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Experimental
Cell Research
112 (1978) 79-87
THE FORMATION OF SEVERAL MITOCHONDRIAL ENZYMES DURING THE CELL CYCLE IN HEAT-SYNCHRONIZED TETRAHYMENA PYRIFORMIS ST A. E. COWAN and P. G. YOUNG Department
of Biology,
Queen’s
University,
Kingston,
Ont. K7L 3N6,
Canada
SUMMARY The formation of a number of mitochondrial marker enzymes has been studied during the cell cycle in heat-synchronized Tetrahymena pyriformis ST. Markers for the inner mitochondrial membrane (succinic dehydrogenase and succinate-cytochrome c reductase) and for the matrix (malate dehydrogenase) were found to increase exponentially through the heat-shocked cell cycle. Their specific activity remained constant throughout the cycle. An outer membrane and endoplasmic reticulum marker (rotenone-insensitive NADH-cytochrome c reductase) increased stepwise in close parallel to the time of increase in cell number under these conditions. During free-running divisions, following the cessation of heat shocks, all four enzymes retained the same mode of increase which they displayed during heat-shocked divisions. The step for rotenone-insensitive NADH-cytochrome c reductase, however, retained the timing characteristic of the heat-shocked cycle and increased independently of cell division. These results are correlated with available morphometric information on the growth and division of mitochondria in this system.
The vast majority of mitochondrial enzymes are synthesized on cytoplasmic ribosomes and are under the control of the nucleus. Several mitochondrial components, however, are coded for by mitochondrial DNA (for reviews see [l, 3, 28, 291). The co-ordination of the activities of these two separate genomes within the cell is poorly understood. Under conditions of balanced growth all mitochondrial components should double during the course of each cell cycle. Information relating to the manner in which they do so, however, is fragmentary and often contradictory. A number of mitochondrial enzyme activities have been followed through the cell cycle including some which are completely coded for in the nucleus (e.g. malate dehydrogenase, fumarase, succinic dehydro6-771809
genase [5, 8, 9, 12, 24, 25, 311) and some which require the assembly of polypeptides synthesized by both genomes (e.g. succinate cytochrome c reductase, cytochrome oxidase [5, 6, 9, 15, 24-26, 311). No consistent picture emerges, however, in terms of whether or not the synthesis of a particular enzyme is continuous or periodic during the cell cycle or, if periodic, whether it presents a step or a peak pattern. Difliculties occur in comparing the various systems due to differences in cell type and in synchronization procedure. In addition, a number of publications present insufficient data points to be certain of the cell cycle pattern. It appears to be impractical to generalize among these various systems at the present time. Recently the growth and division of mitoExpcrllres
112 (1978)
Cowan
80
and Young
IO< 1
60-
ous within the cell. In this communication we have undertaken to correlate the synthesis of some mitochondrial enzymes with this synchronous mitochondrial division. Portions of this work have appeared in abstract form [7].
60 60
40
20
i0
IO 0 IO 0 0 ‘,I40
0
- 120 ~100 -80 -60 -40
METHODS
AND
MATERIALS
Cells and synchronization procedures Tetrahymena pyriformis ST was maintained at 28°C in Neff s medium [22]. Synchrony was induced using the one heat shock per generation technique of Zeuthen [32]. Heat shocks of 34°C for 39 min (time is nominal machine switching time, not the time at 34°C in the flask) were administered at intervals of 174 min, to give an overall cycle of 213 min. This system achieved division indices as high as 80 % at about 85-95 min following the heat shock. Cells were normally used for experimental purposes following the sixth or seventh heat shock.
Harvesting of cells
-20
Repeated differential washing of the cell sample was found to give variable recovery. The following method was instituted as an easy, rapid and quantitative washing procedure for Tetruhymena cells. An aliquot I 200 300 400 0 100 of 10-30 ml of cell culture was overlain onto a 1.5ml cushion of ice-cold homogenization medium (0.35 M Fig. I. Abscissa: time after the sixth heat shock sucrose, 0.001 M ethylenediaminetetraacetic acid (min); ordinate: (left, inner, upper) division index (%) 0.01 M Tris(hydroxymethy1) aminomethane, (0-O); (right, inner, upper) no. of cellslmlx 10m3 (EDTA), pH 7) in a 50 ml conical centrifuge tube. The cells (Cm); (left, outer, upper) MDH act. (A-A); (right, were centrifuged at 800 g for 3 min in an International outer) SDH act. (X-X); (left, inner, lower) sucdnatecentrifuge (Model V). The medium and washing solucytochrome c reductase act. (0-O); (right, inner, tion were then withdrawn by suction. Cells prepared lower) rotenone-insensitive NADH-cytochrome c rein this way are effectively freed of culture medium by ductase act. (A-A); (left, outer, lower) mg cellular passage through the cushion and require no further protein/ 10 ml culture (+-+). washes. Patterns of mitochondrial enzyme activity through the cell cycle during heat-shock synchrony. All enzyme activities are expressed as nmoles/min/lO ml Homogenization culture. Most points represent the average of duplicate Small cell samples were transferred to a 1 ml tuberdeterminations. Solid blocks indicate the time of the culin syringe and the volume made up to the 1 ml heat shocks. mark with homogenization medium (actually equivalent to 1.05 ml allowing for the volume within the needle). The cells were then broken by passage chondria in heat-synchronized Tetrahythrough a 26-gauge syringe needle at WC. Complete breakage required 5-6 passes. The homogenate was menu has been studied utilizing morphothen sampled for enzyme assays. Coupled with the metric techniques [ 131. The mitochondria washing procedure this system is rapid and simple, were found to develop synchronously and and it allowed for a quantitative recovery of cellular materials. It enabled frequent samples to be taken durto divide during the heat shock. This sys- ing the cell cycle. For the preparation of mitochondria from larger volumes of culture the cells were tem seems to be excellent for studying mitohomogenized using a hand emulsifier (Fisher).
chondrial cell cycle events due to the facility with which synchrony can be induced and the knowledge that growth and division of the mitochondria are synchronEXP cd
res I12
(1978)
Enzyme assays Aliquots of crude homogenates were assayed spectrophotometrically (Gilford) for the following enzyme ac-
Mitochondria
501 o-
40 O60 55 50 !5 20
6 O-
5 O-
4 O-6 i0
i0
in Tetrahymena
81
Lloyd et al. [16]. The reaction mixture contained 0.3 mg/ml cytochrome c (equine, Sigma), 10 mM KCN, 0.19 mM NADH, 2.5 PM rotenone and 20 mM potassium phosphate buffer, pH 7.4. The reaction was initiated with NADH and the increase in absorbance for cytochrome c at 550 nm was measured. An extinction coefficient of 18500 cm-’ mole-’ liter was used. Succinic dehydrogenase (EC 1.3.99.1; succinate: (acceptor) oxidoreductase) was assayed according to Redfearn & Dixon [27] as modified by Muller et al. [21]. The reaction mixture contained 10 mM succinic acid (pH 7.4 with KOH), 32 I.~M dichlorophenolindophenol (DCPIP), 1 mM phenazine methosulphate (PMS) and 20 mM potassium phosphate buffer, pH 7.4. DCPIP was added for a 15 min incubation and then PMS was added to intiate the reaction. The decrease in absorbance for DCPIP at 600 nm was monitored (extinction coefftcient, 15 600 cm-’ mole-’ liter). Samples used for this assay had been quick frozen in dry ice-acetone and stored at -20°C for several days. Succinate-cytochrome c reductase (EC 1.3.99.-; succinate: cytochrome c oxidoreductase) was assayed according to Mahler [ 181 as modified by Turner et al. [30]. The reaction contained 0.3 mg/ml equine cytochrome c, IO mM succinate, 10 mM KCN and 20
10
50 L
I
0
50
1
100
8
IS0
I
200
B
_6.0
J
Fig. 2. Abscissa: time after the heat shock (min); ordinate: (left, upper) MDH (A-A); (right, upper) succinate-cytochrome c reductase (O-Cl); (left, lower) SDH (X-X); (right, lower) rotenone-insensitive NADH-cytochrome c reductase (A-A). Specific activity of mitochondrial enzymes through the heat-shocked cell cycle. All specific activities are expressed as nmole/min/mg total cellular protein. Error range bars indicate 1 SE; solid blocks, heat shocks.
+ - 4.0 ,J +----+-+ 1 D
2
tivities by the method indicated. Initial rates were utilized in all cases. All assays were tested for their dependence on substrate and their response to specific inhibitors as indicated. Background rates, if any, were subtracted in all cases. The assays were tested for response to increasing enzyme concentration and utilized in the linear portion of their range. The observed specific activities of the various enzymes are within the ranges reported in the literature [21, 301. Malate dehydrogenase (EC 1.1.1.37; L-malate: NAD oxidoreductase) was assayed according to Ochoa [23] as modified by Poole et al. [26]. The reaction mixture contained 37 mM MgCI,, 8.3 mM oxaloacetic acid, 32 PM NADH and 20 mM potassium phosphate buffer pH 7.4. The reaction was initiated by the addition of the oxaloacetic acid and monitored by the loss of absorbance for NADH at 340 nm. An extinction coefficient of 6 200 cm-’ mole-’ liter was utilized. Rotenone-insensitive NADH-cytochrome c reductase (EC 1.6.2.2; NADH,: cytochrome b, oxidoreductase) was assayed according to Mahler [I81 and
- 2.0
K+
_220 - 180 / -Lx-x_,. 140 -120
F
-220 _ 180 160
+ 0
/y ..-.-.IO
k-r zoo
I20 300
Fig. S. Abscissa: time after the sixth heat shock (min); ordinate: (A) (right) division index (%) (0-O); (left) no. of cellslmlx 10e3 (Cm); (B) mg total cellular protein/IO ml culture; (C) succinate
Experimental
Cell Research
112 (1978) 79-87
THE FORMATION OF SEVERAL MITOCHONDRIAL ENZYMES DURING THE CELL CYCLE IN HEAT-SYNCHRONIZED TETRAHYMENA PYRIFORMIS ST A. E. COWAN and P. G. YOUNG Department
of Biology,
Queen’s
University,
Kingston,
Ont. K7L 3N6,
Canada
SUMMARY The formation of a number of mitochondrial marker enzymes has been studied during the cell cycle in heat-synchronized Tetrahymena pyriformis ST. Markers for the inner mitochondrial membrane (succinic dehydrogenase and succinate-cytochrome c reductase) and for the matrix (malate dehydrogenase) were found to increase exponentially through the heat-shocked cell cycle. Their specific activity remained constant throughout the cycle. An outer membrane and endoplasmic reticulum marker (rotenone-insensitive NADH-cytochrome c reductase) increased stepwise in close parallel to the time of increase in cell number under these conditions. During free-running divisions, following the cessation of heat shocks, all four enzymes retained the same mode of increase which they displayed during heat-shocked divisions. The step for rotenone-insensitive NADH-cytochrome c reductase, however, retained the timing characteristic of the heat-shocked cycle and increased independently of cell division. These results are correlated with available morphometric information on the growth and division of mitochondria in this system.
The vast majority of mitochondrial enzymes are synthesized on cytoplasmic ribosomes and are under the control of the nucleus. Several mitochondrial components, however, are coded for by mitochondrial DNA (for reviews see [l, 3, 28, 291). The co-ordination of the activities of these two separate genomes within the cell is poorly understood. Under conditions of balanced growth all mitochondrial components should double during the course of each cell cycle. Information relating to the manner in which they do so, however, is fragmentary and often contradictory. A number of mitochondrial enzyme activities have been followed through the cell cycle including some which are completely coded for in the nucleus (e.g. malate dehydrogenase, fumarase, succinic dehydro6-771809
genase [5, 8, 9, 12, 24, 25, 311) and some which require the assembly of polypeptides synthesized by both genomes (e.g. succinate cytochrome c reductase, cytochrome oxidase [5, 6, 9, 15, 24-26, 311). No consistent picture emerges, however, in terms of whether or not the synthesis of a particular enzyme is continuous or periodic during the cell cycle or, if periodic, whether it presents a step or a peak pattern. Difliculties occur in comparing the various systems due to differences in cell type and in synchronization procedure. In addition, a number of publications present insufficient data points to be certain of the cell cycle pattern. It appears to be impractical to generalize among these various systems at the present time. Recently the growth and division of mitoExpcrllres
112 (1978)
Cowan
80
and Young
IO< 1
60-
ous within the cell. In this communication we have undertaken to correlate the synthesis of some mitochondrial enzymes with this synchronous mitochondrial division. Portions of this work have appeared in abstract form [7].
60 60
40
20
i0
IO 0 IO 0 0 ‘,I40
0
- 120 ~100 -80 -60 -40
METHODS
AND
MATERIALS
Cells and synchronization procedures Tetrahymena pyriformis ST was maintained at 28°C in Neff s medium [22]. Synchrony was induced using the one heat shock per generation technique of Zeuthen [32]. Heat shocks of 34°C for 39 min (time is nominal machine switching time, not the time at 34°C in the flask) were administered at intervals of 174 min, to give an overall cycle of 213 min. This system achieved division indices as high as 80 % at about 85-95 min following the heat shock. Cells were normally used for experimental purposes following the sixth or seventh heat shock.
Harvesting of cells
-20
Repeated differential washing of the cell sample was found to give variable recovery. The following method was instituted as an easy, rapid and quantitative washing procedure for Tetruhymena cells. An aliquot I 200 300 400 0 100 of 10-30 ml of cell culture was overlain onto a 1.5ml cushion of ice-cold homogenization medium (0.35 M Fig. I. Abscissa: time after the sixth heat shock sucrose, 0.001 M ethylenediaminetetraacetic acid (min); ordinate: (left, inner, upper) division index (%) 0.01 M Tris(hydroxymethy1) aminomethane, (0-O); (right, inner, upper) no. of cellslmlx 10m3 (EDTA), pH 7) in a 50 ml conical centrifuge tube. The cells (Cm); (left, outer, upper) MDH act. (A-A); (right, were centrifuged at 800 g for 3 min in an International outer) SDH act. (X-X); (left, inner, lower) sucdnatecentrifuge (Model V). The medium and washing solucytochrome c reductase act. (0-O); (right, inner, tion were then withdrawn by suction. Cells prepared lower) rotenone-insensitive NADH-cytochrome c rein this way are effectively freed of culture medium by ductase act. (A-A); (left, outer, lower) mg cellular passage through the cushion and require no further protein/ 10 ml culture (+-+). washes. Patterns of mitochondrial enzyme activity through the cell cycle during heat-shock synchrony. All enzyme activities are expressed as nmoles/min/lO ml Homogenization culture. Most points represent the average of duplicate Small cell samples were transferred to a 1 ml tuberdeterminations. Solid blocks indicate the time of the culin syringe and the volume made up to the 1 ml heat shocks. mark with homogenization medium (actually equivalent to 1.05 ml allowing for the volume within the needle). The cells were then broken by passage chondria in heat-synchronized Tetrahythrough a 26-gauge syringe needle at WC. Complete breakage required 5-6 passes. The homogenate was menu has been studied utilizing morphothen sampled for enzyme assays. Coupled with the metric techniques [ 131. The mitochondria washing procedure this system is rapid and simple, were found to develop synchronously and and it allowed for a quantitative recovery of cellular materials. It enabled frequent samples to be taken durto divide during the heat shock. This sys- ing the cell cycle. For the preparation of mitochondria from larger volumes of culture the cells were tem seems to be excellent for studying mitohomogenized using a hand emulsifier (Fisher).
chondrial cell cycle events due to the facility with which synchrony can be induced and the knowledge that growth and division of the mitochondria are synchronEXP cd
res I12
(1978)
Enzyme assays Aliquots of crude homogenates were assayed spectrophotometrically (Gilford) for the following enzyme ac-
Mitochondria
501 o-
40 O60 55 50 !5 20
6 O-
5 O-
4 O-6 i0
i0
in Tetrahymena
81
Lloyd et al. [16]. The reaction mixture contained 0.3 mg/ml cytochrome c (equine, Sigma), 10 mM KCN, 0.19 mM NADH, 2.5 PM rotenone and 20 mM potassium phosphate buffer, pH 7.4. The reaction was initiated with NADH and the increase in absorbance for cytochrome c at 550 nm was measured. An extinction coefficient of 18500 cm-’ mole-’ liter was used. Succinic dehydrogenase (EC 1.3.99.1; succinate: (acceptor) oxidoreductase) was assayed according to Redfearn & Dixon [27] as modified by Muller et al. [21]. The reaction mixture contained 10 mM succinic acid (pH 7.4 with KOH), 32 I.~M dichlorophenolindophenol (DCPIP), 1 mM phenazine methosulphate (PMS) and 20 mM potassium phosphate buffer, pH 7.4. DCPIP was added for a 15 min incubation and then PMS was added to intiate the reaction. The decrease in absorbance for DCPIP at 600 nm was monitored (extinction coefftcient, 15 600 cm-’ mole-’ liter). Samples used for this assay had been quick frozen in dry ice-acetone and stored at -20°C for several days. Succinate-cytochrome c reductase (EC 1.3.99.-; succinate: cytochrome c oxidoreductase) was assayed according to Mahler [ 181 as modified by Turner et al. [30]. The reaction contained 0.3 mg/ml equine cytochrome c, IO mM succinate, 10 mM KCN and 20
10
50 L
I
0
50
1
100
8
IS0
I
200
B
_6.0
J
Fig. 2. Abscissa: time after the heat shock (min); ordinate: (left, upper) MDH (A-A); (right, upper) succinate-cytochrome c reductase (O-Cl); (left, lower) SDH (X-X); (right, lower) rotenone-insensitive NADH-cytochrome c reductase (A-A). Specific activity of mitochondrial enzymes through the heat-shocked cell cycle. All specific activities are expressed as nmole/min/mg total cellular protein. Error range bars indicate 1 SE; solid blocks, heat shocks.
+ - 4.0 ,J +----+-+ 1 D
2
tivities by the method indicated. Initial rates were utilized in all cases. All assays were tested for their dependence on substrate and their response to specific inhibitors as indicated. Background rates, if any, were subtracted in all cases. The assays were tested for response to increasing enzyme concentration and utilized in the linear portion of their range. The observed specific activities of the various enzymes are within the ranges reported in the literature [21, 301. Malate dehydrogenase (EC 1.1.1.37; L-malate: NAD oxidoreductase) was assayed according to Ochoa [23] as modified by Poole et al. [26]. The reaction mixture contained 37 mM MgCI,, 8.3 mM oxaloacetic acid, 32 PM NADH and 20 mM potassium phosphate buffer pH 7.4. The reaction was initiated by the addition of the oxaloacetic acid and monitored by the loss of absorbance for NADH at 340 nm. An extinction coefficient of 6 200 cm-’ mole-’ liter was utilized. Rotenone-insensitive NADH-cytochrome c reductase (EC 1.6.2.2; NADH,: cytochrome b, oxidoreductase) was assayed according to Mahler [I81 and
- 2.0
K+
_220 - 180 / -Lx-x_,. 140 -120
F
-220 _ 180 160
+ 0
/y ..-.-.IO
k-r zoo
I20 300
Fig. S. Abscissa: time after the sixth heat shock (min); ordinate: (A) (right) division index (%) (0-O); (left) no. of cellslmlx 10e3 (Cm); (B) mg total cellular protein/IO ml culture; (C) succinate
