THE ENERGETICS OF MAMMALIAN CELL GROWTH

Size: px

Start display at page:

Download "THE ENERGETICS OF MAMMALIAN CELL GROWTH"

Hortense Butler
5 years ago
Views:

1 J. Cell Sci. 4, (1969) 645 Printed in Great Britain THE ENERGETICS OF MAMMALIAN CELL GROWTH D. G. KILBURN*, M. D. LILLY AND F. C. WEBB Biochemical Engineering Section, Department of Chemical Engineering, University College, London, England SUMMARY Data from batch growth curves of mouse LS cells cultivated at controlled dissolved oxygen partial pressures were used to calculate the weight of cells produced per mole of adenosine triphosphate generated (Y Arv ). These values agree well with those reported for bacteria. A theoretical relationship was developed which allowed the biosynthetic and maintenance energy requirements to be estimated. The biosynthesis of LS cells required i-6 x io" 11 moles of ATP/cell. The maintenance energy, which is a function of growth rate, was 2-9 x io"" 11 moles ATP/new cell when the mean generation time was 1-15 days. The proportion of the total energy used for maintenance under these conditions was 65 %. This corresponds to a value of less than 10% for bacterial maintenance when the organisms are grown at near their maximum rate. A comparison of biosynthetic energy requirements indicates that bacteria and moulds require about 4 times as much energy as animal cells to generate the same weight of cell material. Possible explanations of this difference are discussed. INTRODUCTION In recent years attempts have been made to relate the mass of bacterial cells produced to the number of moles of adenosine triphosphate (ATP) estimated to be formed by the utilization of the energy-producing substrate (Bauchop & Elsden, i960; Gunsalus & Shuster, 1961). The rationale behind this approach was outlined by Senez (1962), who argued that since ATP is apparently used directly or indirectly in all the major energy-consuming reactions of the cell, it represents the best estimate of the useful free energy available to the cell. When bacteria were grown in complex media containing the amino acids and other monomers needed for growth, and the oxidizable substrate served only as an energy source, it was found that 1 mole of ATP yielded an average of 10-5 g dry weight of cells. Some workers have considered ATP yield (Y A1:P ) t0 be a biological constant but in fact it must depend on the conditions of growth. The biosynthetic requirements of the cell are thought to account mainly for Y ATP values and most of the reported values have been determined under growth conditions in which maintenance energy has been almost negligible. But Y ATP would be expected to be different if the maintenance energy (i.e. energy consumption not associated with growth) represented a large proportion of the total energy requirements. In animal cells the maintenance energy is thought to be substantial; Paul (1965) Present address: Department of Microbiology, University of British Columbia, Vancouver 8, B.C., Canada.

2 646 D. G. Kilburn, M. D. Lilly and F. C. Webb has estimated that the sodium pump alone consumes 20% of the energy available to the cell. If bacteria and animal cells have the same efficiency of biosynthesis one would expect the Y ATP f animal cells to be lower than 10 g dry weight per mole ATP. Our studies on the growth of mouse LS cells at controlled dissolved oxygen partial pressures (j>0 2 ) have permitted us to estimate independently both Y ATP and maintenance energy. This has led to the paradoxical conclusion that although the maintenance energy of animal cells is about 10 times that of bacteria (both growing at maximum rates) the Y ATP f these organisms is identical. These results, the methods used to obtain them and a discussion of the significance of the findings form the subject of this paper. THEORY Determination of A TP yield The Y ATF value of 10-5 g cells per mole ATP for bacteria was determined from anaerobically grown cultures in which the amount of ATP generated by fermentation of the energy-producing substrate is known (Bauchop & Elsden, i960). For aerobic growth of bacteria, however, there is some difficulty in calculating Y. KTP because the phosphorylation ratio (P/O) is not definitely known. This problem does not prevent an analysis of the energetics of animal cell growth, because oxidative phosphorylation in mammalian mitochondria has been studied extensively and a P/O ratio of 3 for NADH oxidation seems well documented (Mahler & Cordes, 1966). In animal cells, ATP is produced primarily in the conversion of glucose to pyruvate and in the oxidation of pyruvate to CO 2 and water via the tricarboxylic acid (TCA) cycle (i.e. aerobic metabolism). During anaerobic metabolism pyruvate is reduced to lactate, and ATP is produced only by substrate-level phosphorylation. In general the total energy production is the sum of the contributions from both aerobic and anaerobic processes, and the following stoichiometric relationships are generally accepted glucose + 6O ADP + 38P -> 6CO 2 + 6H 2 O + 38ATP glucose + 2ADP + 2P -> 2 lactate + 2ATP. Provided that other decarboxylation reactions are negligible, the ATP generated during cultivation can be calculated from the CO 2 and lactate produced ATP formed (moles) = -- CO 2 (moles) + lactate (moles). Determination of maintenance energy The energy utilized by a microbial cell can be considered to consist of two components: (i) a steady rate required to maintain concentration gradients, internal organization, repair of degraded molecules and like functions, i.e. maintenance energy; and (ii) a rate associated with the biosynthesis of new cell material required for multiplication, i.e. biosynthetic energy; the first will depend on the number

3 Energetics of mammalian cell growth 647 (or weight) of cells present, while the second will depend upon the rate at which the cells are growing. It is assumed that the rate of energy generation in terms of ATP is equivalent to its rate of utilization. The over-all rate of energy utilization can be expressed by the equation de Adx - = mx + A^, (1) where dejdt = the total rate of energy expenditure (moles ATP/litre of culture/day), m = specific maintenance energy rate (moles ATP/cell/day), x = cell concentration (no. of cells/litre of culture, provided that the average weight per cell is not changing), A = specific biosynthetic energy rate (moles ATP/cell). This expression forms the basis of the techniques used by Marr, Nilson & Clark (1963) and by Pirt (1965) to estimate the maintenance energy of bacteria. These workers formulated the equation in terms of energy-producing substrate consumed rather than ATP generated. During the exponential phase of cell growth where /.(, = specific growth rate. Integrating equation (2), J t =»X, (2) In* = pt+c, x = Ce>". (3) Applying the boundary conditions, x = x 0 at t = o, x = x o e>». (4) Substituting for dx/dt from equation (2) and for x from equation (4) in the energy rate equation (1), -j t = mx + Afix = (m + A/i)x, = (m + A/i)x o e" 1. (5) Integrating this expression, E = (m + A/.*) {x o {fi) e' a + C. (6) Applying the boundary conditions E = o at t = o, then C = -(tn + A/i)(x o lfi). Therefore, E = (m + Afi)(x o lfc)(e' u -1). (7) The integrated form of equation (1) expresses the relationship between energy generation and time during the exponential phase of a batch culture. A plot of E against (e''' i) should give a straight line passing through the origin with slope = (m + A/,t)(x Q l/.i), provided that at the arbitrary point chosen for E = o, and t = o, the culture is in exponential growth. It is possible by growing cultures at different controlled po 2 levels to alter the

4 648 D. G. Kilburn, M. D. Lilly and F. C. Webb exponential growth rate /t (Kilburn, Lilly, Self & Webb, 1968). An analysis of the rate of ATP generation in several such batch cultures allows the values of m and A to be estimated. PROCEDURE All experiments utilized 3-I. suspension cultures grown at C in Eagle's minimal essential medium, supplemented with 2-5 g/1. lactalbumin hydrolysate, 1 g/1. carboxymethyl cellulose and 2% (v/v) horse serum. The glucose concentration of the medium was increased to 2 g/1. The ph was controlled at by the automatic addition of 0-5 M NaOH. Liquid phase po 2 was measured by an oxygen electrode and controlled by admitting pulses of oxygen to the gas space of the vessel. Details of the culture vessel, the control systems for ph and po 2, the measurement of liquid and gas phase pco 2 and the general culture method have been presented elsewhere (Kilburn & Webb, 1968). The measured CO 2 output was found to agree well with oxygen uptake (Kilburn et al. 1968), indicating that decarboxylation reactions other than those associated with energy metabolism were negligible. RESULTS ATP yield In Table 1 the cumulative amount of ATP produced during a batch culture without po z control is compared with the dry weight of cells produced (sample dry weight minus initial dry weight after inoculation). The figures for ATP produced were calculated from the concentrations of CO 2 and lactate. Table 1. Calculated -values of F ATP during a batch culture without po^ control Sample Net dry wt. (g/1.) Cumulative ATP (mole/1.) ^ATP = g dry wt./ mole ATP O-II O O-OI2 O-O O-O Table 2. Calculated values of Y A1:F at the maximum cell count for batch cultures at controlled po z Controlled po 2 (mmhg) i-6 Y ATP, g dry wt./mole ATP 8-o These values of Y ATP agree well with the average value of 10-5 g/mole ATP reported for bacteria. The dry weights for the samples were based on the measured dry weight of sample 6, but since the size of cell decreases slightly during the growth cycle, the estimated dry weights of the initial samples may be slightly low.

5 Energetics of mammalian cell growth 649 Table 2 shows Y ATP for the batch cultures at controlled values of po 2 calculated as previously, but only at the peak of the growth curve, where the dry weight was determined. These values for Y AT1? of LS cells are similar to those reported for bacterial cells. Energy utilization Equation (7) was derived to express the relationship between energy generated and time during the exponential growth phase of a batch culture. A distinction was made between the energy used for biosynthesis and that used for maintenance, or more precisely between growth-associated and non-growth-associated energy utilization. According to equation (7) a graph of ATP generated (the cumulative value calculated from the lactate and CO 2 production) against e' 1 ' 1 for cells growing at different rates should still give a straight line passing through the origin with the slope = (m + A/i)(x o lii). Thus, rearranging (slope) = m + /ua. (8) Fig. 1. Energy produced in batch cultures at controlled dissolved oxygen partial pressures plotted against ef' i:, po 2 = 16 mmhg, x 0 = 27x10' cells/1., /t = 0-30 day- 1 ;, po., = i6ommhg, x 0 = 2-8 xio 8 cells/1., fi = 0-53 day" 1 ; D, po t = 96 mmhg, x 0 = 2-4 x io 8 cells/1., /i = o-6o day" 1 ; A, po., = 12 mmhg, #o = i-6 x io 8 cells/1., /* = 0-65 day" Cell Sci. 4

6 650 D. G. Kilburn, M. D. Lilly and F. C. Webb In each case x 0 and /i are known, If the values of the left-hand side of the equation are calculated and plotted against fi a straight line should be obtained, having a slope equal to A. The value of m may be obtained by extrapolation to /t = o. Figure 1 shows values of ATP generated plotted against (e' u 1) for cultures grown at four different dissolved oxygen partial pressures. In this way the term /t/.v 0 (slope) in (8) was obtained for six cultures and plotted against [i (Fig. 2). From this graph as described above, m = 17 x icr 11 moles ATP/cell day and A = 2-3 x moles ATP/cell Specific growth rate (/t, day- 1 ) 08 Fig. 2. Plot of specific growth rate, fi, against the term fi/x 0 (slope) from equation (8). The value of the slope is taken from the lines in Fig. i ; x 0 is the cell concentration at the beginning of exponential growth. An independent estimate of m can be obtained from the rate of ATP production during the stationary phase of the growth cycle. However the stationary phase, except at 320 mmhg, was too short for accurate measurement of ATP production. Thus during the period over which the measurements shown in Table 3 were made there was usually a decrease in viable cell count. Such estimates cannot be accepted without reservation, because it is suspected that at this stage in a culture energy generation may be partially uncoupled from NADH oxidation (Paul, 1965). Neverthe-

7 Energetics of mammalian cell growth 651 less it can be seen that the value of m obtained by solving the rate equation for the exponential growth phase is only slightly lower than the values calculated from the rate of ATP production during the stationary phase. The value of m for the 320 mmhg po % culture is a special case, made up of a true m and a biosynthetic energy rate constant. Cell division was inhibited during the interval selected, but the cell size increased. It is also possible that the actual maintenance requirement of the cell was increased by the need to maintain itself in a partially reduced state in a highly oxidizing atmosphere. For the subsequent discussion, it will be assumed that m = 1*7 x io~ u moles ATP/cell/day, A = 2-3 x moles ATP/cell. Table 3. Estimates of the maintenance energy rate constant (m) from stationary growth phase energy generation Culture po a (mmhg) i-6 Time interval (days) 2-0 io" u moles ATP/cell o-8 2-O i6o O 3 no po % control i-o 2-0 DISCUSSION It has been found that the Y ATr of LS cells closely approximates the values reported for bacterial cells. This observation indicates that the over-all efficiency of energy utilization is identical in widely different organisms which would seem to illustrate similarities underlying biological processes as a whole. However, when the distribution of energy between biosynthesis and maintenance is considered and the absolute values are estimated, it can be shown that the correspondence of Y ATP values for bacterial and animal cells is probably fortuitous and, rather than illustrating the unity of metabolic processes, it hides some significant differences. Energy required for cell syntjiesis In equation (5) the term fia is the specific rate of energy utilization for biosynthesis. The average time for the synthesis of a new cell is the mean generation time which is numerically equal to In2//t. Thus the mean value for the energy required to synthesize one cell is /ia\r\2j/j. = A In2 = i-6 x io~ u moles of ATP/cell. Paul (1965) estimated that the minimum ATP requirement for the synthesis of a single L cell was 0-87 x io~ u moles (including turnover of protein and nucleic acid, but assuming that messenger RNA is conserved). Paul's value is increased to 1-15 x io~ n moles ATP/cell if his calculation is repeated using the cell dry weight found in the present work (i.e. 6-6 x io" 10 g/cell rather than 5-0 x io" 10 g/cell). Hence there is good agreement between the purely theoretical figure and that derived from the experimental data. The energy required to synthesize rog dry weight of cells is the biosynthetic 41-2

8 652 D. G. Kilburn, M. D. Lilly and F. C. Webb energy per cell times the number of cells per g, i.e. (i-6 x io~ u )(i'5 x io 9 ) = moles ATP. The maximum possible Y ATP ^ no energy is used for maintenance is thus 1/0-024 = 42 g cells/mole ATP. Maintenance energy If it is assumed that the weight of a single growing cell increases linearly and that the maintenance energy is proportional to the cell weight, then during the time in which a new cell is synthesized maintenance energy must be provided on the average for 1-5 cells. If the doubling time is 1-15 days, the maintenance expenditure in the net synthesis of one cell is ( I# S) ( I>I S) ( I- 7 x JO" 11 ) = 2-9 x io~ u moles ATP. On this basis the net synthesis of 1 g dry weight of cells requires moles ATP for maintenance functions. Over-all cell yield The total ATP required during the synthesis of 1 g dry weight of cells is the sum of the maintenance energy and biosynthetic energy consumed during the time taken for synthesis, i.e = moles ATP/g cells. The Y ATP is therefore 1/0-068 = 14-7 g cells/mole ATP, which is consistent with the experimental values (Tables 1, 2). Table 4. Comparison of maintenance energy requirements moles ATP/g Assumed moles Organism dry wt./day ATP/mole glucose Reference Aerobacter aerogenes Pirt (1965) Pemcillium chrysogenum Mouse LS cells o-n Righelato et al. (1968) Present work In Table 4 the specific maintenance energy requirement of mouse LS cells is compared with values given in the literature for Aerobacter aerogenes and Penicillium chrysogenum. The maintenance energy of the bacterium is about 4 times that of the mould and about 10 times that of the animal cell. This seems reasonable considering the higher surface area per g of bacteria which would increase the energy needed to maintain concentration gradients. Although the absolute value of the maintenance energy for bacteria is higher than for animal cells, bacteria use a smaller proportion of their total energy for maintenance. The proportion of the total energy generated that is expended in maintenance will depend on the specific growth rate, and will be smallest at the highest /*. From the values calculated earlier for LS cells growing at /.i = o-6o day" 1, the maintenance energy represents ^t(ioo) = 65% of the total energy expenditure.

Energetics of mammalian cell growth 653 From the information given by Pirt (1965), it can be calculated that at a specific growth rate of 0-5 h" 1 (i.e. near the maximum), the maintenance energy of Aerobacter cloacae represents 7 % of the total energy consumption.

9 Energetics of mammalian cell growth 653 From the information given by Pirt (1965), it can be calculated that at a specific growth rate of 0-5 h" 1 (i.e. near the maximum), the maintenance energy of Aerobacter cloacae represents 7 % of the total energy consumption. The reason for this difference in maintenance expenditure between animal cells and bacteria arises, of course, from the higher growth rate of bacteria. Although the magnitude of the animal cell maintenance energy is smaller, it must be expended over a much longer time. If the growth rate of a bacterium could be slowed to that of an animal cell, its maintenance energy would consume almost 90% of the available energy. The biosynthetic energy requirement of bacteria can be calculated from the Y ATP (i.e g dry wt/mole ATP). Assuming that 90% of the Y ATV is biosynthetic energy, the ATP (biosynthesis) is mole ATP/g dry wt. Righelato, Trinci, Pirt & Peat (1968) determined the biosynthetic component of Penicillium chrysogenum in terms of oxygen uptake. If a phosphorylation ratio (P/O) of 3 is assumed, this is equivalent to 0-12 moles ATP per g dry weight or approximately the same value as calculated for bacteria. For LS cells, the ATP (biosynthesis) is mole ATP/g dry wt. Thus bacteria and moulds require about 4 times the energy used by animal cells to synthesize cell material. In view of this it is surprising that the F ATP values are almost identical. It appears that the higher maintenance expenditure of animal cells compensates for their energy advantage in biosynthesis. The apparent inefficiency of biosynthesis in bacteria and moulds might arise from a higher turnover rate of constituents such as m-rna. Messengers in higher organisms are thought to be quite stable; once the m-rna ribosome complex is formed, proteins may be turned out continuously perhaps for days. The biosynthetic energy requirement found for LS cells is consistent with m-rna conservation. In bacteria, however, m-rna is probably very unstable with a half-life of at most several minutes (Mahler & Cordes, 1966). While at first sight this seems to account for the inconsistency between the biosynthetic requirements of lower and higher organisms, the work of Salser, Janin & Levinthal (1968) indicates that despite their short life bacterial messengers are read repeatedly (30 60 times) and their resynthesis represents only a small part of the total energy expended in protein synthesis. One must thus seek some other explanation for the large biosynthetic energy requirement of bacteria. It is conceivable that bacteria waste more ATP than animal cells because of some growth-associated, but unproductive, ATPase system. Alternatively, bacteria may expend a substantial amount of energy on active transport of monomers which, because of the favourable concentration gradients and low rate of demand, can enter animal cells by diffusion without the need of energy expenditure. REFERENCES BAUCHOP, T. & ELSDEN, S. R. (i960). The growth of microorganisms in relation to their energy supply. J. gen. Microbiol. 23, GUNSALUS, I. C. & SHUSTER, C. U. (1961). Energy-yielding metabolism in bacteria. In The Bacteria, vol. 2 (ed. I. C. Gunsalus & R. Y. Stanier), pp New York: Academic Press.

10 654 D - G - Kilburn, M. D. Lilly and F. C. Webb KILBURN, D. G., LILLY, M D. SELF. D. A. & WEBB, F. C. (1968). The effect of dissolved oxygen partial pressure on the growth and carbohydrate metabolism of mouse LS cells. J. 'CM Sci. 4, KILBURN, D. G. & WEBB, F. C. (1968). The cultivation of animal cells at controlled dissolved oxygen partial pressure. Biotedinol. Bioeng 10, MAHLER, H. R. & CORDES, E. H. (1966). Biological Chemistry, pp New York: Harper & Row. MARR, A. G., NILSON, E. H. & CLARK, D. J. (1963). The maintenance requirement of Escherichia coli. Ann. N.Y. Acad. Sci. 102, PAUL, J. (1965). Carbohydrate and energy metabolism. In Cells and Tissues in Culture, vol. 1 (ed. E. N. Willmer), pp New York: Academic Press. PIRT, S. J. (1965). The maintenance energy of bacteria in growing cultures. Proc. R. Soc. B 163, RICHELATO, R. C, TRINCI, A. P. J., PIRT, S. J. & PEAT, A. (1968). The influence of maintenance energy and growth rate on the metabolic activity, morphology and conidiation of Penicillium chrysogenum. J. gen. Microbiol. 50, SALSER, W., JANIN, J. & LEVINTHAL, C. (1968). Measurement of the unstable RNA in exponentially growing cultures of Bacillus subtilis and Escherichia coli. J. vwlec. Biol. 31, SENEZ, J. C. (1962). Some considerations on the energetics of bacterial growth. Bad. Rev. 26, 9S-IO7- (Received 10 October 1968)

In glycolysis, glucose is converted to pyruvate. If the pyruvate is reduced to lactate, the pathway does not require O 2 and is called anaerobic

Glycolysis 1 In glycolysis, glucose is converted to pyruvate. If the pyruvate is reduced to lactate, the pathway does not require O 2 and is called anaerobic glycolysis. If this pyruvate is converted instead