BIOCHIMICA ET BIOPHYSICA ACTA 601 BBA 56028 LIPIDS ASSOCIATED WITH CYTOCHROME OXIDASE DERIVED FROM YEAST MITOCHONDRIA E. D. THOMPSON AND L. W. PARKS Dcpavtmmt of Microbiology, Oregon state university, Coroalhs, oveg. 9733I (U.S.A.) (Received November qrd, 1971) SUMMARY The association of lipids with cytochrome oxidase has been studied. The enzyme has been purified from yeast mitochondria using a freezethaw disruption technique, cholate solubilization, and (NH&SO, fractionation. Concomitant ptication of ergo- sterol with cytochrome oxidase activity was noted, and enzyme activity was shown to be dependent on the presence of lipid. The most effective activators of cytochrome oxidase were C,, and &, unsaturated fatty acids. Sterol is not essential for in vitro cytochrome oxidase activity. INTRODUCTION The purification and characterization of yeast cytochrome oxidase recently has been an area of extensive research’-8, yet little attention has been paid to the precise lipid component(s) required for activity. In yeast, ergosterol and oleic acid are ne- cessary for maintenance of respiratory competency’, and the synthesis of ergosterol, oleic acid, and cytochrome oxidase occurs concurrently on aeration of anerobically grown cultures6-7. Upon extensive purification cytochrome oxidase requires the addi- tion of a crude phospholipid mixture for activitya. However, to date the exact lipids required for cytochrome oxidase activity have not been determined. The present communication describes a method for obtaining highly purified preparations of cytochrome oxidase for study of the precise lipid requirements for enzymic activity. EXPERIMENTAL. PROCEDURES Organism and c&Ural conditions Saccharomyces cerevisiae, strain 80 BM-I, a haploid methionine-requiring or- ganism was cultured in a 1% tryptone, 0.5% yeast extract broth to which r.9% ethanol was added following sterilization. The yeast was grown in 30-l batches in a New Brunswick Fermacell Fermentor (Model CF-50) with stirring set at 300 rev./n& Biochhn. Biopkys. Ada, 260 (1972) 601-607
Transcript
BIOCHIMICA ET BIOPHYSICA ACTA 601
BBA 56028
LIPIDS ASSOCIATED WITH CYTOCHROME OXIDASE DERIVED FROM
YEAST MITOCHONDRIA
E. D. THOMPSON AND L. W. PARKS
Dcpavtmmt of Microbiology, Oregon state university, Coroalhs, oveg. 9733I (U.S.A.)
(Received November qrd, 1971)
SUMMARY
The association of lipids with cytochrome oxidase has been studied. The enzyme has been purified from yeast mitochondria using a freezethaw disruption technique, cholate solubilization, and (NH&SO, fractionation. Concomitant ptication of ergo- sterol with cytochrome oxidase activity was noted, and enzyme activity was shown to be dependent on the presence of lipid. The most effective activators of cytochrome oxidase were C,, and &, unsaturated fatty acids. Sterol is not essential for in vitro cytochrome oxidase activity.
INTRODUCTION
The purification and characterization of yeast cytochrome oxidase recently has been an area of extensive research’-8, yet little attention has been paid to the precise lipid component(s) required for activity. In yeast, ergosterol and oleic acid are ne- cessary for maintenance of respiratory competency’, and the synthesis of ergosterol, oleic acid, and cytochrome oxidase occurs concurrently on aeration of anerobically grown cultures6-7. Upon extensive purification cytochrome oxidase requires the addi- tion of a crude phospholipid mixture for activitya. However, to date the exact lipids required for cytochrome oxidase activity have not been determined.
The present communication describes a method for obtaining highly purified preparations of cytochrome oxidase for study of the precise lipid requirements for enzymic activity.
EXPERIMENTAL. PROCEDURES
Organism and c&Ural conditions Saccharomyces cerevisiae, strain 80 BM-I, a haploid methionine-requiring or-
ganism was cultured in a 1% tryptone, 0.5% yeast extract broth to which r.9% ethanol was added following sterilization. The yeast was grown in 30-l batches in a New Brunswick Fermacell Fermentor (Model CF-50) with stirring set at 300 rev./n&
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602 E. D. THOMPSON, L. W. PARKS
and vessel aeration at 2 l/min. A 15% (v/v) inoculum (late log phase cells) was added, and early stationary phase was reached in 18-20 h. Cells were harvested rapidly in a Sharples centrifuge, washed twice in distilled water, and resuspended in 0.25 M sucrose (1.0 ml per g wet wt). Final yield from a 30-l batch was approx. 300-400 g wet
wt.
Assay procedure for cytochrome oxidase Cytochrome oxidase was assayed by following the oxidation of ferrocytochrome
c spectrophotometrically at 550 nm on a PMQ II Zeiss spectrophotometer. Reduced cytochrome c was prepared as previously described by Duncan and Macklerl. The reaction mixture contained IOO ,umoles of phosphate buffer, pH 7.0, sufficient cyto- chrome c to yield an initial absorbance of 0.9, and water to a final volume of 3.0 ml. The reaction was initiated by the addition of the protein and was followed for 30 s. Activity is reported as change in absorbance per min per ml of extract and specific activity is defined as change in absorbance per min per mg protein. Protein was de- termined by the method of Lowry et aLa.
Pwification of cytochrome oxidase The sucrose-suspended cells were broken with a 45-s burst of a‘Bronwil1 MSK
Cell Homogenizer using o.25-mm glass beads. Unbroken cells and other cell fragments were removed by centrifugation at 2500xg for 30 min. The supernatant was then centrifuged at 25 ooo xg for 20 min to pellet the mitochondria. The protein super- natant was discarded. The mitochondrial pellet was resuspended in a volume of 0.25 M sucrose equal to that of the discarded protein, and washed three more times in the sucrose. Following the final centrifugation, the mitochondrial pellet was re- suspended in one half the volume of sucrose used for each wash and disrupted by freezing the suspension overnight. The frozen mitochondrial preparation was thawed and subjected to centrifugation (105000 xg) for 30 min and the supernatant fraction discarded. The pellet was resuspended in an equal volume 1.33% deoxycholate-o.67% cholate-o.or M Tris-HCl buffer (pH 7.4) using a glass tissue homogenizer. 5 mg of sodium dithionite was added and the preparation was allowed to stir for 20 min. Solid (NH&SO, was added to 18% saturation with stirring, and the mixture was allowed to stir for 20 min. The preparation was then centrifuged at 25 ooo xg for 20 min and the pellet discarded. The supernatant portion was taken to 29% saturation with solid (NH&SO, and after 20 min stirring was again centrifuged at 25 ooo xg for 20 min. The supernatant portion was discarded and the pellet was resuspended in a small volume (15-20 ml) of 3% deoxycholate-r”/o cholate-o.or M Tris-HCl buffer, pH 8.5. The suspension was centrifuged for I h at 105000 xg and the pellet discarded. Absorption spectra (oxidized and reduced) of the purified cytochrome oxidase pre- paration showed typical absorbance maxima for cytochrome oxidase as described by Myers. The absence of absorbance maxima at 500 and 560 nm indicate cytochromes c,c, and b were not present in the preparation. Table I shows the results of a typical purification.
Separation of lipid components from purijed cytochrome oxidase Purified cytochrome oxidase (5.0 ml) was placed on a Sephadex G-50 column
(2.5 cmx 20 cm) and eluted with 3% deoxycholate-1% cholate-o.or M Tris-HCl
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LIPIDS OF CYTOCHROME OXIDASE 603
buffer, pH. 8.5. Two peaks were obtained as determined by absorbance at 280 nm. The contents of the second peak were pooled and the pH lowered to 5.0 by the drop- wise addition of 6 M HCl. The white precipitate was collected by centrifugation and extracted three times with n-hexane. The hexane was dried under N, and the residual lipid mixture resuspended in a minimal volume of hexane.
Fractionation of the lipid components into 8 discreet classes (saturated hydro- carbons, unsaturated hydrocarbons, sterol esters, triglycerides, non-sterified sterols, diglycerides, monoglycerides and phospholipids) was accomplished by the stepwise elution of I .o ml of the lipid mixture through a I .5 cm x 20 cm column of Bio-Sil HA (325 mesh) as previously described”.
Lipid anaJysis Ergosterol was isolated by “methanolic pyrogallol saponification” as previously
described by Adams and Park.+. The non-saponifiable lipids were extracted from the mixture with n-hexane, dried under N,, and resuspended in chloroform. Total sterol was determined by a modified Liebermann-Burchard reactions. Identification of ergo- sterol was based on the Liebermann-Burchard reaction, thin-layer chromatography using AgNO,-impregnated Silica Gel-G, and ultraviolet scans as previously described’*. Inorganic phosphate was determined by the procedure of Chen et al.la. Fatty acid analysis was performed by converting the fatty acids to their methyl esters14 which were identified gas chromatographically by their retention times relative to authentic standards as described by Madyastha and Parksls. Quantitative determination of the fatty acids was accomplished by integrating the area under the respective peaks using a Hewlett-Packard Integrator model 3370 A.
Reactivatiolz of cytochrome oxidase Activation experiments were performedby mixing portions of the inactive cyto-
chrome oxidase protein (Peak I from the Sephadex G-50 column; see Fig. I) with the desired lipid component and incubating for 5 min at 30 “C. All control experiments were diluted in the 3% deoxycholate-1% cholate-o.or M Tris-HClbuffer, pH 8.5, and incubated for an equivalent period. All stimulation values are based on the activity of the inactive cytochrome oxidase protein.
Materials Cytochrome c (Grade III), sodium deoxycholate, fatty acid methyl esters and
cholate were purchased from Sigma Chemical Co. All phospholipids and fatty acids were purchased from Calbiochem. Bio-Sil HA was purchased from Bio-Rad Laborato- ries, and Sephadex was obtained from Pharmacia Fine Chemicals Inc. Tryptone and yeast extract were purchased from Difco. AU other compounds used were analytical reagent grade.
RESULTS
The method used to purified cytochrome oxidase results in an approx. 8o-fold increase in specific activity with the final preparation containing 7-S% of the initial mitochondrial protein and 18-30~~ of the total mitochondrial ergosterol (Table I). Cytochrome oxidase of higher specific activity may be obtained by an additional
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604 E. D. THOMPSON, L. W. PARKS
TABLE I
PROCEDURE USED TO PURIFY CYTOCHROME OXIDASE FROM YEAST MITOCHONDRIA
Purification values are based on intact mitochondria and not whole cells.
Fraction Volume Protein Speci$c Total Sterol Total (mglml) activity activity (pglml) sterol (pg)
(NH&SO, fractionation (35% saturation) in the 4% cholate buffer. However, since this step also removes the desired lipid fraction from the protein, it was not employed in this research.
That ergosterol is truly associated with cytochrome oxidase activity can be demonstrated by applying a portion of the enzyme purified through the (NH&SO, fraction steps but resuspended in 1.33% deoxycholate-o.67% cholate-o.or M Tris- HCl buffer, pH 7.4, on a Sephadex G-50 column and eluting with the same buffer. Cytochrome oxidase activity and ergosterol elute simultaneously immediately after the void volume. Similar results are obtained with Sephadex G-zoo column chromato-
graphy. However, if the purified enzyme is resuspended in the 3% deoxycholate-1%
cholate-o.or M Tris-HCl buffer at pH 8.5 and chromatographed on a Sephadex G-50 column, drastically different results are obtained. Fig. I shows that two distinct peaks are detected by absorbance at 280 nm. The first peak contains protein and about
i 3 5 TUBE NTUYI)ER
i II 13
Fig. I. Elution profile of purified cytochrome oxidaae from a Sephadex G-5o~column (a.5 cm x 20
cm) using 3% deoxycholater% cholate-o.or M Tris-HCl buffer, pH 8.5. See Experimental pro- cedure for description of puiified cytochrome oxidase pre$at!ion, sterol, and proteifi detennina- tions.
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LIPIDS OF CYTOCHROME OXIDASE 605
~-IO% of the original enzyme activity. The second peak contains neither protein nor enzyme activity, rather the bulk of the ergosterol and other lipid components are pre- sent. If equal portions of the two peaks are mixed back together and incubated at 30 “C, essentially full activity can be restored to the enzyme.
The lipid activator was completely precipitated by the acid treatment of the second peak since the resulting supematant fraction was devoid of stimulatory activity. Fractionation of the lipid activator from the acid precipitated portion of the second peak into various components as described in Experimental procedure showed two types of sterols present in approximately equal amounts (Table II). The first
TABLE II
ANALYSIS OF THE FRACTIONS OBTAINED BY SEPARATION OF THE ACTIVATOR FRACTION BY BIO-SIL
HA COLUMN CHROMATOGRAPI-IY
Fraction number refers to each discreet class of lipid obtained (see text for description!. Each fraction was taken to dryness under Ns and resuspended in hexane to a final volume of IO ml. I ml of each fraction was dried under N, then resuspended with the “inactive cytochrome oxidase” protein obtained from the first peak of the Sephadex G-go column shown in Fig. I. The enzyme- lipid mixture was allowed to incubate 3 min at 30 “C prior to assaying. Concentrations are based on units/ml of the resuspended lipid fractions following column chromatography.
sterol elutes from the Rio-W column in Fraction 2, is not precipitated by digitoninl6, and has an RF value of 0.74 on thin-layer chromatography. The second sterol fraction is precipitable with digitonin, has an RF value of 0.14 and exhibits an ultraviolet spec- trumidentical to ergosteroP*. Based on these facts, the sterol in Fraction 5 is believed to be free ergosterol.
Fatty acid content of the acid precipitated activator fraction was limited to two species (oleic and palmitoleic) which were present in three different fractions (Table II). All three of these fractions significantly stimulated cytochrome oxidase activity. Maximal stimulation of cytochrome oxidase activity was achieved with 1.0 ml of
either Fractions 4 or 8, corresponding to approx. 1.0 ,umole of the fatty acid compo- nents per mg protein. Fraction 5 was not soluble in the cholate buffer at these con-
centrations, and therefore could not be similarily tested. Because previous reports-had indicated that phospholipids were necessary for
cytochrome oxidase activitya, and the results obtained here showed that the phos- pholipid fraction was stimulatory, attempts were made to activate the protein with a variety of purif$ed~phospholipids. Table III shows that @itied phospholipids were
not as stimrilatory as the activator fraction itself. When the fatty acid moieties of these phospholipids were, analyzed, ,it was found that only C;, or smaller fatty acids were present. As shown in Table &III, fatty acids of 15 carbons or less are very poor
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606 E. D. THOMPSON, L. W. PARKS
TABLE III
EFFECTS OF SEVRRAL LIPID COMPONENTS AS POSSIBLE ACTIVATORS OF THE CYTOCHROME OXIDASE
PROTEIN
Lipid concentrations were in excess of that required for maximal enzyme stimulation. Phospho- lipid concentrations are expressed as yg phospholipid per mg protein and fatty acid concentrations are in pmoles per mg protein. The protein fraction was derived from the first peak eluted from a Sephadex G-50 column with the 4% deoxycholate-cholate buffer, pH 8.5 (Fig. I). The activator fraction was derived from the second peak eluted from the same column.
Component tested Concentration of Activity Stimulation (-fold) component tested (AA jmin per ml)
* Prepared from egg. * * Prepared from bovine brain.
* * * Synthetic. t Components first solubilized with polysaccharide sterol solubilizing agent”.
activators of cytochrome oxidase. The lecithin used was known to be of the /?,y-dipal- mityl form, and it was subsequently shown that free palmitic acid was as stimulatory as the intact phospholipid (Table III). Since it was known that only oleic and palmit- oleic acids were present in the cytochrome oxidase activator fraction, it was decided to test these as possible activators of the enzyme. Table III shows that both oleic and palmitoleic acids were extremely efficient activators of cytochrome ox&se. Sub- sequent experiments have shown that insufficient amounts of the activator fraction were used in these experiments to give full activation of the enzyme. When the con- centration of this fraction was increased, stimulation equal to that for the free fatty acids was obtained.
DISCUSSION
Of notable interest in the purification procedure is the dramatic increase in the total enzyme units. We believe this to be a direct result of using the freeze-thaw me- thod of mitochondrial disruption in place of the classical sonication procedure pre- viously employed. This phenomenon is probably a result of increased accessibility of the large substrate (Mr IZOOO) to the enzyme due to removal of hindering membrane fragments during the purification.
The involvement of fatty acids in cytochrome oxidase activity is of interest because it has been previously demonstrated that fatty acids (especially oleic acid) when added to the growth medium retard the loss of respiratory comp&ency at high temperatures‘. Additionally, it demonstrates that enzyme stimulation is primarily a
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LIPIDS OF CYTOCHROME OXIDASE 607
function of the fatty acid, and relatively specific for the C,, and C,, unsaturated species. Although phospholipids are stimulatory, there is at least one other component of the activator fraction that is equally effective. Of significance is the fact that maximal stimulation of cytochrome oxidase is attained at equivalent fatty acid con- centrations with both fractions.
Although sterols are not directly involved in cytochrome oxidase activity, it is apparent that at least two types of sterols are present in the activator fraction. Of interest is the fact that the sterols in Fraction 2 are not precipitated by digitonin, indicating either the sterol is not of the 3-/?-hydroxy configuration, or that it is esteri- fiedl’. However, treatment with BF, did not yield any detectable methylated fatty acids, making the latter possibility doubtful. Because Paltauf and Schatzl’ could not detect fatty acids of C,, or less in yeast mitochondria, and since Madyastha and ParksI did not detect sterol esters of less than 13 carbons, it appears that the sterol component(s) present in Fraction 2 may indeed be novel.
The association of ergosterol with cytochrome oxidase during the purification may be simply fortuitous. However, the possibility exists that the sterol is necessary during respiratory adaptation to provide structural integration of the various en- zymic components in the inner mitochondrial membrane. The role of sterols during respirational maturation remains a subject of continuing investigation in our laboratory.
ACKNOWLEDGMENTS
This research was supported by grants from the National Science Foundation (GB-31119) and the U.S. Public Health Service (AM-ogrgo-II). Oregon Agricultural Experiment Station technical paper number 3136. The excellent technical assistance of Mrs Elizabeth MacDonald during the early phases of this work is gratefully acknowledged.
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