Mitochondrial - NCBI

Plant Physiol. (1991) 95, 157-163 0032-0889/91/95/01 57/07/$01 .00/0

Received for publication June 21, 1990 Accepted September 20, 1990

Effects of Polyamines on the Oxidation of Exogenous NADH by Jerusalem Artichoke (Helianthus tuberosus)

Mitochondrial Michela Rugolo, Fabiana Antognoni, Alberto Flamigni, and Davide Zannoni* Department of Biology, Biochemistry Laboratory, University of Bologna, 1-40126 Bologna, Italy teins, resulting in an altered catalytic potential of the electron transport chain ( 19). By using the fluorescent amine 9-AA2 as a probe for determining the extent of electrostatic screening of fixed charges on the surface of thylakoid and mitochondrial membranes (4, 16), it has been demonstrated that Arum mitochondria contain fewer negative charges per mg of protein than do JAM (16). Because Arum mitochondria oxidize exogenous NADH at a rate which is 10 times faster than the corresponding activity in JAM, a correlation between the inhibitory effect produced by the presence of negative charges on oxidation of external NADH, has also been suggested (16). The above data strongly indicate that the formation of membrane complexes, by bridging and shielding the surface charges, is expected to reduce the repulsive forces between negatively charged membrane components with a great effect on membrane-bound enzymes such as those involved in the oxidation of external NADH. Accordingly, the interaction of organic polycations, such as putrescine, spermidine, and spermine, with acidic membrane phospholipids or negatively charged residues of membrane-bound proteins, is expected to affect the oxidation of exogenous NADH, as previously shown for inorganic cations such as Mg2" and Ca2" (4). However, because the polyamine positive charges are distributed at fixed lengths along a conformationally flexible carbon chain, polyamines are not point-localized charges and are able to bridge only critical distances (27). These features allow specific interactions and functions, which are not shared by metal

ABSTRACT The effect of polyamines (putrescine, spermine, and spermidine) on the oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L. cv. OBI) mitochondria, have been studied. Addition of spermine and/or spermidine to a suspension of mitochondria in a low-cation medium (2 millimolar-K+) caused a decrease in the apparent Km and an increase in the apparent Vm.x for the oxidation of exogenous NADH. These polycations released by screening effect the mitochondrially induced quenching of 9-aminoacridine fluorescence, their efficiency being dependent on the valency of the cation (C4+ > C3+). Conversely, putrescine only slightly affected both kinetic parameters of exogenous NADH oxidation and the number of fixed charges on the membranes. Spermine and spermidine, but not putrescine, decreased the apparent Km for Ca2+ from about 1 to about 0.2 micromolar, required to activate external NADH oxidation in a high-cation medium, containing physiological concentrations of Pi, Mg2+ and K . The results are interpreted as evidence for a role of spermine and spermidine in the modulation of exogenous NADH oxidation by plant mitochondria in vivo.

In contrast to mammalian tissues, in plant cells, cytosolic NADH may donate electrons directly to the mitochondrial respiratory chain by means of an externally located NADH dehydrogenase without entering the matrix or using shuttle mechanisms (7). This enzyme is located on the outer surface of the inner membrane and donates reducing equivalents into the bc 1 complex of the respiratory chain (7). The oxidation of exogenous NADH by plant mitochondria is specifically activated by Ca" ions and unspecifically enhanced by cations (reviewed in ref. 18). From the experiments of Cowley and Palmer (5) and Moore and Akerman (21), it was calculated that exogenous NADH oxidation requires about 1 ,uM free Ca2 for maximum activity. The nonspecificity of the cations and the relative effectiveness of cations of different valencies (C3+ > C2+ > C+) was consistent with the concept that they act by screening the fixed charges associated with the surface of the membranes (10). The screening affects not only the ability of NADH to reach the active site of the membrane-bound exogenous NADH dehydrogenase (10) but also appears to affect the lateral diffusion of membrane pro-

cations. Polyamines are ubiquitous polycationic metabolites in prokaryotic and eukaryotic cells (reviewed in ref. 27). Effects of polyamines on membranes were among the earliest documented in the polyamine field, e.g. the stabilizing action of spermine on bacterial membranes (13, 30). Since then, numerous publications have indicated that polyamines might influence, if not modulate, membrane properties and functions. However, the mode of action at a molecular level of polyamines is still matter of speculation (27). The present work shows the influence of putrescine, spermidine, and spermine on the kinetic (Km and Vmax) parameters of the oxidation of exogenous NADH in Jerusalem artichoke (Helianthus tuberosus) mitochondria. In addition, we tested 2Abbreviations: 9-AA, 9-aminoacridine; FCCP, carbonylcyanidep-trifluoromethoxy phenylhydrazone; JAM, Jerusalem artichoke mitochondria.

' Research work supported by Consiglio Nazionale delle Ricerche

and Ministero della Pubblica Istruzione of Italy. 157

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the possibility that in the presence of micromolar concentrations of polyamines the concentration of free Ca2" required to activate the oxidation of exogenous NADH might be different from the currently accepted value of about 1 uM (5, 21). The results were interpreted to show that spermine and spermidine, but not putrescine, can affect not only the capacity of the external NADH dehydrogenase to catalyze NADH oxidation at a higher rate by screening of fixed charges on the membrane, but also appear to affect the affinity of the catalytic site for Ca2" ions.

MATERIALS AND METHODS Plant Material

Helianthus tuberosus L. cv OB 1 (Jerusalem artichoke) was grown and vegetatively propagated in the Botanical Garden of the University of Bologna. The tubers were harvested at the beginning of dormancy in November and stored in moist sand at 4°C. Isolation of Purified Mitochondria Crude mitochondria were isolated according to Douce et al. (6) using a chilled (4°C) isolation medium (0.3 M mannitol, 10 mm Hepes buffer (pH 7.8), 1 mm EDTA, mM EGTA) and then squeezed through four layers of cheesecloth. The final pellet was resuspended in 0.3 M mannitol, 2 mM Hepes buffer (pH 7.2), to be immediately used for purification. Crude mitochondria were purified on a 21 % (v/v) continuous Percoll gradient according to Liden and M0ller (11). As described by Douce et al. (6), the integrity of the external mitochondrial membrane was assayed by testing the antimycin A-sensitivity of the NADH-Cyt c oxidoreductase activities in both intact and osmotically burst mitochondria. The media and the conditions used were as follows: purified mitochondria were resuspended in 5 mM Hepes buffer (pH 7.2) containing mm KCN, 50 gM horse heart Cyt c, and 0.3 M mannitol, in the presence or absence of antimycin A (1 A M). Mannitol was excluded from the assay of burst mitochondria. The reaction was initiated with mM NADH. Cyt c reduction was measured at 550 nm (E = 21 mM-' cm-') in an Hitachi 100-40 spectrophotometer. The percentage of broken mitochondria was given by the ratio between the antimycin Asensitive rates in intact and burst mitochondria. In all preparations the percentage of mitochondrial membrane integrity was about 85% with P/O ratios of 1.6. In all experiments, purified mitochondria were suspended in a low-salt medium, i.e. 0.3 M mannitol, 2 mM Hepes buffer (pH 7.2)/0.1% bovine serum albumin, to minimize the number of cations present in the final preparation.

Hepes buffer/0. 1% bovine serum albumin [pH 7.2]) and highsalt medium (0.3 M mannitol, 10 mM Hepes buffer/0.1% bovine serum albumin, 1 mM MgCl2, 20 mM KCl, 5 mM KH2PO4 [pH 7.2]). The free Ca2+ concentration of the medium in the presence of EGTA/Ca2' buffer system was calculated as described by Bers (3). According to Nicchitta and Williamson (22) polyamines (s0.4 mM) do not change the free Ca2+ concentration.

Measurements of 9-AA Fluorescence The fluorescence of 9-AA (20 ,M) was measured in low-salt resuspension medium (see above) at a room temperature in a Jasco FP-550 spectrofluorimeter. The excitation wavelength was 398 nm (slitwidth, 5 nm) and the emission was at 457 nm (slitwidth, 5 nm). The c'/2 value for a given cation (salt or polyamine), i.e. the concentration of the cation needed to give a release of quenching of 9-AA fluorescence equal to half that given by 20 mm MgCl2 (26) was determined as described previously by M0ller et al. (16). Protein Assay

Proteins were determined according to Lowry et al. (12) with bovine serum albumin as the standard. RESULTS Effect of Inorganic and Organic Cations on the Apparent Km and Vmax of NADH Oxidation Figure 1 shows the Lineweaver-Burke plots of the oxidation of exogenous NADH by JAM under conditions of low and high ionic strength. It is apparent that mitochondria suspended in a low-salt medium oxidize NADH at a Vmax which is lower than that measured in high-salt medium. In line with a previous report (19), the Km for NADH were 56 and 117 ,M under high- and low-salt conditions, respectively. In Figure la, the effect of the trivalent cation, spermidine (Spm3+), and of the tetravalent cation, spermine (Sm4+), on the apparent Vmax and Km of NADH oxidation, is also shown. Interestingly, both polycations (1 mM) show an increase in the Vmax (see also below, Fig. 2) and a decrease in the apparent Km (from 100 to 50 jM) of NADH oxidation, when added to a low-salt medium. Conversely, in mitochondria suspended in high-salt medium the kinetic parameters of NADH oxidation were basically not affected by addition of Sm4+ only/or Spd3+ (shown in Fig. lb, closed symbols). Notably, 1 mM putrescine had little effect on the apparent Km for NADH oxidation (from 117 to 100 jAM) by mitochondria suspended in low-salt medium (data not shown). NADH Oxidation and Cations

Oxidation of NADH NADH oxidation was measured either as 02 consumption in a Yellow Springs model 5331 oxygen electrode (1.5 mL, 26°C) or as the disappearance of NADH measured at 340 nm in an Hitachi 100-40 spectrophotometer (1 mL, room temperature). Experiments were performed by using two suspension media, namely: low-salt medium (0.3 M mannitol, 2 mM

The data of Figure 2 demonstrate that in a low-salt medium the rate of exogenous NADH oxidation may be enhanced by the addition of KCl, CaCl2, putrescine, spermidine, and spermine. It can be seen that the percent activation of the rate of NADH oxidation varies between the different cations tested; in addition, the cation concentration at which NADH oxidation was half-maximally stimulated occurred over dif-

EFFECTS OF POLYAMINES ON THE EXTERNAL NADH DEHYDROGENASE

159

Figure 1. Lineweaver-Burk plots of the effect of spermine and spermidine on the kinetic parameters of NADH oxidation. NADH oxidation was measured at 340 nm (90 Ag of mitochondnal protein/mL) either in the low-salt (a) or in the high-salt (b) medium as described in "Materials and Methods," in the absence (open symbols) or in the presence (closed symbols) of polyamines. Rates are given as nmol of NADH (consumed)min- mg-1 of protein. V,, for low and high cation conditions were 56 and 125 nmol min-1 mg-1 of protein, respectively. Abbreviations: Spd (spermidine); Sm (spermine). Symbols: control (0); Spd (A); Sm (A).

5OpM 117pM

1/S [pM]

1/S [PM]

56pM

ferent concentration ranges, i.e. 50 AM for Sm4+ (spermine), 90 AM for Spm3+ (spermidine), 0.17 mm for Ca2+, 0.3 mm for Pu2" (putrescine), and 30 mm for K+. These results are consistent with previous observations made using monovalent, divalent, and trivalent inorganic cations (10) and support the concept that the stimulation can simply be due to an electrostatic screening of fixed charges associated with the membrane surface. As previously suggested, the ultimate effect could be the removal of electrostatic restrictions on diffusion ratelimited steps in electron transport (19). It has been reported (20), that NADH oxidation activities in mitochondria isolated from Arum spadices and Jerusalem artichoke tubers and suspended in low-cation media, were 20% stimulated and 40% inhibited, respectively, by 60 AM LaCl3. Thus, the trivalent cation La" might cause some inhibitory effect on external NADH oxidation. This latter conclusion was not confirmed by the experiments of Figure 3 showing the effect of La" on the oxidation of exogenous

NADH under different ionic strength conditions. Closed symbols indicate experiments in which mitochondria were suspended in a low-salt medium in the presence of different polyamines, whereas the open symbols refer to experiments with mitochondria suspended in low-salt medium (control) or in high-salt medium (HSM). It is apparent that La" had little effect on NADH oxidation under low-ionic strength in the presence or absence of putrescine; conversely, a strong inhibitory effect of La3+ was clearly seen in the presence of spermine or spermidine. The data of Figure 3 tend to demonstrate that the capacity of La" to inhibit the oxidation of external NADH is induced by electrostatic screening of fixed charges on the membrane surface and se is strongly enhanced by spermine and spermidine. Effect of La3+, Spermine, Spermidine, and Putrescine on 9-Aminoacridine Fluorescence 9-Aminoacridine is a fluorescent, monovalent amine which shows a decrease in fluorescence ("quenching") when it is

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Figure 2. Stimulation by salts (CaCl2, KCI) and polyamines (putrescine, spermine, spermidine) of the oxidation of exogenous NADH by JAM. The stimulation is expressed as the percent of increase over the control containing no added cations. Mitochondria (0.1 mg/mL) were suspended in low-salt medium. Each point represent a separate determination. The rate of NADH oxidation in the absence of cation was 70 nmol min-' mg-1 of protein. Abbreviations as for Figure 1. Symbols: Sm4+ (U); Spd3+ (A); Ca2+ (0); Pu2+ (putrescine) ( ); K+ (Q).

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RUGOLO ET AL.

,gM for the divalent cations Ca2" and Mg", respectively. These

values were approximately 30 times lower than the value of c1/2 obtained with the divalent polyamine putrescine at the same concentration of mitochondrial proteins (cl/2 = 0.75 mM) (not shown).

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La3+ [M] Figure 3. Effect of LaCI3 on the oxidation of NADH by mitochondria suspended in high-salt medium (HSM) and in low-salt medium in the presence (closed symbols) or in the absence (open symbols) of putrescine, spermine, and spermidine. Identification symbols, control rate, and experimental conditions as in Figure 2.

bound to biological membranes (26). 9-AA is attracted into the diffuse layer associated with the membrane negative charges. The addition of cations will cause an increase in fluorescence ("quenching release") due to a displacement of 9-AA from the diffuse layer (26). The concentration of the salt needed to release the quenched fluorescence of 9-AA to half that achieved by 20 mm MgCl2 was defined by Searle et al. (26), as cl/2. A previous report indicated that in plant mitochondria the valency of the cation was more important than its chemical specificity in determining the cl/2 values, i.e. cl/2 for La" is 103 lower than for K+ and 102 lower than for Mg2> (16). It was also shown that the cl/2 value, for all cations irrespective of valency, increased in parallel with increasing concentration of mitochondrial proteins (16). This phenomenon is in line with the Gouy-Chapman theory predicting an increase in the c½/2 value with increasing area of negatively charged membranes per unit volume of suspension (16). In Figure 4, the dependence of the response of 9-AA fluorescence to La3+, spermidine (Spd3+), and spermine (Sm4+) on the concentration of JAM is shown. It is apparent that the cl/2 values, at a given protein concentration (>0.05 mg/mL), were dependent on the cation (C) valency; the order of efficiency in releasing the 9-AA quenching was C4' > C3+. Notably, although both the trivalent cations La>3 and Spd3+ released the 9-AA quenching at micromolar concentrations, spermidine was less effective than La3" over the protein concentration range tested (0.05-0.25 mg/mL). A previous report (16) indicated that JAM suspended at a concentration of 0.08 mg of protein/mL, resulted in cl/2 values of 26 and 25

Ca2+ Dependency of NADH Oxidation A previous report demonstrated that oxidation of exogenous NADH by plant mitochondria depends on Ca2 (17) which is normally found bound to the mitochondrial membrane in sufficient amount to ensure maximal activity (17). Indeed, when a chelator such as EGTA was used to remove Ca2 ion an inhibition was observed, this phenomenon being reversed by free Ca2 concentrations close to 1 ,uM (17, 21). The control experiments of Figure 5 (traces in a, b, and c) show that in both low- and high-salt media (see "Materials and Methods") NADH oxidation by JAM was markedly inhibited by EGTA (1 mM), but as the Ca` activity of the incubation medium was increased stepwise from 0.03 gM to about 1.5 uM free Ca2+ concentration, NADH oxidation via the externally located NADH dehydrogenase is enhanced (Fig. 5, trace in c). Figure 6 (a and b) show the rate of NADH oxidation as a function of free Ca2+ concentrations under uncoupled state 3 and state 4 conditions. In both cases, maximal activity was obtained between 1 and 1.4 uM free Ca>2. The effect of spermine (0.4 mM) on the free Ca2>

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mg of protein ml-1 Figure 4. Effect of La3+, spermine, and spermidine on the release of quenching of 9-AA fluorescence as a function of the concentration of mitochondrial protein. The c1/2 value for the effect of a given cation was taken to be the concentration of the cation needed to release the quenched fluorescence of 9-AA (20 gM) to half that achieved by addition of 20 mm MgCI2 at the end of each titration curve (not shown). Mitochondria (0.05-0.025 mg/mL) were suspended in lowsalt medium. All measurements were performed on one preparation of mitochondria.


(a)

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Low cation

Low cation

161

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Figure 5. Oxidation of exogenous NADH by JAM suspended in low-salt and/or high-salt media as a function of different EGTA/Ca2' ratios. Mitochondria (1.1 mg/mL) were suspended in low-salt (a) and high-salt (b and c) media. Additions (small arrows): 1 mm EGTA; 1 mm NADH; 100 tLM Ca2+ (each time) (see text for further details). The values above the traces denote the rates of respiration and were obtained polarographically (nmol of 02 min-' mg-1 of protein). All measurements were performed under uncoupled conditions (+0.5 tLM FCCP).

High cation High

cation

concentration required to reach maximal activity of the NADH oxidation, is also shown. It is apparent that concentrations of 0.2 and 0.3 uM free Ca2" were sufficient to ensure maximal activity under uncoupled state 3 and state 4, respectively. Notably, putrescine (0.4 mM) had no effect on the apparent Km for Ca2+ whereas the trivalent cation spermidine showed an effect on the apparent Km for Ca2+ similar to that induced by spermine (not shown).

DISCUSSION AND CONCLUSION The results presented in this study demonstrate that organic polycations such as spermine, spermidine, and putrescine affect the oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus) mitochondria isolated in a lowcation medium, by screening of fixed negative membrane charges (10). Although the double layer theory (14) predicts that the effectiveness of the cation in charge shielding is determined by its charge, and not by the chemical nature of the cation, the data presented suggest that some specificity exists among the polyamines tested. Indeed, physiological concentrations (0.1-0.4 mM) of spermine (Sm4+) and spermidine (Spd3+) were seen to be efficient activators of the external NADH oxidation whereas their precursor, putrescine (Pu21) was 2 to 3 times less effective even at concentrations one order of magnitude higher (2-3 mM) (see Fig. 2). Additional support to the concept that spermine and spermidine show a specific effect on NADH oxidation was given by the results obtained with the trivalent cation La". Both La3+ and Spd3+ released quite efficiently the quenching of 9-AA (Fig. 4), thus suggesting analogous charge screening effects. However, La" did not stimulate NADH oxidation, as Spd3+ did, but instead a progressive inhibition of NADH oxidation was observed as a function of the ionic strength, this phenomenon being particularly evident in the presence of Sm4+ and Spd3+ (Fig. 3). This finding suggests that La" does not inhibit by itself but requires a screening effect which is particularly evident in the presence of tri- and tetravalent polyamines (Fig. 3). The mechanism by which spermine and spermidine considerably increase the inhibitory effect of La3+ on the external NADH oxidation, is presently unclear. This phenomenon might be related to a conformational change of the dehydro-

genase and/or a possible effect of polyamines on the phospholipid region close to the enzyme, facilitating the access of La3+ to its inhibitory binding site(s). It is therefore noteworthy that spermine (50 ,M) reduces the "threshold concentration" of Ca" for the fusion of lipid vesicles (23). Effects of cations and polyamines on the aggregation and fusion of vesicles of phosphatidylserine and phosphatidylcholine mixed with different acidic phospholipid, have been reported (15, 23, 3 1). It was concluded that there is a good correlation between the degree of membrane fusion and that of the increased surface tension of the membrane. La3+ (2 10 FM), was the most effective cation in causing fusion of the membranes whereas polyamines showed little or no effect, in spite of their chargeneutralizing activity. It has also been shown that Sm4+ can induce massive aggregation of acidic phospholipid vesicles even at low (40 uM) concentrations (23, 31), suggesting that polyamines are unable to penetrate into lipid polar groups upon their binding to membrane surfaces (however, see refs. 24, 32). The conclusion is that spermidine and spermine are only adsorbed on the phospholipid polar groups whereas La3+ can penetrate into the lipid layer to induce membrane fusion. The role that Ca" plays in activating exogenous NADH oxidation seems to be related to the interaction ofthe external dehydrogenase with the respiratory chain at the ubiquinone level (NADH dehydrogenase donor site) (18, 29). The fact that chelators inhibit NADH oxidation more when added before the substrate than after, led to the suggestion that oxidation of NADH affects the membrane enzyme region in such a way that Ca" is locked into the active site (17; see also Fig. 5). Conversely, the lag which is observed before maximal linear rate of NADH oxidation can be achieved (17; see Fig. 5), was suggested to represent the time- and respirationdependent phase required to mobilize free matrix Ca>2 and/ or Ca>2 bound on the outer surface of the inner membrane (17). Polyamines compete with Ca>2 for binding to anionic phospholipids (15), mitochondria (1), and to various specific complexes (25) thereby increasing the Ca>2 dissociation constant (1). Because one of the main effects of spermine and spermidine on mitochondrial external NADH oxidation is to decrease its apparent Km for external free Ca2+ (Fig. 6), it appears that these organic polycations enhance the interaction between Ca>2 and its binding site. This effect cannot be due to simple electrostatic interaction with the membrane since

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RUGOLO ET AL.

the positively charged polyamines should reduce the surface negative charge density and thus increase the apparent Km for free Ca2" rather than decrease it. In addition, our data were obtained with mitochondria suspended in high-salt media where most of the negative membrane charges are likely to be screened by inorganic cations. Under this latter condition, Sm4+ and Spd3+ did not affect the apparent Km and the Vmax of NADH oxidation (Fig. 1). Thus, the effect of polyamines must be more specific.

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The rate of binding of ligands (or ions) with membrane receptors at very low concentrations depends on their surface diffusion rate (2). Thus, if binding groups for an ion exist on the membrane surface, the rate of the ions surface diffusion would be inhibited. Since Ca2' binds strongly to anionic phospholipids, the presence of these groups should impede surface diffusion of Ca2' at low Ca2+ concentrations, i.e. 6 1 AM. These considerations provide a possible explanation for the effect of Sm4` and Spd3` on the Km for Ca2+ of the NADH oxidation, namely: at low external free Ca2+ the formation of the Ca2+ membrane-complex is limited by surface diffusion of Ca2+ ion since it is slowed down by Ca2+ binding to anionic phospholipid. Polyamines interact with anionic phospholipids so to enhance the Ca2' surface diffusion, the Ca2+ dissociation constant, and the formation of the Ca2+ membrane-complex at low free Ca2+ concentrations. Our finding that spermine and spermidine affect the oxidation of exogenous NADH through a decrease of the apparent Km for Ca2+ might suggest some physiological significance. In vivo, polyamines are likely to be mostly bound, and consequently may affect the cytosolic level of free Ca2` (which is estimated to be about 0.1-0.2 Mm) (8). This latter value is close to the in vitro free Ca2+ concentration required to activate the oxidation of exogenous NADH in the presence of spermine and/or spermidine (this work). Temporal and spatial oscillations of polyamines induced by variations of the ornithine decarboxylase activity, have been observed (9). Polyamines have also been reported to inhibit phospholipase C (33) and to enhance inositol phospholipid synthesis (28), thus also modulating the activity of the inositol-lipid second messenger system. Combining the latter effects with the effects of polyamines on Ca2+ sequestration by mitochondria and with mitochondrial polyamine uptake (24, 32), it is plausible to conclude that spermine and spermidine may be important cytoplasmic factors affecting mitochondrial oxidation of exogenous NADH in vivo.

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ACKNOWLEDGMENTS

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We wish to thank Dr. M. Bonini for skilful assistance during the course of the mitochondrial isolation procedure. We also wish to thank Prof. B. A. Melandri (University of Bologna, I) for helpful discussions and Dr. A. L. Moore (University of Sussex, Brighton,

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U.K.) for critically reading the manuscript.

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[Ca2+], M Figure 6. (a and b). NADH oxidation by JAM suspended in high-salt medium in the presence (closed symbols) or in the absence (open symbols) of spermine (0.4 mM) as a function of different free Ca2+ concentrations (see text for further details). The NADH consumption was measured polarographically in the presence (state 3 unc, a) or in the absence (state 4, b) of 0.5 gM FCCP. Conditions as in Figure 5c.

4.

5.

6.

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