Isolation and Comparative Studies of Mitochondrial F1-ATPase from Rat Testis and Beef Heart

Comp. Biochem. Physiol. Vol. 116B, No. 3, pp. 303–309, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0305-0491/97/$17.00 PII S0305-0491(96)00207-6

Isolation and Comparative Studies of Mitochondrial F1-ATPase from Rat Testis and Beef Heart M.E. Va´zquez-Memije,* C. Beltra´n† and M. Tuena de Go´mez-Puyou ‡ *Unidad de Investigacio´n Me´dica en Gene´tica Humana, Centro Me´dico Nacional, IMSS, Apdo. Postal 73032, Me´xico, D.F. 03020; † Instituto de Biotecnologi´a, Universidad Nacional Auto´noma de Me´xico, Cuernavaca Mor.; and ‡ Instituto de Fisiologi´a Celular, Universidad Nacional Auto´noma de Me´xico, Apdo. Postal 70-600, Me´xico, D.F. 04510; Me´xico ABSTRACT. The isolation and properties of F1-mitochondrial ATPase from rat testis are described. The isolation medium involves a chloroform extraction, and it is suitable even with small amounts of starting material that have a relatively low specific activity as in the case of rat testis submitochondrial particles. The isolated enzyme from rat testis had a specific activity of 30–45 µmol Pi/min/mg protein, which could be increased up to 90 µmol Pi/min/mg protein only in the presence of bicarbonate and maleate. The isolated enzyme represented less than 0.6% of the initial membrane proteins. It exhibited a typical five-band pattern in sodium dodecyl sulfate gel electrophoresis. However, it showed a ratio of subunits α : β higher than the heart enzyme; its significance is unknown. The purified enzyme was cold labile and inhibited by natural ATPase inhibitor protein from bovine heart mitochondria and by dicyclohexylcarbodiimide. The results presented suggest that the low ATPase activity of testis submitochondrial particles is due to a reduced content of the F1-ATPase. comp biochem physiol 116B; 3:303–309, 1997.  1997 Elsevier Science Inc. KEY WORDS. β-subunit, F1-ATPase, heart, testis, submitochondrial particles

INTRODUCTION The production of most of ATP in normal cells occurs within mitochondria by the process of oxidative phosphorylation. However, rat testis and many tumor tissues may rely on a high level of aerobic glycolysis for their source of ATP (23,29). It has been shown that adult rat testis mitochondrial ATPase had a lower sensitivity both to oligomycin and N,N’Dicyclohexylcarbodiimide (41); this ATPase is not stimulated by either DNP or FCCP. DNP scarcely enhances state 4 respiration, and the mitochondria are poorly coupled (42). However, when testis mitochondria are isolated in the presence of 1% BSA, ATPase is stimulated by DNP as well as by FCCP (42). In spite of this, testis mitochondria isolated in the latter conditions show quantitative differences in respect to the liver system (42); whether these differences are related to structural and/or functional alterations of this mitochondrial ATPase complex is an open question. In tumor mitochondria the possibility of a structurally altered ATPase has been confirmed. In fact, SDS-PAGE patterns Address reprint requests to: M. E. Va´zquez-Memije, Unidad de Investigacio´n en Gene´tica Humana, Centro Me´dico Nacional, IMSS, Apdo. Postal 73032, Me´ xico, D.F. 03020, Me´xico. Tel. (525) 627-6941; Fax (525) 7610952. Abbreviations–BSA, bovine serum albumin; DCCD, N,N′dicyclohexylcarbodiimide; DNP, 2,4-dinitrophenol; FCCP, carbonylcyanide pmethoxyphenylhydrazone; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TEA, triethanolamine. Received 19 February 1996; accepted 14 July 1996.

of the proteins of the inner mitochondrial membrane from rat tumor (35), hepatoma 7800 (28), and that of human mitochondria with a T . G mutation at position 8993 of mtDNA (18) show an altered stoichiometry, particularly in relation to α and β or other subunits of F1-ATPase. Also in this respect it has been shown that during early hepatic regeneration, the mitochondria are uncoupled due to the absence of a large proportion of the catalytic β-F1 subunit of F0F1-ATPsynthase (15); this results in an uncontrolled proton flux through non-specific associated F0 moities (6). Testis germinal epithelium consists mostly of meiotic (spermatocytes) and differentiating (spermatids) cells. During spermatogenesis, mitochondria proliferate and undergo dramatic morphological changes (2,10,14). Thus it is a unique model for the study of organelle differentiation in a mammalian tissue. In this work, a simple procedure of isolating pure mitochondrial F1-ATPase from rat testis is described. Comparative studies on the molecular and enzymatic properties of the purified ATPases have been carried out in order to explore if there are possible structural and/ or functional differences between the heart and the testis enzymes.

MATERIALS AND METHODS Adult male rats of the Sprague-Dawley strain were used. Rat testis mitochondria were isolated in 250 mM sucrose, 3 mM

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TABLE 1. Comparative characteristics of submitochondrial

particles from rat testis and beef heart Rat testis ATPase (µmol Pi/min/mg protein) Cytochrome a1a3 content (nmol hemo/mg protein) ATPase/nmol a1a3

Beef heart

2.54 6 0.519

9.8 6 0.863

0.254 6 0.006 10.00

0.379 6 0.042 25.22

TEA, 1 mM EDTA, pH 7.4, by the method described (42) and stored in 250 mM sucrose at 15–20 mg/ml at 270°C. The beef heart mitochondria were prepared as in (25) and stored similarly. Beef heart mitochondrial F1 was prepared as in (3). Submitochondrial particles from both tissues were prepared by sonication of a mitochondrial suspension (75 mM sucrose, 30 mM Tris–SO4, pH 8.0, 2 mM EDTA, 250 mM KCl, 12–15 mg/ml of mitochondrial protein) for four 60-sec periods at 0–4°C in a MSE sonifier. The sonicate was centrifuged at 10,000 g for 10 min. The supernatant was incubated at 37°C and centrifuged at 105,000 g for 30 min; the pellet was washed once with 0.25 M sucrose. Inhibitor protein from beef heart mitochondria was purified as in (19). ATP hydrolysis was assayed at 25°C by the spectrophotometric method in which ATP is regenerated through the action of pyruvate kinase and phosphoenolpyruvate (33). In some experiments ATP hydrolysis was measured by determining Pi released from ATP (38). SDS-PAGE was performed according to the procedure of Laemmli (21), with a 5% polyacrylamide stacking gel and 13.5% polyacrylamide separating gel. Native 6% PAGE was carried out as in (30) and acid gel according to (1). The staining used was Coomassie Blue. The gels were scanned in a Beckman DU64 scanner at 560 nm. Cytochrome a 1 a3 content was measured spectrophotometrically as in (9). Protein was estimated either by the Biuret method (13) or according to Lowry (26). RESULTS Testis submitochondrial particles were exposed to various conditions that bring about activation of the ATPase activity of heart submitochondrial particles, i.e. incubation with phosphate at 38°C (4), and high concentrations of KCl (19). It was found that under all conditions the activity reached was around 2.5 µmol Pi /min/mg protein. In heart submitochondrial particles an activity of 9.8 µmol Pi/min/ mg protein was found. In more than 15 preparations the reproducibility was higher than 95%. When the ATPase of both types of particles were referred to the content of cytochrome a 1 a3 , the values were 10 µmol Pi/min/nmol a 1 a3 for testis and 25 µmol/Pi/min/nmol a 1 a3 for heart submitochondrial particles (Table 1). For the purification of F1 from testis, submitochondrial

particles that had been activated by the KCl method were the starting material. The chloroform treatment of submitochondrial particles described by Beechey (3) has been extensively used for the isolation of F1 . This method was used as follows: submitochondrial particles were suspended in a solution containing 0.25 M sucrose, 10 mM Tris–SO4 and 1 mM EDTA pH 8.2 to give a final protein concentration of 5 mg/ml. ATP was added to a final concentration of 5 mM and the suspension was vigorously mixed with 0.5 volume chloroform at room temperature for 20 sec. Following centrifugation in glass tubes at 12,000 g for 7 min, water phase was centrifuged at 105,000 g for 60 min at 24°C. The supernatant contained the ATPase activity. Fractionation was performed with saturated ammonium sulfate solution. The fraction obtained at 0–35% saturation of ammonium sulfate was discarded, and a second fraction, collected between 35 and 50% saturation exhibited the ATPase activity. The suspension was stored at 4°C with no loss of the enzyme activity for at least 4 weeks. Table 2 shows results of a typical isolation procedure described above. The yields of total ATPase activity were 13–18% and 0.6% of ATPase protein using testis submitochondrial particles as the starting material. The specific activity at pH 8.2 (pH optimum) of the purified enzyme was 30–45 µmol Pi/min/mg protein for testis and 50–60 µmol Pi/min/mg protein for heart tissue. ELECTROPHORETIC PATTERNS OF PURIFIED F1 -ATPASE.

The enzyme isolated from beef heart exhibited the characteristic five bands (α,β,γ,δ,e) (Fig. 1A). The patterns also showed that the isolated enzyme was practically free of contaminating proteins. The purified F1-ATPase isolated from rat testis had the same pattern on SDS-gels (Fig. 1B). In native-PAGE the electrophoretic mobility of rat testis F1 was similar to that of beef heart; interestingly both preparations showed a similar contaminating protein (Fig. 1E). In order to evaluate the stoichiometry of α : β subunits in F1-ATPase from testis, an acid gel was prepared. The densitometric tracings of acid gel (Fig. 1C,D) showed a ratio α : β of 0.965 6 0.088 for heart (n 5 12) and 1.30 6 0.28 for testis (n 5 5). We obtained P , 0.02 according to MannWhitney U-test (36). These data suggest a diminished content of the catalytic β subunit in the F1-ATPase from rat testis. HYDROLYTIC ACTIVITY OF F1 FROM TESTIS. The Km value of the F1-ATPase from testis for ATP was 0.280 mM. This observation is consistent with the Km of F1 isolated from bovine heart mitochondria (7,31). A well-known property of purified mitochondrial ATPase is its inactivation by low temperatures (16). F1 from testis showed the same property (Fig. 2). Moreover, the inactivation curves of both testis and heart F1-ATPase were very similar. The results of different preparations of the isolated F1-ATPase showed that 50% cold inactivation was reached within 25–30 min. However,

Properties of F1-ATPase from Testis Mitochondria

305

TABLE 2. Isolation of mitochondrial F1-ATPases from rat testis and beef heart

Rat testis Fraction SMP** Chloroform extract

Protein (mg) 62 0.40

Beef heart

Specific activity*

Total ATPase

Yield (%)

2.22 6 0.11

138

–

45.40 6 10.10

18

13

Protein (mg) 45 0.80

Specific activity*

Total ATPase

Yield (%)

5.68 6 0.88

256

–

60.45 6 3.83

48

19

*µmol Pi/min/mg protein. **Submitochondrial particles.

the F1 factor was fairly stable at room temperature (22°C); in 3 hr, only 10–20% of the activity was lost. The ATPase activity of F1-ATPase from testis increased as the temperature of incubation was raised from 15 to 50°C (Fig. 3A). A similar response was found for heart enzyme, except that maximum activity occurred at 45°C. In an Arrhenius plot, the data showed a break at 30°C for both enzymes. Below the transition temperature the calculated activation energies were 14,800 and 10,990 cal/mol for heart and testis respectively.

FIG. 1. Electrophoretic pattern of F1-ATPase. SDS-Poly-

acrylamide gel electrophoresis of 10 mg beef heart F1 (A) and 35 mg of rat testis F1 (B). In this gel a mixture of seven different proteins of 66-14 KDa (dalton Mark VII-L, Sigma) were used as standards. Acid gel of 10 mg beef heart F1 (C) and rat testis F1 (D). (E) Native polyacrylamide gel electrophoresis of 10 mg beef heart F1 (line 1) and rat testis F1 (line 2).

INHIBITION AND ACTIVATION OF F1 -ATPASE FROM TESTIS.

The study of the inhibition of F1-ATPase by several agents could be useful to find possible differences between the F1 factor from testis and F1 factors from other sources. It is not known if testis mitochondria have a natural ATPase inhibitor protein similar to that of heart mitochondria (34). Nevertheless considering that the inhibitor protein from heart inhibits F1-ATPases from several sources (39,43) it was explored, if the F1-ATPase from testis was inhibited by the inhibitor protein from heart. This proved to be the case; as shown in Fig. 4, the inhibition of F1 ATPase from rat testis by beef heart inhibitor protein was similar to that of heart F1-ATPase. The amount of inhibitor protein required to inhibit 50% of the activity was 0.05 µg IP/µg F1 . The soluble F1-ATPase isolated from bacteria, chloroplast

FIG. 2. Cold lability response of F1-ATPase from rat testis.

The enzyme sample was divided into two portions, one of which was held at 0° and the other at room temperature for the time indicated prior to the assay. Incubation media: 0.25 M sucrose, 10 mM Tris–SO4 pH 8.0. ATPase activity was measured by determining Pi released from ATP. (s) 22°C; (d) 0°C.

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FIG. 3. Effect of temperature on F1-ATPase activity (A), and Arrhenius plot (B). The ATPase activity was measured as in

Fig. 2. (d) Rat testis; (s) bovine heart.

and mammalian mitochondria are inactivated by the binding of one DCCD molecule to one of the three β-subunits of F1 (11,32). F1-ATPase from testis was inhibited by DCCD through a process that depended on the concentration of DCCD and the time of incubation (Fig. 5). Half-maximal inhibition was obtained with 50 µM DCCD in a 60-min period, or in 20 min with 150 µM DCCD. It is known (22) that anions such as bicarbonate, maleate and sulfate activate the ATPase activity of both soluble and membrane-bound ATPases. Here it was found that chloroform-released rat testis ATPase was activated 3.0–3.5-fold (Fig. 6) by bicarbonate, and 2.5-fold by maleate. Acetate, chloride and sulfate had no effect. A comparison of the effects of various activators and inhibitors of testis and F1-ATPases from other sources is shown in Table 3. Apparently there are no significant differences between the two enzymes. DISCUSSION The experiments reported here describe the purification of ATPase from mitochondria of rat testis in a relatively pure form. The results of comparative studies of the properties of the ATPases including specific activity, K m, cold lability, effect of DCCD and anions indicate a marked similarity between the enzymes isolated from rat testis and beef heart tissues. The kinetic properties of isolated ATPase of testis mitochondria are according to those described for tissues with distinct metabolic patterns, i.e. differentiating tissues (15), brown adipose tissue (17,40) and tumours (24). Moreover, the SDS-gel patterns of the testis and heart enzymes were indistinguishable. The rat testis enzyme differs from beef heart ATPase only in the apparent lower content of β

FIG. 4. Inhibition of rat testis F1-ATPase by the inhibitor pro-

tein from beef heart mitochondria. The incubation medium was: 250 msucrose, 10 mM MOPS-HCl pH 6.6, 2 mM MgATP 10 mg F1, and the indicated amounts of inhibitor protein in a final volume of 0.1 ml for 15 min. At this time ATPase activity was assayed spectrophotometrically in the presence of an ATP regenerating system. Rat testis (d); beef heart (s).

Properties of F1-ATPase from Testis Mitochondria

307

FIG. 5. Inhibition of F1-ATPase by DCCD. F1 (0.3 mg/ml)

was preincubated during 60 min at 24°C in a series of tubes containing 0.1 ml of a medium made of: 250 mM sucrose, 50 mM MOPS–Tris, 2 mM EDTA, 4 mM ATP, final pH 7.0 and increasing concentrations of DCCD in methanol. Controls were run with methanol alone (final concentration: 2%). ATPase activity was assayed as in Fig. 4. The insert shows the plot of the time course of inactivation by 150 mM DCCD. Rat testis (d); beef heart (s).

subunit, as inferred from the acid gel data. Its significance is unknown, and the existence in vivo of this ratio cannot be ascertained. The fact that inhibitor protein from heart very effectively inhibited F1-ATPase from rat testis, indicates that both F1-ATPase from testis and heart possess similar sites for the inhibitor protein. From the ratio ATPase activity : cyt a 1 a3, our results suggest that there is a decrease in ATPase activity in testis submitochondrial particles that is related to a diminution in F1 content. In addition it was found that the yield of F1 from testis submitochondrial particles was lower than from heart particles (Table 2). In subsequent and preliminary experiments it has been found that, in fact, testis mitochondria contain less F1 than rat liver or beef heart mitochondria. Changes in activity and F1 content have been reported in several differentiating tissues such as regenerating rat liver (15), Zajdela hepatoma (20), Morris hepatoma (37) and various tissues of aged rats (8). Spermatogenesis is a complex series of events starting with a relatively undifferentiated stem cell, the primitive type A spermatogonia, and ending with a highly differentiated cell, the spermatozoa. Reported evidence has shown that the synthesis of unique gene products such as lactate dehydrogenase X (27), testis-

FIG. 6. The effect of anions on rat testis mitochondrial F1-

ATPase. F1 was incubated (0.001 mg/ml) in the buffer: 30 mM Tris–HCl, pH 8.0; 5 mM MgCl2, 250 mM sucrose, 5 mM ATP, with the indicated amounts of salts. Substituting Tris–HCl with Tris–SO4 gave identical results. The assay tubes were incubated 3 min at 30°C and the reaction arrested by the addition of 0.2 ml of 30% CCl3COOH. The specific activity of the control was 30 mmol Pi/min/mg protein. (s) Bicarbonate; (n) maleate; (m) sulfate; (d) acetate; (h) chloride.

TABLE 3. Sensitivity of the chloroform released mitochon-

drial ATPase to various activators and inhibitors ATPase activity (%) Additions 30 40 40 40 40

Rat testis

Rat liver

310 250 90 110 107

250 a 100 a 90 a 90 a 110 a Beef heart 50 50

mM bicarbonate mM maleate mM sulfate mM acetate mM chloride

150 µM DCCD 0.05 µg IP/µg F1 a

Eur. J. Biochem. 22:355–363; 1971.

50 50

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specific histones (5), and testis-specific cytochrome c in mouse (12) occur at a specific stage of spermatogenesis. Accordingly, spermatogenesis represents a dynamic system in which the normal order of assembly of the various subunits of a protein complex may be altered, leading to observable functional and structural anomalies. It might be speculated that the reduced amount of ATPase in testis mitochondria, in addition to our previous observations (41,42), could represent one of the factors accounting for the peculiarities in energy metabolism observed in testis, and that the lower phosphorylation capacity of this tissue is related to a low content of F1 in the membrane. We thank Dr. A. Go´mez-Puyou for critically reading the manuscript and Dr. Graciela Delhumeau for her helpful advice and discussion during this work. This study was supported in part by grant P228CCOX880562 from the Consejo Nacional de Ciencia y Tecnologia (CONACyT) Me´xico, and was performed at Instituto de Fisiologı´a Celular, Universidad Nacional Auto´noma de Mexico, Me´xico, D.F.

13. 14.

15.

16.

17.

18.

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