ANALYTICAL BIOCHEMISTRY 70, 86--93
(1976)
Sedimentation Coefficient, Buoyant Density, Morphology, and Optimal Recovery of Mitochondria from the Ileal Mucosa of Guinea Pigs ERIK SLINDE, 1 KNUT-JAN ANDERSEN, LARS MORKRID AND HARALD KRYVI2 Department of Biochemistry and Institute of Anatomy. University of Bergen, 5000 Bergen, and Section for Clinical Research, Medical Department .4, University of Bergen, 5016 Haukeland Sykehus, Norway Received June 5, 1975; accepted August 5, 1975 Marker enzymes have been used to determine the average sedimentation coefficient (g-value) of mitochondria from the ileal mucosa of guinea pigs in isotonic sucrose medium. From the determined g-value, 5590 + 320S, a procedure has been developed allowing optimal recovery of these mitochondria by differential centrifugation. Transmission electron micrographs of the isolated mitochondria show well-preserved mitochondria. Scanning electron microscopy reveals a homogenous population with low average diameters, 0.61 -+0.09/xm (short axis) and 0.68 + 0.09/xm(long axis). These values are consistent with the low g-value and the low measured buoyant density, D2020= 1.167 g/cm3. Procedures used for the isolation of subcellular c o m p o n e n t s o f the ileal m u c o s a ( 1 - 5 ) have been based on those originally described for liver (6,7). T h e isolation of ileal mitochondria, however, is complicated by the presence o f brush b o r d e r fragments, filamentous material (8,9), mucin (10,11), and proteolytic e n z y m e s (12,13). F u r t h e r m o r e , for the study o f transport processes in mitochondria the isolation has to be p e r f o r m e d in isotonic sucrose (14). It has been reported (15) that mitochondria from different m a m m a l i a n tissues show variation in their average sedimentation coefficient (g-value), and it is therefore far from optimal to apply the same fractionation procedure to mitochondria of different tissues. The present study reports the average sedimentation coefficient in an isotonic sucrose medium, b u o y a n t density, size, and m o r p h o l o g y of the mitochondria from the ileal m u c o s a of guinea pigs. F r o m the g-value determined, a fractionation p r o c e d u r e has been designed allowing optimal r e c o v e r y o f such mitochondria by differential centrifugation.
MATERIALS AND METHODS Animals, preparation o f the " 1 0 0 % h o m o g e n a t e " and determination o f g-values. Male guinea pigs ( H a r t l e y strain, 300-400 g body weight) were 1 Department of Biochemistry. 2 Institute of Anatomy. 86 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved
ILEAL M I T O C H O N D R I A
87
fasted for 24 hr. The ileum was washed through with 80 ml of 0.25 M sucrose, 1 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid), 5 mM E D T A (pH 7.2), and was everted, and mucosal scrapings (approx 3 g/animal) were homogenized in 40 ml of the buffer (containing only 0.2 mM EDTA) at 400 rpm in a Teflon-glass homogenizer. Determination of g-values was performed as previously described (16). When cytochrome c oxidase was used as the marker enzyme of the mitochondrial inner membrane, the homogenate was centrifuged in a swinging-bucket rotor (H B-4) of a Sorvall RC2-B refrigerated centrifuge at a time integral of either 9" 107 min -1 or 19.107 min -1 (t = 10 min, Rmin = 6.2 cm, Rm~x = 14.4 cm). When malate dehydrogenase was used as the marker enzyme of the mitochondrial matrix, the homogenate was filtered through acid washed glass-wool to get rid of filamentous material as well as mucus present (8,11), and then subjected to centrifugation at a time integral of 110" 107 min -1 This was done to remove proteolytic enzymes as well as cytoplasmic malate dehydrogenase. The pellet was rehomogenized and the nuclear fraction sedimented at 11.107 min-L The supernatants were diluted to approximately 2 mg of protein/ml and termed "100% homogenate." All operations were carried out in a cold room at 4 ___0.5°C.
Determination of ft rpm2dt and the buoyant density of the isolated mitochondria. Total time integrals of rpm 2 were determined by an integrator with a precision of 3% (17). The buoyant density of isolated mitochondria was determined as previously described (18). Enzymic assays. Cytochrome c oxidase (EC 1.9.3.1) and acid phosphatase (EC 3.1.3.2) were determined as previously described (16). Malate dehydrogenase (EC 1.1.1.37) was measured according to Bergmeyer (19). Protein. Protein was determined by the method of Lowry et al. (20). Morphological examinations. Fixations for electron microscopy were performed in 1%, followed by 2.5%, glutaraldehyde and postfixed in 1% osmium tetroxide. Specimens for scanning electron microscopy were dehydrated, critical-point dried with CO2 (21), coated with carbon and gold, examined in a J E O L scanning electron microscope, JSM-U3, at 25 kV, and tilted at an angle of 45°. Specimens for transmission electron microscopy were dehydrated and embedded in Epon. The blocks were sectioned on a LKB Ultrotome III, and 50-nm sections stained in uranyl acetate (22), followed by lead citrate (23), were studied in a Siemens Elmiskop 1.
RESULTS Sedimentation of cytochrome c oxidase, malate dehydrogenase, and acid phosphatase. From the g-values determined (Table 1), which all fall within the experimental error, the average sedimentation coefficient of the ileal mitochondria has been calculated to be 5590 +_ 320S. No significant
88
SLINDE ET AL.
TABLE 1 DETERMINATION OF THE AVERAGE SEDIMENTATION COEFFICIENT OF MITOCHONDRIA AND LYSOSOMES FROM THE ILEAL MUCOSA OF GUINEA PIGS
Marker e n z y m e
Protein concentration in sedimentation medium (mg/ml)
Cytochrome c b oxidase Acid phosphatase b Cytochrome c c oxidase Malate dehydrogenase
0.6 0.6 0.5 0.4
Estimated g-value (g4,B) a (S) 5260 5810 5880 5640
_+ 500 +-- 510 -+ 480 _+ 340
a g4,B represents the average sedimentation coefficient at 4°C in the suspending medium, 0.25 ~i sucrose, 1 mM H E P E S , 0.2 mM E D T A (pH 7.2). g-value determined by leastsquares regression analysis. b The nuclear fraction was sedimented at a time integral of 9" 107 min -1. c The nuclear fraction w a s sedimented at a time integral of 19' 10 r rain -1.
influences of filamentous material, brush border fragments, mucus, or any other heavy material were observed since the change in centrifugal effect from 9" 107 to 19" 107 min -I did not change the g-value (Table 1) or the recovery of the mitochondria. The convergence values (16) were found to be 73 and 71%, respectively. When malate dehydrogenase was used as the mitochondrial marker enzyme, the average sedimentation coefficient was 5640 _+ 340S as shown in Fig. 1. At the convergence level, 90% of the malate 000 -004 - 008 -012
-016 ~:a- -020 i
-024
g
-028
o
-032 -036 040
CONVERGENCE I
50
"
1
I
100
~ I
150 200 t rpm2dt (xlO-Tmln-1)
I
250
300
350
o
Fxc. 1. Determination of the average sedimentation coefficient of ileal mitochondria of guinea pigs. The protein concentration was 0.4 mg/ml, and malate dehydrogenase was used as the marker e n z y m e . From the slope of the curve the g-value was determined: g4,B = 5640 _+ 340S. The small deviation from origo is due to the hemispherical shape of the centrifuge tube (16). The experiment was carried out in a HB-4 rotor of a Sorvall RC2-B c e n t r i f u g e (Rmax = 14.4 cm and Rmln = 6.2 cm). For details see the text.
89
ILEAL MITOCHONDRIA
dehydrogenase was sedimented, indicating that fragmentation of mitochondria upon resuspension was small. The sedimentation properties of lysosomes are similar to those of mitochondria, and the average sedimentation coefficient was determined to be 5810 --- 510S when acid phosphatase was used as the lysosomal marker enzyme. The lysosomal convergence level was reached at 50%, indicating a high degree of fragmentation of these particles. Isolation and morphology of ileal mitochondria. For optimal recovery of the ileal mitochondria of guinea pigs, the fractionation procedure shown in Table 2 has been established based on the determined g-value. The homogenate, prepared as described under Materials and Methods, was stirred gently with acid-washed glass wool, decanted, and sedimented at a centrifugal effect of 227.10 r min -~ in order to remove proteolytic activity in the homogenate. The pellet (P1) was rehomogenized in 40 ml of buffer by one stroke, restirred with glass wool, and nuclei and brush borders were TABLE
2
PROCEDURE FOR THE OPTIMAL RECOVERY OF ILEAL MITOCHONDR1A OF GUINEA PIGS (g = 5590S) BY DIFFERENTIAL CENTRIFUGATION USING THE H B - 4 ROTOR OF THE SORVALL R C 2 - B REFRIGERATED CENTRIFUGE (Rmin = 6.2 c m and Rmax ~c 14.4 cm) a Tissue homogenate, 40 ml, 0 75 g glass wool, filtered.
1 Sedimented
rpm2dt = 227.107 mm ~ (Sm~ = 5590S, approx 10.0t)fi rpm, 20 mln)
Jo
[
1
Sediment PI resuspended, 40 ml, 0.75 g glass wool, filtered.
1
Supernatant St (discarded)
Sedlmented
t
Supernatant $2 (saved)
fl rP~2dt=27
107 mln-l ( S ~ = 50,000S, approx 7000 rpm, 5 min)
Sediment P2 resuspended, 40 ml, 0.75 g glass wool, filtered ft
Sedlmented J0
rpm2dt= 27. lfir m(n 1. I
Supernatant $3 (saved)
Sediment P~ Idiscarded)
I $2 + $3 sedlmented
rpm2dt = 227 I0T min -1
1
Supernatant $4 (discarded)
I Sediment P4 resuspended. Sedlmented f l rpm2dt = 227 107 min -~
I Supernatant $5 (discarded) a For detads, see the text
Sediment Ps mitochondria
FIG. 2. (A) Transmission electron micrograph of the isolated mitochondria, 42,000x. For details see the text. (B,C) Scanning electron micrographs of the isolated mitochondria. (B) Low magnification (6500x); notice the homogeneity of size and shape. (C) The higher magnification (33,000x) shows only modest shape distortion of the mitochondria. (D) A scanning electron micrograph, with a mitochondrion that appears to divide (arrow); 33,000 ×. 90
91
ILEAL MITOCHONDRIA
then sedimented at a centrifugal effect of 27.10 r min -1. The supernatant SE was saved. The pellet P2 was treated and sedimented as above to enrich the supernatant with regard to mitochondria (15). The supernatants $2 and $3 were combined and subjected to a centrifugal effect of 227.10 r min -1, and the pellet P4 was washed by resuspension and resedimentation at the same centrifugal effect. The pellet, Ps, is termed mitochondria. The centrifuge should be used without the brake (16). Electron microscopic studies (Fig. 2A) revealed that the fraction consisted almost exclusively of mitochondria, with only very few lysosomes and fragments of microvilli present. The mitochondria were mostly in a condensed form with an apparently intact outer membrane, and the major part of the population had a pattern corresponding to the orthodox configuration described by Hackenbrock (24). A few dividing mitochondria were also observed (Fig. 2D), in agreement with the facts that the ileal epithelium is a rapidly proliferating tissue (5) and that mitochondria divide by fission (25). Buoyant density and size o f the isolated ileal mitochondria. The buoyant density of the isolated ileal mitochondria was found to be D2o2° = 1.167 g/cm~ (Fig. 3) as compared to the value 1.200 g/cm 3 for isolated rat liver mitochondria under similar sedimentation conditions (18). Assuming that all the particles isolated were mitochondria, the average diameters measured from scanning electron micrographs (Figs. 2B and C) were found to be 0.68 + 0.09 /~m (long axis) and 0.61 + 0.09 /.,m (short axis) as compared to 0.764 _+ 0.207/~m for rat liver mitochondria (26).
DISCUSSION Mitochondria from the ileal mucosa are difficult to obtain in the native form due to proteolytic attack by lysosomal enzymes as well as by 4
08
b >
cE 0 6 © (N llq
3
*5O4
2
o _o o~
u o ~
02
-
O0
*5 0
122
120
118
116
5
114
Density (D~ ° )
FIG. 3. D e t e r m i n a t i o n of the buoyant density of ileal mitochondria of guinea pigs. T h e isolated mitochondria were layered on a linear sucrose gradient containing 5 mM H E P E S buffer (pH 7.4).
92
SLINDE ET AL.
pancreatic enzymes adsorbed to the surface of the intestinal mucosa (12,13). Prolonged exposure to these proteolytic enzymes may damage the mitochondria, and the first sedimentation of the differential centrifugation procedure (Table 2) was therefore performed at the high centrifugal effect (227.107 min -1) to clear out all the intact mitochondria. Another problem is the contamination by mucus (11) as well as brush border fragments. While glass wool (Table 2) was found to adsorb mucus (8), a rather high centrifugal effect (Smi, = 50,000 S) 3 was selected to sediment brush border fragments together with nuclei, since the g-value of the mitochondria is much lower than that of the brush border fragments present. The low standard deviation of the average diameters measured from electron micrographs (Figs. 2B and C) demonstrates a very homogeneous population of the mitochondria. Although the sedimentation coefficient of lysosomes is close to that of the mitochondria, only a few lysosomes were observed by transmission electron microscopy (Fig. 2A). This is explained by the high degree of fragmentation observed. The low g-value determined for the ileal mitochondria as compared to that of rat liver, g4,B° = 11,820 -----760S (16), is in agreement with the observations that both the radius (the shape factor (27), f ~ 1) and the buoyant density are smaller than that of rat liver. If both rat liver and ileal mitochondria are assumed to be spherical particles, with a radius of 0.382 /.~m (26) and 0.32 /zm, respectively, the calculated volume of the ileal mitochondria is only 59% of that of the rat liver mitochondria. The fact that the buoyant density as well as the radius or the volume of these mitochondria is smaller than that of rat liver mitochondria, explains the observed difference in sedimentation coefficient.
ACKNOWLEDGMENTS We are grateful to Professor T. Flatmark for his stimulating interest and discussions of this study and to Mrs. Aud Utheim for skillful technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Porteous, J. W., and Clark, B. (1965) Biochem. J. 96, 159-171. Hfibscher, G., West, G. R., and Brindley, D. N. (1965) Biochem. J. 97, 629-642. Berg, G. G., and Chapman, B. (1965) J. Cell. Comp. Physiol, 65, 361-372. Iemhoff, W. G. J., Van Den Berg, J. W. O., De Pijper, A. M., and Htilsmann, W. C. (1970) Biochim. Biophys. Acta 215, 229-241. Iemhoff, W. G. J., and Hiilsmann, W. C. (1971) Eur. J. Biochem. 23, 429-434. Schneider, W. C. (1948)J. Biol. Chem. 176, 259-266. De Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R., and Appelmans, F. (1955) Biochem. J. 60, 604-617. Forstner, G. G., Sabesin, S. M., and Isselbacher, K. J. (1968)Biochem. J. 106, 381-390.
3 g4~, The average sedimentation coefficient determined in buffered sucrose medium; g4.a°, the g4,B value at infinite dilution (zero protein concentration); gm~, the lightest particle to be completely sedimented at the actual sedimentation conditions.
ILEAL M I T O C H O N D R I A
93
9. Eichholz, A., and Crane, R. K. (1974) In Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 31, pp. 123-124, Academic Press, New York. 10. Jennings, M. A., and Florey, H. W. (1956) Quart. J. Exp. Physiol. 41, 131-152. 11. Bush, H., Davis, J. R., and Anderson, D. C. (1958) Cancer Res. 18, 916-926. 12. Pelot, D., and Grossman, M. I. (1962)Amer. J. Physiol. 202, 285-288. 13. Ugolev, A. M. (1972) Gut 13, 735-747. 14. Azzone, G. F., and Massari, S. (1975) Biochim. Biophys. Acta 301, 195-226. 15. Slinde, E., Morild, E., and Flatmark, T. (1975)Anal. Biochem. 66, 151-158. 16. Slinde, E., and Flatmark, T. (1973)Anal. Biochem. 56, 324-340. 17. Slinde, E.. Storetvedt, H., Strand, J., and Flatmark, T. (1974) Anal. Biochem. 58, 170-174. 18. Slinde, E., Pedersen, J. I., and Flatmark, T. (1975)Anal. Biochem. 65, 581-585. 19. Bergmeyer, H. U. (1970) In Methoden der Enzymatischen Analyse, 2. Aufl., Band 1 (Bergmeyer, H. U., ed.), pp. 575-579, Verlag Chemie, Weinheim. 20. Lowry, O. A., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem. 193, 265-275. 21. Nemanic, M. K. (1972)In Scanning Electron Microscopy 1972 (Johari, O., and Corvin, I., eds.), pp. 298-303, l i T Research Institute, Chicago. 22. Watson, M. L. W. (1958) J. Biophys. Biochem. Cytol. 4, 475-478. 23. Reynolds, E. S. (1963) J. Cell Biol. 17, 208-212. 24. Hackenbrock, C. R. (1972)Ann. N . Y . Acad. Sci. 195, 492-505. 25. Tandler, B., and Hoppel, C. L. (1972) Mitochondria, Academic Press, London and New York. 26. Baudhuin, P., and Berthet, J. (1967)J. Cell Biol. 35, 631-648. 27. De Duve, C., Berthet, J., and Beaufay, H. (1959)Progr. Biophys. Mol. Biol. 9,326-369.
(1976)
Sedimentation Coefficient, Buoyant Density, Morphology, and Optimal Recovery of Mitochondria from the Ileal Mucosa of Guinea Pigs ERIK SLINDE, 1 KNUT-JAN ANDERSEN, LARS MORKRID AND HARALD KRYVI2 Department of Biochemistry and Institute of Anatomy. University of Bergen, 5000 Bergen, and Section for Clinical Research, Medical Department .4, University of Bergen, 5016 Haukeland Sykehus, Norway Received June 5, 1975; accepted August 5, 1975 Marker enzymes have been used to determine the average sedimentation coefficient (g-value) of mitochondria from the ileal mucosa of guinea pigs in isotonic sucrose medium. From the determined g-value, 5590 + 320S, a procedure has been developed allowing optimal recovery of these mitochondria by differential centrifugation. Transmission electron micrographs of the isolated mitochondria show well-preserved mitochondria. Scanning electron microscopy reveals a homogenous population with low average diameters, 0.61 -+0.09/xm (short axis) and 0.68 + 0.09/xm(long axis). These values are consistent with the low g-value and the low measured buoyant density, D2020= 1.167 g/cm3. Procedures used for the isolation of subcellular c o m p o n e n t s o f the ileal m u c o s a ( 1 - 5 ) have been based on those originally described for liver (6,7). T h e isolation of ileal mitochondria, however, is complicated by the presence o f brush b o r d e r fragments, filamentous material (8,9), mucin (10,11), and proteolytic e n z y m e s (12,13). F u r t h e r m o r e , for the study o f transport processes in mitochondria the isolation has to be p e r f o r m e d in isotonic sucrose (14). It has been reported (15) that mitochondria from different m a m m a l i a n tissues show variation in their average sedimentation coefficient (g-value), and it is therefore far from optimal to apply the same fractionation procedure to mitochondria of different tissues. The present study reports the average sedimentation coefficient in an isotonic sucrose medium, b u o y a n t density, size, and m o r p h o l o g y of the mitochondria from the ileal m u c o s a of guinea pigs. F r o m the g-value determined, a fractionation p r o c e d u r e has been designed allowing optimal r e c o v e r y o f such mitochondria by differential centrifugation.
MATERIALS AND METHODS Animals, preparation o f the " 1 0 0 % h o m o g e n a t e " and determination o f g-values. Male guinea pigs ( H a r t l e y strain, 300-400 g body weight) were 1 Department of Biochemistry. 2 Institute of Anatomy. 86 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved
ILEAL M I T O C H O N D R I A
87
fasted for 24 hr. The ileum was washed through with 80 ml of 0.25 M sucrose, 1 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid), 5 mM E D T A (pH 7.2), and was everted, and mucosal scrapings (approx 3 g/animal) were homogenized in 40 ml of the buffer (containing only 0.2 mM EDTA) at 400 rpm in a Teflon-glass homogenizer. Determination of g-values was performed as previously described (16). When cytochrome c oxidase was used as the marker enzyme of the mitochondrial inner membrane, the homogenate was centrifuged in a swinging-bucket rotor (H B-4) of a Sorvall RC2-B refrigerated centrifuge at a time integral of either 9" 107 min -1 or 19.107 min -1 (t = 10 min, Rmin = 6.2 cm, Rm~x = 14.4 cm). When malate dehydrogenase was used as the marker enzyme of the mitochondrial matrix, the homogenate was filtered through acid washed glass-wool to get rid of filamentous material as well as mucus present (8,11), and then subjected to centrifugation at a time integral of 110" 107 min -1 This was done to remove proteolytic enzymes as well as cytoplasmic malate dehydrogenase. The pellet was rehomogenized and the nuclear fraction sedimented at 11.107 min-L The supernatants were diluted to approximately 2 mg of protein/ml and termed "100% homogenate." All operations were carried out in a cold room at 4 ___0.5°C.
Determination of ft rpm2dt and the buoyant density of the isolated mitochondria. Total time integrals of rpm 2 were determined by an integrator with a precision of 3% (17). The buoyant density of isolated mitochondria was determined as previously described (18). Enzymic assays. Cytochrome c oxidase (EC 1.9.3.1) and acid phosphatase (EC 3.1.3.2) were determined as previously described (16). Malate dehydrogenase (EC 1.1.1.37) was measured according to Bergmeyer (19). Protein. Protein was determined by the method of Lowry et al. (20). Morphological examinations. Fixations for electron microscopy were performed in 1%, followed by 2.5%, glutaraldehyde and postfixed in 1% osmium tetroxide. Specimens for scanning electron microscopy were dehydrated, critical-point dried with CO2 (21), coated with carbon and gold, examined in a J E O L scanning electron microscope, JSM-U3, at 25 kV, and tilted at an angle of 45°. Specimens for transmission electron microscopy were dehydrated and embedded in Epon. The blocks were sectioned on a LKB Ultrotome III, and 50-nm sections stained in uranyl acetate (22), followed by lead citrate (23), were studied in a Siemens Elmiskop 1.
RESULTS Sedimentation of cytochrome c oxidase, malate dehydrogenase, and acid phosphatase. From the g-values determined (Table 1), which all fall within the experimental error, the average sedimentation coefficient of the ileal mitochondria has been calculated to be 5590 +_ 320S. No significant
88
SLINDE ET AL.
TABLE 1 DETERMINATION OF THE AVERAGE SEDIMENTATION COEFFICIENT OF MITOCHONDRIA AND LYSOSOMES FROM THE ILEAL MUCOSA OF GUINEA PIGS
Marker e n z y m e
Protein concentration in sedimentation medium (mg/ml)
Cytochrome c b oxidase Acid phosphatase b Cytochrome c c oxidase Malate dehydrogenase
0.6 0.6 0.5 0.4
Estimated g-value (g4,B) a (S) 5260 5810 5880 5640
_+ 500 +-- 510 -+ 480 _+ 340
a g4,B represents the average sedimentation coefficient at 4°C in the suspending medium, 0.25 ~i sucrose, 1 mM H E P E S , 0.2 mM E D T A (pH 7.2). g-value determined by leastsquares regression analysis. b The nuclear fraction was sedimented at a time integral of 9" 107 min -1. c The nuclear fraction w a s sedimented at a time integral of 19' 10 r rain -1.
influences of filamentous material, brush border fragments, mucus, or any other heavy material were observed since the change in centrifugal effect from 9" 107 to 19" 107 min -I did not change the g-value (Table 1) or the recovery of the mitochondria. The convergence values (16) were found to be 73 and 71%, respectively. When malate dehydrogenase was used as the mitochondrial marker enzyme, the average sedimentation coefficient was 5640 _+ 340S as shown in Fig. 1. At the convergence level, 90% of the malate 000 -004 - 008 -012
-016 ~:a- -020 i
-024
g
-028
o
-032 -036 040
CONVERGENCE I
50
"
1
I
100
~ I
150 200 t rpm2dt (xlO-Tmln-1)
I
250
300
350
o
Fxc. 1. Determination of the average sedimentation coefficient of ileal mitochondria of guinea pigs. The protein concentration was 0.4 mg/ml, and malate dehydrogenase was used as the marker e n z y m e . From the slope of the curve the g-value was determined: g4,B = 5640 _+ 340S. The small deviation from origo is due to the hemispherical shape of the centrifuge tube (16). The experiment was carried out in a HB-4 rotor of a Sorvall RC2-B c e n t r i f u g e (Rmax = 14.4 cm and Rmln = 6.2 cm). For details see the text.
89
ILEAL MITOCHONDRIA
dehydrogenase was sedimented, indicating that fragmentation of mitochondria upon resuspension was small. The sedimentation properties of lysosomes are similar to those of mitochondria, and the average sedimentation coefficient was determined to be 5810 --- 510S when acid phosphatase was used as the lysosomal marker enzyme. The lysosomal convergence level was reached at 50%, indicating a high degree of fragmentation of these particles. Isolation and morphology of ileal mitochondria. For optimal recovery of the ileal mitochondria of guinea pigs, the fractionation procedure shown in Table 2 has been established based on the determined g-value. The homogenate, prepared as described under Materials and Methods, was stirred gently with acid-washed glass wool, decanted, and sedimented at a centrifugal effect of 227.10 r min -~ in order to remove proteolytic activity in the homogenate. The pellet (P1) was rehomogenized in 40 ml of buffer by one stroke, restirred with glass wool, and nuclei and brush borders were TABLE
2
PROCEDURE FOR THE OPTIMAL RECOVERY OF ILEAL MITOCHONDR1A OF GUINEA PIGS (g = 5590S) BY DIFFERENTIAL CENTRIFUGATION USING THE H B - 4 ROTOR OF THE SORVALL R C 2 - B REFRIGERATED CENTRIFUGE (Rmin = 6.2 c m and Rmax ~c 14.4 cm) a Tissue homogenate, 40 ml, 0 75 g glass wool, filtered.
1 Sedimented
rpm2dt = 227.107 mm ~ (Sm~ = 5590S, approx 10.0t)fi rpm, 20 mln)
Jo
[
1
Sediment PI resuspended, 40 ml, 0.75 g glass wool, filtered.
1
Supernatant St (discarded)
Sedlmented
t
Supernatant $2 (saved)
fl rP~2dt=27
107 mln-l ( S ~ = 50,000S, approx 7000 rpm, 5 min)
Sediment P2 resuspended, 40 ml, 0.75 g glass wool, filtered ft
Sedlmented J0
rpm2dt= 27. lfir m(n 1. I
Supernatant $3 (saved)
Sediment P~ Idiscarded)
I $2 + $3 sedlmented
rpm2dt = 227 I0T min -1
1
Supernatant $4 (discarded)
I Sediment P4 resuspended. Sedlmented f l rpm2dt = 227 107 min -~
I Supernatant $5 (discarded) a For detads, see the text
Sediment Ps mitochondria
FIG. 2. (A) Transmission electron micrograph of the isolated mitochondria, 42,000x. For details see the text. (B,C) Scanning electron micrographs of the isolated mitochondria. (B) Low magnification (6500x); notice the homogeneity of size and shape. (C) The higher magnification (33,000x) shows only modest shape distortion of the mitochondria. (D) A scanning electron micrograph, with a mitochondrion that appears to divide (arrow); 33,000 ×. 90
91
ILEAL MITOCHONDRIA
then sedimented at a centrifugal effect of 27.10 r min -1. The supernatant SE was saved. The pellet P2 was treated and sedimented as above to enrich the supernatant with regard to mitochondria (15). The supernatants $2 and $3 were combined and subjected to a centrifugal effect of 227.10 r min -1, and the pellet P4 was washed by resuspension and resedimentation at the same centrifugal effect. The pellet, Ps, is termed mitochondria. The centrifuge should be used without the brake (16). Electron microscopic studies (Fig. 2A) revealed that the fraction consisted almost exclusively of mitochondria, with only very few lysosomes and fragments of microvilli present. The mitochondria were mostly in a condensed form with an apparently intact outer membrane, and the major part of the population had a pattern corresponding to the orthodox configuration described by Hackenbrock (24). A few dividing mitochondria were also observed (Fig. 2D), in agreement with the facts that the ileal epithelium is a rapidly proliferating tissue (5) and that mitochondria divide by fission (25). Buoyant density and size o f the isolated ileal mitochondria. The buoyant density of the isolated ileal mitochondria was found to be D2o2° = 1.167 g/cm~ (Fig. 3) as compared to the value 1.200 g/cm 3 for isolated rat liver mitochondria under similar sedimentation conditions (18). Assuming that all the particles isolated were mitochondria, the average diameters measured from scanning electron micrographs (Figs. 2B and C) were found to be 0.68 + 0.09 /~m (long axis) and 0.61 + 0.09 /.,m (short axis) as compared to 0.764 _+ 0.207/~m for rat liver mitochondria (26).
DISCUSSION Mitochondria from the ileal mucosa are difficult to obtain in the native form due to proteolytic attack by lysosomal enzymes as well as by 4
08
b >
cE 0 6 © (N llq
3
*5O4
2
o _o o~
u o ~
02
-
O0
*5 0
122
120
118
116
5
114
Density (D~ ° )
FIG. 3. D e t e r m i n a t i o n of the buoyant density of ileal mitochondria of guinea pigs. T h e isolated mitochondria were layered on a linear sucrose gradient containing 5 mM H E P E S buffer (pH 7.4).
92
SLINDE ET AL.
pancreatic enzymes adsorbed to the surface of the intestinal mucosa (12,13). Prolonged exposure to these proteolytic enzymes may damage the mitochondria, and the first sedimentation of the differential centrifugation procedure (Table 2) was therefore performed at the high centrifugal effect (227.107 min -1) to clear out all the intact mitochondria. Another problem is the contamination by mucus (11) as well as brush border fragments. While glass wool (Table 2) was found to adsorb mucus (8), a rather high centrifugal effect (Smi, = 50,000 S) 3 was selected to sediment brush border fragments together with nuclei, since the g-value of the mitochondria is much lower than that of the brush border fragments present. The low standard deviation of the average diameters measured from electron micrographs (Figs. 2B and C) demonstrates a very homogeneous population of the mitochondria. Although the sedimentation coefficient of lysosomes is close to that of the mitochondria, only a few lysosomes were observed by transmission electron microscopy (Fig. 2A). This is explained by the high degree of fragmentation observed. The low g-value determined for the ileal mitochondria as compared to that of rat liver, g4,B° = 11,820 -----760S (16), is in agreement with the observations that both the radius (the shape factor (27), f ~ 1) and the buoyant density are smaller than that of rat liver. If both rat liver and ileal mitochondria are assumed to be spherical particles, with a radius of 0.382 /.~m (26) and 0.32 /zm, respectively, the calculated volume of the ileal mitochondria is only 59% of that of the rat liver mitochondria. The fact that the buoyant density as well as the radius or the volume of these mitochondria is smaller than that of rat liver mitochondria, explains the observed difference in sedimentation coefficient.
ACKNOWLEDGMENTS We are grateful to Professor T. Flatmark for his stimulating interest and discussions of this study and to Mrs. Aud Utheim for skillful technical assistance.
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3 g4~, The average sedimentation coefficient determined in buffered sucrose medium; g4.a°, the g4,B value at infinite dilution (zero protein concentration); gm~, the lightest particle to be completely sedimented at the actual sedimentation conditions.
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