Isolation and comparative studies of mitochondrial F1-ATPase from Morris hepatoma and rat liver

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 195, No. 1, June, pp. 136-144, 1979

Isolation

and Comparative Studies of Mitochondrial Morris Hepatoma and Rat Liver

VITALY

F,-ATPase

from

L. SPITSBERG, H. P. MORRIS,’ AND SAMUEL H. P. CHAN

Biological

Research Laboratories,

Department

of Biology,

Syracuse

University,

Syracuse, New York 13210 Received December 27, 1978; revised February 12, 1979 A simple method of isolating mitochondrial ATPase from rat liver and Morris hepatoma cell lines by chloroform extraction and chromatography on DEAE-Sephadex is described. This method is suitable even when small amounts of starting material with relatively low specific ATPase activity (in the case of hepatoma mitochondria and submitochondrial particles) are available. The isolated enzyme from both rat liver and hepatomas had a high specific activity, was similarly activated by bicarbonate and 2,4-dinitrophenol, and had a typical five-band pattern in sodium dodecyl sulfate electrophoresis. Prior to DEAE-Sephadex chromatography, an additional protein band which migrates between the Sand Esubunits in the tumor F,-ATPase preparation was observed. The purified enzymes were cold labile and restored oxidative phosphorylation function of F,-ATPase depleted submitochondrial particles prepared from rat liver. The ATPase activity of the isolated enzymes was inhibited by mitochondrial ATPase inhibitor protein. The apparent stoichiometry of the inhibitor protein to the purified ATPase was extrapolated to be 2:l.

The production of ATP in most normal animal cells occurs within mitochondria by the process of oxidative phosphorylation. In contrast, many tumor tissues including some Morris hepatomas with a wide range of growth rates, may rely on higher level of aerobic glycolysis for their source of ATP (1). Whether this energetic imbalance in these tumor cells is directly linked to structural and/or functional alterations of the mitochondrial ATPase complex is an open question. Pedersen and Morris (2) using Morris hepatoma lines confirmed and extented earlier observations (3, 4) that, in contrast to normal tissue mitochondrial ATPase, tumor mitochondrial ATPase was not activated by uncouplers. However, subsequently Kaschnitz et al. (5) reported that when tumor mitochondria were isolated from the same hepatoma lines in the presence of high levels of serum albumin, mitochondrial 1 Present address: Cancer Research Unit, Department of Biochemistry, Howard University, Washington, D. C. 20059.

ATPase could be activated by 2,4-dinitrophenol (DNP)2 similar to the case of normal liver mitochondria. Recently Pedersen (1) confirmed the results of Kaschnitz et al. (5) and pointed out the necessity of preincubating the isolated tumor mitochondria with ATP prior the addition of DNP in order to elicit the normal uncoupler-stimulated ATPase activity. Nevertheless, the possibility of a structurally altered uncoupler binding site in tumor mitochondrial ATPase has not been ruled out (6). Furthermore, SDSgel electrophoretic patterns of the proteins of the inner mitochondrial membrane isolated from a mouse carcinoma (7) and Morris hepatoma (1, 8) showed some possibly altered protein subunit compositions, particularly in the range of molecular weights close to a! andlor j3 subunits of F,-ATPase. In this communication, a simple proce2 Abbreviations used: A-particles, alkaline submitochondrial particles prepared at pH 9.2, SMP, submitochontrial particles; CCl,COOH, trichloroacetic acid; DNP, 2,4-dinitrophenol; UA-particles, A-particles treated with 3 M urea; SDS, sodium dodecyl sulfate.

136 0003-9861/79/070136-09$02.00/O Copyright 0 1979 by AcademicPress, Inc. All rights

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in any form reserved.

MITOCHONDRIAL

ATPase FROM MORRIS HEPATOMA AND RAT LIVER

137

RESULTS dure of isolating pure mitochondrial F,ATPase from normal rat liver and Morris Purification of chloroform-released hepatoma cell lines is described. ComparaATPase from rat liver and Morris hepative studies on the molecular and enzymatic toma. Beechy et al. (15) introduced the chloproperties of the purified ATPases have roform treatment of submitochondrial parbeen carried out in an attempt to elucidate ticles for the release of F,-ATPase. Subsepossible structural and/or functional differquently chloroform treatment was used ences between the normal and tumor tissue successfully for the isolation of mitochondrial enzymes. F,-ATPase from several different sources (16, 17). In the present studies the release MATERIALS AND METHODS of F,-ATPase from liver and tumor SMP by chloroform treatment was used and followed Adult, male Buffalo strain rats were obtained from with DEAE-Sephadex chromatography. Simonsen Laboratories, Gilroy, Calif. Tumor-bearing The DEAE-Sephadex purification step was rats of the same strain were mailed from Dr. H. P. previously used by Spitsberg and Blair (18) Morris, Howard University, Washington, D. C. Morris for preparing F,-ATPase from beef heart hepatomas 16 (slow growth rate, highly differentiated), 7800 and 77948 (intermediate growth rate, well dif- SMP. SMP were prepared by sonication of a ferentiated), and 7777 (rapid growth rate, poorly mitochondrial suspension (0.25 M sucrose, differentiated) were used. The tumor-bearing rats 10 mM T&-SO,, pH 7.4-7.6,l InM EDTA, were sacrificed when the tumors grew to appropriate size (about l-2 cm in diameter). Rat liver mitochondria lo-20 mg of mitochondrial protein/ml) for were isolated in 0.25 M sucrose, 10 mM Tris-SO1, 1 3 x 45 s at 0-4°C using a setting of 7 on a mM EDTA (STE medium), pH 7.4, by the method of Branson sonifier. After removing the unJohnson and Lardy (9) and stored in STE medium at broken mitochondria by a 10 min centrifugaprotein concentrations of 20-30 mg/ml at -70°C. The tion at 15,OOOg,the SMP were sedimented yield of mitochondria usually was about 2-3%. The by centrifugation for 40 min at 105,OOOg. tumor mitochondria were prepared and stored simiThe SMP were suspended in 0.25 M sucrose, larly. The tumor tissues before the homogenization were rinsed with a small amount of ice-cold STE me- 10 mM Tris-SO,, and 1 InM EDTA, pH 7.6, dium. The yield of the tumor mitochondria was usually at a protein concentration of lo-15 mg/ml, the pH of the suspension was increased to about 1%. ATPase activity was determined by the release of 8.0-8.2 by adding 1 M Tris at 4”C, and the inorganic phosphate according to the procedure of suspension was centrifuged at 105,OOOg for Senior and Brooks (IO). The assay mixture (final vol- 40 min (“washing step”). ume, 1 ml) contained 30 mM Tris-HCl, 5 mM MgCI,, The SMP were again suspended in 0.25 M 30 mM sodium bicarbonate, pH 8.5, and 5 to 200 pg sucrose, 10 mM Tris-SO,, 1 mM EDTA, pH of protein. The reaction was started by the addition of 7.6, buffer at a protein concentration of lo0.1 ml of ATP (0.1 M disodium salt, pH 7.4) and followed for 1 or 2 min at 30°C. The reaction was stopped 15 mg/ml, and the pH was adjusted to pH by the addition of 0.5 ml of 5% CCl,COOH, and the 8.5 with 1 M Tris. ATP was added to make a inorganic phosphate was determined by the procedure final concentration to 5 MM, and the susof Horstman and Racker (11). One unit of ATPase is pension of SMP was gently mixed with 0.5 defined as the amount of enzyme which catalyzes the volume chloroform at room temperature for release of 1 pm01 of Pi per minute at 30°C. 15 s. Following centrifugation in glass tubes SDS-gel electrophoresis was performed according at 10,00&~ for 5 min, the supernatant was to the procedure of Dunker and Rueckert (12). The centrifuged at 100,OOOg for 30 min at 20°C. bovine heart and rat liver mitochondrial ATPase in- Saturated ammonium sulfate solution at hibitor proteins were prepared according to the proroom temperature containing 1 mM EDTA cedures of Horstman and Racker (11) and Chan and Barbour (13), respectively. Protein was determined by and 2 mM ATP was added to the supernatant the method of Ohnishi and Barr (14), using Folin and to make a 55% saturated solution. The preBiuret reagents. The carrier-free inorganic [32P]phos- cipitate was then collected by centrifugation for 10 min at 0-5°C and dissolved at room phate was obtained from the Amersham, Chicago. All other chemical reagents were analytical grade. temperature in l-2 ml of 40 InM Tris-Cl,

138

SPITSBERG, MORRIS, AND GHAN TABLE I ISOLATIONOF MITOCHONDRIALF,-ATPASES FROMRAT LIVER AND MORRISHEPATOMAS Rat liver

Fraction Sonicated mitochondria SMP Chloroform extract DEAE-Sephadex A-50

Protein (mg)

Specific activity (unitslmg)

Hepatoma 7794A

Hepatoma 7777

Yield (%)

Protein (mg)

Specific activity (units/mg)

Yield (%)

Protein (mg)

Specific activity Yield (units/mg) (%)

355 127”

3.7 6.0

100 58

350 62.5”

2.3 3.3

100 25.5

275.3 87”

10

50.0

38

4.8

60-65

36-39

3.4

75

44

2.0

100-110

25-27

1.5

110

28

4.0

90-120

27-36.5

2.14 3.75

100 57

a Washed submitochondrial particles. * Unwashed submitochondrial particles.

4 mM ATP, 1 InM EDTA, pH 7.4 (ATPcontaining buffer). Fractionation was performed with saturated ammonium sulfate solution. The first fraction at O-35% saturation of ammonium sulfate was discarded, and a second fraction, collected between 35 and 55% saturation, contained the ATPase. The protein precipitate was again collected by centrifugation for 10 min at 0-5°C and then dissolved at room temperature in a small volume (1-2 ml) of ATP-containing buffer and dialyzed against the same buffer for 2-3 h at room temperature. The dialyzed solution was applied to a column (1 x 2-3 cm) of DEAE-Sephadex A-50 equilibrated with the ATP-containing buffer. The flow rate was adjusted to l-2 ml/min. The column was washed first with 15 ml of ATPcontaining buffer, and then with 15 ml of ATP-containing buffer which contained 0.12 M NaCl. The enzyme was eluted with the ATP-containing buffer which also contained 0.3 M NaCl. The active fractions were combined, and to it saturated ammonium sulfate solution containing 1 IIIM EDTA and 4 mM ATP was slowly added to give 55% saturation. The suspension was stored at 4°C with no loss of the enzyme activity for at least a few weeks. Table I shows results of a typical isolation as described above. The yield usually was about 30% using the sonicated mitochondria as the starting material. The procedures for the isolation of F,-

ATPase from Morris hepatoma (cell lines 16, 7800,7794A, and 7777) were similar to those described above except that the washing step was omitted and that the chloroform extraction step was at pH 7.5 and also in the presence of 5 IIIM ATP. Table I also shows results for ATPase isolated from two Morris hepatoma lines. Yields of the enzyme were 25-30% using sonicated mitochondria as the TABLE II EFFECTOF CHLOROFORMTREATMENTONTHE YIELD OF ATPASE FROMRAT LIVER AND MORRISHEPATOMA SUBMITOCHONDRIAL PARTICLES Tissue source Rat liver

Hepatoma 7794A Hepatoma 16 Hepatoma 7800

Total enzyme units

Yield (%I

SMP Chloroform extract

786

100

580

74

SMP Chloroform extract

202

100

288-315

142 156

102

100

137

134

86

100

91

106

Fractions

SMP Chloroform extract SMP Chloroform extract

MITOCHONDRIAL TABLE

ATPase FROM MORRIS

III

RATIO OF ATPASE ACTIVITIES OF PURIFIED F,-ATPASES DETERMINED IN THE PRESENCE AND ABSENCE OF BICARBONATE Ratio of activities and without bicarbonate”

with

Source of purified F,-ATPase

At pH 8.0

At pH 8.5

Rat liver Morris hepatoma 7800 Morris hepatoma 7777 Morris hepatoma 16 Morris hepatoma 7794A

1.5-1.9” 1.50 1.70 1.50 1.60

1.45-2.0” 1.40 1.70 1.40 1.74

0 Buffer system used was 30 mM Tris-HCI, pH 8.0 or 8.5, in the presence or absence of 30 mM NaHCO,. Substituting Tris-HCl with Tris-SO, gave identical results. The specific activity of F,-ATPase from each source was 1.2 to 1.5-fold higher at pH 8.5 than at pH 8.0. ti The range was obtained using different preparations of F,-ATPase from rat liver with specific activities of 90-120 units/mg.

HEPATOMA

AND

RAT LIVER

139

effect of F,-ATPase activity of anions, such as bicarbonate and DNP, is well established (19). Chloroform-released rat liver and tumor ATPases were similarly activated 1.5- to 2.0-fold (Table III) by bicarbonate anion when the assay was performed at pH 8.0 to 8.5 as it was for the rat liver enzyme prepared previously (19). The uncoupler-stimulated ATPase activities are presented in Fig. 1. It is clear that the activity of all isolated mitochondrial F,-ATPases was stimulated by DNP in a similar way with maximal activation occurring at a 1 mM concentration of the uncoupler (19). SDS-gel electrophoretic patterns of putijied F ,-ATPases. The enzyme isolated from

normal rat liver contains the characteristic five bands (a, /3, y, 6, E) as indicated by the SDS-gel electrophoresis pattern (Fig. 2A). The pattern also shows that the isolated enzyme was practically free of contaminating proteins. The purified F,-ATPases isolated from all four lines of Morris hepatoma also had similar pattern on SDS-gels (Fig. 2C). In Fig. 2B, the gel pattern of starting material. The specific activity at the same tumor F,-ATPase prior to the pH 8.5 of the purified enzyme in the pres- DEAE-Sephadex column step is shown. As ence of 30 mM bicarbonate was 85 to 120 seen in this case, in addition to the charunits/mg protein for the rat liver and 90- 110 acteristic five-band pattern, there is one adunits/mg for the tumor tissues. Table II summarizes the ATPase activity 1 of submitochondrial particles and the chloroform extracts of three tumor lines and rat liver. The results shown in the table illustrate the effect of chloroform on the yield of ATPase from the tumor tissues compared with that of the normal rat liver submitochondrial particles. In contrast to the 75% yield for the normal tissue, yields greater than 100% (in fact, up to 156%) of enzyme were obtained for the tumor submitochondrial particles. This observation may reflect activation of the masked ATPase in the latter cases.

Effect of bicarbonate and uncoupler on the purified chloroform-released ATPase.

Results reported by Lambeth and Lardy (19) showed that the activity of the F,-ATPase may be determined by the type of anion at the binding site and its influence on the hydrolysis of ATP at the active site(s). The

FIG. 1. The effect of 2,4-dinitrophenol on the activity of purified ATPases from rat liver and Morris hepatomas. Buffer used: 30 mM Tris-SO,, pH 8.0. The enzyme (5 @g/assay) was preincubated with indicated amounts of DNP for 3 min prior to the addition of ATP.

140

SPITSBERG,

MORRIS,

AND CHAN

ditional band with a molecular weight of about 10,000, i.e., between the 6 and Ebands. This protein band is a characteristic of ATPases isolated from all four Morris hepatoma lines examined, and the amount of it varied in different cell lines. The electrophoretic position of this band coincides with that of the F,-ATPase inhibitor protein (13, 20). Whether this additional protein is identical to the inhibitor protein in tumor mitochondria has yet to be determined. FIG. 3. The effect of low temperature (0°C) on the activity of F,-ATPase from rat liver and Morris hepatoma. Buffer used: 10 mM Tris-HCl, 0.25 M sucrose containing 2 mM ATP.

Inactivation of F,-ATPase at low temperature. A characteristic property of puri-

fied mitochondrial F,-ATPase is its inactivation at low temperature. The loss of activity has been correlated with its dissociation into subunits (21). The rate of cold inactivation under controlled conditions is often a dependable criterion to examine the structural differences between the related multisubunit enzymes. The kinetic results of the cold inactivation of mitochondrial F,-ATPase purified from rat liver and Morris hepatoma (7’777) are shown in Fig. 3. F,-ATPases of identical concentrations (0.5 mg/ml) were cold-inactivated (O’C) in 0.25 M sucrose with 2 mM ATP. As seen in Fig. 3, the coldinactivated curves of the normal and tumor F,-ATPase are very similar, although the normal F, was inactivated at a slightly faster rate. The cold-inactivation curves for the other preparations of the tumor F,-ATPase were similar to the curves shown in Fig. 3. Analysis of the results of the cold-inactivation of the different preparations of the isolated F,-ATPases showed that 50% coldinactivation was reached within 30-40 min, regardless of the source of F,-ATPase. FIG. 2. Densitometric tracings of 10% sodium dodecyl sulfate-gel electrophoresis of rat liver and tumor (Morris 7800) ATPase isolated by the chloroform extraction procedure. Electrophoresis was carried out for 3.5 h at 45 V at room temperature. The dye front was marked with a metal wire prior to staining. (A) ATPase purified from rat liver mitochondria, (B) and (C) ATPase from Morris hepatoma 7800 before and after DEAE-Sephadex column, respectively.

The interactions of the mitochondrial inhibitor protein (IF,) with purified F,-ATPuse. It has been shown that mitochondrial

membrane contains a low molecular weight polypeptide (IF,) which inhibits the F,ATPase in the presence of Mg*+ . ATP (11, 13); although the exact role of this protein in oxidative phosphorylation is unknown, it has been proposed that it has a regulatory function (22).

MITOCHONDRIAL

ATPase FROM MORRIS

Since the interaction between IF, and F,-ATPase is quite specific and this interaction can be characterized by an apparent inhibition constant (Ki) and also the number of the binding sites (N) on the F,-ATPase (23), we believed that the investigation of the inhibition of F,-ATPase by IF, could be useful to find possible structural differences between rat liver F,-ATPase and the F,-ATPase isolated from the Morris hepatoma lines. For the quantitative characterization of F,-ATPase from different sources, the use of a highly purified inhibitor preparation is essential. For this study the IF, isolated from beef heart mitochondria (11) was used for the following reason. This inhibitor, as shown previously (13), could strongly inhibit the rat liver F,-ATPase at a concentration lower than that of the inhibitor prepared from rat liver. To ascertain a homogeneous preparation for stoichiometry studies, an additional step of passing the IF, through a CM-Sephadex column was included. The inhibition titration curves of the F,-ATPase activities are shown in Fig. 4A. From the Easson-Stedman plot (Fig. 4B) the Ki and N varied within a small range, namely, 4.0-8.0 x IO-” M and 1.5-2.0, respectively. The inhibition of F,-ATPase from the Morris hepatoma 7777 (shown) and hepatoma 16 (not shown) by beef heart IF, was similar to the inhibition of rat liver F,-ATPase, with similar values for Ki and N. These data

pg OF INHIBITOR PROTEIN

HEPATOMA

AND RAT LIVER

141

also showed that the maximal number of binding sites for the IFI, on the F,-ATPase is probably 2.0. Rat liver IF, prepared by the method of Chan and Barbour (13) also inhibited F,ATPase isolated from both rat liver and Morris hepatomas. Reconstitution of oxidative phosphorylaisolated F,-ATPases and membranes from liver. A recent report described

tion u&ng

the successful reconstitution of oxidative phosphorylation in the rat liver mitochondrial system (24) using F,-ATPase isolated by a previously described procedure (25) and depleted membrane prepared by 3 M urea treatment. The procedure of preparing 3 M urea particles by Pedersen and Hullihen (24) was followed in this study except that instead of using digitonin particles as the starting material, A-particles of Fessenden and Racker (26) were used since these particles exhibited an oligomycin-induced respiratory control ratio of about 2.0. However, the preparation of A-particles required the presence of bovine serum albumin during the preparation of mitochondria (27). As shown in Table IV, both rat liver F,-ATPase and tumor FL-ATPases (Morris hepatoma lines 16 and 7777) were readily bound to the UA-particles in an oligomycin-sensitive fashion (membrane bound ATPase is more than 70% inhibited by oligomycin) and synthesized ATP by oxidative phosphorylation. (P/O ratios were

e ‘/l-i

FIG. 4. (A) Inhibition of rat liver F,-ATPase (10 pg) by the inhibitor protein from beef heart mitochondria. (B) Easson and Stedman plots of rat liver (O), and tumor 7777 (m) F,-ATPases by the inhibitor protein (IF,). The values extrapolated on the ordinate axis gave the number N of binding sites (N = 2.0 and 1.5 for the rat liver and tumor, respectively) and the slope of the linear portion the apparent inhibitor constant, K, (Ki = 7.3 x lo-$ M for the rat liver, and 4.5 x 10ey M for the tumor). (IF,){ is given in micrograms; i represents the degree of inhibition.

142

SPITSBERG,

MORRIS, TABLE

AND CHAN

IV

RESTORATION OF OLIGOMYCIN-SENSITIVE ATPASE AND P/O RATIO OF 3 M UREA-TREATED SUBMITOCHONDRIAL PARTICLES FROM RAT LIVER WITH PURIFIED F,” Oligomycin-sensitive ATPase (units/mg)

Experiment 1. 2. 3. 4. 5.

Particles Particles Same as Particles Particles

alone + F, from rat liver 2. Preincubate at O”C, 2 h + F, from hepatoma 16 + F, from hepatoma ‘7777

P/O* with NADH

0.5-1.0 6.95 6.90 6.40 8.60

0.001 0.88 0.32 0.636 N.D.

Succinate 0.001 N.D.’ N.D. 0.362 0.370

(1One to two milligrams of 3 M urea-treated (24) alkaline submitochondrial particles (26) were preincubated with 300 to 600 pg of F, in 0.35 ml of Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer, containing 0.6 ITIM ATP at room temperature for 2 h. Particles were then centrifuged at 15O,OOOg,30 min and resuspended in the Hepes buffer at room temperature for enzymatic assays. b P/O ratio measurements were by the procedure of Tzagoloff et al. (30). c N.D. = not determined.

increased from 0.001 to 0.64-0.88 for NADH as substrate and from 0.001 to 0.37 for succinate.) It is interesting to note that during the reconstitution, the membrane-bound ATPase activity was stable to cold inactivation at 0°C for 2 h, while the ATP synthesis activities were diminished two- to threefold. A similar observation was also reported by Pedersen and Hullihen (24), i.e., that incubation at 0°C of the reconstituted membranes inactivated the ATP synthetic capability of the system. The reconstitution experiments with UA-particles prepared from tumor mitochondria also indicated that these particles were able to bind purified F,-ATPase from both normal and

tumor tissues in an oligomycin-sensitive fashion (Table V). However, in this case the synthesis of ATP was not restored, nor was oligomycininduced respiratory control observed. Probably the F, components of the ATPase complex in the membrane were damaged during the preparative procedure. DISCUSSION

The possibility of finding structural and/or functional differences between F,-ATPases from tumor and normal tissues has not been ruled out in spite of the recent observations (1, 5, 28) that tumor mitochondria isolated under special conditions exhibited good

TABLE

V

THE BINDING OF F,-ATPASE FROM RAT LIVER TO 3 M UREA-TREATED SUBMITOCHONDRIAL PARTICLESOFMORRIS HEPATOMAS" Specific ATPase activity (units/mg)

Experiment 1. 2. 3. 4. 5. 6.

Particles Particles Particles Same as Particles Same as

a Preparation

from hepatoma 16 from hepatoma 7794A from hepatoma 16 + F, from rat liver in 3. Preincubate at 0°C for 1.5 h from hepatoma 7794A + F, from rat liver in 5. Preincubate at 0°C for 1.5 h

of urea-treated

particles

and conditions

0.70 0.90 10.86 9.18 6.63 6.40

Percentage inhibition by oligomycin 95 95 70 90 77 90

for ATPase assays were the same as in Table IV.

MITOCHONDRIAL

ATPase FROM MORRIS

respiratory control, normal uncoupler-stimulated ATPase activity, and oxidative phosphorylation. Comparative studies on purified mitochondrial F,-ATPases from tumor and normal tissues were not possible without a simple procedure of isolating very highly purified enzyme. This procedure must also be applicable when only small amounts of starting material are available. Beechey et al. (15) first introduced the use of chloroform for the release of F,-ATPase from SMP of beef heart and pointed out that the method could be used when only small amounts of mitochondria were available. Subsequently, Spitsberg and Blair (18) provided evidence that the chloroformreleased F,-ATPase could be further purified with DEAE-Sephadex chromatography and that the purified enzyme has the usual five subunits and was competent to restore oxidative phosphorylation in mitochondrial membranes. In this report, the chloroform extraction procedure and the DEAE-Sephadex chromatography step were modified so that pure F,-ATPases from rat liver and four Morris hepatoma lines were successfully isolated. It was shown that the maximal extraction of ATPase from submitochondrial particles of rat liver was at pH 8.0-8.5, and for the tumor ATPase, it was at pH 7.5. In both cases, 5 mM ATP was required for maximal release of the ATPase from the membranes, probably due to the protective effect against denaturation of ATP on the enzyme during the chloroform treatment. The difference observed between the optimal release of the enzyme from the normal and tumor mitochondrial membrane may reflect differences in the binding site(s) in the F, components of the ATPase complex especially concerning the membrane lipid components. The results on the comparative study of the properties of the ATPases including specific activity, SDS-gel electrophoretic pattern, effects of bicarbonate, DNP, and low temperature, the interaction with the specific inhibitor protein, and the restoration of oxidative phosphorylation on ATPasedepleted SMP isolated from normal tissue all indicated similarity between the enzymes isolated from the normal and tumor tissues. In fact, for example, not only were the

HEPATOMA

AND RAT LIVER

143

SDS-gel patterns of the two enzymes indistinguishable, but the molecular weights of each of the five subunits were exactly the sameas those from the liver mitochondrial ATPase isolated by a previous method (19) or from the beef heart enzyme (18) in agreement with Senior and Brooks (29). The results of the present study show a lower specific ATPase activity in both mitochondria and submitochondria particles from the tumor tissues, indicating a decreased amount of the F,-ATPase in these tumor mitochondria. This decreased level of F,-ATPase could be due to the following two reasons. First, the mitochondria were isolated in a medium without bovine serum albumin, and the tumors were larger than those used in a previous study (5); and second, a large percentage of the ATPase in the tumor mitochondria could be in the masked form, namely, tightly complexed by the specific inhibitor protein (13, 24, 28). The additional protein band in the F,-ATPase preparation from tumor mitochondria prior to DEAE-Sephadex chromatography (Fig. 2B) might also verify this point especially if this additional protein is identified as the specific inhibitor protein. Attempts to activate the masked form of the ATPase in tumor submitochondrial particles were not successful. It seemed that the procedures used (washing and/or sonication at alkaline pH) for normal tissue submitochondrial particles were too harsh for the tumor tissues, resulting in partially damaged membrane preparations with lower specific ATPase activity than the starting material. After the present investigation was completed, a paper appeared in the literature reporting the presence of significant amounts of antigenically active F,-ATPase components in the postribosomal fraction of Zajdela hepatoma (32), whereas the specific antigenic determinants were not detected in the corresponding fraction of rat liver. This observation may reflect an impairment in the assembly of a mitochondrial ATPase in Zajdela hepatoma. When we compared our studies of F,-ATPase from Morris hepatomas with that reported for the Zajdela hepatoma, the yield was significantly higher in our case than for the Zajdela hepatoma (31).

144

SPITSBERG,

MORRIS,

ACKNOWLEDGMENTS

This study was supported in part by Grant CA 20454 from the National Cancer Institute and by the Syracuse University Senate Research Fund, and a grant from the American Heart Association, New York Upstate Chapter. REFERENCES 1. PEDERSEN, P. L. (1978) in Progress in Experimental Tumor Research (Wallach, D. F. H., ed.), Vol. 22, pp. 190-274, Karger, Base]. 2. PEDERSEN, P. L., AND MORRIS, H. P. (1974) J. Biol. Chem. 249, 3327-3334. 3. DEVLIN, T. M., AND PRIJSS, M. P. (1962) Proc. Amer. Assoc. Cancer Res. 3, 315. 4. KOLAROV, J., KUZELA, s., KEMPASK*, V., AND UJHAZY, V. (1973) Biochem. Biophys. Res. Commun. 55, 1173-1179. 5. KASCHNITZ, R. M., HATEFI, Y., AND MORRIS, H. P. (1976) Biochim. Biophys. Acta 449, 224-235. 6. SENIOR, A. E., AND TOMETSKO, A. M. (1975) in

Electron Transfer Chains and Oxidative Phosphorylation (Quagliariello et al., eds.), pp. 155-160, North-Holland, Amsterdam. 7. SENIOR, A. E., MCCOWAN, S. E., AND HILF, R. (1975) Cancer Res. 35, 2051-2067. 8. PEDERSEN, P. L., ESKA, T., MORRIS, H. P., AND CATTERALL, W. A. (1971) Proc. Nat. Acad. Ski. USA 68, 1079-1082. 9. JOHNSON, D., AND LARDY, H. A. (1967) in

Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 94-96, Academic Press, New York. 10. SENIOR, A.

E., AND BROOKS, J. C. Arch. Biochem. Biophys. 140, 257-266. 11. HORSTMAN, L. L., AND RACKER, E. J. Biol. Chem. 245, 1336-1344. 12. DUNKER, A. K., AND RUECKERT, R. R. J. Biol. Chem. 244, 5074-5080. 13. CHAN, S. H. P., AND BARBOUR, R. L. Biochim. Biophys. Acta 430, 426-433.

(1970) (1970)

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