oxygen electrode cuvettes were permeabilized with digitonin and incubated for 15 min at 37°C with cytosol from untreated Jurkat cells or Jurkat cells treated for 120 min with anti-Fas antibody (100 ng/ml) as described in the text. Ascorbate/TMPD was then added and oxygen uptake was measured to assess electron transport through cytochrome oxidase. In the experiments in which the effect of cytochrome c was examined, the cytochrome (100 M final concentration) was added to the reaction mixture in the oxygen electrode after measuring initial rate. Numerical values represent azide-sensitive oxygen consumption (ng-atoms O 2 /min/3 ϫ 10 7 cells).