Characterization of mitochondrial nitric oxide synthase

Characterization of mitochondrial nitric oxide synthase

[31] MITOCHONDRIALNO SYNTHASE 339 TABLE IV PHARMACOL(~ICALREGULATIONOF MITOCHONDRIALNITRIC OXIDE SYNTHASEACTIVITY Drug Daily dose/treatment Orga...

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TABLE IV PHARMACOL(~ICALREGULATIONOF MITOCHONDRIALNITRIC OXIDE SYNTHASEACTIVITY

Drug

Daily dose/treatment

Organ

Effect on mtNOS (% of control activity)

Haloperidol Chlorpromazine Enalapril Enalapril Enalapril Losartan Thyroxine (T4) Methylprednisolone

2 mg/kg mouse, 1 day 10 mg/kg mouse, 1 hr 30 mg/kg rat, 15 days 30 mg/kg rat, 15 days 10 mg/kg rat, 6 months 10 mg/kg rat, 6 months 0.2 mg/kg rat, 2 weeks 3/zg/ml medium, 30 min

Brain Brain Liver, heart Kidney Heart, liver Heart Liver Thymocytes

-54 -48 313-330 890 105-120 95 -45 450

anti-nNOS directed against the amino and carboxy terminus of the polypeptide chain. Chlorpromazine treatment has no effect on the band intensity of the protein reacting with the anti-nNOS (amino terminus) antibody, despite the measured decrease in enzymatic activity (Fig. 2 and Table III) indicating a direct inhibitory effect of chlorpromazine on the enzymatic protein. P h a r m a c o l o g i c a l R e g u l a t i o n of m t N O S - 1 a n d m t N O S - 2 Table IV lists a series of in vivo treatments involving the use of five pharmacological substances used commonly that affect both mtNOS-I and mtNOS-2 activity. Both increases and decreases of mtNOS activity are described. The in vitro effect of methylprednisolone in thymocytes is included, considering its pharmacological relevance.

[3 i] Characterization of Mitochondrial Nitric Oxide S y n t h a s e By PEDRAM

GHAFOURIFAR

The diverse roles of nitric oxide (NO) in biology have been studied broadly in the last two decades. NO reacts with hemoproteins, thiols, and ions, such as superoxide anion (02-). Many of the actions of NO are due to its ability to react with soluble guanylate cyclase and subsequently increase cyclic GMP levels, However, a considerable portion of the biological functions of NO are cyclic GMP independent. Mitochondria, the lipoprotein cytoplasmic particles of eukaryotic cells,

METHODSIN ENZYMOLOGY,VOL.359

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possess many hemoproteins and thiols and are the main cellular sources of 02-. Thus, mitochondria play an important role in mediating cGMP-independent effects of NO. Mitochondria are heavily compartmentalized organelles. The mitochondrial matrix, inner membrane, and intermembrane space differ in a number of ways, such as in electrochemistry, redox state, and pH, as well as radical and protein content. Thus NO and its metabolites interact with these different mitochondrial components in distinct manners. For example, cytochrome oxidase, an abundant hemoprotein embedded in the mitochondrial inner membrane, interacts with NO in a transient, reversible, and 02 concentration-dependent manner. Physiologically relevant concentrations of NO inhibit cytochrome oxidase in a manner resembling a pharmacological competitive antagonism with oxygen. 1 NO can also react with reduced thiols to produce nitrosothiols. This reaction is also reversible, however, it is redox and pH sensitive. Although S-nitrosylation is generally fast and S-nitrosylated proteins are relatively stable in vitro, their formation and stabilization are not favored in certain biological compartments. For example, inorganic phosphate, which is abundant in the mitochondria matrix, inhibits the S-nitrosylation reaction. 2 Also, some enzymes present in the matrix, such as glutathione peroxidase3 or thioredoxin reductase, 4 can decompose S-nitrosothiols. In contrast to the matrix, the mitochondrial intermembrane space seems the preferred site for S-nitrosylation. To date, S-nitrosylation of a mitochondrial intermembrane space protein caspase-3 has been demonstrated. 5 Caspase-3 is S-nitrosylated as long as it is within the organelle, rendering it apoptotically silent. This is likely a mechanism for protecting mitochondria against the protease activity of the caspase. Nitric oxide can also react with O2- to produce the powerful oxidizing agent, peroxynitrite (ONOO-). This reaction is stoichiometric, diffusion limited, and requires relatively high pH, which is provided in the mitochondrial matrix. In intact, tightly coupled succinate-energized mitochondria, the pH is in the range of 7.5 to 7.8 in the matrix6 and 6.9 to 7.0 in the intermembrane space. 7 Thus, the mitochondrial matrix environment can favor the formation and reactions of ONOO-. In contrast to the reversible reactions of NO with rnitochondrial proteins, the ones of ONOO- are mostly irreversible. A direct measurement of ONOOin biological samples is difficult, however, biomarkers such as nitrotyrosine or lipid peroxides are good indicators. Tyrosine nitration of mitochondrial matrix 1 p. Ghafourifar, U. Bringold, S. D. Klein, and C. Richter, BioL Sign. Recep. 10, 57 (2000). 2 E. G. DeMaster, B. J. Quast, and R. A. Mitchell, Biochem. Pharmacol. 53, 581 (1997). 3 y. Hou, Z. Guo, J. Li, and R G. Wang, Biochem. Biophys. Res. Commun. 228, 88 (1996). 4 D. Nikitovic and A. Holmgren, J. Biol. Chem. 271, 19180 (1996). 5 j. B. Mannick, C. Schonhoff, N. Papeta, R Ghafourifar, M. Szibor, K. Fang, and B. Gaston, J. Cell Biol. 154, 1111 (2001). 6 E Bemardi, J. Biol. Chem. 267, 8834 (1992). 7 j. D. Cortese, A. L. Voglino, and C. R. Hackenbrock, Biochim. Biophys. Acta 1100, 189 (1992).

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proteins, e.g., MnSOD 8 or succinyl-CoA:3-oxoacid CoA-transferase (SCOT), 9 has been reported. With the exception of cytochrome c all of the mitochondrial respiratory chain components have matrix faces. Peroxynitrite-induced inactivation of mitochondrial respiratory chain complexes I to IV has been reported by many groups (reviewed in Ghafourifar and Richterl°). Although tyrosine nitration of cytochrome c, which is located in the low pH intermembrane environment, does not seem favorable in biology, ONOO- can dissociate cytochrome c from mitochondfia. T M How S-nitrosothiols or ONOO- are formed within mitochondria has not yet been fully elucidated. However, evidence suggests that the newly characterized mitochondrial NO synthase (mtNOS) might be the hitherto undetected source of NO and its congeners within mitochondria (Fig. 1). M i t o c h o n d r i a l Nitric O x i d e S y n t h a s e In biology, NO is synthesized by NO synthase (NOS; EC I. 14.13.39) family members. NOS isozymes catalyze oxidation of the terminal guanidino nitrogen of U-arginine to NO and L-citrulline as the coproduct. So far, three distinct isoforms of NOS have been well characterized in mammalian tissues. Although none of these isozymes has a tissue-specific pattern of expression, they are referred to as endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). All presently characterized NOS isozymes are heme-containing proteins that are dimeric under native conditions with a monomer molecular mass of about 126-160 kDa. nNOS and eNOS are expressed constitutively, whereas iNOS is expressed when cells are stimulated by certain stimuli. The constitutive isoforms show a typical Ca 2+calmodulin sensitivity, whereas the activity of iNOS, once expressed, is not altered by increased cytosolic Ca 2+. Several laboratories have investigated the possible presence of a NOS within mitochondria. In 1997, we demonstrated for the first time the activity of a constitutively expressed Ca2+-sensitive NOS within mitochondria (mtNOS)J 3 We also demonstrated that mtNOS is associated with the mitochondrial inner membrane and regulates mitochondrial 02 consumption and AS. These results were later confirmed by other laboratories. 14'15 Since 1997, several laboratories have shown 8 L. A. MacMillan-Crow, J. P. Crow, and J. A. Thompson, Biochemistry 37, 1613 (1998). 9 I. V. Turko, S. Marcondes, and E Murad, Am. J. Physiol. Heart. Circ. Physiol. 281, H2289 (2001). l0 E Ghafourifar and C. Richter, in "Mitochondrial Ubiquinone (Coenzyme Q10): Biochemical, Functional, Medical, and Therapeutical Aspects in Human Health and Disease" (M. Ebadi, J. Marwah, and R. Chopra, eds.), p. 437. Prominent Press, AZ, 2000. 11E Ghafourifar, U. Schenk, S. D. Klein, and C. Richter, J. Biol. Chem. 274, 31185 (1999). 12 V. Borutaite, R. Morkuniene, and G. C. Brown, Biochim. Biophys. Acta 1453, 41 (1999). 13 E Ghafourifar and C. Richter, FEBS Lett. 418, 291 (1997). 14 Z. Lacza, M. Puskar, J. P. Figueroa, J. Zhang, N. Rajapakse, and D. W. Busija, Free Radic. Bio. Med. 31, 1609 (2001). 15 A. J. Kanai, L. L. Pearce, E R. Clemens, L. A. Brider, M. M. VanBibber, S. Y. Choi, W. C. de Groat, and J. Peterson, Proc. Natl. Acad. Sci. U.S.A. 98, 14126 (2001).

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FIG. 1. Mitochondria, highly compartmentalized organelles. The inner membrane (IM), the matrix, and the intermembrane space (IMS) and their distinct composition, electrochemistry, and redox state are illustrated. IM: The respiratory chain complexes are embedded in this compartment. The chain consists of four complexes (I to IV), coenzyme Q (ubiquinone; Q) and cytochrome c (cyto c). The respiratory complexes are arranged functionally in an electrochemical hierarchy that provides a unique broad spectrum of redox potentials. Electrons flow down the chain to complex 1V where they reduce 02 to H20. Coupled to the electron flow, protons are pumped from the matrix into IMS. This proton extrusion establishes a transmembrane potential (A~, negative inside) and an electrochemical gradient (ApH, alkaline inside) across IM. In response to A~p, mitochondria take up Ca 2+, which stimulates mtNOS activity. The produced NO inhibits the complex IV activity, which affects A~p, ApH, and mitochondrial Ca 2+ homeostasis. The mitochondrial respiratory chain is one of the main cellular sources of 02-. Nitric oxide reacts readily with 0 2 - to produce ONOO-. Mitochondrial redox barriers, such as MnSOD, may affect the rate of ONOO- formation. Matrix: Key mitochondrial redox defense barriers, e.g., MnSOD, GSH, or glutathione peroxidase (GPX), are located within the matrix. Some matrix proteins, such as MnSOD and SCOT, are susceptible to ONOO--induced oxidative damage. IMS: Cytochrome c is the only respiratory chian member located in this compartment. Certain mitochondrial apoptogenic proteins, such as caspase 3, are also located in IMS.

interest in performing mtNOS research. Rat liver is the most commonly used source for isolating mitochondria. Mitochondria can be isolated by the conventional differential centrifugation as described. 16 However, the following are practical tips for specifically studying mtNOS in rat liver mitochondria. 16 C. Richter, M e t h o d s E n z y m o l . 105, 435 (1984).

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Procedure Fast the animals one night before the experiment, with water ad libitum. Decapitate and deplete the body of blood to limit the exposure of mitochondria to NO-reacting molecules, such as hemoglobin. Remove the liver quickly, place it in a dish on ice, and remove fat, ducts, connective tissues, and blood clots. Cut the liver into small pieces and wash several times in ice-cold buffer to remove remaining blood. Homogenize the washed pieces in a glass homogenizer with a Teflon pestle. It is highly recommended that the homogenizer, pestle, or centrifuge tubes not be washed with any detergent. Detergents released from the glass, Teflon, or centrifuge tubes can release membrane-associated proteins, such as mtNOS, from mitochondrial membranes. It is important to keep the temperature low during the homogenization, e.g., by placing the glass tube on ice. Do not overhomogenize because heat and the mechanical force produced during homogenization can strongly damage mtNOS. During centrifugation, a red spot in the low-spin pellet may indicate contamination with blood. If there is a fat layer floating above the supernatant of the high-spin steps, remove it with a soft lint-free tissue. Discard the fluffy light brown layer above the high-spin pellet. This layer is not mitochondria. A good rat liver mitochondria preparation provides about 30-40 mg mitochondrial protein per rat liver. Higher protein contents may indicate contamination. There are several methods for assessing the purity of the preparation. We do this routinely by measuring the cytochrome a content spectrophotometrically at 605-630 nrn using the extinction coefficient of 12 M -1 c m - l J 7 D e t e r m i n a t i o n of m t N O S Activity NOS activity can be determined by different techniques. The rather unique structure and composition of mitochondria, however, limit the use of some of these techniques in mtNOS activity determination. Also, it must be noted that the activity of mtNOS, as well as that of many other mitochondrial enzymes, declines rapidly in isolated mitochondria. Thus, it is highly recommended that miNOS-related measurements be performed only with freshly isolated mitochondria, generally within 4 - 6 hr of isolation.

Hemoglobin (Hb) Assay In aqueous solutions, NO reacts rapidly with oxyhemoglobin (oxyHb) to produce methemoglobin (metHb). This reaction is stoichiometric and can be followed spectrophotometricaUy. With minor modifications, the standard Hb assay can be used as a reliable method for quantifying mtNOS activity. However, if the isolated 17R. S. Balaban,V. K. Mootha,and A. Arai,AnalBiochem.237, 274 (1996).

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mitochondria are intact and not contaminated with cytosolic proteins, e.g., cytosolic forms of NOS, no metHb formation is observed. NO produced within mitochondria reacts with several mitochondrial NO traps such as thiols. This prevents intramitochondrially formed NO from reaching the extramitochondrial probe, oxyHb. Additionally, oxyHb cannot enter mitochondria because the mitochondrial inner membrane is impermeable to almost every molecule larger than 100-150 Da (oxyHb is 65 KDa). Thus, formation of metHb from oxyHb within intact freshly prepared mitochondria generally indicates artifacts, such as contamination with extramitochondrial proteins, mtNOS activity can be determined by the Hb assay if mitochondrial membranes are ruptured, i.e., in broken mitochondria (BM). Some investigators, however, have suggested that oxymyoglobin (18 kDa) might be used to detect mtNOS activity in intact mitochondria) 8 Another confounding factor that limits the use of the Hb assay in mitochondria is 02-. Superoxide anion can potently react with NO, and mitochondria are one of the main cellular sources 02-. Thus, relatively high amounts of superoxide dismutase (SOD), e.g., > 1 kU/ml, is highly recommended in this assay. Preparation of Broken Mitochondria. Apply a hypoosmolar shock by adding 2-4 volumes of ice-chilled H20 containing the protease inhibitors (10/zM each) leupeptin, pepstatin A, aprotinin, and phenylmethylsulfonyl fluoride (PMSF). Apply a mild sonification, e.g., 100-150 W, 50% duty cycle, 75 sec, to break the remaining intact mitochondria. To avoid reaction with oxygen, purge the water with N2 for at least 15 min and apply N2 over the suspension during the sonification. Osmolality can be readjusted by the addition of pH-adjusted concentrated buffer (e.g., a 10× buffer) to the BM suspension. Spin the suspension at 10,000g for 10 min. The supernatant is BM. 13 Spectrophotometric Determination of mtNOS Activity. Mitochondria contain sufficient concentrations of the substrate and cofactors NOS requires, and Ca 2+ per se seems sufficient to trigger the mtNOS activity in intact isolated mitochondria. 11,19However, dilution or oxidation of some of these factors during the preparation of BM or submitochondrial particles (SMP) may require the addition of the following substrates or cofactorsl3: 1-10/zg/ml calmodulin: prepare a 1-mg/ml stock solution, make aliquots, and store at - 8 0 ° . Avoid freeze-thawing. 1 mM-L-arginine: prepare a 100 mM stock solution and store at - 2 0 °. 0.2 mM NADPH: prepare a 20 mM stock solution daily. 5/zM FAD: prepare a 0.5 mM stock solution daily. 5/zM FMN: prepare a 0.5 mM stock solution daily. 18 C. Giulivi, J. J. Poderoso, and A. Boveris, J. BioL Chem. 273, 11038 (1998). 19 U. Bringold, P. Ghafourifar, and C. Richter, Free Radic. Biol. Med. 29, 343 (2000).

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10/zM tetrahydrobiopterin (BH4): prepare a 2 mM stock solution in 10 mM HC1 immediately prior to the experiment. It undergoes rapid autoxidation on dilution. > 1 kU/ml SOD: prepare a 100 kU/ml stock solution. Make aliquots and store at - 2 0 ° . Avoid freeze-thawing.

Procedure Add the 37 ° buffer (e.g., 0.1 M HEPES, pH 7.0) to a 37 ° thermostated cuvette and then add the mtNOS substrate(s) and 4 / z M oxyHb. Mix and record the absorbance. Add 0.03 to 0.1 mg of BM or SMP/ml. More than 0.1 mg mitochondrial protein per milliliter can disturb the measurement due to too much light scattering. MetHb formation is shown by a continual increase in optical density. Several wavelengths can be used. 2° For example, at 401 nm the NO formation can be quantified using an E401(metHb-oxyHb) of 49 mM -l cm -1.

Citrulline Assay Determination of radiolabeled L-citrulline produced from radiolabeled Larginine is one of the widely used methods for NOS activity determination. This assay is preferred for intact mitochondria, as the Hb assay cannot be used. However, many of the buffers used traditionally to investigate mitochondrial functions contain high concentrations of Mg 2+, a well-known mitochondrial Ca 2+ uptake blocker. The presence of Mg 2+ decreases mtNOS activity drastically and can abolish its Ca 2+ sensitivity. In the presence of 5 mM Mg 2+, mtNOS may even appear Ca 2+ insensitive. 21

Procedure Incubate 0.1 to 3 mg mitochondrial protein with NOS substrates (as discussed earlier) supplemented with L-[3H]arginine (30,000-50,000 cpm) at 37 ° in a total volume of 100 #1 buffer. Terminate mtNOS activity by adding 1 ml of ice-chilled stop solution containing 2 mM EDTA, 1 mM unlabeled L-citrulline, and 20 mM sodium acetate buffer, pH 5.0. Basal mtNOS activity can be determined by supplementing mitochondria with 30,000-50,000 cpm L-[3H]arginine without NOS substrates. 11,13 Preparation of Cation-Exchange Columns. Preparetheexchangeresin (Dowex 50W x 8, mesh size 200-400, H + form) as described. 22 Load the spin filters with 20 M. Feelisch, D. Kubitzek, and J. Werringloer, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, eds.), p. 455. Wiley, Chichester, 1996. 21 S. French, C. Giulivi, and R. S. Balaban, Am. J. Physiol. Heart Circ. PhysioL 280, H2863 (2001). 22 B. Mayer, P. Klatt, E. R. Werner, and K. Schmidt, Neuropharmacology 33, 1253 (1994).

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FIG. 2. mtNOS and mitochondrial oxygen consumption. Oxygen consumption of 1 mg/ml isolated rat liver mitochondria in 0.1 M HEPES, pH 7.1 (cont), was measured at room temperature with a tightly sealed Clarke-type electrode under continuous stirring. Respiration was supported by 0.4 mM succinate (succ). Ca2+ (100/zM) or L-NMMA (10 mM) was present in the buffer prior to the addition of mitochondria. Reproduced with permission from P. Ghafourifar, U. Schenk, S. D. Klein, and C. Richter, J. Biol. Chem. 274, 31185 (1999).

0.6 to 1 ml of the prepared resin, spin at 5000g for 2 min, and wash with 500/zl H20. Load the colunms with 500/zl of the radiolabeled ruitochondrial samples and spin at 5000g for 2-5 min. Wash the resins twice with 200/zl H20. Transfer the effluent to scintillation vials, add 3 ml of scintillation fluid, and determine the radioactivity. Radioactive material passing through the colunm after mock incubation (no mitochondrial material added) is normally about 1% of the total radioactivity used in the experiment. mtNOS and Mitochondrial Functions

mtNOS and Oxygen Consumption mtNOS regulates mitochondrial oxygen consumption. In intact mitochondria, Ca 2+ per se is sufficient to decrease respiration in an mtNOS-dependent manner (Fig. 2) given that mitochondria are energized, thus they can take up Ca 2+, and Ca 2+ uptake is not blocked, e.g., by Mg 2+.

Procedure Into a tightly sealed thermostated chamber equipped with an oxygen electrode, add the buffer, e.g., 0.1 MHEPES, the desired concentrations of Ca 2+ , and 1 mg/ml of mitochondria or mitochondrial subfractions. If broken mitochondria are used, mtNOS substrates (as mentioned earlier) may also be required. After a 3- to 5-min incubation at room temperature with the mtNOS substrates or inhibitors, energize mitochondria by adding mitochondrial respiratory substrates. Relatively high concentrations of NOS inhibitors may be required to inhibit mtNOS activity. Most

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10 #M safranin. Electron transport was supported by 0.8 mM potassium succinate (A). A~p was measuredby dual-wavelengthspectrophotometryat 533-511 nm. The additionof EGTA(0.5 mM) or CCCP (1/zM) is indicatedby arrows. Where indicated,5 mM L-NMMA was present in the buffer. (A) Ca2+ (40 nmol/mg mitochondrialprotein) was added where indicated. (B) Ca2+ (40 nmol/mg mitochondrialprotein) was present in the buffer prior to the additionof succinate.Reproduced with permissionfrom P. Ghafourifarand C. Richter,J. Biol. Chem. 380, 1025 (1999). of the commonly used NOS inhibitors are competitive L-arginine analogs, and the intramitochondrial L-arginine concentration is in the millimolar range. 23 Additionally, mitochondrial membranes are not very permeable to most of these inhibitors. If longer preincubations are needed, mitochondria can be preincubated with the NOS inhibitor on ice for up to 1 hr. Broken mitochondria or submitochondrial particles normally do not need extensive preincubation with NOS inhibitors. m t N O S and Mitochondrial M e m b r a n e Potential

mtNOS regulates the mitochondrial transmembrane potential (A~p) (Fig. 3). The A~z can be determined by several techniques, such as dual-wavelength spectroscopy. 23M. Dolinskaand J. Albrecht,Neurochem. Int. 33, 233 (1998).

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FIG. 4. mtNOS and mitochondrial Ca 2+ homeostasis. Mitochondria were suspended in 0.1 M HEPES buffer, pH 7.4, containing 50/zM arsenazo III, and the respiratory chain was supported by 0.8 mM potassium succinate (A) in the presence of 5/zM rotenone (A). Mitochondrial Ca 2+ homeostasis was followed by dual-wavelength spectrophotometry at 685-675 nm. Repeated additions of 10 nmol CaZ+/mg mitochondrial protein are shown by dashed arrows and 0.5 mM EGTA by the filled arrow. Where indicated, 5 mM L-NMMA was present in the buffer. Reproduced with permission from P. Ghafoufifar and C. Richter, J. Biol. Chem. 380, 1025 (1999).

Procedure

Incubate 1 mg/ml of mitochondria in buffer (e.g., 0.1 M HEPES, pH 7.35) in the presence of 10/zM safranin T. Energize mitochondria with mitochondrial respiratory substrates, such as 0.8 mM succinate, and record the optical density at 511-533 nm. Inhibition of mtNOS increases both the rate and the magnitude of A~p formation, and its stimulation decreases it. mtNOS and Mitochondrial Ca2+ Homeostasis

Until recently, the importance of mitochondria in cellular Ca 2+ homeostasis was overlooked. However, recent findings indicate the crucial role of mitochondria in phasic Ca 2+ homeostasis. 24 Mitochondria take up Ca 2+ in response to their A~p and release Ca 2+ via distinct pathways, z5 Mitochondrial Ca 2+ homeostasis can be followed by several techniques, such as dual-wavelength spectroscopy at 685-675 nm using Arsenazo III as a probe. Procedure

Suspend 1 mg mitochondria/ml in 0.1 M HEPES buffer, pH 7.4, in the presence of 50/zM Arsenazo III and measure the optical density at 685-675 nm. Support respiration by adding mitochondrial respiratory substrates, such as 0.8 mM potassium succinate, mtNOS is stimulated upon mitochondrial Ca 2+ uptake, and mtNOS inhibition can be achieved by preincubating mitochondria with a NOS inhibitor, as described earlier (Fig. 4). 24 R. Rizzuto, P. Pinton, W. Carrington, E S. Fay, K. E. Fogarty, L. M. Lifshitz, R. A. Tuft, and T. Pozzan, Science 280, 1763 (1998), 25 C. Richter, E Ghafourifar, M. Schweizer, and R. Laffranchi, Biochem. Soc. Trans. 25, 914 (1997).

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FIG. 5. mtNOS and mitochondrial matrix pH. Mitochondria were suspended in the incubation buffer as described in the text, and proton extrusion was supported by 0.8 mM potassium snccinate (A) in the presence of 5/xM rotenone (A). (A) NO was provided exogenously by the repeated addition of 0.5 mM nitrosoglutathione (GSNO); (B) mtNOS activity was stimulated by loading mitoehondria with Ca2+ (40 nmol/mg mitochondrial protein) and was inhibited using L-NMMA (5 mM); (C) rntNOS basal activity was inhibited by 5 mM L-NMMA; and (D) mtNOS basal activity was inhibited by 5 mM L-citrulline (L-cit). Exogenous NO was provided by repeated additions of 0.5 mM GSNO. Reproduced with permission from P. Ghafourifar and C. Richter, J. Biol. Chem. 380, 1025 (1999).

mtNOS and Mitochondrial Matrix pH

Mitochondrial respiratory complexes are functionally arranged in a redox potential (also called midpoint potential) hierarchy. Electrons enter the chain, flow to the downstream complexes, and reduce 02 to water at the terminal respiratory complex, complex IV. Coupled to the electron flow, protons are pumped from the mitochondrial matrix into the mitochondrial intermembrane space. This establishes the ApH, a proton gradient across the coupling membrane that renders the membrane alkaline inside. Inhibition of the mitochondrial electron transport chain, e.g., by intramitochondriaUy formed NO, decreases ApH (Fig. 5). Mitochondrial matrix pH can be determined spectrofluorometrically using 2',7'-bis(carboxyethyl)-5,6carboxyfluorescein (BCECF) as a probe. 26 26 E Ghafourifar and C. Richter, J. Biol. Chem. 380, 1025 (1999).

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Buffers Loading buffer: 250 mM sucrose, 10 mM Tris-HCL, 0.1 mM EGTA-Tris, pH 7.4 Incubation buffer: 200 mM sucrose, 0.5 mM Tris-MOPS, pH 7.4

Procedure Suspend 25 mg mitochondrial protein in 1 ml of the loading buffer containing 10/zg BCECF. Mix and incubate for 20 min at room temperature in the dark (because BCECF is very light sensitive) while shaking very gently. Spin the mitochondria at 10,000g for 10 min at 4 °, wash the pellet with 1 ml ice-cold loading buffer, and recentrifuge. Resuspend the pellet in 1 ml of the ice-cold incubation buffer. Measure the matrix pH of 0.2 mg mitochondrial protein/ml in incubation buffer at room temperature with a spectrofluorometer with an excitation wavelength of 500 nm (4-mm slit) and an emission wavelength of 525 nm (5-mm slit).

Calibration Incubate 0.2 mg of BCECF-loaded mitochondrial protein in 1 ml of calibration buffer containing 100 mM KC1, 30 /zM EGTA-Tris, 2 #M rotenone, 0.8/zg/ml oligomycin, 50 nMcarbonyl cyanide 3-chlorophenylhydrazone (CCCP), 0.25/zg/ml valinomycin, and 10 mM Tris-MOPS, pH ranging from 6.2 to 8.2. Intra- and extramitochondrial pH are equal under these conditions. Measure extramitochondrial pH with a glass electrode and the intramitochondrial fluorescence as explained earlier. Draw the working curve by plotting the fluorescence intensity against pH. Acknowledgment I greatly acknowledge the enormous intellectual freedom that Dr. Christoph Richter provided to me during the time I spent in his laboratory. The extremely valuable discussions with and the critical reading of the manuscript by Dr. Tammy Dugas are greatly appreciated.