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A simple method for the large-scale preparation of mitochondria from microorganisms
ANALYTICAL
BIOCHEMISTRY
A Simple
77, 1lo- 121 (19771
Method for the Large-Scale Preparation Mitochondria from Microorganisms
of
BERND LANG, GERTRAUD BURGER, ILIAS DOXIADIS, DAVID Y. THOMAS, WOLFHARD BANDLOW, AND FRITZ KAUDEWITZ Genetisches
Institut
der Universitiit D-8000 Miinchen
Miinchen, Maria-Ward-Strasse 19, Germany
la,
Received July 15, 1976; accepted August 23, 1976 A simple mechanical procedure that has been developed for the largescale preparation of intact mitochondria from yeast, is also applicable to the extraction of organelles from other organisms having cell walls. A procedure for the isolation of large quantities of pure mitochondrial DNA from these mitochondria is described. In Schizosaccharomyces pombe, further purification of the mitochondria by urograkt isopycnic centrifugation leads to 50% recovery of whole cell respiration activity in a vesicular fraction of respiratory chain enzymes, with NADH ox&se activity usually greater than 10 pmol of electrons/min/mg of protein. The method has the advantage of rapidity and low cost and it is extremely healthy for the operator.
The use of eucaryotic microorganisms such as the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae in organellar biochemistry is not as widespread as one would expect from their sophisticated organellar genetics. In some respects this is because it is easier to prepare large quantities of organelles from heterogenous animal tissues (e.g., beef heart) than from microorganisms having tough cell walls. For the preparation of mitochondria from yeast, the best quality, as measured by phosphorylation activity and integrity of mitDNA,’ has been obtained after enzymatic digestion (1) of the cell walls. This procedure is expensive, usually requires preincubation of cells under potentially deleterious conditions (0.5 M Tris pH 9.3, 0.1 M Nathioglycollate), and cannot be applied to some mutants and to cells grown under certain conditions. Additionally, the lytic enzyme preparations themselves may contain enzymatic activities inimical to the macromolecules purified (e.g., nucleases and peptidases in Helix pomatia digestive juice). Rapid mechanical disintegration methods (e.g., Braun shaker) i Abbreviations used: mitDNA, mitochondrial DNA; FCCP, trihuormethoxyphenylhydrazone; TCA, trichloroacetic acid. 110 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
carbonyl
cyanideg-
MITOCHONDRIA
FROM
111
MICROORGANISMS
applied to yeast lead to preparations of mitochondria with a low content of cytochrome c, no respiratory control, and rather variable, unstable respiratory activities (2). Since these methods of cell disruption are accompanied by the generation of heat, it is necessary to cool the cell suspension during the breakage procedure. In practice, the maintenance of precise temperature proves to be rather difficult. The NADH oxidase of mitochondrial particles prepared from cells broken with a mechanical shaker is never greater than 25% of the anticipated specific activity (calculated from the respiration rate of whole cells). Although about one-third of the estimated amount of cellular mitochondrial protein can be finally recovered, the bulk of enzyme activity is lost. Another problem arises in the characterization of mutants altered in mitochondrial membrane components. Using methods described for the disruption of respiratory-deficient mutants by the Braun homogenizer (3), we often do not find any enzymatic activity in the highly fragile cytochrome bc,-segment in some preparations of mutants of Sch. pombe, though it can be demonstrated spectroscopically to be present in whole cells and active in mitochondria prepared from enzyme digested cells. Therefore, we have developed a new, extremely simple cell breakage method, which leads to homogenous mitochondrial preparations and high respiratory activities which remain stable for some hours at room temperature, even after having been frozen in liquid nitrogen. This method is as convenient for the breakage of many different smallscale samples (e.g., screening of mutants) as it is for the breakage of kilogram quantities of cells for subsequent purification of macromolecules. MATERIALS Strains S. cerevisiae
Sch. pombe
Neurospora Chemicals
crassa
AND METHODS
SM 202 diploid p+ A 364 A haploid p+ 6 (I! haploid p+ 972 hANTR8 (prototrophic isolate from cross 50 ade 7 h- x 972 h+ 74-OR 23-1A
(4)
from L. Hartwell from D. Wilkie from U. Leupold
(5) from F. J. De Serres
and Media
Antimycin was purchased from Nutritional Biochemicals Corp., urograim was from Schering, NADH and horse heart cytochrome c from Boehringer, NaI was from Merck, and Sarcosyl NL-97 was from
112
LANG
ET AL.
Geigy Chemicals Ltd. Ubiquinone-3 was donated by Dr. Solms and FCCP was a gift from Dr. P. G. Heytler. NaI was recrystallized once from water and then stored as a saturated stock solution at room temperature in the presence of a few crystals of sodium sulphite. For preparation of intact mitochondria, cells were broken in a low salt buffer: 10 mM EDTA, 0.6 M sorbitol, pH 6.5. For preparation of mitDNA, cells were broken in a buffer containing 20 mM Tris, pH 8, 10 mM EDTA, and 0.35 M sucrose. Urogratm was diluted in an EDTA buffer (10 mM, pH 7) and was stored at -20°C in brown flasks to avoid decomposition. Growth Conditions Yeast cells were cultivated in a stirred (200 rpm) fermenter (Chemap) with O2 regulation at ca. 80% O2 saturation using air, at 32°C. For preparation of DNA, Neurosporu and yeasts were grown in l-liter bottles and aerated through an aquarium aeration stone. The complete glycerol medium contained 5% glycerol, 1% yeast extract (Difco), and 1% Bacto peptone (Difco) and was adjusted for the growth of Sch. pombe strains with KHzPOl to pH 5.8. For the preparation of DNA, cells were grown in 4% glycerol, 0.1% glucose, 1% yeast extract, and 2% Bacto peptone. Cells were harvested by centrifugation in the early stationary phase. The yield was lo-40 g wet weight per liter of medium. For preparation of DNA, they were washed once in water and once with an equal volume of buffer. Determination
of Enzymatic
Activities
and Cytochrome
Content
Oxygen uptake was measured with a Clark-type electrode. The optimal cell respiration was reached by addition of 1% glucose in the presence of 5-15 PM FCCP. Oxidative phosphorylation of the mitochondria was determined as described earlier (6) in a buffer containing 10 mM Tris, 1 mM EDTA, 0.6 M mannitol, 2.25 mM phosphate, 5 PM serum albumin, and 10 mM maleic acid, pH 6.5. The rate of mitochondrial respiration was measured in a buffer containing 0.1 mM cytochrome c at 25”C, as described by Burger et al. (7). The cytochrome b yield was estimated using an Aminco DW-2 double-beam spectrophotometer at room temperature with the wavelength pair 563-573 nm. Protein Determination In strains where the cytochrome b:protein ratio has been shown previously to be constant, the protein concentration was determined
MITOCHONDRIA
from
the content
FROM MICROORGANISMS
of cytochrome of urogralln
et al. (8) after removal
113
b or by the method of Lowry by washing and centrifugation.
Sodium Iodide Density Gradients
Mitochondria prepared as described in Results were resuspended in 50 mM EDTA, 50 mM Tris pH 8, and 1 M .NaCl and rapidly frozen at -20°C. The suspensions were then thawed rapidly at 60°C in the presence of 2% Sarkosyl NL-97 for 15 min. The lysate was cooled to room temperature and NaI was added to a final density of 1.45 g/ml; l-5 pg/ml of ethidium bromide was added as a visual marker. Centrifugation on a preparative scale (up to 25 mg of DNA) was in a Spinco Ti 60 rotor (8 x 20 ml); initial centrifugation was done at 144,000g overnight at 21°C. The gradients were usually checked under long wavelength (~300 nm) uv illumination to ensure that the DNA bands were forming in the center of the centrifuge tubes. Equilibrium for DNA was achieved after a further centrifugation (87,OOOg, 40 hr, at 21°C). As NaI is a chaotropic agent, it was not appropriate to reduce the viscosity of the solution and thereby the centrifugation time by running it at higher temperatures. Gradients were fractionated by piercing the tubes at the level of the mitDNA band. The mitDNA was repurified by a further NaI centrifugation and NaI was removed by extensive dialysis of DNA against 5 mM Tris, pH 8, and 1 mM M&l*. Sucrose Gradients and Labeling of mitDNA
DNA of S. cerevisiae (A 364 A) was labeled throughout growth with either rH]adenine (IO #Yml, 20 mCi/mmol) or [14CJadenine (0.1 @/ml, 54.2 mCi/mmol). Mitochondria were then either prepared by the manual breakage procedure described (3H-labeled culture), or from spheroplasts prepared by the method of KovaC (1) (14C-labeled culture). Mitochondria were purified by urogratin gradient centrifugation, resuspended in 50 mM EDTA, 5 mM Tris, pH 8, 1 M NaCl, mixed with a twofold volume of water to allow lysis of the mitochondria, and layered on a 36-ml linear sucrose gradient (5-25%), which had been previously overlaid with 1 ml of detergent mix (9). The gradients were left at room temperature for 15 min before centrifugation for 16 hr at 14,000 r-pm in a Spinco SW 27 rotor. Afterwards, the gradients were fractionated by piercing the tubes at the bottom with a needle and l-ml fractions were collected. These were made in 1 M NaOH and incubated at 60°C for 2 hr to hydrolyze the labeled RNA. The fractions were cooled and bovine serum albumin (100 pg) and TCA (10% final concentration) were added. Precipitation was allowed to occur at 0°C for 1 hr. Precipitates were collected by filtration on Schleicher & Schiill No. 9
114
LANG
ET AL.
glass fiber filters, and the filters were washed with 5% TCA, followed by ethanol, and then dried at 60°C for 40 min. Samples were counted in an Isocap liquid scintillation spectrometer using a [3H]/[14C] doublelabel program. Endonuclease Eco RI Cleavage of mitDNA Electrophoresis
and Agarose Gel
S. cerevisiae (6a) mitDNA was isolated by NaI equilibrium density centrifugation as described before. Restriction endonuclease (Eco RI) was isolated from Escherichia coli strain RY-13 (from R. Yoshimori) by an osmotic shock method (J. Davies, personal communication) and purified by published procedures (11,12). The cleavage mix contained 100 mM Tris, pH 7.5, 10 mM MgC12, 30 mM NaCl, 5 mM pmercaptoethanol, and 10% glycerol (final concentrations). The reaction was run at 37°C for 3 hr and the sample was layered on a 0.7% agarose cylindrical gel using the buffer system of Tanaka and Weissblum (10). Electrophoresis was for 15 hr at l-5 V/cm. The gels were soaked for 30 min in ethidium bromide (1 pg/ml) solution and photographed under long wavelength UV (Philips TL 20 W-08) illumination, using a redorange filter and Agfapan 400 ASA film. RESULTS
Disruption
of Cells with Glass Beads
The cells were washed once with an equal volume of ice-cold buffer and were then resuspended at 0.2-0.4 g wet weight of cells/ml of the same buffer. The cell suspension was poured into a glass bottle with a screw cap (l-liter brown glass bottles for acids, etc., supplied by Merck). Per milliliter of suspension, 3-4 g of ice-cold, O.U-mm-diameter, acid-washed glass beads were added. The bottle was then held vertically and vigorously shaken up and down in a vertical motion through about 50 cm, at a frequency of 2 Hz (i.e., 2 cycles/set). A healthy operator can continue this shaking for at least 2 min, during which time the temperature of the buffer rises not more than 4°C. The bottle may be cooled in ice between breakage cycles. The cell breakage normally obtained with our strains was 70-95% (2 min) for S. cerevisiae, 50-90% (4 min) for Sch. pombe, and 90-95% (2 min) for N. crassa, as determined by microscopy. Isolation
of the Crude Mitochondrial
Fraction
The broken cell suspension was decanted from glass beads, and the beads were washed repeatedly with small volumes of buffer. The fractions were combined and centrifuged for 20 min at 46,000g (15 min, 15,OOOg
MITOCHONDRIA
recydemce thmugh &eakage plVC&lre
FROM MICROORGANISMS
115
ccntrlfuge.LBXMxg.20
pellet
FIG. 1. Scheme of the described preparation organisms.
wpemotant
method of mitochondria
from micro-
for oxidative phosphorylation experiments). The fluffy mitochondrial layer overlaying the broken and whole cells was resuspended in a small volume. In cases where the mitochondria were more fragile (e.g., petite or mit- mutants) only 40-50% of the cells were broken and the cell pellet was recycled for a second disintegration step. Purification
of the Mitochondria
by Urograjin Gradient Centrifugation
Further purification of this crude fraction was necessary to (i) separate the mitochondria from other membrane materials and lipids, (ii) increase the stability of the respiratory activity of the mitochondria by removing lytic enzymes, (iii) remove nuclear and cytoplasmic DNA (for isolation of mitDNA), and (iv) to separate the distinct mitochondrial populations found in respiratory deficient mutants in Sch. pombe (13). The mitochondria were either layered on top of a linear urograhn gradient (U-30%) or under a gradient (22-40%) and were centrifuged for 1 hr at
116
LANG ET AL. TABLE
RESPIRATORY
Strain
ACTIVITIES
OF PURIFIED
NADH
1
MITOCHONDRIA
NADH + ubiquinone-3 (26/A
Sch. pombe 972 h-
5.0-9.5
7.0- 13.0
Sch. pombe Ant* 8
8.4-11.0
10.0-15.5
S. cerevisiae SM 202
3.4-6.4
7.1-8.9
N. crassa
4.3-6.2
No stimulation of activity by ubiquinone-3
WITH
VARIOUS
SUBSTRATES’
NADH + succinate About 5% inhibition by succinate 5- 15% inhibition succinate
by
Identical to NADH only 5.5-7.5
a The electron acceptor was oxygen. Specific activities were determined at 25°C and are given as micromol of electrons per minute per milligram of mitochondrial protein.
27,000 rpm in a Spinco SW 27 swing-out rotor at 5°C. In the first case, the mitochondria were resuspended in 10 mM EDTA, pH 7, in the second case in 60% urogratin. In both types of gradients, the mitochondrial fraction moved to the middle of the centrifuge tube and could be seen as a clear red band. The band was isolated and washed once with the preparation buffer to remove the urografin. Flotation gradients of mitochondria proved to be more effective in removing chromosomal DNA contamination. For large scale preparation of mitochondria it was practical to centrifuge the crude mitochondrial suspension through a cushion of 15% urogralm. A scheme of the total preparation procedure is given in Fig. 1. . Enzymatic Properties of the Mitochondria (a) Yield of mitochondria at different stages of purification. The yield was determined by spectroscopy, measuring the content of cytochrome a or b (in a buffer containing 1% Lubrol WX, to clarify turbid suspensions). The values obtained from almost totally disintegrated cell homogenates was taken as 100%. After the first centrifugation of a suspension, with 50430% of the cells broken, 30-50% of the mitochondria can be recovered. (In Sch. pombe, the recovery of cytochromes was in agreement with the recovery of NADH oxidase activity in the cell homogenate and the crude mitochondrial fraction.) By urogralm gradient centrifugation, less than one-tenth of the initial mitochondrial membranes is lost in the wild-type strains of Sch.
MITOCHONDRLA
FROM TABLE
ENZYMATIC
PROPERTIES
Organism
Method of preparation
S. cerevisiae
Handshake+
OF THE MITOCHONDRIA
Growth Log phase
Early stationary
A la KovaC (I)
Sch. pombe
HandshakeV’
state
Early
stationary
Early stationary
phase
phase
phase
117
MICROORGANISMS 2 PREPARED
Substrate0
BY THE DESCRIBED
METHOD
0,uptake’
RC’
P/O’
NADH
0.45-0.61
i.6-2.4
1.1-1.3
Malate/ pyrwate
0.02-0.04
1.6-2.6
1.7-1.9
a-Ketw glutarate
0.02-0.03
3.2-4.5
1.9-3.4
NADH
0.28-0.38
1.2-1.9
0.4-I .2
Malate/ pyruvate
0.02-0.04
1.4-2.1
1.1-1.7
ol-Ketoglutarate
0.02-0.03
2.4-4.2
1.4-2.2
NADH
0.16-0.28
1.8-2.9
1.2-1.9
Malate/ pyruvate
0.04-0.07
1.6-3.0
1.4-2.1
a-Ketoglutarate
0.04-0.06
2.2-5.2
1.7-3.4
NADH
0.34-0.44
1.1-1.3
0.4-0.6
Malatei pyluvate
0.02-0.03
1.1-1.5
0.6-0.8
a-Ketoglutarate
0.01-0.03
1.2-1.7
0.6-0.9
e The concentration of NADH was I mu, malate 5 mu. pyrwate 5 IIIM, and a-ketoglutarate 12 mu. In the cases malateipyruvate. and a-keto-glutante, the mitochondria were preincubated with the substrates anaerobically 0°C for 10 min. b O2 uptake is expressed as micromol of 0, per minute and milligram of protein. c Respiratory control ratios (RC) and ADP:O values (P/O) are calculated from state 3-state 4 transition as defined others ( 15). d Handshaken mitochondria were isolated after 6O-70% breakage in S. cerevisiae (30-90 se4 and 30% breakage Sch. pombe (I20 set).
of at
by in
pombe, S. cerevisiae, and N. crassa. In some respiratory-deficient mutants of Sch. pombe, however, up to 80% of the mitochondria, which were enzymatically less active, were found aggregated at the bottom of the urogralin gradient. In S. pombe 972h- two bands were found in urogratin gradients. The upper band (about 70-90% of the total cytochromes) showed high respiratory activities and was orange and clear. The lower one had about 70% of the activity, was greyish and turbid, and could be increased in yield by prior starvation of the cells for 12 or more hr. (6) Respiration. Assuming that 100 g wet weight of yeast cells maximally yield 0.5- 1 g of purified mitochondria, which is true for most
118
LANG ET AL.
a
b C
3P Wver FIG.
2. Densitogram
1.1
km1 Lower
of NaI gradients; separation of mitDNA
and nuclear DNA of
S. cerevisiue.The gradients were loaded with: (a) 4 x 108 spheroplasts (1); (b) Xl mg of mitochondrial protein prepared by the described hand-shaken method, but not further purified by urografin gradient centrifugation (upper layer, see Fig. 1); (c) 20 mg of mitochondrial protein prepared like (b) but purified by centrifugation through a urogratin gradient. To spheroplasts or mitochondria, 1.45 g/ml of NaI (final density) and ethidium bromide were added. After equilibrium for DNA was reached, gradients were photographed like the gels (see Methods) and the photographs were scanned in a Joyce-Loebel Chromoscan in reflectance mode, using a filter with a 1.5 o.d. for (a) and (b) and a filter with a 2 o.d. for(c).
of our yeast strains, one should expect for uncoupled cell respiration values between 7 and 15 pmol of electrons/min/mg of protein [cf. (7)]. Those values are easily obtained in Sch. pombe but not in S. cerevisiae and N. crassa. The inactivation occurs during the preparation and is primarily caused by the loss of loosely bound cytochrome c. We failed to restore complete activity by addition of 0.1 mM horseheart cytochrome c. The respiratory activities of purified mitochondria prepared by this method from different sources and their reaction on ubiquinone and on simultaneous addition of two substrates (14) are summarized in Table 1. (c) Phosphotylation. It was possible to find respiratory control by ADP and phosphorylation in S. cerevisiae as well as in Sch. pombe. We
MITOCHONDRIA
FROM MICROORGANISMS
119
FIG. 3. Agarose gel electrophoresis of mitDNA, cleaved with Eco RI. mitDNA (12.5 pg) from S. cerevisiae was digested with the restriction endonuclease Eco RI, layered on a 0.7% agarose gel, and 1.5 V/cm was applied for 15 hr. The gels, soaked in ethidium bromide, were photographed under uv illumination as described in Methods.
do not claim this method to be the most useful for studies of energy conservation, but we use it here as a criterion for the intactness of the mitochondria after mechanical disintegration. The results are summarized in Table 2 and show that the P/O ratio and the RC values are higher in the early stationary phase than in the log phase, although the time of shaking had to be extended to break the stationary phase cells. However, all values are considerably lower than one might obtain from log phase cells after enzymatic digestion of the cell walls. Phosphorylation was similar in different Saccharomyces strains. Isolation of DNA
The method has been used successfully for the preparation of large quantities (up to 25 mg) of mitDNA, for restriction endonuclease analysis and DNA-RNA hybridization, and, on a smaller scale, for the preparation of high molecular weight radioactively labeled mitDNA.
120
LANG ET AL.
1
5
10
20 15 25 Fraction Number
30
35
LO
‘0
FIG. 4. Sedimentation of mitDNA in a sucrose gradient. DNA of S. cerevisiae was labeled either with [Tladenine, and mitochondria were prepared from spheroplasts by the Kovac method (1) ( - - -); or with PH]adenine, and mitochondria were prepared by the described hand-shaken method (-). Both types of mitochondria were combined, purified by urogratm gradient centrifugation, lyzed, and layered on a linear sucrose gradient (S-25%) (see Methods).
The separation achieved in NaI gradients for mitDNA and nuclear DNA is shown in Fig. 2. mitDNA prepared by this method and digested with Eco RI restriction endonuclease gave the pattern shown in Fig. 3. The total molecular weight of these fragments in comparison with A phage Eco RI fragments is 52 x IO6 daltons. Note the low fluorescent background, indicating the lack of small pieces of DNA in these preparations. It should be emphasized that this preparation was not fractionated by molecular weight before digestion with Eco RI [cf (16)]. On a smaller scale, a comparison was made between the molecular weight of [3H]adenine-labeled mitDNA from mitochondria prepared by this mechanical procedure and [14C]adenine-labeled mitDNA from mitochondria prepared from spheroplasts by the KovaC procedure (1). A sucrose density gradient of both DNA types (Fig. 4) showed a prominent band of mitDNA at 50 x lo6 daltons (established by comparison with XcIe5, S, phage DNA). There does not appear to be a significantly different distribution of mitDNA prepared by the two methods. DISCUSSION
A new, very simple method for the isolation of organelles from eucaryotic microorganisms is presented. It is surely applicable to other organisms having tough cell walls and possibly to plant tissues as well. No specialized equipment is needed and it is suitable for any scale of breakage from subgram to kilogram quantities of cells. It has also been used for the screening of mutants for mitochondrial enzyme activites (in one instance, up to 100 samples in one day).
MITOCHONDRIA
FROM MICROORGANISMS
121
ACKNOWLEDGMENTS We thank Miss G. D&-r for her skillful assistance. Our work was supported by the Deutsche Forschungsgemeinschaft.
REFERENCES 1. Kovac, L., Groot, G. S. P., and Racker, E. (1972) Biochim. Biophys. Acfa 252, 55-65. 2. Tzagoloff, A. (1971) J. Biol. Chem. 246, 3050-3056. 3. Needleman, R. B., and Tzagoloff, A. (1975) Anal. Biochem. 64, 545-549. 4. Schweyen, R., et al., in preparation. 5. Lang, B., Burger, G., Wolf, K., Bandlow, W., and Kaudewitz, F. (1975) Mol. Gen. Genet. 137, 353-363. 6. Estabrook, R. W. (1%7) in Methods in Enzymology (E&brook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 41-47, Academic Press, New York. 7. Burger, G., Lang, B., Bandlow, W., and Kaudewitz, F. (1975) Biochim. Biophys. Acta 396, 187-201. 8. Lowry, 0. H., Rosebrough, N. .I., Farr, A. L., and Randall, R. J. (1951)3. Biol. Chem. 209, 23-29. 9. Blamire, J. A., Cryer, D. R., Finkelstein, D. B., and Marmur, J. (1972) J. Mol. Biol. 67, 11-24. 10. Tanaka, T., and Weissblum, B. (1975) J. Bacterial. 121, 354-362. 11. de Kloet, S. R., and Andrean, B. A. G. (1971) Biochim. Biophys. Acta 247, 516-527.
12. Scragg, A. H., Eggit, M. J., and Thomas, D. Y. (1976) in Euratom Symposium on Genetics, Biogenesis and Bioenergetic of Mitochondria, Munchen 1975 (Bandlow, W., Schweyen, R., Thomas, D. Y., Wolf, K., and Kaudewitz, F., eds.), de Gruyter, Berlin (in press). 13. Lang, B., Burger, G., Wolf, K., Bandlow, W., and Kaudewitz, F. (1976) in Euratom Symposium on Genetics, Biogenesis and Bioenergetics of Mitochondria, Miinchen 1975 (Bandlow, W., Schweyen, R., Thomas, D. Y., Wolf, K., and Kaudewitz, F., eds.), de Gruyter, Berlin (in press). 14. Lang, B., and Burger, G. (1975) in 10th FEBS Congress, Paris, 1975, Abstract No. 1215. 15. Chance, B., and Williams, G. R. (1955) Adv. Enzyrnol. 17, 65-134. 16. Morimoto, R., Lewin, A., Hsu, H.-J., Rabinowitz, M., and Fukuhara, H. (1975) Proc. Nat. Acad. Sci. USA 72,3868-3872.
BIOCHEMISTRY
A Simple
77, 1lo- 121 (19771
Method for the Large-Scale Preparation Mitochondria from Microorganisms
of
BERND LANG, GERTRAUD BURGER, ILIAS DOXIADIS, DAVID Y. THOMAS, WOLFHARD BANDLOW, AND FRITZ KAUDEWITZ Genetisches
Institut
der Universitiit D-8000 Miinchen
Miinchen, Maria-Ward-Strasse 19, Germany
la,
Received July 15, 1976; accepted August 23, 1976 A simple mechanical procedure that has been developed for the largescale preparation of intact mitochondria from yeast, is also applicable to the extraction of organelles from other organisms having cell walls. A procedure for the isolation of large quantities of pure mitochondrial DNA from these mitochondria is described. In Schizosaccharomyces pombe, further purification of the mitochondria by urograkt isopycnic centrifugation leads to 50% recovery of whole cell respiration activity in a vesicular fraction of respiratory chain enzymes, with NADH ox&se activity usually greater than 10 pmol of electrons/min/mg of protein. The method has the advantage of rapidity and low cost and it is extremely healthy for the operator.
The use of eucaryotic microorganisms such as the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae in organellar biochemistry is not as widespread as one would expect from their sophisticated organellar genetics. In some respects this is because it is easier to prepare large quantities of organelles from heterogenous animal tissues (e.g., beef heart) than from microorganisms having tough cell walls. For the preparation of mitochondria from yeast, the best quality, as measured by phosphorylation activity and integrity of mitDNA,’ has been obtained after enzymatic digestion (1) of the cell walls. This procedure is expensive, usually requires preincubation of cells under potentially deleterious conditions (0.5 M Tris pH 9.3, 0.1 M Nathioglycollate), and cannot be applied to some mutants and to cells grown under certain conditions. Additionally, the lytic enzyme preparations themselves may contain enzymatic activities inimical to the macromolecules purified (e.g., nucleases and peptidases in Helix pomatia digestive juice). Rapid mechanical disintegration methods (e.g., Braun shaker) i Abbreviations used: mitDNA, mitochondrial DNA; FCCP, trihuormethoxyphenylhydrazone; TCA, trichloroacetic acid. 110 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
carbonyl
cyanideg-
MITOCHONDRIA
FROM
111
MICROORGANISMS
applied to yeast lead to preparations of mitochondria with a low content of cytochrome c, no respiratory control, and rather variable, unstable respiratory activities (2). Since these methods of cell disruption are accompanied by the generation of heat, it is necessary to cool the cell suspension during the breakage procedure. In practice, the maintenance of precise temperature proves to be rather difficult. The NADH oxidase of mitochondrial particles prepared from cells broken with a mechanical shaker is never greater than 25% of the anticipated specific activity (calculated from the respiration rate of whole cells). Although about one-third of the estimated amount of cellular mitochondrial protein can be finally recovered, the bulk of enzyme activity is lost. Another problem arises in the characterization of mutants altered in mitochondrial membrane components. Using methods described for the disruption of respiratory-deficient mutants by the Braun homogenizer (3), we often do not find any enzymatic activity in the highly fragile cytochrome bc,-segment in some preparations of mutants of Sch. pombe, though it can be demonstrated spectroscopically to be present in whole cells and active in mitochondria prepared from enzyme digested cells. Therefore, we have developed a new, extremely simple cell breakage method, which leads to homogenous mitochondrial preparations and high respiratory activities which remain stable for some hours at room temperature, even after having been frozen in liquid nitrogen. This method is as convenient for the breakage of many different smallscale samples (e.g., screening of mutants) as it is for the breakage of kilogram quantities of cells for subsequent purification of macromolecules. MATERIALS Strains S. cerevisiae
Sch. pombe
Neurospora Chemicals
crassa
AND METHODS
SM 202 diploid p+ A 364 A haploid p+ 6 (I! haploid p+ 972 hANTR8 (prototrophic isolate from cross 50 ade 7 h- x 972 h+ 74-OR 23-1A
(4)
from L. Hartwell from D. Wilkie from U. Leupold
(5) from F. J. De Serres
and Media
Antimycin was purchased from Nutritional Biochemicals Corp., urograim was from Schering, NADH and horse heart cytochrome c from Boehringer, NaI was from Merck, and Sarcosyl NL-97 was from
112
LANG
ET AL.
Geigy Chemicals Ltd. Ubiquinone-3 was donated by Dr. Solms and FCCP was a gift from Dr. P. G. Heytler. NaI was recrystallized once from water and then stored as a saturated stock solution at room temperature in the presence of a few crystals of sodium sulphite. For preparation of intact mitochondria, cells were broken in a low salt buffer: 10 mM EDTA, 0.6 M sorbitol, pH 6.5. For preparation of mitDNA, cells were broken in a buffer containing 20 mM Tris, pH 8, 10 mM EDTA, and 0.35 M sucrose. Urogratm was diluted in an EDTA buffer (10 mM, pH 7) and was stored at -20°C in brown flasks to avoid decomposition. Growth Conditions Yeast cells were cultivated in a stirred (200 rpm) fermenter (Chemap) with O2 regulation at ca. 80% O2 saturation using air, at 32°C. For preparation of DNA, Neurosporu and yeasts were grown in l-liter bottles and aerated through an aquarium aeration stone. The complete glycerol medium contained 5% glycerol, 1% yeast extract (Difco), and 1% Bacto peptone (Difco) and was adjusted for the growth of Sch. pombe strains with KHzPOl to pH 5.8. For the preparation of DNA, cells were grown in 4% glycerol, 0.1% glucose, 1% yeast extract, and 2% Bacto peptone. Cells were harvested by centrifugation in the early stationary phase. The yield was lo-40 g wet weight per liter of medium. For preparation of DNA, they were washed once in water and once with an equal volume of buffer. Determination
of Enzymatic
Activities
and Cytochrome
Content
Oxygen uptake was measured with a Clark-type electrode. The optimal cell respiration was reached by addition of 1% glucose in the presence of 5-15 PM FCCP. Oxidative phosphorylation of the mitochondria was determined as described earlier (6) in a buffer containing 10 mM Tris, 1 mM EDTA, 0.6 M mannitol, 2.25 mM phosphate, 5 PM serum albumin, and 10 mM maleic acid, pH 6.5. The rate of mitochondrial respiration was measured in a buffer containing 0.1 mM cytochrome c at 25”C, as described by Burger et al. (7). The cytochrome b yield was estimated using an Aminco DW-2 double-beam spectrophotometer at room temperature with the wavelength pair 563-573 nm. Protein Determination In strains where the cytochrome b:protein ratio has been shown previously to be constant, the protein concentration was determined
MITOCHONDRIA
from
the content
FROM MICROORGANISMS
of cytochrome of urogralln
et al. (8) after removal
113
b or by the method of Lowry by washing and centrifugation.
Sodium Iodide Density Gradients
Mitochondria prepared as described in Results were resuspended in 50 mM EDTA, 50 mM Tris pH 8, and 1 M .NaCl and rapidly frozen at -20°C. The suspensions were then thawed rapidly at 60°C in the presence of 2% Sarkosyl NL-97 for 15 min. The lysate was cooled to room temperature and NaI was added to a final density of 1.45 g/ml; l-5 pg/ml of ethidium bromide was added as a visual marker. Centrifugation on a preparative scale (up to 25 mg of DNA) was in a Spinco Ti 60 rotor (8 x 20 ml); initial centrifugation was done at 144,000g overnight at 21°C. The gradients were usually checked under long wavelength (~300 nm) uv illumination to ensure that the DNA bands were forming in the center of the centrifuge tubes. Equilibrium for DNA was achieved after a further centrifugation (87,OOOg, 40 hr, at 21°C). As NaI is a chaotropic agent, it was not appropriate to reduce the viscosity of the solution and thereby the centrifugation time by running it at higher temperatures. Gradients were fractionated by piercing the tubes at the level of the mitDNA band. The mitDNA was repurified by a further NaI centrifugation and NaI was removed by extensive dialysis of DNA against 5 mM Tris, pH 8, and 1 mM M&l*. Sucrose Gradients and Labeling of mitDNA
DNA of S. cerevisiae (A 364 A) was labeled throughout growth with either rH]adenine (IO #Yml, 20 mCi/mmol) or [14CJadenine (0.1 @/ml, 54.2 mCi/mmol). Mitochondria were then either prepared by the manual breakage procedure described (3H-labeled culture), or from spheroplasts prepared by the method of KovaC (1) (14C-labeled culture). Mitochondria were purified by urogratin gradient centrifugation, resuspended in 50 mM EDTA, 5 mM Tris, pH 8, 1 M NaCl, mixed with a twofold volume of water to allow lysis of the mitochondria, and layered on a 36-ml linear sucrose gradient (5-25%), which had been previously overlaid with 1 ml of detergent mix (9). The gradients were left at room temperature for 15 min before centrifugation for 16 hr at 14,000 r-pm in a Spinco SW 27 rotor. Afterwards, the gradients were fractionated by piercing the tubes at the bottom with a needle and l-ml fractions were collected. These were made in 1 M NaOH and incubated at 60°C for 2 hr to hydrolyze the labeled RNA. The fractions were cooled and bovine serum albumin (100 pg) and TCA (10% final concentration) were added. Precipitation was allowed to occur at 0°C for 1 hr. Precipitates were collected by filtration on Schleicher & Schiill No. 9
114
LANG
ET AL.
glass fiber filters, and the filters were washed with 5% TCA, followed by ethanol, and then dried at 60°C for 40 min. Samples were counted in an Isocap liquid scintillation spectrometer using a [3H]/[14C] doublelabel program. Endonuclease Eco RI Cleavage of mitDNA Electrophoresis
and Agarose Gel
S. cerevisiae (6a) mitDNA was isolated by NaI equilibrium density centrifugation as described before. Restriction endonuclease (Eco RI) was isolated from Escherichia coli strain RY-13 (from R. Yoshimori) by an osmotic shock method (J. Davies, personal communication) and purified by published procedures (11,12). The cleavage mix contained 100 mM Tris, pH 7.5, 10 mM MgC12, 30 mM NaCl, 5 mM pmercaptoethanol, and 10% glycerol (final concentrations). The reaction was run at 37°C for 3 hr and the sample was layered on a 0.7% agarose cylindrical gel using the buffer system of Tanaka and Weissblum (10). Electrophoresis was for 15 hr at l-5 V/cm. The gels were soaked for 30 min in ethidium bromide (1 pg/ml) solution and photographed under long wavelength UV (Philips TL 20 W-08) illumination, using a redorange filter and Agfapan 400 ASA film. RESULTS
Disruption
of Cells with Glass Beads
The cells were washed once with an equal volume of ice-cold buffer and were then resuspended at 0.2-0.4 g wet weight of cells/ml of the same buffer. The cell suspension was poured into a glass bottle with a screw cap (l-liter brown glass bottles for acids, etc., supplied by Merck). Per milliliter of suspension, 3-4 g of ice-cold, O.U-mm-diameter, acid-washed glass beads were added. The bottle was then held vertically and vigorously shaken up and down in a vertical motion through about 50 cm, at a frequency of 2 Hz (i.e., 2 cycles/set). A healthy operator can continue this shaking for at least 2 min, during which time the temperature of the buffer rises not more than 4°C. The bottle may be cooled in ice between breakage cycles. The cell breakage normally obtained with our strains was 70-95% (2 min) for S. cerevisiae, 50-90% (4 min) for Sch. pombe, and 90-95% (2 min) for N. crassa, as determined by microscopy. Isolation
of the Crude Mitochondrial
Fraction
The broken cell suspension was decanted from glass beads, and the beads were washed repeatedly with small volumes of buffer. The fractions were combined and centrifuged for 20 min at 46,000g (15 min, 15,OOOg
MITOCHONDRIA
recydemce thmugh &eakage plVC&lre
FROM MICROORGANISMS
115
ccntrlfuge.LBXMxg.20
pellet
FIG. 1. Scheme of the described preparation organisms.
wpemotant
method of mitochondria
from micro-
for oxidative phosphorylation experiments). The fluffy mitochondrial layer overlaying the broken and whole cells was resuspended in a small volume. In cases where the mitochondria were more fragile (e.g., petite or mit- mutants) only 40-50% of the cells were broken and the cell pellet was recycled for a second disintegration step. Purification
of the Mitochondria
by Urograjin Gradient Centrifugation
Further purification of this crude fraction was necessary to (i) separate the mitochondria from other membrane materials and lipids, (ii) increase the stability of the respiratory activity of the mitochondria by removing lytic enzymes, (iii) remove nuclear and cytoplasmic DNA (for isolation of mitDNA), and (iv) to separate the distinct mitochondrial populations found in respiratory deficient mutants in Sch. pombe (13). The mitochondria were either layered on top of a linear urograhn gradient (U-30%) or under a gradient (22-40%) and were centrifuged for 1 hr at
116
LANG ET AL. TABLE
RESPIRATORY
Strain
ACTIVITIES
OF PURIFIED
NADH
1
MITOCHONDRIA
NADH + ubiquinone-3 (26/A
Sch. pombe 972 h-
5.0-9.5
7.0- 13.0
Sch. pombe Ant* 8
8.4-11.0
10.0-15.5
S. cerevisiae SM 202
3.4-6.4
7.1-8.9
N. crassa
4.3-6.2
No stimulation of activity by ubiquinone-3
WITH
VARIOUS
SUBSTRATES’
NADH + succinate About 5% inhibition by succinate 5- 15% inhibition succinate
by
Identical to NADH only 5.5-7.5
a The electron acceptor was oxygen. Specific activities were determined at 25°C and are given as micromol of electrons per minute per milligram of mitochondrial protein.
27,000 rpm in a Spinco SW 27 swing-out rotor at 5°C. In the first case, the mitochondria were resuspended in 10 mM EDTA, pH 7, in the second case in 60% urogratin. In both types of gradients, the mitochondrial fraction moved to the middle of the centrifuge tube and could be seen as a clear red band. The band was isolated and washed once with the preparation buffer to remove the urografin. Flotation gradients of mitochondria proved to be more effective in removing chromosomal DNA contamination. For large scale preparation of mitochondria it was practical to centrifuge the crude mitochondrial suspension through a cushion of 15% urogralm. A scheme of the total preparation procedure is given in Fig. 1. . Enzymatic Properties of the Mitochondria (a) Yield of mitochondria at different stages of purification. The yield was determined by spectroscopy, measuring the content of cytochrome a or b (in a buffer containing 1% Lubrol WX, to clarify turbid suspensions). The values obtained from almost totally disintegrated cell homogenates was taken as 100%. After the first centrifugation of a suspension, with 50430% of the cells broken, 30-50% of the mitochondria can be recovered. (In Sch. pombe, the recovery of cytochromes was in agreement with the recovery of NADH oxidase activity in the cell homogenate and the crude mitochondrial fraction.) By urogralm gradient centrifugation, less than one-tenth of the initial mitochondrial membranes is lost in the wild-type strains of Sch.
MITOCHONDRLA
FROM TABLE
ENZYMATIC
PROPERTIES
Organism
Method of preparation
S. cerevisiae
Handshake+
OF THE MITOCHONDRIA
Growth Log phase
Early stationary
A la KovaC (I)
Sch. pombe
HandshakeV’
state
Early
stationary
Early stationary
phase
phase
phase
117
MICROORGANISMS 2 PREPARED
Substrate0
BY THE DESCRIBED
METHOD
0,uptake’
RC’
P/O’
NADH
0.45-0.61
i.6-2.4
1.1-1.3
Malate/ pyrwate
0.02-0.04
1.6-2.6
1.7-1.9
a-Ketw glutarate
0.02-0.03
3.2-4.5
1.9-3.4
NADH
0.28-0.38
1.2-1.9
0.4-I .2
Malate/ pyruvate
0.02-0.04
1.4-2.1
1.1-1.7
ol-Ketoglutarate
0.02-0.03
2.4-4.2
1.4-2.2
NADH
0.16-0.28
1.8-2.9
1.2-1.9
Malate/ pyruvate
0.04-0.07
1.6-3.0
1.4-2.1
a-Ketoglutarate
0.04-0.06
2.2-5.2
1.7-3.4
NADH
0.34-0.44
1.1-1.3
0.4-0.6
Malatei pyluvate
0.02-0.03
1.1-1.5
0.6-0.8
a-Ketoglutarate
0.01-0.03
1.2-1.7
0.6-0.9
e The concentration of NADH was I mu, malate 5 mu. pyrwate 5 IIIM, and a-ketoglutarate 12 mu. In the cases malateipyruvate. and a-keto-glutante, the mitochondria were preincubated with the substrates anaerobically 0°C for 10 min. b O2 uptake is expressed as micromol of 0, per minute and milligram of protein. c Respiratory control ratios (RC) and ADP:O values (P/O) are calculated from state 3-state 4 transition as defined others ( 15). d Handshaken mitochondria were isolated after 6O-70% breakage in S. cerevisiae (30-90 se4 and 30% breakage Sch. pombe (I20 set).
of at
by in
pombe, S. cerevisiae, and N. crassa. In some respiratory-deficient mutants of Sch. pombe, however, up to 80% of the mitochondria, which were enzymatically less active, were found aggregated at the bottom of the urogralin gradient. In S. pombe 972h- two bands were found in urogratin gradients. The upper band (about 70-90% of the total cytochromes) showed high respiratory activities and was orange and clear. The lower one had about 70% of the activity, was greyish and turbid, and could be increased in yield by prior starvation of the cells for 12 or more hr. (6) Respiration. Assuming that 100 g wet weight of yeast cells maximally yield 0.5- 1 g of purified mitochondria, which is true for most
118
LANG ET AL.
a
b C
3P Wver FIG.
2. Densitogram
1.1
km1 Lower
of NaI gradients; separation of mitDNA
and nuclear DNA of
S. cerevisiue.The gradients were loaded with: (a) 4 x 108 spheroplasts (1); (b) Xl mg of mitochondrial protein prepared by the described hand-shaken method, but not further purified by urografin gradient centrifugation (upper layer, see Fig. 1); (c) 20 mg of mitochondrial protein prepared like (b) but purified by centrifugation through a urogratin gradient. To spheroplasts or mitochondria, 1.45 g/ml of NaI (final density) and ethidium bromide were added. After equilibrium for DNA was reached, gradients were photographed like the gels (see Methods) and the photographs were scanned in a Joyce-Loebel Chromoscan in reflectance mode, using a filter with a 1.5 o.d. for (a) and (b) and a filter with a 2 o.d. for(c).
of our yeast strains, one should expect for uncoupled cell respiration values between 7 and 15 pmol of electrons/min/mg of protein [cf. (7)]. Those values are easily obtained in Sch. pombe but not in S. cerevisiae and N. crassa. The inactivation occurs during the preparation and is primarily caused by the loss of loosely bound cytochrome c. We failed to restore complete activity by addition of 0.1 mM horseheart cytochrome c. The respiratory activities of purified mitochondria prepared by this method from different sources and their reaction on ubiquinone and on simultaneous addition of two substrates (14) are summarized in Table 1. (c) Phosphotylation. It was possible to find respiratory control by ADP and phosphorylation in S. cerevisiae as well as in Sch. pombe. We
MITOCHONDRIA
FROM MICROORGANISMS
119
FIG. 3. Agarose gel electrophoresis of mitDNA, cleaved with Eco RI. mitDNA (12.5 pg) from S. cerevisiae was digested with the restriction endonuclease Eco RI, layered on a 0.7% agarose gel, and 1.5 V/cm was applied for 15 hr. The gels, soaked in ethidium bromide, were photographed under uv illumination as described in Methods.
do not claim this method to be the most useful for studies of energy conservation, but we use it here as a criterion for the intactness of the mitochondria after mechanical disintegration. The results are summarized in Table 2 and show that the P/O ratio and the RC values are higher in the early stationary phase than in the log phase, although the time of shaking had to be extended to break the stationary phase cells. However, all values are considerably lower than one might obtain from log phase cells after enzymatic digestion of the cell walls. Phosphorylation was similar in different Saccharomyces strains. Isolation of DNA
The method has been used successfully for the preparation of large quantities (up to 25 mg) of mitDNA, for restriction endonuclease analysis and DNA-RNA hybridization, and, on a smaller scale, for the preparation of high molecular weight radioactively labeled mitDNA.
120
LANG ET AL.
1
5
10
20 15 25 Fraction Number
30
35
LO
‘0
FIG. 4. Sedimentation of mitDNA in a sucrose gradient. DNA of S. cerevisiae was labeled either with [Tladenine, and mitochondria were prepared from spheroplasts by the Kovac method (1) ( - - -); or with PH]adenine, and mitochondria were prepared by the described hand-shaken method (-). Both types of mitochondria were combined, purified by urogratm gradient centrifugation, lyzed, and layered on a linear sucrose gradient (S-25%) (see Methods).
The separation achieved in NaI gradients for mitDNA and nuclear DNA is shown in Fig. 2. mitDNA prepared by this method and digested with Eco RI restriction endonuclease gave the pattern shown in Fig. 3. The total molecular weight of these fragments in comparison with A phage Eco RI fragments is 52 x IO6 daltons. Note the low fluorescent background, indicating the lack of small pieces of DNA in these preparations. It should be emphasized that this preparation was not fractionated by molecular weight before digestion with Eco RI [cf (16)]. On a smaller scale, a comparison was made between the molecular weight of [3H]adenine-labeled mitDNA from mitochondria prepared by this mechanical procedure and [14C]adenine-labeled mitDNA from mitochondria prepared from spheroplasts by the KovaC procedure (1). A sucrose density gradient of both DNA types (Fig. 4) showed a prominent band of mitDNA at 50 x lo6 daltons (established by comparison with XcIe5, S, phage DNA). There does not appear to be a significantly different distribution of mitDNA prepared by the two methods. DISCUSSION
A new, very simple method for the isolation of organelles from eucaryotic microorganisms is presented. It is surely applicable to other organisms having tough cell walls and possibly to plant tissues as well. No specialized equipment is needed and it is suitable for any scale of breakage from subgram to kilogram quantities of cells. It has also been used for the screening of mutants for mitochondrial enzyme activites (in one instance, up to 100 samples in one day).
MITOCHONDRIA
FROM MICROORGANISMS
121
ACKNOWLEDGMENTS We thank Miss G. D&-r for her skillful assistance. Our work was supported by the Deutsche Forschungsgemeinschaft.
REFERENCES 1. Kovac, L., Groot, G. S. P., and Racker, E. (1972) Biochim. Biophys. Acfa 252, 55-65. 2. Tzagoloff, A. (1971) J. Biol. Chem. 246, 3050-3056. 3. Needleman, R. B., and Tzagoloff, A. (1975) Anal. Biochem. 64, 545-549. 4. Schweyen, R., et al., in preparation. 5. Lang, B., Burger, G., Wolf, K., Bandlow, W., and Kaudewitz, F. (1975) Mol. Gen. Genet. 137, 353-363. 6. Estabrook, R. W. (1%7) in Methods in Enzymology (E&brook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 41-47, Academic Press, New York. 7. Burger, G., Lang, B., Bandlow, W., and Kaudewitz, F. (1975) Biochim. Biophys. Acta 396, 187-201. 8. Lowry, 0. H., Rosebrough, N. .I., Farr, A. L., and Randall, R. J. (1951)3. Biol. Chem. 209, 23-29. 9. Blamire, J. A., Cryer, D. R., Finkelstein, D. B., and Marmur, J. (1972) J. Mol. Biol. 67, 11-24. 10. Tanaka, T., and Weissblum, B. (1975) J. Bacterial. 121, 354-362. 11. de Kloet, S. R., and Andrean, B. A. G. (1971) Biochim. Biophys. Acta 247, 516-527.
12. Scragg, A. H., Eggit, M. J., and Thomas, D. Y. (1976) in Euratom Symposium on Genetics, Biogenesis and Bioenergetic of Mitochondria, Munchen 1975 (Bandlow, W., Schweyen, R., Thomas, D. Y., Wolf, K., and Kaudewitz, F., eds.), de Gruyter, Berlin (in press). 13. Lang, B., Burger, G., Wolf, K., Bandlow, W., and Kaudewitz, F. (1976) in Euratom Symposium on Genetics, Biogenesis and Bioenergetics of Mitochondria, Miinchen 1975 (Bandlow, W., Schweyen, R., Thomas, D. Y., Wolf, K., and Kaudewitz, F., eds.), de Gruyter, Berlin (in press). 14. Lang, B., and Burger, G. (1975) in 10th FEBS Congress, Paris, 1975, Abstract No. 1215. 15. Chance, B., and Williams, G. R. (1955) Adv. Enzyrnol. 17, 65-134. 16. Morimoto, R., Lewin, A., Hsu, H.-J., Rabinowitz, M., and Fukuhara, H. (1975) Proc. Nat. Acad. Sci. USA 72,3868-3872.