Cytochrome c oxidase

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

114, 50-55 (1966)

Cytochrome

c Oxidase

VIII. Lipid XARTIN Department

MORRISON, of 3~~che~~~tr~,

JOHN

Content’

BRIGHT,

AND

GEORGE

City of Hope MedicaE Center, Medical Duarte, California

ROUSER

Research Imtitute,

Received June 24, 1965 -4 study of the lipids contained in purified preparations of cytoehrome c oxidase has been made. Other than the cholate which had been added as a solubilising agent, only about 0.05 mg of lipid per milligram of protein was present. The phospholipid content did not exceed 9 fig per milligram protein in any preparation analyzed. For the most part, the lipids could be identified with the degradation products of naturally occurring lipid. These results suggest that in the isolated cytochrome c oxidase system, although solubilizing agents are required to achieve high enzyme activity, there is little or no specificity required for phospholipid. ing the rate of oxidation of reduced cytochrome c spectrophotometrieally. The biuret procedure was used to determine protein (10, 12). Purification of choZate. Cholic acid obtained from Nutritional Biochemicals Co. was recrgstallized three times from ethanol. The material was converted to the ammonium salt for use in the enzyme purification. In order to accomplish this, 500 mg of cholic acid was dissolved in 400 ml of chloroform-methanol (2:l v/v), which was saturabed with concentrated ammonium hydroxide. This solution of cholate was then t,aken to dryness under a vacuum by means of a rotary evaporator at room temperat~~re. To insure that all of the cholate had been converted to t,he ammonium salt, the last step was repeated with an additional 100 ml of solvent cont,aining ammonium hydroxide. The ammonium salt thus prepared was analyzed by thin-layer chromatography (TLC) and shown t,o be homogeneous. ~x~ra~ion of lipid. A cytochrome c oxidase preparation was made employing the pure ammonium cholate described above and was then analyzed for its lipid content. In a typical experiment, 4.1 ml of a concentrated cytochrome c oxidase preparation containing 154 mg of protein was extracted with 82 ml of the chloroform-methanol (2:l) by homogenization in a Waring Blender. The insoluble residue was removed by filtration in a medium porosity sintered glass funnel. The residue remaining was returned to the blender

A number of reports have suggested that phospholipid plays an important role in the cytochrome c oxidase activity. These conclusions have been based on studies which show that (a) purified preparations of eytoc~ome c oxidase are rich in phospho~pids (l-7), (b) eytochrome e oxidase can be activated by a variety of phospholipids (l-9), and (c) that cytochrome c itself can be prepared in a highly active form which is complexed with phospholipid @,9>The present paper is a continuation of our efforts to define the components necessary for cytochrome c oxidase activity (10, 11). The purpose of this report is to present the procedures and results obtained in an investigation of the lipids of our purified preparation of cytochrome c oxidase. METHODS The cytochrome c oxidase was isolated and purified as previously described (10). The eytochrome e oxidase activity was assayed by follow1 This inveEtig&tion was supported in part by research grants GM-10214, NB-01847, and CA03134 from the United States Public Health Service, and the Thomas C. Lynch Fund. 50

LIPID

CONTENT

OF A PURIFIED

CYTOCHRO~E

and the extraction procedure was repeated with 40 ml of chloroform-met,hanol. It was then filtered and the extraction was repeated a third time with the same solvent. Finally, the residue was extraot,ed in a similar manner with 40 ml of chloroform-methanol (7:l) saturated with 28y0 aqueous ammonium hydroxide. The combined chloroformmethanol extracts were evaporated to dryness on a rotary evaporator under reduced pressure. The residue obtained in this way was then extracted three times with 30 ml of chloroform-methanol (2:l). These chloroform extract~s were coneentrated t,o a small volume by evaporation under nitrogen and dissolved in chloroform-methanol (19:l) saturated with water. This was then placed on a Sephadex G-25 column prepared as described by Siakotos and Rouser (13). Small volumes of the same solvent mixture were used to wash the flask and the column. The lipid was then eluted from the column with 1 liter of chloroform-methanol (19:l) saturated with water to yield Fraction 1. Fraction 2 was obtained by eluting the column with 1 liter of methanol-water (l:l). Both fractions were then evaporated under nitrogen to obtain appropriate solutions of lipids for thinlayer chromatography. Excess eholate ooufd be removed from the preparation before extraction of lipid by adding sufficient saturated ammonium sulfate to make the solution 35y0 saturated in order to precipitate the enzyme. The precipitated cytochrome c oxidase was centrifuged at 10,000 g for 1 hour. In some experiments, the cytochrome preparation was dissolved and precipitated in this way two additional times to lower the cholate eoncentration even further. A modified procedure was employed on one occasion for simultaneous extraction and removal of salts. After excess cholate was removed as described above, the protein precipitate of preparation 2 was suspended in 15 ml of 0.1 M phosphate buffer (pH 7.4) to give a protein content of approximately 10 mg per milliliter. Fifteen ml of enzyme was then extracted with 60 ml of chloroform-methanol (2:1, v/v). The chloroform phase was separated from the aqueous phase containing the precipitated protein as well as salts, and the extraction was repeat,ed with 16 ml of chloroform-methanol. The procedure was repeated a third time and the combined chloroform-methanol phases were evaporated to dryness on a rotary evaporator. The dry residue was dissolved in chloroform-methanol (9:1, v/v) for DEAE chromatography. ~ie~~~lu~i~oe~~~l g~up~ie sep~r~~~~.

(DEAE)

cellulose

C~TO~U~-

A chloroform-methanol solution (9:l) of lipid was applied to a 2.5 X 20 cm column of diethylaminoethyl (DEAE) cellulose

c OXIDASE

PREPARATION

51

in the acetate form prepared as described by Rouser et aE. (M-16). The lipid fractions were then eluted using the sequence: chloroform-methanol, 9:l; methanol; chloroform-acetic acid, 3:l; glacial acetic acid; and chloroform-methanol (4: 1) containing 20 ml of 28% (by weight) aqueous ammonium hydroxide per liter and 0.01 M ammonium acetate. One liter of each of the solvents was allowed bo percolate through the column and collected as a single fraction at a flow rate of 3 ml per minute. Solids were recovered from each fraction by evaporation to dryness on a rotary evaporator and were dried to constant weight over KOH in a vacuum dessieator flushed with pure nitrogen (15). Each fraction was analyzed by one- and two-dimensional TLC (18). Lipids were localized as charred spots after spraying with a sulfuric acid-potassium dichromate spray (17), or as fluorescent, spots after spraying with alkaline rhodamine 6 G (15). The amount of phosphorus in fractions was determined by a modification of the procedure of Chen et aE. (17). RESULTS

Thin-layer ehron~a~graphy of commercial preparations of cholic acid and sodium cholate showed that these mate~als were not homogeneous. On chromatography, several extraneous substances which were only partially removed by recrystallization from ethanol could be seen. These components appeared to be formed when sodium cholate was prepared from pure cholic acid by neutralization with sodium hydroxide. Even when TLC indicated the presence of only traces of t,hese contaminants, they were detectable in the final cytochrome c oxidase preparation, su~esting that the contaminants in the sodium cholate were being concentrated in the enzyme preparation. This was not observed when ammonium cholate was used. Table I shows the analysis of the lipid extracted from our cytochrome c oxidase preparations by two different procedures. Sephadex column chromatography is useful for the separation of all lipids, including cholate, from water-soluble nonlipid substances (13). The maximum phospholipid content of the lipid fraction obtained from Sephadex can be calculated on the basis of phosphorus analysis. DEAE column chromatography provides a means for the separation of cholate from phospholipids as well

52

MORRISON,

BRIGHT,

AND

ROUSER

chromatogram (Silica gel G as adsorbent.) developed with chloroFIG. 1. Thin-layer form-metha~~ol-barer (65:25:4), sprayed with alkaline rhodamine 6 G, and photographed under ultraviolet light. Application (1) 1OOpg of total ~hlorofo~-~~ethanol extract from the cytoehroae c oxidase preparation. Applications (2) through (6) contained 100, 50, 20, 100, and 100 pg, respectively, of DEAE-cellulose column fractions eluted with chloroformmethanol, 9: 1 (2) ; methanol (3) ; chloroform-acetic acid, 3: 1 (4) ; glacial acetic acid (5) ; and chloroform-methanol-ammonia-ammonium acetate (6). The diffuse cholate spots in the original preparation (1), the chloroform-acetic acid (4), and acetic acid (5) fractions are due to spreading aft,er spraying with the strongly alkaline rhodamine reagent‘. This spreading phenomenon facilitat,es recognition of the cholate spot. Application (7) was 80 ~2 of beef brain chloroform-methanol extract showing from above downward cholesterol (at the solvent, front), three spots from cerebrosides, phosphatidyl ethanolamine, lecithin, sphingomyelin, a very light spot of phosphatidyl inositol, and phosphatidyl serine just off the point of application. Comparison of the spots from the DEAE fractions indicates the presence of lecithin (applicalion 2) and traces of phosphatidyl ethanolamine (applieation 3) and cardiolipin (appl~catio~ls 5 and 6). The other component,s in the fractions are not found in mitochondria and may be decomposition products of t,he major nat~urally occurring lipids of mitochondria. FIG. 2. Two-dimensional TLC of the total chloroform-methanol extract of a cytochrome c oxidase preparation. The sample (800 pg) was applied to the lower right corner. The adsorbent was silica gel plain (9 parts) plus magnesium silicate (1 part) developed first (vertical direction) with chloroform-methanol-28~ aqueous ammonia (65:35:5) followed by drying in air for 10 minutes and development (horizont,al direct,ion) with chloroformacetone-methanol-acetic acid-water (5:2:1:1:0.5). These spots were localized by spraying with 559& sulfuric acid containing 0.6% potassium dichromate and heating to 180” for 30 minutes. Ch, cholesterol; FA, fatty acid; PE, phosphatidyl ethanolamine; L, lecithin; C, cholate; X, uncharacterized.

as the separation

of phospholipids into characteristic groups, The difference in t!he quantity of total lipid obtained by the two procedures is largely a reflection of the amount of cholate removed by ammonium sulfate precipitation prior to extraction. On the other hand, t,he difference in the amount of phospholipid reflects the vari-

ations in preparations. Phosphorus anaIysis by both methods shows that between 0.1 and 0.2% of the weight of the lipid can be attributed to phospholipid. The value of 0.009 mg phospholipid per milligram protein was the highest value obtained for any preparation analyzed. As shown in Table I, DEAE column

LIPID

CONTENT

OF A PURIFIED

CYTOCIIROME TABLE

LIPID

c OXIDASE

PREPARATION

I

CONTENT 0~ C~TOCHR~ME 0~1~~~33 Preparation 1

content

Total lipid Phosphorus content” Noneholate lipidd Phospholipid+

53

Preparation 2

0.180 0.001 0.048 0.005

0.263 mg/mg proteina 0.0013 mg/mg lipid 0.009 mgfmg protein

mg/mg mg/mg mg/mg mg/mg

proteinb lipid protein protein

a Fraction 1 from Sephadex column obtained as described in the text. b The chloroform:methanol extractable material obtained aa described for the alternate procedure. c Direct analysis of phosphorus as described in text. d Total lipid minus oholate. This value was not determined for preparation 1. 0 Calculated assuming that phosphorus is 4y0 of the phospholipid. TABLE FRACTIONS FROM DEAE

II

CHROMATOGRAPHY OF THE LIPIDS IN A CYTOCHROME c OXIDASE PREPARATION

Eking solvents

Fraction

wt.

(mg)

% of total

lipid

Pho~s&$ipid

% Of in phosphorus fraction --

Chloroform-methanol (9:1, v/v) Methanol Chloroform-a(:etic acid (3:1, v/v) Acetic acid Chloroform-methanol-ammonia. salt Total

1.19 1.52 19.19 1.87 1.87

4.64 5.93 74.8 7.3 7.3

25.646

106.07

0.55 0.15 0.00 0.00 0.95

0.16 0.06 0.00 0.00 0.44 0.66

a Calculated assuming that phosphorus is 470 of the phospholipid. h 25.62 mg sample applied.

chromatography provides a convenient means to separate the lipids from the large amount of added cholate used in the preparation of the enzyme. When the Sephadex procedure was used, no effort was made to remove the excess cholate. Two-dimensional TLC is then particularly useful for detection of even minor lipid components in the presence of a large amount of cholate. In Table II are shown the results of analysis by DEAE column chromatography of the lipid extracted by the alternate procedure. The percentage of the total lipid eluted by each solvent shows that almost 75% of the weight of the total lipid was eluted with chloroform-acet,ic acid (3 : 1) solvent. Thin-layer chromatography (Fig. 1) of the fractions from the column indicated that this component was cholate. Preparations which had not been precipitated repeatedly with ammonium sulfate prior to extraction of lipid contained even more cholate. The amount of the other

components varied for different preparations, though not significantly. Of the three major phospholipids present in mitochondria, TLC (Fig. 1) showed only traces of lecithin (9: 1 chloroform-methanol fraction) phosphatidyl ethanolamine (methanol fraetion) and diphosphatidyl glycerol (cardiolipin present in the chloroform-methanolammonia-salt fraction). These are the major phospholipids of heart mitochondria as deterred by DEAL-cellulose column and thin-layer chromatography (19). The other components of the cytochrome c oxidase preparation appear to be decomposition products and lipids of other parts of the cell, since they are not detectable in mitochondrial lipid extracts. The same type of result was obtained by Sephadex column chromatography, as the two-dimensional TLC (Fig. 2) of Fraction 1 shows. While the phospholipids are present in this fraction (13), it is apparent from the chromatogram that cholate comprises the

54

MORRISON, TABLE

BRIGWT,

III

RELATION OF ACTIVITY AND HEME CONTENT TO PHOSPHOLIPID IN CYTOCHROME c 0x1~~s~~

Activity k = 3.95 see-ijrng proteins Mmoles heme/mg proteinb &moles phosp~olipid/mg proteinc

0.008 pmole/mg 0.012 mole/mg

a Assayed as indicated in text at 22” C. *Extinction coefficient of 21.6 employed for 603 rnp absorption peak of the reduced preparation (21). c Value based on total phosphorus contained in Fraction 1 assuming an average molecular weight of 775 and 4% phosphorus for phospholipid. d Rata taken from analysis of preparation employing Sephadex column,

greatest amount of material present. The other spots are again, for the most part, lipids not detected in the intact mitochondria (19). DISCUSSION

While previous investigations of purified preparations of cytoehrome c oxidase have all indicated the presence of a much higher concentration of lipid, pa~icularly phospholipid, the analysis of our preparation of purified cytochrome c oxidase clearly shows very low lipid content. The results reported here show that between 0.18 and 0.263 mg chloroform-methanol extractable material is present per milligram of protein in the preparation. Only about 1% of the total chloroform-methanol extract can be accounted for as phospholipid. By far the greatest amount of this material could be accounted for as cholate. The differences in the amount of chloroform-methanol extractable material in the two preparatioI~s shown in Table I were due primarily to the amount of cholate present in the two preparations. Much of the excess cholate was removed from preparation 2 prior to extraction. However, the phospholipid content per milli~am of cyto~hrome c oxidase protein was very low regardless of the method of analysis employed. In preliminary studies, the unpurified cholic acid which was being employed for

AND ROUSER

the isolation of the ~ytochrome c oxidase was found to contain other components. It was particularly interesting to note that preparations of the cytochrome c oxidase obtained with impure cholate contained higher concentrations of these contaminants than the starting cholate. It appears, therefore, that the contaminants were concentrated during preparation of the enzyme. Of all the other lipid materials visualized by TLC, only small amounts of components characteristic of heart mitochondria (18, 19) are detectable. Other substances are probably degradation products of the naturally occurring lipids. The decomposition of pure cardiolipin and phosphatidyl ethanolamine gives rise to many spots with both higher and lower Rf values than the unaltered material. The degradation of the lipids of mitochondria and other subcellular particles and the resulting artifacts have been considered in some detail elsewhere (19). As Table III shows, the enzyme activity in the preparation used in this study is typical for purified preparations of cytochrome c oxidase. Since these preparations contain only slightly more than one mole of phospholipid per mole of heme, it is difficult to attribute an obligatory role to phospholipid, at least for in vitro conditions. This is particularly true since much of the phospholipid present in the preparation is altered from the native state. While some investigators have indicated that phospholipids are necessary for enzyme activity, we have routinely used Tween 80, a nonionic detergent, to achieve high activity of the enzyme. While a role for phospholipids in mitochondrial function has been suggested using the basic approach of removal and reinsertion of lipid (22, 23), our preparations of cytochrome e oxidase have been demonstrated to contain a very low phospholipid concentration. Further, they contain not a single phospholipid, but rather a heterogeneous group, a large percentage of which has been altered from the form found in the mi~ehond~a. It is possible that the cholate in our preparations could serve in a manner

LIPID

CONTENT

OF A PURIFIED

CYTOCHROAME

similar to that portrayed for the phospholipid. This possibility, however, only stresses the nonspecificity of the lipid requirement for cytochrome c oxidase activity in vitro. REFERENCES 1. BRIERLEY, G. P., AND MEROLA, A. J., Biochim. Biophys. Acta 64, 205 (1962). 2. COHEN, M.., AND WAINIO, W. W., J. Biol. Chem. 288, 879 (1963). 3. GREENLEES, J., AND WAINIO, W. W., J. Biol. Chem. 234, 658 (1959). 4. GRIFFITHS, D. E., AND WHARTON, D. C., J. Biol. Chem. 236, 1850 (1961). 5. YONETANI, T., J. Bid. Chem. 236, 1680 (1961). 6. IGO, R. P., MACELER, B., DUNCAN, H., RIDYARD, J. N. A., AND HANAHAN, D., Biochim. Biophys. Acta. 42, 55 (1960). 7. MARI~YETTI, G. V., ~ARA~~UZZINO, D. J., AND STOTZ, IL, J. BioL. C&em. 324,819 (1957). 8. DAS, M. L., AND CRANE, F. L., Biochemistry 3, 696 (1964). 9. GREEN, D. E., AND FLEISCHER, S., Biochim. Biophys. Acta ‘70, 554 (1963). 10. HORIE, S., AND MORRISON, M., J. BioE. Chem. 238, 1855 (1963). 11. MORRISON, M., in “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.). Wiley, New York (1965).

c OXIDASE

PREPARATION

55

12. GORNALL, A. G., BARE)AWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). 13. SIAKOTOS, A. N., AND ROUSER, G., J. Am. Oil Chem. Sot. 42, 913 (1965). 14. ROUSER, G., BAUMAN, A. J., KRITCHEVSKY, G., HELLER, D., AND O’BRIEN, R. S., J. Am. Oil Chem. Sot. 38, 544 (1961). 15. ROUSER, G., KRITCHEVSKY, G., KELLER, D., AND LIEBER, E., J. A,m. Oil Chem. Sot. 40, 425 (1963). 16. ROUSER, G., KRITCHEVSKY, G., GALLI, C., AND HELLER, D., J. Am. Oil Chem. Sot. 43, 215 (lQ65). 17. CHEN, P. S., JR., TORIBARA, T. Y., AND WARNER, H., Anal. Chem. 28,1756 (1956). 18. ROUSER, G., GALLI, C., LIEBER, E., BLANK, M. L., AND PRIVETT, 0. S., J. Am. Oil Chem. Sot. 41, 836 (1964). 19. FLEISCKER, S., AND ROUSER, G., J. Am. Oil Chem. Sot. 42. 5% (1965). 20. LEMBERG, R., NEWTON, N., AXD O’HAGAN, J. E., Proc. Roy. Sac. (London), Ser. B 166, 356 (1961). 21. MORRISON, M., AND HORIE, S., Anal. Biochem. 12, 77 (1965). 22. FLEISCHER, S., BRIERLEY, G., KLOVXEN, H., AND SL~~W~RBAC~, D. B., J. Biol. Chem., 237, 3263 (1962). 23. FLEISCHER, S., Sixth Intern. Gong. Biochem. (196’4) New York, Symposium I, Sect. VIII, p. 605.