Comparative biochemical studies of ATPases in cells ... - Springer Link

J. Inher. Metab. Dis. 21 (1998) 829È836 ( SSIEM and Kluwer Academic Publishers. Printed in the Netherlands

Comparative biochemical studies of ATPases in cells from patients with the T8993G or T8993C mitochondrial DNA mutations M. E. VA ZQUEZ-MEMIJE1, S. SHANSKE2, F. M. SANTORELLI2, P. KRANZ-EBLE2, D. C. DEVIVO2 and S. DIMAURO2* 1 Unidad de Investigacio n en Gene tica Humana, Hospital de Pediatr• a, Centro Me dico Nacional-IMSS, Me xico, D.F. ; 2 College of Physicians and Surgeons, Columbia University, New Y ork, USA *Correspondence : Columbia College of Physicians and Surgeons 4-420, 630 W 168th Street, New Y ork, NY 10032, USA MS received 8.1.98

Accepted 15.5.98

Summary : We performed comparative biochemical studies in cultured Ðbroblast mitochondria from patients with the T8993G or the T8993C point mutations in the ATPase 6 gene of mitochondrial DNA. We found that ATP production was much more severely decreased in cells from patients with the T8993G mutation than in those from patients with the T8993C mutation. Kinetic studies suggest that both mutations a†ect only the F sector of the 0 mitochondrial ATPase complex. We conclude that these two mutations, which result in the substitution of di†erent amino acids at the same site of the ATPase, result in an enzyme with di†erent biochemical characteristics. The mitochondrial oxidative phosphorylation system consists of the electron transport chain (complexes IÈIV) and the F F -ATPase (complex V) and is the primary 1 0 source of ATP production in most tissues (HateÐ 1993). Genetic alterations a†ecting the components of this very complex enzymatic system can lead to insufficient energy supply and result in various mitochondrial disorders. Mitochondrial ATP synthase consists of two functional domains, F and F 1 0 (Boyer 1993). F protrudes into the matrix, is hydrophilic, and contains Ðve subunits 1 (3a, 3b, c, d, e) and the inhibitor protein (Abrahams et al 1994 ; Amzel et al 1992). F 0 is hydrophobic, is embedded in the mitochondrial inner membrane, and contains subunits a, b, c, d, e, f, g, F , OSCP and A6L (Fillingame 1992). F is connected to 6 1 F by a stalk that contains the subunits OSCP, F , b and d (Walker and Collinson 0 6 1994). Subunits 6 and A6L are encoded by the mitochondrial genome (Anderson et al 1981), whereas all of the other subunits are encoded by nuclear genes. ATP synthase deÐciency can therefore be due to mutations of either nuclear or mitochondrial genes. While the number of mitochondrial diseases due to impairment of 829

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V a zquez-Memije et al

oxidative phosphorylation is increasing, speciÐc defects of mitochondrial ATP synthase appear to be relatively rare. So far, only one patient has been described in whom ATP synthase deÐciency was caused by a mutation in one of the nuclearencoded subunits of the enzyme (Holme et al 1992). On the other hand, several mutations a†ecting the ATPase 6 gene in the mitochondrial genome have now been reported. A T ] G point mutation at nt 8993 in mitochondrial DNA (mtDNA) resulting in the replacement of a leucine by arginine in the ATPase 6 gene is associated with neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) (Holt et al 1990). When this mutation is present in high percentage (more than 95%), the phenotype is maternally inherited Leigh syndrome (LS) (Tatuch et al 1992). Patients with LS and the T8993G mutation are severely a†ected and usually die in infancy. In a previous study, we found that ATP synthesis in mitochondria isolated from Ðbroblasts of patients with the T8993G point mutation was decreased by 55% compared to normal controls (Vazquez-Memije et al 1996). Subsequently, a T ] C mutation at the same position (resulting in the replacement of leucine by proline) was described in patients with milder clinical manifestations (deVries et al 1993 ; Santorelli et al 1994). We have carried out comparative biochemical studies of ATPase in mitochondria isolated from Ðbroblasts of patients with the T8993G and the T8993C point mutations.

MATERIALS AND METHODS We studied 5 patients with the T8993G mutation, 2 patients with T8993C mutation and 6 normal controls. In cells from patients, standard PCR/RFLP analyses and quantitation showed that the T ] G or T ] C mutations were present in very high proportions (more than 95%). Human Ðbroblasts were grown from skin biopsies and cultured in EagleÏs minimal essential medium supplemented with MEM nonessential amino acids, 2 mmol/L L-glutamine, vitamin solution, and 0.2% sodium bicarbonate. Mitochondria were isolated by scraping monolayer cultures from three culture dishes (5 ] 106 cells) in 30 ml of an isolation bu†er consisting of 0.27 mol/L mannitol, 0.1 mmol/L EDTA, 0.05% bovine serum albumin (BSA) and 10 mmol/L Tris-HCl pH 7.3, according to Millis and Pious (1973). EDTA submitochondrial particles (EDTA-SMP) were prepared by sonication of mitochondria (10 mg/ml) in 2 mmol/L EDTA and 0.25 mol/L sucrose, pH 8.6 (with Tris solid) for 1 min. The sonicate was centrifuged at 11 000g for 10 min. The supernatant was centrifuged at 105 000g for 30 min at 4¡C and the resulting pellet was resuspended in 0.25 mol/L sucrose, 10 mmol/L Tris-HCl pH 7.5 at 10È15 mg of protein/ml, according to Lee et al (1964). ATP synthesis during oxidative phosphorylation was assayed by measuring the phosphorylation of ADP by inorganic phosphate (32P ) in the presence of i the myokinase inhibitor adenosine pentaphosphate (Tuena de Gomez-Puyou et al 1984). ATP hydrolysis was assayed at 25¡C by a spectrophotometric method in which ATP is regenerated through the action of pyruvate kinase in the presence of J. Inher. Metab. Dis. 21 (1998)

AT Pases in patients with mtDNA mutations

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excess phosphoenol pyruvate (Pullman et al 1960). In some experiments, ATP hydrolysis was measured by determining P released from ATP (Summer 1944). i To study oxidative phosphorylation, mitochondria were suspended at a concentration of 2.5È4.0 mg protein/ml in respiratory bu†er consisting of 0.25 mol/L mannitol, 0.2 mmol/L EDTA, 1 mmol/L MgCl , 10 mmol/L KCl, and 10 mmol/L 2 potassium phosphate (pH 7.2). A Gilson oxygraph Ðtted with a Clark electrode was used to measure the respiratory rate according to Estabrook (1967). Protein was determined by the Lowry method using bovine serum albumin as standard. RESULTS ATP synthesis and ATPase activities were assayed in Ðbroblast mitochondria from patients with the T8993G or T8993C point mutations and compared to normal controls. The ATP synthase activity in mitochondria isolated from cells with the T ] G mutation was decreased by more than 50% as compared to control with all substrates tested (Table 1), similarly to what we had previously observed (VazquezMemije et al 1996). In contrast, mitochondria from cells of patients with the T ] C point mutation showed a much milder decrease in ATP synthase activity (an average of 25%) with all the substrates tested. The hydrolytic activity, measured as oligomycin-sensitive ATPase, was similar in the three groups. Polarographic measurements of the overall activity of the mitochondrial respiratory chain, including respiratory controls, give a more general assessment of mitochondrial metabolism. Compared to controls, cells with the T ] G point mutation showed similar state 3, state 4 and uncoupled respiratory rates in the presence of succinate or malate as substrates (Table 2). The ATP synthase activity calculated from state 3 was 65% and 85% of the control with succinate and malate, respectively. In patients with the T ] C point mutation, we observed a slight decrease in the respiratory control index due to a decrease of respiratory rate state 3. However, the ATP synthase activity obtained from state 3 with succinate and malate as substrates was similar to that of the control. The ADP/O ratios were similar in all cases. Oligomycin inhibits the synthesis of ATP by speciÐcally blocking the proton conduction through F (Fillingame 1980). The site of action of oligomycin is the mem0 brane part (F ) of the ATPase system, to which oligomycin binds in a noncovalent, 0 Table 1 ATP synthesis in Ðbroblast mitochondria from patients with the T8993G or T8993C point mutation Succinate Control T8993G T8993C

2283 ^ 408 1029 ^ 214 1723 ^ 81

Malate a-Ketoglutarate (nmol AT P/h per mg protein) 2051 ^ 78 657 ^ 99 1345 ^ 185

1195 ^ 219 445 ^ 17 929 ^ 3

Pyruvate/malate AT Pasea 406 ^ 57 180 ^ 21 339 ^ 13

36 ^ 7.1 38 ^ 6.7 40 ^ 5.8

a Oligomycin sensitive ATPase, expressed as nmol Pi/min per mg protein Values represent the mean ^ SD of a minimum of 4 experiments (each in duplicate) for 5 patients with the T8993G and 2 patients with the T8993C mutations. Control value are the mean ^ SD of 20 normal cell lines

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Table 2 Oxidative phosphorylation in Ðbroblast mitochondria from patients with the T8993G or T8993C mutations ADP/O Control

Succinate Malate

2.10 ^ 0.02 2.61 ^ 0.03

R.C.b State 3 State 4 (nmol O consumed/min per mg protein) 2 2.76 ^ 0.13 42.31 ^ 1.3 15.32 ^ 1.50 2.79 ^ 0.15 27.14 ^ 1.5 9.74 ^ 1.26

T8993G

Succinate Malate

2.38 ^ 0.03 2.62 ^ 0.03

1.92 ^ 0.27 2.14 ^ 0.24

34.36 ^ 2.3 25.47 ^ 2.7

17.92 ^ 2.7 11.92 ^ 2.9

36.00 ^ 1.8 25.68 ^ 2.4

70 ^ 4.3 68 ^ 4.8

T8993C

Succinate Malate

2.75 ^ 0.03 3.40 ^ 0.03

1.82 ^ 0.46 1.25 ^ 0.25

26.33 ^ 3.1 12.46 ^ 2.8

14.43 ^ 3.2 10.00 ^ 4.1

25.58 ^ 3.6 12.50 ^ 3.3

94.4 ^ 10.0 83.3 ^ 6.6

Substratea

DNP AT P synthasec 41.30 ^ 2.0 27.27 ^ 2.9

108 ^ 6.0 80 ^ 10.0

a 0.3È0.6 mg/ml of mitochondrial protein and 6 mmol/L oxidizable substrate b Respiratory Control Index : state 3/state 4 c ATP synthase activity was calculated from the duration of state 3 and expressed as nmol of ADP added (150È300 nmol) per min per mg protein DNP, 2,4-dinitrophenol (100 kmol/L)



833

reversible manner (Glaser et al 1982). It has also been proposed that oligomycin inhibits ATPase activity by causing a conformational change in the F portion of 0 the complex that is transmitted to F , resulting in impaired binding of substrate to 1 the catalytic sites (Penefsky 1985). We studied the e†ect of oligomycin on ATP synthesis in cells with the T ] G or T ] C point mutations (Figure 1). At a concentration of 25 ng oligomycin/mg, ATP synthase activity was only mildly inhibited (10%) in control cells, whereas in the T ] G cells the activity was inhibited by 70% and in the T ] C cells by 85%. Thus, at low concentrations of the inhibitor (less than 75 ng/mg protein), there was a signiÐcantly higher sensitivity to oligomycin in mitochondria from cells with either point mutation and there was also an indication of a di†erence between the two mutations. Mitochondrial ATPase is a lipid-dependent membrane enzyme whose kinetics are a†ected by various perturbations of the membrane such as changes of cholesterol content (Calanni et al 1986) or the action of organic solvents (Zanotti et al 1992). In order to investigate whether the T8993G or T8993C point mutations a†ect the kinetic parameters of the F -ATPase, we measured K and V in EDTA sub1 m max mitochondrial particles (EDTA-SMP) prepared from Ðbroblast mitochondria. The ATP hydrolase activity of EDTA-SMP from controls or patients was calculated from saturation curves. No signiÐcant di†erence was observed in the K m

Figure 1 E†ect of oligomycin on the rate of ATP synthesis in intact mitochondria from patients with the T8993G or T8993C point mutations. Mitochondria (200È300 kg protein) were incubated in 0.3 ml of 6 mmol/L succinate, 1 mmol/L ADP, 10 mmol/L Pi-Tris (10È 15 000 cpm 32Pi/nmol Pi), 20 mmol/L glucose, 10 mmol MgCl , 20 U hexokinase, 30 mmol/L 2 Tris-acetate pH 7.2, and the indicated concentrations of oligomycin. Incubation was for 40 min at 30¡C



834

Table 3 Kinetic features of EDTA submitochondrial particles with the T8993G or T8993C point mutation K m ) (mmol/L Control T8993G T8993C

0.857 ^ 0.200 0.458 ^ 0.060 0.580 ^ 0.096

V max mg protein) (kmol P /min per i 1.800 ^ 0.028 0.976 ^ 0.034 1.170 ^ 0.165

Submitochondrial particles (50È100 kg) prepared in the presence of 2 mmol/L EDTA, were incubated in 1 mmol/L phosphoenolpyruvate, 10 U pyruvate kinase, 50 mmol/L sucrose, 50 mmol/L Tris-acetate pH 7.4, 30 mmol/L potassium acetate, 3 mmol/L magnesium acetate and ATP. pH of the incubation mixture was 8.2, temperature 30¡C, Ðnal volume 1.0 ml. ATP hydrolysis was measured by P released in the i presence of an ATP regenerating system. The ATP concentration ranged from 38 kmol/L to 2.5 mmol/L. K and V values were obtained from double-reciprocal plots of ATPase activity m max

values for ATP between EDTA submitochondrial particles from control and mutated cells, whereas the V value for ATP hydrolysis was lower in EDTA-SMP max from cells with the point mutations than in control particles (Table 3). The e†ect of oligomycin on the ATPase activity in these particles was 57% inhibition for the mutant cells and 70% for the control particles. Furthermore, in these submitochondrial particles we detected a higher ATPase activity in the supernatant of the mutated submitochondrial particles than in control cells (data not shown). DISCUSSION Our Ðndings suggest that the mitochondrial F F -ATP synthase in patients with 1 0 point mutations at nt 8993 in the ATPase 6 gene of mtDNA is structurally and functionally altered. We found that ATP production was severely decreased in cells from patients with the T8993G mutation, owing to a defect in the proton channel and P/O coupling of the ATP synthase. This is in agreement with data from Tatuch and Robinson (1993). In contrast, no signiÐcant impairment appears to occur in the presence of the T8993C point mutation, since ATP synthesis was not severely a†ected. ATP hydrolysis, measured as oligomycin-sensitive ATPase, was similar in the three groups (control and both mutants). This is not surprising since it has been established that blocking proton translocation does not inhibit F ATP hydrolysis 1 (Cain and Simoni 1988). The binding of oligomycin seems to be inÑuenced by the functional state of the ATPase complex (Glaser et al 1982). Penefsky (1985) has proposed that a change in the state of ionization of one or more charged amino acid residues in F results in a 0 conformational change in F . An attractive explanation for our results would be 1 that the mutation causes a conformational change in subunit 6 that is reÑected in the ATP production and sensitivity to oligomycin. The conformational state of the enzyme will depend on whether the amino acid change introduces a positively charged amino acid (arginine, T ] G mutation) or a non-charged amino acid (proline, T ] C mutation). This interpretation agrees with data of Cain and Simoni (1989), who observed di†erent e†ects on ATP synthesis activity in E. coli depending upon the amino acid substituted at the same site of the enzyme. J. Inher. Metab. Dis. 21 (1998)


835

Kinetic analysis showed a discrete change in the V of the hydrolytic activity of max the ATP synthase complex, with no change of K , in mitochondria from Ðbroblasts m with either mutation. A decreased V could be due either to a low content of F or max 1 to a catalytic deÐciency of F -ATPase. In our case, the latter can be discarded since 1 the affinity for ATP of the F -ATPase, as measured by the V /K ratio, was very 1 max m similar in the three samples. It has been reported that the distribution of F between 1 the cytosolic and membranous cell fraction can be indicative of assembly of the F F -ATP synthase (Cain and Simoni 1988). Our results suggest that the catalytic 1 0 subunit F is loosely bound to the membrane sector F because the hydrolytic 1 0 activity in submitochondrial particles was not fully sensitive to the speciÐc F inhib0 itor oligomycin and because we detected an oligomycin-insensitive ATPase activity in the supernatant of the SMP. This is in agreement with data from Houstek et al (1995), who described a markedly decreased content of the F subunit b in muscle 1 mitochondria from patients with the T ] G point mutation, and increased content of the F subunit c. There is considerable evidence that energy transmission from F 0 0 to F is indirect and is mediated by long-range conformational changes induced by 1 proton transport through the stalk (Boyer 1993). Thus, if the stalk is disrupted by physical or chemicals means, intact F -ATPase is released. 1 In summary, our data suggest that the substitution of di†erent amino acids at the same site of the ATPase produces changes in the F conformation that a†ect the 0 F ÈF interaction. Thus, both the T8993G and T8993C mutations a†ect only the F 1 0 0 sector of the mitochondrial ATPase complex directly, but also indirectly inÑuence the F catalytic portion of the enzyme. These di†erent amino acid substitutions 1 might result in an enzyme with di†erent biochemical characteristics which, in turn, might a†ect ATP production in di†erent ways, thereby resulting in distinct clinical phenotypes. ACKNOWLEDGEMENTS This work was supported by National Institutes of Health grant NS 11766 and by a grant from the Muscular Dystrophy Association. M. E. Vazquez-Memije was supported by a grant from the Unidad de Investigacion en Genetica Humana, Centro Medico Nacional-IMSS, Mexico, D.F. F. M. Santorelli was supported by a scholarship from the Telethon Italia. REFERENCES Abrahams JP, Leslie AGW, Lutter R, Walker JE (1994) Structure at 2.8 Ó resolution of F1ATPase from bovine heart mitochondria. Nature 370 : 621È628. Amzel LM, Bianchet MA, Pedersen PL (1992) Quaternary structure of ATP synthases : symmetry and asymmetry in the F1 moiety. J Bioenerg Biomembr 24 : 429È433. Anderson S, Bankier AT, Barrel BG, et al (1981) Sequence and organization of the human mitochondrial genome. Nature 290 : 457È465. Boyer PD (1993) The binding change mechanism for ATP synthaseÈsome probabilities and possibilities. Biochim Biophys Acta 1140 : 215È250. Cain BD, Simoni RD (1988) Interaction between Glu-219 and His-245 within the a subunit of F F -ATPase in Escherichia coli. J Biol Chem 263 : 6606È6612. 1 0 J. Inher. Metab. Dis. 21 (1998)

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