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Energy metabolism in developing Ascaris lumbricoides eggs
DEVELOPMENTAL
BIOLOGY
Energy
42, 188-195 (1975)
Metabolism II. The Steady
in Developing State Content
Ascaris
lumbricoides
of Intermediary
Eggs
Metabolites
I.BEIS AND J. BARRETT’ Department
of Zoology, Accepted
University
of Oxford,
September
Oxford, England
16, 1974
The steady state levels of intermediary metabolites were measured in freeze clamped, developing, dormant, and activated infective Ascaris lumbricoides eggs. The [ATPy[ADP] ratio is low in the developmental stages and rises sharply in the dormant egg; on activation of the dormant egg the [ATP]/[ADP] ratio falls. The levels of the phosphorylated glycolytic intermediates of acetyl-CoA and of isocitrate do not change markedly during development, but the levels of lactate, citrate, P-oxoglutarate, glutamate, succinate, and malate all show significant changes in the developing, dormant, and activated egg. The dormant egg also appears to be characterized by a low cytoplasmic redox potential. INTRODUCTION
The changes in metabolic rate which occur during the development of the eggs of Ascaris lum bricoides cannot be adequately explained by changes in the maximum catalytic capacities of the major pathways of lipid or carbohydrate catabolism (Barrett and Beis, 1975). The changes in QO, during development could be due to the inhibition of one or more of the key regulatory enzymes of lipid or carbohydrate catabolism. Inhibition of the regulatory enzymes might be brought about by the production of a specific inhibitory compound or by changes in the levels of the intracellular metabolites; alternatively, the properties of the regulatory enzymes themselves might change during development such that in the infective egg the metabolic pathways were inhibited by metabolite levels which did not affect the earlier developmental stages. In this paper the steady state levels of intermediary metabolites have been measured in freeze clamped developing, dormant, and activated infective Ascaris eggs. MATERIALS
Ascaris
AND
METHODS
eggs were prepared
and em-
’ Present address, Department of Zoology, University College of Wales, Aberystwyth, Wales. 188 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved,
bryonated as described by Ward and Fairbairn (1970). The eggs were incubated at 30°C and all times refer to development at this temperature. The dormant infective eggs were hatched (activated) as described by Fairbairn (1961). Metabolite
Assays
Egg suspensions were freeze clamped between aluminum blocks cooled in liquid nitrogen (Wollenberger et al., 1960). The frozen tissue was pulverized in a percussion mortar and a neutral perchlorate extract prepared (Adam, 1963a). The metabolites were assayed enzymatically with a Gilford Model 240 recording spectrophotometer, using the following methods: ATP, Lamprecht and Trautschold (1963); ADP and AMP, Adam (1963b); glucose-6-phosphate and fructose-6-phosphate, Hohorst (1963a); dihydroxyacetone phosphate and fructose-1,6-diphosphate, Biicher and Hohorst (1963); 3-phosphoglycerate, Czok and Eckert (1963); pyruvate, Biicher et al. (1963) (phosphoenolpyruvate was measured in the same cuvette by the addition of ADP and pyruvate kinase); malate and lactate, Hohorst (1963b,c); citrate, Dagley (1963); isocitrate, Siebert (1963); glutamate, Bernt and Bergmeyer (1963); Z-oxoglutarate, Bergmeyer and Bernt (1963);
BEIS AND BARRETT
Energy Metabolism
acetyl-CoA, Decker (1963); succinate, Kmetic (1966); arginine phosphate was determined as for creatine phosphate, but using arginine kinase instead of creatine kinase (Lamprecht and Stein, 1963). Arylamine transacetylase was prepared from pigeon livers by the method of Wieland et al. (1972); arginine kinase (prepared from lobster muscle) was the gift of Dr. A. R. Leech; all other coupling enzymes were purchased from the Boehringer Corp. Ltd. Inorganic phosphate (Pi) was measured in unneutralized perchlorate extracts by the method of Allen (1940). Enzyme Assays
The enzyme assays were performed at 30°C in a Gilford Model 240 recording spectrophotometer, using 1 ml semimicro cells. The eggs were homogenized in 0.1 M Tris-Cl buffer, pH 7.6; 0.09 M KCl; 0.028 M MgSO,, using a l-ml ground glass homogenizer. The homogenizer was cooled in ice and the progress of homogenization was checked under the microscope. The homogenate was centrifuged for 5 min at 700 g at 2°C and the supernatant fraction used for enzyme assays. Adenylate kinase (EC 2.7.4.3), nucleosidediphosphate kinase (EC 2.7.4.6), and arginine kinase (EC 2.7.3.3) were assayed as described by Barrett (1973). The total ATPase activity of the homogenate was estimated from the rate of activity in the nucleosidediphosphate kinase assay before the addition of dGDP. All enzyme activities are expressed as nmoles/min/mg protein. Protein was measured by the method of Lowry et al. (1951); the protein precipitates from the perchlorate extracts were dissolved by heating in 1 N NaOH. Standard errors were calculated as described by Dean and Dixon (1951). Respiratory
Rates
Oxygen uptake was measured in a Yellow Springs Model 53 Oxygen monitor, using a final volume of 4 ml. Rat liver and
in Ascaris Eggs. II
189
Ascaris eggs were homogenized in 0.25 M
sucrose, 1 mM EDTA, pH 7.4; rat liver mitochondria were prepared in isotonic sucrose by the method of Schneider (1957). All additions were made in neutral solution, with the exception of the 2,4-dinitrophenol experiments where the eggs were suspended in 0.01 N H,SO,. This enabled the 2,4-dinitrophenol to penetrate the vitelline membrane, which is impermeable to ionized compounds. RESULTS
During the development of Ascaris eggs there are considerable changes in the steady state levels of the metabolic intermediates. One of the most important group of metabolites from the point of view of energy metabolism are the adenylate nucleotides. The steady state levels of ATP, ADP, AMP, and inorganic phosphate (Pi) in developing Ascaris eggs are summarized in Table 1 and the changes in the [ATP y [ADP] ratio in Fig. 1. The attainment of infectivity in Ascaris eggs is accompanied by a sharp rise in the [ATPy[ADP] ratio; in the developing stages the ratio is less than 1 and increases to 5 by the time the infective stage is reached. When the dormant infective eggs are given a hatching stimulus, there is a drop in the [ATPy [ADP] ratio to about 1.7. The changes in the adenylate charge (Atkinson and Walton, 1967) parallel the changes in the [ATPj/[ADP] ratio (Table 1). The levels of inorganic phosphate are the reverse of the [ATP y[ADP] ratio, being high in the developing egg, dropping in the dormant egg, and increasing again on activation. The increase in the [ATPj/[ADP] ratio in the dormant egg is not accompanied by a drop in ATPase activity. The total ATPase activity of egg homogenates increases during development, as does the activity of adenylate kinase and nucleosidediphosphate kinase (Table 2). Neither arginine kinase nor arginine phosphate could be detected by spectrophotometric methods
190
DEVELOPMENTAL
BIOLOGY
TABLE STEADY STATE CONTENT
OF ADENINE
lumbricoides
VOLUME 42. 1975
1
NUCLEOTIDES AND INORGANIC PHOSPHATE IN FREEZE CLAMPED EGGS AT DIFFERENT STAGES OF DEVELOPMENT
Ascaris
flmoles/lOO mg protein”
Age (days)
ATP 0 1 3 5 8 10 14 18 22 50 (dormant) 50 (activated)
1.00 1.55 1.50 0.95 1.00 1.57 2.00 2.80 2.60 1.60 0.71
* * * * * * * * rt i i
ADP
0.05 0.20 0.20 0.15 0.05 0.40 0.24 0.40 0.10 0.20 0.06
1.20 1.70 2.10 2.00 1.70 0.75 0.67 0.70 0.50 0.33 0.41
AMP
* 0.30 + 0.60 * 0.10 * 0.30 * 0.30 * 0.04 z+z0.08 * 0.10 * 0.07 * 0.04 + 0.06
0.06 0.07 0.08 0.08 0.05 0.04 0.05 0.06 0.02 0.06 0.10
+ * + + * * * + * i *
0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.02
Pi
Adenylateb charge
8.1 zt 0.8 8.8 zt 0.4 7.4 * 0.2 4.9 * 0.5 5.0 + 0.8 5.7 * 0.5 4.5 i 0.7 5.2 + 0.5 5.5 zt 0.6 5.0.1 0.9 10.0 * 0.4
0.70 0.72 0.69 0.64 0.67 0.82 0.85 0.88 0.91 0.89 0.75
” Mean + SEM (10 determinations). o Atkinson and Walton (1967).
0
FIG. 1. The [ATPy[ADP] (N = 10).
I 5
I 10
I I5
I 20 DAYS EMBAVONATEO
ratio in developing Ascaris lumbricoides
in Ascaris eggs, at any stage of development. The rate of respiration of infective 22day-old Ascaris eggs was found to be stimulated by 2,4-dinitrophenol. At lo-” and 10m5 M the stimulation was 8 and 25% respectively, at low3 M, 2,4-dinitrophenol inhibited respiration by 25%. The ability of 2,4-dinitrophenol to stimulate respiration
eggs. The vertical
lines represent + SEM
in the dormant infective egg indicates that the maximum capacities of the metabolic pathways are not rate limiting. There is no evidence for any general metabolic inhibitor in the dormant infective egg of Ascaris. Homogenates of dormant eggs do not affect the rate of respiration of homogenates of lo-day Ascaris eggs in the presence or absence of substrates (10
Energy Metabolism
BEIS AND BARRETT
mM glucose or 10 mA4 succinate). Nor do homogenates of dormant eggs affect the rate of oxygen uptake of rat liver homogenates or of isolated rat liver mitochondria in the presence or absence of exogenous substrate (10 mA4 glucose or 10 mM succinate, respectively). The steady state levels of glycolytic and TCA cycle intermediates in freeze clamped Ascaris eggs are shown in Tables 3 and 4, respectively. The developmental stages TABLE
2
SPECIFIC ACTIVITIES OF ADENYLATE KINASE, NUCLEOSIDEDIPHOSPHATEKINASE AND AIIENOSINE TRIPHOSPHATASEIN Ascaris lumbricoides EGGS AT DIFFERENT STAGES OF DEVELOPMENT Age (days)
T
nmoleslmin/mg protein” Adenylate kinase
0 1 3 5 8 10 14 18 22 50 (dormant) 50 (activated)
200 205 260 240 285 300 361 364 376 350
Nucleosidediphosphate kinase
i 18.0 i 9.4 zt 7.5 zt 12.6 zt 13.0 * 17.0 zt 25.0 + 18.0 + 17.0 * 7.0
361 + 15.0
508 632 634 651 638 615 612 612 626 611
i 19 zt 25 zt 12 + 24 zt 28 * 15 + 20 * 24 z+z31 zt 31
594 zt 23
ATPase 7.5 8.3 7.0 8.0 9.6 13.0 17.0 25.0 21.0 25.0
i 0.9 + 1.1 f 0.6 f 1.2 zt 0.8 * 0.5 f 0.8 + 1.4 * 1.3 =+2.0
30.0 f 3.0
L
n Mean + SEM (10 determinations).
chosen were 1 day, when the rate of respiration is low, 10 days when the respiratory rate is maximal, and 22 days, which is the fully developed dormant infective egg (Fairbairn, 1961; Passey and Fairbairn, 1955). The glycolytic intermediates can be divided into four groups; those which remain fairly constant throughout development; those which decrease in the dormant egg and increase again on activation; those which decrease in the dormant egg, but do not increase again on activation; and finally those which increase in the dormant egg and decrease on activation. The levels of fructose-1,6-diphosphate and dihydroxyacetone phosphate remain more or less constant throughout development. In contrast glucose-6-phosphate and fructose6-phosphate levels both increase between days 1 and 10 and then fall in the dormant infective egg; on receipt of the hatching stimulus, the levels of the sugar phosphates increase again. The levels of phosphoenolpyruvate and 3-phosphoglycerate remain fairly constant between day 1 and 10 but decrease in both the dormant and activated infective egg. The levels of pyruvate also drop during development and are low in both the dormant and activated infective egg; in contrast, lactate increases slightly between day 1 and 10, is very high
TABLE
3
STEADY STATE CONTENT OF GLYCOLYTICINTERMEDIATESIN FREEZE CLAMPED Ascaris lumbricoides DIFFERENT STAGES OF DEVELOPMENT
l-Day
a Mean * SEM (eight determinations).
EGGS AT
nmoles/lOO mg protein”
Metabolite
Glucose-6-phosphate Fructose-6-phosphate Fructose-1,6-diphosphate Dihydroxyacetone phosphate 3-Phosphoglycerate Phosphoenolpyruvate Pyruvate Lactate
191
in Ascaris Eggs. ZZ
154 38 15 98 60 134 320 1337
f 15 + 5 * 2 i 20 + 10 19 i 41 f 90
lo-Day 191 45 16 113 58 173 207 1639
i f + * + i i +
18 7 3 20 10 18 27 191
22-Day (dormant) 66 25 18 103 37 89 188 4517
i 6 i 5 zt 2 * 9 * 11 + 9 i 16 i 298
22-Day (activated) 189 47 17 114 38 52 193 1469
17 *3 12 f 50 zt 12 +4 * 30 i 341
192
DEVELOPMENTALBIOLOGY
VOLUME 42, 1975
TABLE 4 STEADY STATE CONTENT OF TCA CYCLE INTERMEDIATESIN FREEZE CLAMPED Ascaris lumbricoides EGGSAT DIFFERENT STAGESOF DEVELOPMENT
Metabolite
nmoles/lOQ mg protein” l-Day
Citrate Isocitrate 2-oxoglutarate Glutamate Succinate Malate Acetyl Co-A
702 zt 35 11 13 36 zt 6 349 * 14 161 f 11 785 + 44 2.3 zt 1.0
lo-Day 994 + 130 21 12 43 * 12 362 + 17 497 zt 185 748 + 71 5.0 * 1.0
22-Day (dormant)
22-Day (activated)
3795 f 311 21 * 2 80 zt 14 410 f 15 363 + 93 217 A 57 2.0 l 1.2
2963 zt 314 26 f 5 50 f 10 341 * 21
555 * 113 285 + 30 3.8 + 1.5
a Mean i- SEM (eight determinations).
in the dormant egg, and drops again on activation. If it can be assumed that lactate and pyruvate are equally distributed throughout the tissues, the lactate dehydrogenase system can be used to calculate the redox state of the free [NAD+ j/ [NADH] couple in the cytoplasm of the developing eggs (Williamson et al., 1967). In the l-day egg the free [NAD+j/[NADH] ratio in the cytoplasm is 2156:l; this drops to 1137:l in the lo-day egg and falls to 3751 in the dormant infective egg. In the activated infective egg, the free [NAD+J/ [NADH] ratio in the cytoplasm again increases to 1183:l. So the dormant infective egg would appear to be characterized by a low cytoplasmic redox potential. The TCA cycle intermediates (Table 4) can be divided into the same four groups as the glycolytic intermediates. The levels of citrate, 2-oxoglutarate, and glutamate increase slightly between day 1 and 10, rise sharply in the dormant egg, and drop again on activation. Malate shows the reverse pattern, decreasing in the dormant egg and increasing slightly on activation. Succinate levels were difficult to measure in Ascaris eggs due to the presence of interfering compounds in the neutral perchlorate extracts, and the results may be unreliable. The succinate levels appear to increase from day 1 to 10 and are then similar to malate in decreasing in the
dormant egg and increasing again on activation. In contrast to the changes in citrate levels in developing eggs, the levels of isocitrate, after increasing between day 1 and 10, remain unchanged in the dormant and activated infective eggs. The levels of acetyl-CoA are extremely low throughout development, and are at the limits of the assay method used. DISCUSSION
The changes in the metabolic rate of developing, dormant, and activated infective Ascaris eggs are accompanied by changes in the steady state levels of the intermediary metabolites. In particular the dormant egg is characterized by a high [ATPv[ADP] ratio and a low cytoplasmic free [NAD+j/[NADH] ratio. The high [ATP1/[ADP] ratio in the dormant egg may at first appear unusual, since it might ’ have been expected that the ATP levels would have been low in a dormant organism. A high [ATPj/[ADP] level is, however, consistent with a low metabolic rate, since phosphofructokinase which is a key regulatory enzyme of glycolysis is inhibited by ATP and this inhibition is released by ADP; isocitrate dehydrogenase is also activated by ADP and both ,&oxidation and oxidative and substrate phosphorylation are tightly coupled to the availability of
BEIS AND
BARRETT
Energy Metabolism
ADP and Pi (Newsholme and Start, 1973). There is no simple overall explanation for the metabolite changes found in the developing, dormant, and activated infective eggs of Ascaris. The changes observed in the levels of the glycolytic intermediates can be grouped into four types (see Results). However, the glycolytic intermediates show no clear-cut crossover points between the active and dormant egg (Williamson, 1965). The same four patterns of change are found in the TCA cycle intermediates. The changes in the levels of citrate, 2-oxoglutarate, and glutamate may be the result of changes in the relative activities of the different catabolic pathways. The increase in the importance of lipid metabolism in the mature egg, combined with a decrease in the capacity of the TCA cycle, could lead to a rise in the levels of intermediates at the beginning of the TCA cycle and to an increase in the levels of lactate. Citrate may also have a regulatory function, since in mammalian systems at least phosphofructokinase is inhibited by high levels of citrate (Garland et al., 1963). The decrease in malate in the dormant egg could again be due to a decrease in the catalytic capacity of the TCA cycle and a relative increase in the activity of the reverse TCA cycle (Barrett and Beis, 1974), although if the reverse TCA cycle were active in the dormant egg it might have been expected that the levels of succinate would have risen at this stage and not fallen. Considerable changes take place in the isoenzyme patterns of malate dehydrogenase from Ascaris eggs at about the time they become dormant (Barrett and Fairbairn, 1971), and this may be related to the changes in metabolite levels. There are no corresponding changes in the isoenzyme pattern of lactate dehydrogenase in the developing Ascaris egg (Barrett, unpublished). An interesting problem is posed by isocitrate which remains virtually constant in the lo-day, dormant and activated Ascaris egg. In mammalian tissues aconitase is an
in Ascaris Eggs. II
193
equilibrium enzyme, so the ratio of citrate:isocitrate should be constant throughout development. However, the levels of citrate and isocitrate are not in equilibrium (Table 4), and so it must be assumed that citrate is being accumulated in a separate cellular compartment from isocitrate. The levels of aconitase are decreasing rapidly during this period of development (Ward and Fairbairn, 1970), and it is possible that these changes reflect the disappearance of aconitase activity from one or other of the cellular compartments. The measurement of metabolite levels in complex tissues such as developing Ascaris eggs suffers from the same limitations as enzyme assays on whole homogenates, namely, that variations between different tissues are lost (Barrett and Beis, 1974). Such measurements do, however, give some indication of the overall changes which are occurring, particularly if they can be correlated with other metabolic events. The onset of dormancy in infective Ascaris eggs and the subsequent activation of the dormant egg is accompanied by changes in the steady state levels of intermediary metabolites. However, it is not clear whether these changes in metabolite levels are the prime cause or merely the result of dormancy. The use of whole organisms means that it can be by no means certain that similar metabolite changes are occurring in all tissues. Also, because of the intracellular compartmentation of metabolites, it is not known if the observed metabolic changes occur throughout the cell, or whether they are restricted to one or other of the cellular compartments; in the case of citrate and isocitrate, the results indicate that the changes are in fact taking place in different cellular compartments. Also, these experiments would not detect changes in the intracellular distribution of metabolites, if the total metabolite level remained unchanged. However, with the known properties of
194
DEVELOPMENTALBIOLOC:Y
the regulatory enzymes of carbohydrate and lipid catabolism, the observed changes in the levels of the intracellular metabolites and in particular the changes in the [ATPVIADP] ratio would be sufficient to account for the drop in QO, in the dormant infective egg (Passey and Fairbairn, 1955), and its subsequent reactivation upon receipt of the hatching stimulus. This work was supported Wellcome Trust.
by grants
from
the
REFERENCES ADAM, H. (1963a). Adenosine5’triphosphate. Determination with phosphoglycerate kinase. In “Methods of Enzymatic Analysis”-(H. U. Bergmeyer, ed.), pp. 539-543. Academic Press, New York. ADAM, H. (1963b). Adenosine-5’-diphosphate and adenosine-5’-monophosphate. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 573-577. Academic Press, New York. ALLEN, R. J. L. (1940). The estimation of phosphorus. Biochem. J. 34, 858-865. ATKINSON, D. E., and WALTON, G. M. (1967). Adenosine triphosphate conservation in metabolic regulation. J. Biol. Chem. 242, 3239-3241. BARRETT, J. (1973). Nucleoside triphosphate metabolism in the muscle tissue of Ascaris lumbricoides (Nematoda). Int. J. Parasitol. 3, 393-400. BARRETT, J., and BEIS, I. (1975). Energy metabolism in developing Ascaris lumbricoides eggs. I. The glycolytic enzymes. Develop. Biol. 42, 181-187. BARRE?T, J., and FAIRBAIRN, D. (1971). Effects of temperature on the kinetics of malate dehydrogenases in the developing eggs and adult muscle of Ascaris lumbricoides (Nematoda). J. Exp. Zool. 176, 169-178. BERGMEYER,H. U., and BERNT, E. (1963). a-oxoglutarate. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 324-327. Academic Press, New York. BERNT, E., and BERGMEYER, H. U. (1963). L-Glutamate. Determination with glutamic dehydrogenase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 384-388. Academic Press, New York. BUTCHER,T., and HOHORST, H. J. (1963). Dihydroxyacetone phosphate, fructose-1,6-diphosphate and n-glyceraldehyde-3-phosphate. Determination with glycerol-l-phosphate dehydrogenase, aldolase and triosephosphate isomerase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 246-252. Academic Press, New York. B&HER, T., CZOK, R., LAMPRECHT, W., and LATZKO, E.
VOLUME 42, 1975
(1963). Pyruvate. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 253-259. Academic Press, New York. CZOK, R., and ECKERT, L. (1963). n3-phosphoglyn-2-phosphoglycerate, cerate, phosphoenolpyruvate. In “Methods of Enzymatic Analysis” (H.U. Bergmeyer, ed.), pp. 224-228. Academic Press, New York. DAGLEY, S. (1963). Citrate. Determination with citrase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 313-317. Academic Press, New York. DEAN, R. B., and DIXON, W. J. (1951). Simplified statistics for small numbers of observations. Anal. Chem. 23, 636-638. DECKER, K. (1963). Acetyl coenzyme-A. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 419-424. Academic Press, New York. FAIRBAIRN, D. (1961). The in vitro hatching of Ascaris lumbricoides eggs. Can. J. Zool. 39, 153-162. GARLAND, P. B., RANDLE, P. J., and NEWSHOLME, E. A. (1963). Citrate as an intermediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes and starvation. Natwe (London) 200, 169-170. HOHORST, H. J. (1963a). o-glucose-6-phosphate and n-fructose-6-phosphate. Determination with glucose-6-phosphate dehydrogenase and phosphoglucose isomerase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 134-138. Academic Press, New York. HOHORST, H. J. (1963b). L-(-)-Malate. Determination with malic dehydrogenase and DPN. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 328-332. Academic Press, New York. HOHORST, H. J. (1963c). ~-(+)-Lactate. Determination with lactic dehydrogenase and DPN. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 266-270. Academic Press, New York. KMVIETIC,E. (1966). Spectrophotometric method for the enzymic microdetermination of succinic acid. Anal. Biochem. 16, 474-480. LAMPRECHT, W., and STEIN, P. (1963). Creatine phosphate. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 610-616. Academic Press, New York. LAMPRECHT, W., and TRAUTSCHOLD, I. (1963). Adenosine-5’-triphosphate. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 543-551. Academic Press, New York. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. NEWSHOLME,E. A., and START, C. (1973). “Regulation
BEIS AND BARRETT
in Metabolism.”
Energy Metabolism
Wiley, London. D. (1955). The respiration of Ascaris lumbricoides eggs. Can. J. Biochem. Physiol. 33, 1033-1046. SCHNEIDER, W. C. (1957). Methods for the isolation of particulate components of the cell. In “Manometric Techniques” (W. W. Umbreit, R. H. Burris, and J. F. Stauffer, eds.), pp. 188201. Burgess, Minneapolis, MN. SIEBERT, G. (1963). Citrate and isocitrate. Determination with aconitase and isocitric dehydrogenase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 318-323. Academic Press, New York. WARD, C. W., and FAIRBAIRN, D. (1970). Enzymes of @oxidation and their function during development of Ascaris lumbricoides eggs. Develop. Biol. 22, PASSEY, R. F., and FAIRBAIRN,
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366-387. 0. H., PATZELT, C., and L~FFLER, G. (1972). Active and inactive forms of pyruvate dehydrogenase in rat liver. Eur. J. Biochem. 26, 426-433. WILLIAMSON, D. H. LUND, P., and KREBS, H. A. (1967). The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem. J. 103, 514-527. WILLIAMSON, J. R. (1965). Metabolic control in the perfused rat heart. In “Control of Energy Metabolism” (B. Chance, R. W. Estabrook, and J. R. Williamson, eds.), pp. 333-346. Academic Press, New York. WOLLENBERGER, A., RISTAU, O., and SCHOFFA, G. (1960). Eine einfache Technik der extrem schnellen Abkiihlung grSsserer Gewebestiicke. Pfltigers Arch. Gesamte Physiol. Menschen. Tiere 270.399-412. WIELAND,
BIOLOGY
Energy
42, 188-195 (1975)
Metabolism II. The Steady
in Developing State Content
Ascaris
lumbricoides
of Intermediary
Eggs
Metabolites
I.BEIS AND J. BARRETT’ Department
of Zoology, Accepted
University
of Oxford,
September
Oxford, England
16, 1974
The steady state levels of intermediary metabolites were measured in freeze clamped, developing, dormant, and activated infective Ascaris lumbricoides eggs. The [ATPy[ADP] ratio is low in the developmental stages and rises sharply in the dormant egg; on activation of the dormant egg the [ATP]/[ADP] ratio falls. The levels of the phosphorylated glycolytic intermediates of acetyl-CoA and of isocitrate do not change markedly during development, but the levels of lactate, citrate, P-oxoglutarate, glutamate, succinate, and malate all show significant changes in the developing, dormant, and activated egg. The dormant egg also appears to be characterized by a low cytoplasmic redox potential. INTRODUCTION
The changes in metabolic rate which occur during the development of the eggs of Ascaris lum bricoides cannot be adequately explained by changes in the maximum catalytic capacities of the major pathways of lipid or carbohydrate catabolism (Barrett and Beis, 1975). The changes in QO, during development could be due to the inhibition of one or more of the key regulatory enzymes of lipid or carbohydrate catabolism. Inhibition of the regulatory enzymes might be brought about by the production of a specific inhibitory compound or by changes in the levels of the intracellular metabolites; alternatively, the properties of the regulatory enzymes themselves might change during development such that in the infective egg the metabolic pathways were inhibited by metabolite levels which did not affect the earlier developmental stages. In this paper the steady state levels of intermediary metabolites have been measured in freeze clamped developing, dormant, and activated infective Ascaris eggs. MATERIALS
Ascaris
AND
METHODS
eggs were prepared
and em-
’ Present address, Department of Zoology, University College of Wales, Aberystwyth, Wales. 188 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved,
bryonated as described by Ward and Fairbairn (1970). The eggs were incubated at 30°C and all times refer to development at this temperature. The dormant infective eggs were hatched (activated) as described by Fairbairn (1961). Metabolite
Assays
Egg suspensions were freeze clamped between aluminum blocks cooled in liquid nitrogen (Wollenberger et al., 1960). The frozen tissue was pulverized in a percussion mortar and a neutral perchlorate extract prepared (Adam, 1963a). The metabolites were assayed enzymatically with a Gilford Model 240 recording spectrophotometer, using the following methods: ATP, Lamprecht and Trautschold (1963); ADP and AMP, Adam (1963b); glucose-6-phosphate and fructose-6-phosphate, Hohorst (1963a); dihydroxyacetone phosphate and fructose-1,6-diphosphate, Biicher and Hohorst (1963); 3-phosphoglycerate, Czok and Eckert (1963); pyruvate, Biicher et al. (1963) (phosphoenolpyruvate was measured in the same cuvette by the addition of ADP and pyruvate kinase); malate and lactate, Hohorst (1963b,c); citrate, Dagley (1963); isocitrate, Siebert (1963); glutamate, Bernt and Bergmeyer (1963); Z-oxoglutarate, Bergmeyer and Bernt (1963);
BEIS AND BARRETT
Energy Metabolism
acetyl-CoA, Decker (1963); succinate, Kmetic (1966); arginine phosphate was determined as for creatine phosphate, but using arginine kinase instead of creatine kinase (Lamprecht and Stein, 1963). Arylamine transacetylase was prepared from pigeon livers by the method of Wieland et al. (1972); arginine kinase (prepared from lobster muscle) was the gift of Dr. A. R. Leech; all other coupling enzymes were purchased from the Boehringer Corp. Ltd. Inorganic phosphate (Pi) was measured in unneutralized perchlorate extracts by the method of Allen (1940). Enzyme Assays
The enzyme assays were performed at 30°C in a Gilford Model 240 recording spectrophotometer, using 1 ml semimicro cells. The eggs were homogenized in 0.1 M Tris-Cl buffer, pH 7.6; 0.09 M KCl; 0.028 M MgSO,, using a l-ml ground glass homogenizer. The homogenizer was cooled in ice and the progress of homogenization was checked under the microscope. The homogenate was centrifuged for 5 min at 700 g at 2°C and the supernatant fraction used for enzyme assays. Adenylate kinase (EC 2.7.4.3), nucleosidediphosphate kinase (EC 2.7.4.6), and arginine kinase (EC 2.7.3.3) were assayed as described by Barrett (1973). The total ATPase activity of the homogenate was estimated from the rate of activity in the nucleosidediphosphate kinase assay before the addition of dGDP. All enzyme activities are expressed as nmoles/min/mg protein. Protein was measured by the method of Lowry et al. (1951); the protein precipitates from the perchlorate extracts were dissolved by heating in 1 N NaOH. Standard errors were calculated as described by Dean and Dixon (1951). Respiratory
Rates
Oxygen uptake was measured in a Yellow Springs Model 53 Oxygen monitor, using a final volume of 4 ml. Rat liver and
in Ascaris Eggs. II
189
Ascaris eggs were homogenized in 0.25 M
sucrose, 1 mM EDTA, pH 7.4; rat liver mitochondria were prepared in isotonic sucrose by the method of Schneider (1957). All additions were made in neutral solution, with the exception of the 2,4-dinitrophenol experiments where the eggs were suspended in 0.01 N H,SO,. This enabled the 2,4-dinitrophenol to penetrate the vitelline membrane, which is impermeable to ionized compounds. RESULTS
During the development of Ascaris eggs there are considerable changes in the steady state levels of the metabolic intermediates. One of the most important group of metabolites from the point of view of energy metabolism are the adenylate nucleotides. The steady state levels of ATP, ADP, AMP, and inorganic phosphate (Pi) in developing Ascaris eggs are summarized in Table 1 and the changes in the [ATP y [ADP] ratio in Fig. 1. The attainment of infectivity in Ascaris eggs is accompanied by a sharp rise in the [ATPy[ADP] ratio; in the developing stages the ratio is less than 1 and increases to 5 by the time the infective stage is reached. When the dormant infective eggs are given a hatching stimulus, there is a drop in the [ATPy [ADP] ratio to about 1.7. The changes in the adenylate charge (Atkinson and Walton, 1967) parallel the changes in the [ATPj/[ADP] ratio (Table 1). The levels of inorganic phosphate are the reverse of the [ATP y[ADP] ratio, being high in the developing egg, dropping in the dormant egg, and increasing again on activation. The increase in the [ATPj/[ADP] ratio in the dormant egg is not accompanied by a drop in ATPase activity. The total ATPase activity of egg homogenates increases during development, as does the activity of adenylate kinase and nucleosidediphosphate kinase (Table 2). Neither arginine kinase nor arginine phosphate could be detected by spectrophotometric methods
190
DEVELOPMENTAL
BIOLOGY
TABLE STEADY STATE CONTENT
OF ADENINE
lumbricoides
VOLUME 42. 1975
1
NUCLEOTIDES AND INORGANIC PHOSPHATE IN FREEZE CLAMPED EGGS AT DIFFERENT STAGES OF DEVELOPMENT
Ascaris
flmoles/lOO mg protein”
Age (days)
ATP 0 1 3 5 8 10 14 18 22 50 (dormant) 50 (activated)
1.00 1.55 1.50 0.95 1.00 1.57 2.00 2.80 2.60 1.60 0.71
* * * * * * * * rt i i
ADP
0.05 0.20 0.20 0.15 0.05 0.40 0.24 0.40 0.10 0.20 0.06
1.20 1.70 2.10 2.00 1.70 0.75 0.67 0.70 0.50 0.33 0.41
AMP
* 0.30 + 0.60 * 0.10 * 0.30 * 0.30 * 0.04 z+z0.08 * 0.10 * 0.07 * 0.04 + 0.06
0.06 0.07 0.08 0.08 0.05 0.04 0.05 0.06 0.02 0.06 0.10
+ * + + * * * + * i *
0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.02
Pi
Adenylateb charge
8.1 zt 0.8 8.8 zt 0.4 7.4 * 0.2 4.9 * 0.5 5.0 + 0.8 5.7 * 0.5 4.5 i 0.7 5.2 + 0.5 5.5 zt 0.6 5.0.1 0.9 10.0 * 0.4
0.70 0.72 0.69 0.64 0.67 0.82 0.85 0.88 0.91 0.89 0.75
” Mean + SEM (10 determinations). o Atkinson and Walton (1967).
0
FIG. 1. The [ATPy[ADP] (N = 10).
I 5
I 10
I I5
I 20 DAYS EMBAVONATEO
ratio in developing Ascaris lumbricoides
in Ascaris eggs, at any stage of development. The rate of respiration of infective 22day-old Ascaris eggs was found to be stimulated by 2,4-dinitrophenol. At lo-” and 10m5 M the stimulation was 8 and 25% respectively, at low3 M, 2,4-dinitrophenol inhibited respiration by 25%. The ability of 2,4-dinitrophenol to stimulate respiration
eggs. The vertical
lines represent + SEM
in the dormant infective egg indicates that the maximum capacities of the metabolic pathways are not rate limiting. There is no evidence for any general metabolic inhibitor in the dormant infective egg of Ascaris. Homogenates of dormant eggs do not affect the rate of respiration of homogenates of lo-day Ascaris eggs in the presence or absence of substrates (10
Energy Metabolism
BEIS AND BARRETT
mM glucose or 10 mA4 succinate). Nor do homogenates of dormant eggs affect the rate of oxygen uptake of rat liver homogenates or of isolated rat liver mitochondria in the presence or absence of exogenous substrate (10 mA4 glucose or 10 mM succinate, respectively). The steady state levels of glycolytic and TCA cycle intermediates in freeze clamped Ascaris eggs are shown in Tables 3 and 4, respectively. The developmental stages TABLE
2
SPECIFIC ACTIVITIES OF ADENYLATE KINASE, NUCLEOSIDEDIPHOSPHATEKINASE AND AIIENOSINE TRIPHOSPHATASEIN Ascaris lumbricoides EGGS AT DIFFERENT STAGES OF DEVELOPMENT Age (days)
T
nmoleslmin/mg protein” Adenylate kinase
0 1 3 5 8 10 14 18 22 50 (dormant) 50 (activated)
200 205 260 240 285 300 361 364 376 350
Nucleosidediphosphate kinase
i 18.0 i 9.4 zt 7.5 zt 12.6 zt 13.0 * 17.0 zt 25.0 + 18.0 + 17.0 * 7.0
361 + 15.0
508 632 634 651 638 615 612 612 626 611
i 19 zt 25 zt 12 + 24 zt 28 * 15 + 20 * 24 z+z31 zt 31
594 zt 23
ATPase 7.5 8.3 7.0 8.0 9.6 13.0 17.0 25.0 21.0 25.0
i 0.9 + 1.1 f 0.6 f 1.2 zt 0.8 * 0.5 f 0.8 + 1.4 * 1.3 =+2.0
30.0 f 3.0
L
n Mean + SEM (10 determinations).
chosen were 1 day, when the rate of respiration is low, 10 days when the respiratory rate is maximal, and 22 days, which is the fully developed dormant infective egg (Fairbairn, 1961; Passey and Fairbairn, 1955). The glycolytic intermediates can be divided into four groups; those which remain fairly constant throughout development; those which decrease in the dormant egg and increase again on activation; those which decrease in the dormant egg, but do not increase again on activation; and finally those which increase in the dormant egg and decrease on activation. The levels of fructose-1,6-diphosphate and dihydroxyacetone phosphate remain more or less constant throughout development. In contrast glucose-6-phosphate and fructose6-phosphate levels both increase between days 1 and 10 and then fall in the dormant infective egg; on receipt of the hatching stimulus, the levels of the sugar phosphates increase again. The levels of phosphoenolpyruvate and 3-phosphoglycerate remain fairly constant between day 1 and 10 but decrease in both the dormant and activated infective egg. The levels of pyruvate also drop during development and are low in both the dormant and activated infective egg; in contrast, lactate increases slightly between day 1 and 10, is very high
TABLE
3
STEADY STATE CONTENT OF GLYCOLYTICINTERMEDIATESIN FREEZE CLAMPED Ascaris lumbricoides DIFFERENT STAGES OF DEVELOPMENT
l-Day
a Mean * SEM (eight determinations).
EGGS AT
nmoles/lOO mg protein”
Metabolite
Glucose-6-phosphate Fructose-6-phosphate Fructose-1,6-diphosphate Dihydroxyacetone phosphate 3-Phosphoglycerate Phosphoenolpyruvate Pyruvate Lactate
191
in Ascaris Eggs. ZZ
154 38 15 98 60 134 320 1337
f 15 + 5 * 2 i 20 + 10 19 i 41 f 90
lo-Day 191 45 16 113 58 173 207 1639
i f + * + i i +
18 7 3 20 10 18 27 191
22-Day (dormant) 66 25 18 103 37 89 188 4517
i 6 i 5 zt 2 * 9 * 11 + 9 i 16 i 298
22-Day (activated) 189 47 17 114 38 52 193 1469
17 *3 12 f 50 zt 12 +4 * 30 i 341
192
DEVELOPMENTALBIOLOGY
VOLUME 42, 1975
TABLE 4 STEADY STATE CONTENT OF TCA CYCLE INTERMEDIATESIN FREEZE CLAMPED Ascaris lumbricoides EGGSAT DIFFERENT STAGESOF DEVELOPMENT
Metabolite
nmoles/lOQ mg protein” l-Day
Citrate Isocitrate 2-oxoglutarate Glutamate Succinate Malate Acetyl Co-A
702 zt 35 11 13 36 zt 6 349 * 14 161 f 11 785 + 44 2.3 zt 1.0
lo-Day 994 + 130 21 12 43 * 12 362 + 17 497 zt 185 748 + 71 5.0 * 1.0
22-Day (dormant)
22-Day (activated)
3795 f 311 21 * 2 80 zt 14 410 f 15 363 + 93 217 A 57 2.0 l 1.2
2963 zt 314 26 f 5 50 f 10 341 * 21
555 * 113 285 + 30 3.8 + 1.5
a Mean i- SEM (eight determinations).
in the dormant egg, and drops again on activation. If it can be assumed that lactate and pyruvate are equally distributed throughout the tissues, the lactate dehydrogenase system can be used to calculate the redox state of the free [NAD+ j/ [NADH] couple in the cytoplasm of the developing eggs (Williamson et al., 1967). In the l-day egg the free [NAD+j/[NADH] ratio in the cytoplasm is 2156:l; this drops to 1137:l in the lo-day egg and falls to 3751 in the dormant infective egg. In the activated infective egg, the free [NAD+J/ [NADH] ratio in the cytoplasm again increases to 1183:l. So the dormant infective egg would appear to be characterized by a low cytoplasmic redox potential. The TCA cycle intermediates (Table 4) can be divided into the same four groups as the glycolytic intermediates. The levels of citrate, 2-oxoglutarate, and glutamate increase slightly between day 1 and 10, rise sharply in the dormant egg, and drop again on activation. Malate shows the reverse pattern, decreasing in the dormant egg and increasing slightly on activation. Succinate levels were difficult to measure in Ascaris eggs due to the presence of interfering compounds in the neutral perchlorate extracts, and the results may be unreliable. The succinate levels appear to increase from day 1 to 10 and are then similar to malate in decreasing in the
dormant egg and increasing again on activation. In contrast to the changes in citrate levels in developing eggs, the levels of isocitrate, after increasing between day 1 and 10, remain unchanged in the dormant and activated infective eggs. The levels of acetyl-CoA are extremely low throughout development, and are at the limits of the assay method used. DISCUSSION
The changes in the metabolic rate of developing, dormant, and activated infective Ascaris eggs are accompanied by changes in the steady state levels of the intermediary metabolites. In particular the dormant egg is characterized by a high [ATPv[ADP] ratio and a low cytoplasmic free [NAD+j/[NADH] ratio. The high [ATP1/[ADP] ratio in the dormant egg may at first appear unusual, since it might ’ have been expected that the ATP levels would have been low in a dormant organism. A high [ATPj/[ADP] level is, however, consistent with a low metabolic rate, since phosphofructokinase which is a key regulatory enzyme of glycolysis is inhibited by ATP and this inhibition is released by ADP; isocitrate dehydrogenase is also activated by ADP and both ,&oxidation and oxidative and substrate phosphorylation are tightly coupled to the availability of
BEIS AND
BARRETT
Energy Metabolism
ADP and Pi (Newsholme and Start, 1973). There is no simple overall explanation for the metabolite changes found in the developing, dormant, and activated infective eggs of Ascaris. The changes observed in the levels of the glycolytic intermediates can be grouped into four types (see Results). However, the glycolytic intermediates show no clear-cut crossover points between the active and dormant egg (Williamson, 1965). The same four patterns of change are found in the TCA cycle intermediates. The changes in the levels of citrate, 2-oxoglutarate, and glutamate may be the result of changes in the relative activities of the different catabolic pathways. The increase in the importance of lipid metabolism in the mature egg, combined with a decrease in the capacity of the TCA cycle, could lead to a rise in the levels of intermediates at the beginning of the TCA cycle and to an increase in the levels of lactate. Citrate may also have a regulatory function, since in mammalian systems at least phosphofructokinase is inhibited by high levels of citrate (Garland et al., 1963). The decrease in malate in the dormant egg could again be due to a decrease in the catalytic capacity of the TCA cycle and a relative increase in the activity of the reverse TCA cycle (Barrett and Beis, 1974), although if the reverse TCA cycle were active in the dormant egg it might have been expected that the levels of succinate would have risen at this stage and not fallen. Considerable changes take place in the isoenzyme patterns of malate dehydrogenase from Ascaris eggs at about the time they become dormant (Barrett and Fairbairn, 1971), and this may be related to the changes in metabolite levels. There are no corresponding changes in the isoenzyme pattern of lactate dehydrogenase in the developing Ascaris egg (Barrett, unpublished). An interesting problem is posed by isocitrate which remains virtually constant in the lo-day, dormant and activated Ascaris egg. In mammalian tissues aconitase is an
in Ascaris Eggs. II
193
equilibrium enzyme, so the ratio of citrate:isocitrate should be constant throughout development. However, the levels of citrate and isocitrate are not in equilibrium (Table 4), and so it must be assumed that citrate is being accumulated in a separate cellular compartment from isocitrate. The levels of aconitase are decreasing rapidly during this period of development (Ward and Fairbairn, 1970), and it is possible that these changes reflect the disappearance of aconitase activity from one or other of the cellular compartments. The measurement of metabolite levels in complex tissues such as developing Ascaris eggs suffers from the same limitations as enzyme assays on whole homogenates, namely, that variations between different tissues are lost (Barrett and Beis, 1974). Such measurements do, however, give some indication of the overall changes which are occurring, particularly if they can be correlated with other metabolic events. The onset of dormancy in infective Ascaris eggs and the subsequent activation of the dormant egg is accompanied by changes in the steady state levels of intermediary metabolites. However, it is not clear whether these changes in metabolite levels are the prime cause or merely the result of dormancy. The use of whole organisms means that it can be by no means certain that similar metabolite changes are occurring in all tissues. Also, because of the intracellular compartmentation of metabolites, it is not known if the observed metabolic changes occur throughout the cell, or whether they are restricted to one or other of the cellular compartments; in the case of citrate and isocitrate, the results indicate that the changes are in fact taking place in different cellular compartments. Also, these experiments would not detect changes in the intracellular distribution of metabolites, if the total metabolite level remained unchanged. However, with the known properties of
194
DEVELOPMENTALBIOLOC:Y
the regulatory enzymes of carbohydrate and lipid catabolism, the observed changes in the levels of the intracellular metabolites and in particular the changes in the [ATPVIADP] ratio would be sufficient to account for the drop in QO, in the dormant infective egg (Passey and Fairbairn, 1955), and its subsequent reactivation upon receipt of the hatching stimulus. This work was supported Wellcome Trust.
by grants
from
the
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(1963). Pyruvate. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 253-259. Academic Press, New York. CZOK, R., and ECKERT, L. (1963). n3-phosphoglyn-2-phosphoglycerate, cerate, phosphoenolpyruvate. In “Methods of Enzymatic Analysis” (H.U. Bergmeyer, ed.), pp. 224-228. Academic Press, New York. DAGLEY, S. (1963). Citrate. Determination with citrase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 313-317. Academic Press, New York. DEAN, R. B., and DIXON, W. J. (1951). Simplified statistics for small numbers of observations. Anal. Chem. 23, 636-638. DECKER, K. (1963). Acetyl coenzyme-A. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 419-424. Academic Press, New York. FAIRBAIRN, D. (1961). The in vitro hatching of Ascaris lumbricoides eggs. Can. J. Zool. 39, 153-162. GARLAND, P. B., RANDLE, P. J., and NEWSHOLME, E. A. (1963). Citrate as an intermediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes and starvation. Natwe (London) 200, 169-170. HOHORST, H. J. (1963a). o-glucose-6-phosphate and n-fructose-6-phosphate. Determination with glucose-6-phosphate dehydrogenase and phosphoglucose isomerase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 134-138. Academic Press, New York. HOHORST, H. J. (1963b). L-(-)-Malate. Determination with malic dehydrogenase and DPN. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 328-332. Academic Press, New York. HOHORST, H. J. (1963c). ~-(+)-Lactate. Determination with lactic dehydrogenase and DPN. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 266-270. Academic Press, New York. KMVIETIC,E. (1966). Spectrophotometric method for the enzymic microdetermination of succinic acid. Anal. Biochem. 16, 474-480. LAMPRECHT, W., and STEIN, P. (1963). Creatine phosphate. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 610-616. Academic Press, New York. LAMPRECHT, W., and TRAUTSCHOLD, I. (1963). Adenosine-5’-triphosphate. Determination with hexokinase and glucose-6-phosphate dehydrogenase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), pp. 543-551. Academic Press, New York. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. NEWSHOLME,E. A., and START, C. (1973). “Regulation
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