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A method for the determination of changes of glycolytic metabolites in yeast on a subsecond time scale using extraction at neutral pH
ANALYTICAL
204,
BIOCHEMISTRY
118-123
(1992)
A Method for the determination of Changes of Gtycolytic Metabolites in Yeast on a Subsecond Time Scale Using Extraction at Neutral pH Wim
de Koning’
and Karel
van Darn’
E. C. Slater Institute for Biochemical Research, Biotechnology Center, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands
Received
January
of
Amsterdam,
13, 1992
Glucose metabolism in yeast can be stopped within 0.1 s by spraying the cells in 60% methanol at -40°C. With this procedure the integrity of the cells is not damaged. Using stopped-flow equipment for the incubation with glucose, major changes within a second are shown to occur in intracellular glucose-g-phosphate whereas the fructose-1,6-bisphosphate concentration remains constant. After quenching, the cells can be separated from the medium, washed with cold methanol when required, and extracted using chloroform at -40°C at neutral pH, ensuring minimal degradation of labile metabolites. With partly automated enzymatic methods, a large variety of metabolites, including all glycolytic intermediates, can be determined in the neutral extracts. During the first second after addition of glucose, a significant increase in free intracellular glucose is found. 10 1992 Academic Press, Inc. -
In the study of a metabolic pathway of an organism, information about the concentration of intermediates can be of crucial interest. Together with information about kinetic properties of the enzymes involved, metabolite levels can be used to investigate the control of specific components of the pathway. More than enzyme activities, metabolite concentrations are very prone to changes induced by (unnoticed) variation in the environment of the cell. For baker’s yeast, growing on glucose, it can be deduced from the
* Present address: Laboratorium voor ~oleculaire Celbiologie, Instituut voor Plantkunde, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium. ’ To whom correspondence should be addressed. 118
University
flux through the pathway and the intracellular metabolite concentrations that a typical metabolite’s half-life is on the order of a second or less (cytosolic glucose is converted at approximately 1 InM s-i). This implies that after a pulse of glucose to a carbon-starved culture, the first significant changes can occur within a second. Study on this time scale therefore is of interest. Furthermore, the high turnover of metabolites makes it imperative that, whenever intracellular glycolytic metabolite concentrations are to be determined, extreme care be taken to stop metabolism very rapidly. Although not all handling of a culture may result in significant changes of metabolites within a second, it is important to be aware of this problem. Direct quenching of the incubation mixture is in principle possible, but many protocols for metabolite determination include a rapid filtration step followed by freezing in liquid nitrogen. Two important advantages are that most of the incubation medium is removed from the sample and that, due to the concentration step, levels of metabolites in the sample are strongly increased. The major disadvantages are: (a) metabolite levels can change during filtration (because the cells are flushed with incubation mixture on the filter, no major changes in the environment are expected to occur during ~ltration, as long as the filter is not running dry) and (b) incubation times shorter than 10 s are impossible to obtain, due to the time required for filtration. Depending on the metabolite(s) of interest, cells are extracted using, for example, perchloric acid or NaOH. With these methods not all metabolites are quantitatively extracted, due to instability of some metabolites at extreme pH. When both NAD’ and NADH are to be measured, usually an acid and an alkaline extraction are performed on separate samples. A number of com0003.2697/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
EXTRACTION
ANALYSIS
OF
pounds are, however, unstable at both high and low pH (dihydroxyacetone phosphate3 (DHAP) and phospho~enol)pyruvate (PEP) are known examples). Especially for these compounds the extraction must be a compromise between completeness (asking for longer incubation times) and prevention of degradation (asking for short times at extreme pH). In this paper a method that overcomes the problems mentioned above is described. It allows quenching of metabolism in yeast in less than 0.1 s. After the quenching the cells can be concentrated and washed, when uptake of components of the medium is investigated. The concentrated cells are extracted at neutral pH, allowing determination of a large variety of metabolites, including nearly all glycolytic intermediates in a single extract. MATERIALS Organism
AND
METHODS
and Preincubation
Experiments were performed with fresh baker’s yeast (Gist-brocades, Delft, The Netherlands). Upon arrival of the pressed yeast cake (usually within 2 days after production), a 1:l (w/w) suspension in Millipore water was made, which was stored at 4°C. Experiments were performed with yeast not older than 14 days. Before an experiment, the yeast suspension was mixed with 1 vol of 100 mM Mes, pH 5.7, flushed with nitrogen, and stored on ice overnight. Thirty minutes before incubation with glucose was started, 6 ml of the yeast suspension (still on ice) was diluted anaerobically with 9 ml of 50 mM Mes, pH 5.7, at 30°C. (The anaerobic conditions used in these experiments were not essential for the methods described but rather were chosen for reasons outside the scope of this paper.) Glucose Incubations Incubations with glucose for less than 10 s were performed with a freeze-quench apparatus (1) with a fourjet tangential mixer (2) under an atmosphere of argon at 30°C. Standard conditions during incubation were 50 mM Mes, pH 5.7, and 50 mM glucose at a cell density of 50 mg wet weight ml-‘. The apparatus was equipped with two l&ml stainless steel syringes and worked with a flow rate of approximately 1 ml s-’ after mixing. The internal volume of the incubation tubing was varied to change the incubation time (from 15 ms to 5 s). The actual flow rate was determined from the speed of the ram in each incubation and used to calculate the exact
3 Abbreviations used: G6P, glucose-6-phosphate; F6P, fructose-6 phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; 3PGA, 3-phosphoglycerate; PPGA, 2-phosphoglycerate; PEP, phospho(enol)pyruvate; Pipes, l,i’-piperazineethanesulfonic acid; Mes, Mo~hoIineethanesuifonic acid.
GLYCOLYTIC
METABOLITES
119
incubation time. Incubations longer than 10 s were performed manually using a syringe without needle to spray the samples in the cold methanol (see below). Quenching Ten- to fifteen-milliliter samples of (incubated) yeast suspension were sprayed in a stirred solution (60 ml) of 60% methanol kept at -40°C by a circulating methanol bath. After the mixture was cooled for approximately 5 min the cells were spun down at 5000 rpm for 5 min at the lowest possible temperature (usually -20°C). Unless stated otherwise, all further manipulations were done at -40°C. The carefully drained pellet (excess remaining water prevents proper mixing with chloroform and increases the freezing point of the sample) was suspended in 2.5 ml of 100% methanol and transferred to precooled 30-ml thick-walled screwcap glass tubes. Twenty microliters of 200 mM EDTA, pH 7, was added under vigorous vortexing of the suspension to inhibit Mg’+-dependent, partly chloroform-resistant enzyme activities (mainly myokinase). After addition of 1 ml of precooled chloroform the tubes were frozen in liquid nitrogen and stored at -70°C. When internal glucose was measured, 25 ml of the quenched suspension was filtered over a cellulose acetate filter (ST 69, Schleicher & Schuell, Dassel, Germany; pore size, 1.2 pm; 25 mm diameter). The cells were washed three times with 5 ml of cold 60% methanol. The filter was transferred quickly to a precooled tube containing 2.5 ml of 100% methanol. After suspension of the cells the filter was removed and EDTA and chloroform were added. Extraction
of Metabolites
Unless stated otherwise, samples were kept at -40°C. To each tube 4 ml of cold chloroform was added. Under vigorous vortexing 2 ml of ice-cold 2 mM Pipes pH 7.2, was added. To achieve complete permeabilization and extraction of the cells the tubes were shaken for 45 min at -35”C, 2200 rpm (in a regulated liquid nitrogencooled incubator; Cryoson TRA 11, Middenbeemster, The Netherlands), in horizontal position on an Ika Vibrax VXR (Janke & Kunkel, Staufen, Germany) using a homemade adaptor. The cells were pelleted on the chloroform phase by centrifugation at 13OOg for 15 min in a swing-out rotor at the lowest possible temperature (-20°C using adaptors for the glass tubes that were precooled at -40°C). The supernatant was carefully collected using an aspiration setup. To the remaining content of the tubes 2 ml methanol was added and, after mixing, 2 ml ice-cold 2 mM Pipes was added under vortexing. After the cells were vortexed for 20 s two times the samples were centrifuged again and the supernatant was added to the first extract. The combined extracts were extracted once with 15 ml diethyl ether to remove traces of lipids.
120
DE
KONING
AND
VAN
DAM
The volume was reduced by evaporation under vacuum (<50 Pa, to ensure sufficient cooling of the samples) in a Speedvac (Savant Instruments, Hicksville, NY) to approximately 0.5-l ml without additional heating. To prevent excessive boiling due to the remaining ether, the evaporation was started with frozen samples. After the volume was measured, the samples were stored at -70°C. Shortly before the determination of metabolites, the samples were thawed on ice, mixed, and centrifuged in order to remove possible small precipitates. After the chloroform was carefully decanted, the cell pellet in the extraction tubes was dried under a stream of air. Five milliliters of 1 N NaOH was added and, after boiling and centrifugation, the total amount of protein in the supernatant was determined according to Lowry et al. (3). This served as a measure for the amount of biomass that was extracted. We prefer to express the amounts of intermediates in terms of cytosolic concentrations for later use in the calculation of metabolic fluxes. To calculate these concentrations, a yeast cytosolic volume of 3.75 ~1 per milligram of protein was assumed. If necessary, this can be converted to dry weight using the conversion that 1 mg protein equals 2.5 mg dry weight (Dr. M. A. Herweijer, personal communication). For the present study it was not essential to determine these values more accurately.
dition of 0.7 U ml-’ glycerol-3-phosphate dehydrogenase and 20 U ml-l triosephosphate isomerase + 0.14 U ml-’ aldolase, respectively. Pyruvate, PEP, 2-phosphoglycerate (BPGA), and 3phosphoglycerate (3PGA) were measured in the presence of 100 mM KCl, 10 mM MgSO,, and 0.15 mM NADH by the addition of 1.1 U ml-’ lactate dehydrogenase, 1.6 U ml-’ pyruvate kinase + 1 mM ADP, 0.6 U ml-’ enolase, and 2 U ml-’ phosphoglycerate mutase + 20 pM 2,3-phosphoglycerate, respectively. ADP and AMP were measured in the presence of 100 mM KCl, 10 mM MgSO,, and 0.15 mM NADH by the addition of 1.1 U ml-l lactate dehydrogenase (pyruvate determination), 1.6 U ml-’ pyruvate kinase + 0.5 mM PEP, and 1.4 U ml-’ myokinase + 0.1 mM ATP, respectively. NADH was measured using 0.5 mM DHAP and 0.4 U glycerol-3-phosphate dehydrogenase. NAD+ was measured using four-times-diluted commercial glycine buffer containing a trapping reagent (Sigma, No. 332-9) with 50 mM ethanol by adding 10 U ml-’ alcohol dehydrogenase. Phosphate was measured according to Bencini et al. (7) from the change in absorbance at 350 nm after the addition of molybdate reagent (11.25 mM ammonium molybdate, 75 mM zinc acetate, pH 5.0).
Analytical
Chemicals
Determinations
All analyses were performed on a Cobas Bio Autoanalyser (Roche, Basel, Switzerland) that was connected with a MS/DOS computer for datalogging. The machine protocols used and the spreadsheet-based dataanalyzing programs are available upon request. Volumes in published methods were adapted to the working volume of this apparatus (0.25 ml). Chemical determinations were done at 25°C and enzymatic determinations at 37°C. Enzymatic determinations of metabolites were performed in 50 mM triethanolamine, pH 7.6, following the changes in NAD(P)H at 340 nm (tNADu,)n = 6.22 cm-’ mM-l), essentially as described by Bergmeyer (4,5). Samples were added so that the amount of metabolite in the assay was always below 0.05 mM. Enzymes were used without desalting and diluted in buffer. Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), ATP, or glucose were determined in the presence of 5 mM MgSO, and 0.4 mM NADP+ by the addition of glucose-6-phosphate dehydrogenase (0.7 U ml-‘), phosphoglucose isomerase (2.8 U ml-‘), and hexokinase (1.1 U ml-‘) + 5 mM fructose. In the case of glucose determination after the addition of glucose-6-phosphate dehydrogenase, hexokinase (1.1 U ml-l) + 0.5 mM ATP was added. DHAP and fructose-1,6-bisphosphate (FBP) were measured in the presence of 0.15 mM NADH by the ad-
Methanol was of technical quality. The pH of each batch was checked to be around 7 when diluted to 60% with Millipore water. Technical-quality ether was acidic; therefore, in this case an analytical-grade (Merck) quality was used. Other chemicals were of analytical grade and used as supplied. Enzymes and biochemicals were from Boehringer-Mannheim or Sigma. Buffers were brought to pH with KOH or HCl. RESULTS
Metabolite
Determinations
Our enzymatic determinations include no new methods, but the use of an automated analyzer greatly facilitated measurements, improved the reproducibility, and also reduced the amount of extract needed. Although the accuracy was somewhat dependent on the quality of the sample and also on the metabolite investigated, computer-assisted manual control of each determination allowed a routine detection level of approximately 1 pM in the sample (=lOO pmol) for NAD(P)H-based assays. For concentrations of metabolites in the sample above 150 pM, differences between duplicates were less than 3%. Variation between different extractions of the same incubation was usually within 10%. Different batches of yeast cells gave occasionally larger variations.
EXTRACTION
ANALYSIS
OF GLYCOLYTIC
TABLE
Quenching Method The method employed to stop metabolism in the yeast cells is partly based on the idea of Saez and Lagunas (6), who advocated washing of yeasts with 50% methanol of -40°C after filtering. In our experiments cells are sprayed in 5 vol of 60% methanol at -40°C and subsequently separated from the methanol either by centrifugation or by filtration at low temperature. This method was tested for two essential properties: (a) cells do not leak intermediates until extracted and (b) sufficient block of the metabolism. Leakage of cells was checked by measuring in the supernat.ant the concentration of metabolites that are present in signi~cant amounts in the cells. The supernatant was concentrated under vacuum to remove the methanol, which interferes with some enzymatic assays, and to increase the sensitivity. Very small amounts of pyruvate were detectable, never exceeding 10% of the pyruvate detected intracellularly (Table 1). Comparison with the amount of pyruvate in a filtrate showed that this pyruvate is most likely already excreted by the cells before quenching. The amount of extracellular pyruvate did not increase when the cells were kept for 30 min at -40°C before centrifugation. Other metabolites were below the detection level (approximately 1 pM, depending somewhat on the metabolite) in the supernatant, independent of the residence time in the methanol. In agreement with these observations is the constant level of intracellular metabolites after quenching. From Table 1 it can be concluded t.hat cells do not leak metabolites when quenched in 60% methanol at -4O’C. The complete block of the metabolism is also evident from these results, but more critical experiments are described in the following paragraphs. The speed of quenching is tested by investigation of whether changes in intermediates can be detected on a short time scale. Using freeze-quench equipment, yeast was incubated with 50 RIM glucose for periods varying from 0 (no glucose added) to 1 s. Figure 1 shows that internal glucose and G6P start to increase as soon as glucose is added, whereas other metabolites are virtually constant during the first second after glucose addition. Note that the first point after glucose addition (15 ms) coincides within experimental error for all metabolites with c = 0, where the cells are not incubated with glucose. This is a strong indication that the quenching of the metabolism is sufficient on the time scale shown. Another possibility is that metabolism starts after a delay that just equals the dead time of the quenching. This seems rather unlikely.
The two important requirements for any extraction method are that no changes in the metabolites of inter-
121
M~TABOLIT~S 1
Resistance to Leakage of Saccharomyces cerevisiae Suspended in 50% Methanol at -40°C Amount of metabolites found intr~cellularIy
Amount of metabolites found extracell~lari~ (nmollml)
fnmol/ml~ Tilne in methanol -.
G6P F6P FBP DHAP 3PGA ZPGA PEP Pyruvate NAD NADH ATP ADP AMP Phosphate
Time in
(min)
supernatant (min)
8
30
25.0 4.9 18.R 12.4 1.2 0.1 0.0 13.6 23.9 7.1 1.2 12.9 102 I.030
24.7 5.0 14.9 12.0 1.3 0.1 0.1 1:x3 23.7 6.0 1.2 13.3 104 lox
30
8 0.7 0.9 - -0.3 - -1.0 0.2 - -0.8 - -0.3 1.2 0.1 0.0 0.5
+ 0.3 I 0.2 rt 0.1 rt- 0.7 -c 0.0 t 0.3 rt 0.1 I?Z0.6 _+ 0.1 ir 0.3 i- 0.1
NA NA 0.2 r 0.0
0.4 1.1 PO.2 -0.4 0.4 -0.8 -0.5 1.3 0.0 0.2 0.8
2 + i f i 2 t k + 2 k
Filtrate 0.3 0.5 0.1 0.5 0.2 0.3 0.6 0.6 0.3 0.4 0.4
NA NA 0.1 IO.1
0.3 0.2 -0.2 -0.2 0.6 --0.3 0.4 2.0 -0.1 -4.1 0.2
NA NA 0.2
Note. After incubation with glucose for 1 min under anaerobic conditions, 20 ml of a cell suspension (5% wet weight) was sprayed in a stirred solution of 60% methanol at -40°C (100 ml). After the indicated time, l&ml portions were spun down at 13OOg, for 10 min at, -2072, and ext,racted as described under Materials and Methods. Supernatants were concentrated under vacuum to approximately 1 ml. Extracts and supernatant were assayed for metabolites as described under Materials and Methods. Both intracellular and extracellular data are expressed as the amount present in 1 ml of incubation mix just before quenching (the cytosolic volume was approximately 3% of the volume of the incubation mixture). For comparison, the sixth column gives the concentrations from a filtrate, obtained after incubation for 1 min with glucose. Measurements are averages of three extracts (average standard deviation, 5%) or supernatants, whereas one filtrate is measured. Negative values can occur due to a combination of the extraction of blanks and a small drift in the baseline in extracts (usually less than 1% in 30 min). NA, not. available.
est occur during extraction (either chemical or enzymatic) and that the metabolites be extracted completely. Several solvents were tested for their ability to permeabilize yeast cells at -4O”C, but only with chloroform was a sufficient permeabilization without interference of the enzymatic assays afterward obtained. The rather strong protein-denaturating property of chloroform is also a major advantage. To test for enzymatic or chemical conversions, a number of metabolites were added at the start of an extraction of cells. Enzymatic conversions up to 70% were initially observed when, after chloroform was added, the extraction was performed at 4’C. Lowering the temperature to -35°C together with the addition of EDTA reduced this problem to an usually acceptable level of less
122
DE
KONING
0.6
FBP V.”
-~ 0.0
0.2
0.4
0.6 time
0.8
1.0
(8)
FIG, 1. Changes in metabolite levels upon addition of glucose. Succharomyces cerevisiae was preincubated anaerobically and incubated with 50 mM glucose for 0 to 1 s using freeze-quench equipment. Quenching, extraction, and determination of metabolites are described under Materials and Methods: 0, glucose; A, G6P; l , FBP; v, DHAP.
than 10% loss (Table 2). Because the observed reactions are affecting metabolites that are considered to be not far from equilibrium in viuo, the conversion rate in normal extracts, in which the concentrations are lower also, will be less. When the amount of conversion as observed in this experiment causes significant problems, the use of an extreme pH (depending on the metabolite) may be the solution. The extraction protocol was tested for completeness by comparison with perchloric acid extraction. When frozen cells (incubated with glucose for 20 min) were shaken with glass beads in the presence of 5% perchloric acid for 20 min, for most metabolites, the same concentrations were found (data not shown). DISCUSSION
In this article we describe methods for quenching metabolism quickly and for extracting metabolites from yeast cells. These methods allow investigation of the metabolism on a very short time scale. The rapid quenching method is recommendable whenever metabolites that have high turnover rates are measured. Alternatives, for example, direct quenching in perchloric acid, have the large disadvantage that cells are not separated from the medium, resulting in very dilute samples. Furthermore, in pilot experiments, we found that yeast cells are rather resistant to 5% perchloric acid: after 5 min of incubation only a small percentage of the metabolites was extracted. This calls into question the suitability of perchloric acid for the inhibition of metabolism on a subsecond time scale.
AND
VAN
DAM
In contrast to the paper by Saez and Lagunas (6), in which washing of yeast ceils on a filter with cold methanol was described, we did not observe leakage of pyruvate from the cells. The small amount present in the medium was already excreted before the quenching of the metabolism. The reason for the discrepancy with the work of Saez and Lagunas is not known. Because cooling is essential to stop metabolism, it is of utmost importance to work at the lowest possible temperature during extraction, even after the addition of chloroform. The procedure is therefore fairly tedious to perform but has the advantage that a rather complete picture of all metabolites can be obtained. Apart from the metabolites mentioned, a number of other related components (CAMP, 2,3-phosphoglycerate, fructose-2,6-bisphosphate, glycerol, glycerol-3-phosphate, NADP+, NADPH, citrate) have been measured and no serious problems are expected with other low-molecular-weight metabolites as long as their solubility in water/methanol is much higher than that in chloroform and diethyl ether. The fairly large amounts of cells that must be extracted are essential to obtain a cell pellet on the chloroform phase that is thick enough to be stable with the tube diameter used; otherwise, quantitative removal of the water phase is difficult. The extracts contain suflicient material to measure all metabolites of interest, using spectrophotometric methods. An automatic ana-
TABLE
2
Recovery of ~e~abolites after Extraction in the Presence of Yeast Cells Metabolite measured in control cell extract (nmol))
Amount added
Metabolite measured in extract with metabolites added before extraction (nmol)
Recovery (So)
F6P G6P
0.3 1.8
147
136 13
92
ADP AMP ATP
69 160 7.0
279
329 183 14
95
4.2 0.2
88
DHAP FBP NADH NAD
11 49
123
93 0.7 133 61
101 99
Note. F6P, ATP, NADH, and DHAP were added just before a suspension of yeast cells in 100% methanol was extracted at -40°C. Protein content of the extracted cells was 12 mg per sample. Extraction and metabolite determination were performed as described under Materials and Methods. Data are expressed in nanomoles per sample and are averages of two different extractions. For each metabolite, values for some metabolites that can be expected to be formed are shown also. Added metabolites are given in italics.
EXTRACTION
ANALYSIS
OF
lyzer (Cobas Bio) greatly facilitated this analysis: 24 samples could be analyzed for 20 metabolites in 1 day. A standard error of less than 10% between different extracts is obtainable for most metabolites. When the same experiments are performed on different batches of yeast cells, occasionally a larger variation is observed. This stresses the time- andcondition-dependent character of metabolite levels in living cells. The method described in this paper presents the opportunity to study changes in yeast metabolism on a very short time scale. In Fig. 1 intracellular accumulation of glucose and G6P within 1 s is clear, whereas other metabolites do not change on this time scale. Measurements of changes in metabolite levels within seconds after glucose addition can be used to elucidate the way in which the glycolytic flux in yeast is regulated by its metabolite concentrations. A paper about such an investigation is in preparation. A further study of the mechanism of glucose uptake is in progress. It may be noted that the method of quenching in cold methanol might be used also for other organisms (other yeast species and bacteria, plant, and animal cell cultures), leaving their membranes intact, although we did not test this. It might even be a useful method in related
GLYCOLYTIC
123
METABOLITES
fields like the study of rapid protein modifications example, phosphorylation by protein kinases).
(for
ACKNOWLEDGMENTS The work was supported in part by a grant from Gist-brocades, The Netherlands. The technical support of Frits Mol and Karel Jaspers is greatly appreciated. We are indebted Jan Berden, Bert Oehlen, and Pieter Postma for valuable discussions and careful reading of the manuscript.
REFERENCES 1. Bray,
2.
B. (7. t 1964) in Rapid Mixing and Sampling Techniques in Biochemistry (Chance, B., Eisenhardt, R. H., Gibson, Q. H., and Louberg-Holm, K. K., Eds.), pp 195-203, Academic Press, New York. Ballou, D. P., and Palmer, G. A. (1974) Anal. Chem. 46, 124% 12<53.
3< Lowry,
Randall,
R. J.
4.
Analysis,
Vol.
0. H., Rosebrough, N. J., Farr, A. L., and (1951) J. Biol. Chem. 193, 265-275. Bergmeyer, H. U. (1985a) Methods of Enzymatic VI, VCH Publishers, Deerfield Beach, FL.
5I Bergmeyer,
H. U. (1985b) Methods of Enzymatic Analysis, Vol. VII, VCH Publishers, Deerfield Beach, FL. Saez, M. J., and Lagunas, R. (1976) Mol. Cell. Biochem. 13,73-78.
6. 7. Bencini,
Biochem.
D. A., Wild,
132,254-X8.
J. R., and
O’Donovan,
G. A. (1983)
Anal.
204,
BIOCHEMISTRY
118-123
(1992)
A Method for the determination of Changes of Gtycolytic Metabolites in Yeast on a Subsecond Time Scale Using Extraction at Neutral pH Wim
de Koning’
and Karel
van Darn’
E. C. Slater Institute for Biochemical Research, Biotechnology Center, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands
Received
January
of
Amsterdam,
13, 1992
Glucose metabolism in yeast can be stopped within 0.1 s by spraying the cells in 60% methanol at -40°C. With this procedure the integrity of the cells is not damaged. Using stopped-flow equipment for the incubation with glucose, major changes within a second are shown to occur in intracellular glucose-g-phosphate whereas the fructose-1,6-bisphosphate concentration remains constant. After quenching, the cells can be separated from the medium, washed with cold methanol when required, and extracted using chloroform at -40°C at neutral pH, ensuring minimal degradation of labile metabolites. With partly automated enzymatic methods, a large variety of metabolites, including all glycolytic intermediates, can be determined in the neutral extracts. During the first second after addition of glucose, a significant increase in free intracellular glucose is found. 10 1992 Academic Press, Inc. -
In the study of a metabolic pathway of an organism, information about the concentration of intermediates can be of crucial interest. Together with information about kinetic properties of the enzymes involved, metabolite levels can be used to investigate the control of specific components of the pathway. More than enzyme activities, metabolite concentrations are very prone to changes induced by (unnoticed) variation in the environment of the cell. For baker’s yeast, growing on glucose, it can be deduced from the
* Present address: Laboratorium voor ~oleculaire Celbiologie, Instituut voor Plantkunde, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium. ’ To whom correspondence should be addressed. 118
University
flux through the pathway and the intracellular metabolite concentrations that a typical metabolite’s half-life is on the order of a second or less (cytosolic glucose is converted at approximately 1 InM s-i). This implies that after a pulse of glucose to a carbon-starved culture, the first significant changes can occur within a second. Study on this time scale therefore is of interest. Furthermore, the high turnover of metabolites makes it imperative that, whenever intracellular glycolytic metabolite concentrations are to be determined, extreme care be taken to stop metabolism very rapidly. Although not all handling of a culture may result in significant changes of metabolites within a second, it is important to be aware of this problem. Direct quenching of the incubation mixture is in principle possible, but many protocols for metabolite determination include a rapid filtration step followed by freezing in liquid nitrogen. Two important advantages are that most of the incubation medium is removed from the sample and that, due to the concentration step, levels of metabolites in the sample are strongly increased. The major disadvantages are: (a) metabolite levels can change during filtration (because the cells are flushed with incubation mixture on the filter, no major changes in the environment are expected to occur during ~ltration, as long as the filter is not running dry) and (b) incubation times shorter than 10 s are impossible to obtain, due to the time required for filtration. Depending on the metabolite(s) of interest, cells are extracted using, for example, perchloric acid or NaOH. With these methods not all metabolites are quantitatively extracted, due to instability of some metabolites at extreme pH. When both NAD’ and NADH are to be measured, usually an acid and an alkaline extraction are performed on separate samples. A number of com0003.2697/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
EXTRACTION
ANALYSIS
OF
pounds are, however, unstable at both high and low pH (dihydroxyacetone phosphate3 (DHAP) and phospho~enol)pyruvate (PEP) are known examples). Especially for these compounds the extraction must be a compromise between completeness (asking for longer incubation times) and prevention of degradation (asking for short times at extreme pH). In this paper a method that overcomes the problems mentioned above is described. It allows quenching of metabolism in yeast in less than 0.1 s. After the quenching the cells can be concentrated and washed, when uptake of components of the medium is investigated. The concentrated cells are extracted at neutral pH, allowing determination of a large variety of metabolites, including nearly all glycolytic intermediates in a single extract. MATERIALS Organism
AND
METHODS
and Preincubation
Experiments were performed with fresh baker’s yeast (Gist-brocades, Delft, The Netherlands). Upon arrival of the pressed yeast cake (usually within 2 days after production), a 1:l (w/w) suspension in Millipore water was made, which was stored at 4°C. Experiments were performed with yeast not older than 14 days. Before an experiment, the yeast suspension was mixed with 1 vol of 100 mM Mes, pH 5.7, flushed with nitrogen, and stored on ice overnight. Thirty minutes before incubation with glucose was started, 6 ml of the yeast suspension (still on ice) was diluted anaerobically with 9 ml of 50 mM Mes, pH 5.7, at 30°C. (The anaerobic conditions used in these experiments were not essential for the methods described but rather were chosen for reasons outside the scope of this paper.) Glucose Incubations Incubations with glucose for less than 10 s were performed with a freeze-quench apparatus (1) with a fourjet tangential mixer (2) under an atmosphere of argon at 30°C. Standard conditions during incubation were 50 mM Mes, pH 5.7, and 50 mM glucose at a cell density of 50 mg wet weight ml-‘. The apparatus was equipped with two l&ml stainless steel syringes and worked with a flow rate of approximately 1 ml s-’ after mixing. The internal volume of the incubation tubing was varied to change the incubation time (from 15 ms to 5 s). The actual flow rate was determined from the speed of the ram in each incubation and used to calculate the exact
3 Abbreviations used: G6P, glucose-6-phosphate; F6P, fructose-6 phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; 3PGA, 3-phosphoglycerate; PPGA, 2-phosphoglycerate; PEP, phospho(enol)pyruvate; Pipes, l,i’-piperazineethanesulfonic acid; Mes, Mo~hoIineethanesuifonic acid.
GLYCOLYTIC
METABOLITES
119
incubation time. Incubations longer than 10 s were performed manually using a syringe without needle to spray the samples in the cold methanol (see below). Quenching Ten- to fifteen-milliliter samples of (incubated) yeast suspension were sprayed in a stirred solution (60 ml) of 60% methanol kept at -40°C by a circulating methanol bath. After the mixture was cooled for approximately 5 min the cells were spun down at 5000 rpm for 5 min at the lowest possible temperature (usually -20°C). Unless stated otherwise, all further manipulations were done at -40°C. The carefully drained pellet (excess remaining water prevents proper mixing with chloroform and increases the freezing point of the sample) was suspended in 2.5 ml of 100% methanol and transferred to precooled 30-ml thick-walled screwcap glass tubes. Twenty microliters of 200 mM EDTA, pH 7, was added under vigorous vortexing of the suspension to inhibit Mg’+-dependent, partly chloroform-resistant enzyme activities (mainly myokinase). After addition of 1 ml of precooled chloroform the tubes were frozen in liquid nitrogen and stored at -70°C. When internal glucose was measured, 25 ml of the quenched suspension was filtered over a cellulose acetate filter (ST 69, Schleicher & Schuell, Dassel, Germany; pore size, 1.2 pm; 25 mm diameter). The cells were washed three times with 5 ml of cold 60% methanol. The filter was transferred quickly to a precooled tube containing 2.5 ml of 100% methanol. After suspension of the cells the filter was removed and EDTA and chloroform were added. Extraction
of Metabolites
Unless stated otherwise, samples were kept at -40°C. To each tube 4 ml of cold chloroform was added. Under vigorous vortexing 2 ml of ice-cold 2 mM Pipes pH 7.2, was added. To achieve complete permeabilization and extraction of the cells the tubes were shaken for 45 min at -35”C, 2200 rpm (in a regulated liquid nitrogencooled incubator; Cryoson TRA 11, Middenbeemster, The Netherlands), in horizontal position on an Ika Vibrax VXR (Janke & Kunkel, Staufen, Germany) using a homemade adaptor. The cells were pelleted on the chloroform phase by centrifugation at 13OOg for 15 min in a swing-out rotor at the lowest possible temperature (-20°C using adaptors for the glass tubes that were precooled at -40°C). The supernatant was carefully collected using an aspiration setup. To the remaining content of the tubes 2 ml methanol was added and, after mixing, 2 ml ice-cold 2 mM Pipes was added under vortexing. After the cells were vortexed for 20 s two times the samples were centrifuged again and the supernatant was added to the first extract. The combined extracts were extracted once with 15 ml diethyl ether to remove traces of lipids.
120
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The volume was reduced by evaporation under vacuum (<50 Pa, to ensure sufficient cooling of the samples) in a Speedvac (Savant Instruments, Hicksville, NY) to approximately 0.5-l ml without additional heating. To prevent excessive boiling due to the remaining ether, the evaporation was started with frozen samples. After the volume was measured, the samples were stored at -70°C. Shortly before the determination of metabolites, the samples were thawed on ice, mixed, and centrifuged in order to remove possible small precipitates. After the chloroform was carefully decanted, the cell pellet in the extraction tubes was dried under a stream of air. Five milliliters of 1 N NaOH was added and, after boiling and centrifugation, the total amount of protein in the supernatant was determined according to Lowry et al. (3). This served as a measure for the amount of biomass that was extracted. We prefer to express the amounts of intermediates in terms of cytosolic concentrations for later use in the calculation of metabolic fluxes. To calculate these concentrations, a yeast cytosolic volume of 3.75 ~1 per milligram of protein was assumed. If necessary, this can be converted to dry weight using the conversion that 1 mg protein equals 2.5 mg dry weight (Dr. M. A. Herweijer, personal communication). For the present study it was not essential to determine these values more accurately.
dition of 0.7 U ml-’ glycerol-3-phosphate dehydrogenase and 20 U ml-l triosephosphate isomerase + 0.14 U ml-’ aldolase, respectively. Pyruvate, PEP, 2-phosphoglycerate (BPGA), and 3phosphoglycerate (3PGA) were measured in the presence of 100 mM KCl, 10 mM MgSO,, and 0.15 mM NADH by the addition of 1.1 U ml-’ lactate dehydrogenase, 1.6 U ml-’ pyruvate kinase + 1 mM ADP, 0.6 U ml-’ enolase, and 2 U ml-’ phosphoglycerate mutase + 20 pM 2,3-phosphoglycerate, respectively. ADP and AMP were measured in the presence of 100 mM KCl, 10 mM MgSO,, and 0.15 mM NADH by the addition of 1.1 U ml-l lactate dehydrogenase (pyruvate determination), 1.6 U ml-’ pyruvate kinase + 0.5 mM PEP, and 1.4 U ml-’ myokinase + 0.1 mM ATP, respectively. NADH was measured using 0.5 mM DHAP and 0.4 U glycerol-3-phosphate dehydrogenase. NAD+ was measured using four-times-diluted commercial glycine buffer containing a trapping reagent (Sigma, No. 332-9) with 50 mM ethanol by adding 10 U ml-’ alcohol dehydrogenase. Phosphate was measured according to Bencini et al. (7) from the change in absorbance at 350 nm after the addition of molybdate reagent (11.25 mM ammonium molybdate, 75 mM zinc acetate, pH 5.0).
Analytical
Chemicals
Determinations
All analyses were performed on a Cobas Bio Autoanalyser (Roche, Basel, Switzerland) that was connected with a MS/DOS computer for datalogging. The machine protocols used and the spreadsheet-based dataanalyzing programs are available upon request. Volumes in published methods were adapted to the working volume of this apparatus (0.25 ml). Chemical determinations were done at 25°C and enzymatic determinations at 37°C. Enzymatic determinations of metabolites were performed in 50 mM triethanolamine, pH 7.6, following the changes in NAD(P)H at 340 nm (tNADu,)n = 6.22 cm-’ mM-l), essentially as described by Bergmeyer (4,5). Samples were added so that the amount of metabolite in the assay was always below 0.05 mM. Enzymes were used without desalting and diluted in buffer. Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), ATP, or glucose were determined in the presence of 5 mM MgSO, and 0.4 mM NADP+ by the addition of glucose-6-phosphate dehydrogenase (0.7 U ml-‘), phosphoglucose isomerase (2.8 U ml-‘), and hexokinase (1.1 U ml-‘) + 5 mM fructose. In the case of glucose determination after the addition of glucose-6-phosphate dehydrogenase, hexokinase (1.1 U ml-l) + 0.5 mM ATP was added. DHAP and fructose-1,6-bisphosphate (FBP) were measured in the presence of 0.15 mM NADH by the ad-
Methanol was of technical quality. The pH of each batch was checked to be around 7 when diluted to 60% with Millipore water. Technical-quality ether was acidic; therefore, in this case an analytical-grade (Merck) quality was used. Other chemicals were of analytical grade and used as supplied. Enzymes and biochemicals were from Boehringer-Mannheim or Sigma. Buffers were brought to pH with KOH or HCl. RESULTS
Metabolite
Determinations
Our enzymatic determinations include no new methods, but the use of an automated analyzer greatly facilitated measurements, improved the reproducibility, and also reduced the amount of extract needed. Although the accuracy was somewhat dependent on the quality of the sample and also on the metabolite investigated, computer-assisted manual control of each determination allowed a routine detection level of approximately 1 pM in the sample (=lOO pmol) for NAD(P)H-based assays. For concentrations of metabolites in the sample above 150 pM, differences between duplicates were less than 3%. Variation between different extractions of the same incubation was usually within 10%. Different batches of yeast cells gave occasionally larger variations.
EXTRACTION
ANALYSIS
OF GLYCOLYTIC
TABLE
Quenching Method The method employed to stop metabolism in the yeast cells is partly based on the idea of Saez and Lagunas (6), who advocated washing of yeasts with 50% methanol of -40°C after filtering. In our experiments cells are sprayed in 5 vol of 60% methanol at -40°C and subsequently separated from the methanol either by centrifugation or by filtration at low temperature. This method was tested for two essential properties: (a) cells do not leak intermediates until extracted and (b) sufficient block of the metabolism. Leakage of cells was checked by measuring in the supernat.ant the concentration of metabolites that are present in signi~cant amounts in the cells. The supernatant was concentrated under vacuum to remove the methanol, which interferes with some enzymatic assays, and to increase the sensitivity. Very small amounts of pyruvate were detectable, never exceeding 10% of the pyruvate detected intracellularly (Table 1). Comparison with the amount of pyruvate in a filtrate showed that this pyruvate is most likely already excreted by the cells before quenching. The amount of extracellular pyruvate did not increase when the cells were kept for 30 min at -40°C before centrifugation. Other metabolites were below the detection level (approximately 1 pM, depending somewhat on the metabolite) in the supernatant, independent of the residence time in the methanol. In agreement with these observations is the constant level of intracellular metabolites after quenching. From Table 1 it can be concluded t.hat cells do not leak metabolites when quenched in 60% methanol at -4O’C. The complete block of the metabolism is also evident from these results, but more critical experiments are described in the following paragraphs. The speed of quenching is tested by investigation of whether changes in intermediates can be detected on a short time scale. Using freeze-quench equipment, yeast was incubated with 50 RIM glucose for periods varying from 0 (no glucose added) to 1 s. Figure 1 shows that internal glucose and G6P start to increase as soon as glucose is added, whereas other metabolites are virtually constant during the first second after glucose addition. Note that the first point after glucose addition (15 ms) coincides within experimental error for all metabolites with c = 0, where the cells are not incubated with glucose. This is a strong indication that the quenching of the metabolism is sufficient on the time scale shown. Another possibility is that metabolism starts after a delay that just equals the dead time of the quenching. This seems rather unlikely.
The two important requirements for any extraction method are that no changes in the metabolites of inter-
121
M~TABOLIT~S 1
Resistance to Leakage of Saccharomyces cerevisiae Suspended in 50% Methanol at -40°C Amount of metabolites found intr~cellularIy
Amount of metabolites found extracell~lari~ (nmollml)
fnmol/ml~ Tilne in methanol -.
G6P F6P FBP DHAP 3PGA ZPGA PEP Pyruvate NAD NADH ATP ADP AMP Phosphate
Time in
(min)
supernatant (min)
8
30
25.0 4.9 18.R 12.4 1.2 0.1 0.0 13.6 23.9 7.1 1.2 12.9 102 I.030
24.7 5.0 14.9 12.0 1.3 0.1 0.1 1:x3 23.7 6.0 1.2 13.3 104 lox
30
8 0.7 0.9 - -0.3 - -1.0 0.2 - -0.8 - -0.3 1.2 0.1 0.0 0.5
+ 0.3 I 0.2 rt 0.1 rt- 0.7 -c 0.0 t 0.3 rt 0.1 I?Z0.6 _+ 0.1 ir 0.3 i- 0.1
NA NA 0.2 r 0.0
0.4 1.1 PO.2 -0.4 0.4 -0.8 -0.5 1.3 0.0 0.2 0.8
2 + i f i 2 t k + 2 k
Filtrate 0.3 0.5 0.1 0.5 0.2 0.3 0.6 0.6 0.3 0.4 0.4
NA NA 0.1 IO.1
0.3 0.2 -0.2 -0.2 0.6 --0.3 0.4 2.0 -0.1 -4.1 0.2
NA NA 0.2
Note. After incubation with glucose for 1 min under anaerobic conditions, 20 ml of a cell suspension (5% wet weight) was sprayed in a stirred solution of 60% methanol at -40°C (100 ml). After the indicated time, l&ml portions were spun down at 13OOg, for 10 min at, -2072, and ext,racted as described under Materials and Methods. Supernatants were concentrated under vacuum to approximately 1 ml. Extracts and supernatant were assayed for metabolites as described under Materials and Methods. Both intracellular and extracellular data are expressed as the amount present in 1 ml of incubation mix just before quenching (the cytosolic volume was approximately 3% of the volume of the incubation mixture). For comparison, the sixth column gives the concentrations from a filtrate, obtained after incubation for 1 min with glucose. Measurements are averages of three extracts (average standard deviation, 5%) or supernatants, whereas one filtrate is measured. Negative values can occur due to a combination of the extraction of blanks and a small drift in the baseline in extracts (usually less than 1% in 30 min). NA, not. available.
est occur during extraction (either chemical or enzymatic) and that the metabolites be extracted completely. Several solvents were tested for their ability to permeabilize yeast cells at -4O”C, but only with chloroform was a sufficient permeabilization without interference of the enzymatic assays afterward obtained. The rather strong protein-denaturating property of chloroform is also a major advantage. To test for enzymatic or chemical conversions, a number of metabolites were added at the start of an extraction of cells. Enzymatic conversions up to 70% were initially observed when, after chloroform was added, the extraction was performed at 4’C. Lowering the temperature to -35°C together with the addition of EDTA reduced this problem to an usually acceptable level of less
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0.6
FBP V.”
-~ 0.0
0.2
0.4
0.6 time
0.8
1.0
(8)
FIG, 1. Changes in metabolite levels upon addition of glucose. Succharomyces cerevisiae was preincubated anaerobically and incubated with 50 mM glucose for 0 to 1 s using freeze-quench equipment. Quenching, extraction, and determination of metabolites are described under Materials and Methods: 0, glucose; A, G6P; l , FBP; v, DHAP.
than 10% loss (Table 2). Because the observed reactions are affecting metabolites that are considered to be not far from equilibrium in viuo, the conversion rate in normal extracts, in which the concentrations are lower also, will be less. When the amount of conversion as observed in this experiment causes significant problems, the use of an extreme pH (depending on the metabolite) may be the solution. The extraction protocol was tested for completeness by comparison with perchloric acid extraction. When frozen cells (incubated with glucose for 20 min) were shaken with glass beads in the presence of 5% perchloric acid for 20 min, for most metabolites, the same concentrations were found (data not shown). DISCUSSION
In this article we describe methods for quenching metabolism quickly and for extracting metabolites from yeast cells. These methods allow investigation of the metabolism on a very short time scale. The rapid quenching method is recommendable whenever metabolites that have high turnover rates are measured. Alternatives, for example, direct quenching in perchloric acid, have the large disadvantage that cells are not separated from the medium, resulting in very dilute samples. Furthermore, in pilot experiments, we found that yeast cells are rather resistant to 5% perchloric acid: after 5 min of incubation only a small percentage of the metabolites was extracted. This calls into question the suitability of perchloric acid for the inhibition of metabolism on a subsecond time scale.
AND
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In contrast to the paper by Saez and Lagunas (6), in which washing of yeast ceils on a filter with cold methanol was described, we did not observe leakage of pyruvate from the cells. The small amount present in the medium was already excreted before the quenching of the metabolism. The reason for the discrepancy with the work of Saez and Lagunas is not known. Because cooling is essential to stop metabolism, it is of utmost importance to work at the lowest possible temperature during extraction, even after the addition of chloroform. The procedure is therefore fairly tedious to perform but has the advantage that a rather complete picture of all metabolites can be obtained. Apart from the metabolites mentioned, a number of other related components (CAMP, 2,3-phosphoglycerate, fructose-2,6-bisphosphate, glycerol, glycerol-3-phosphate, NADP+, NADPH, citrate) have been measured and no serious problems are expected with other low-molecular-weight metabolites as long as their solubility in water/methanol is much higher than that in chloroform and diethyl ether. The fairly large amounts of cells that must be extracted are essential to obtain a cell pellet on the chloroform phase that is thick enough to be stable with the tube diameter used; otherwise, quantitative removal of the water phase is difficult. The extracts contain suflicient material to measure all metabolites of interest, using spectrophotometric methods. An automatic ana-
TABLE
2
Recovery of ~e~abolites after Extraction in the Presence of Yeast Cells Metabolite measured in control cell extract (nmol))
Amount added
Metabolite measured in extract with metabolites added before extraction (nmol)
Recovery (So)
F6P G6P
0.3 1.8
147
136 13
92
ADP AMP ATP
69 160 7.0
279
329 183 14
95
4.2 0.2
88
DHAP FBP NADH NAD
11 49
123
93 0.7 133 61
101 99
Note. F6P, ATP, NADH, and DHAP were added just before a suspension of yeast cells in 100% methanol was extracted at -40°C. Protein content of the extracted cells was 12 mg per sample. Extraction and metabolite determination were performed as described under Materials and Methods. Data are expressed in nanomoles per sample and are averages of two different extractions. For each metabolite, values for some metabolites that can be expected to be formed are shown also. Added metabolites are given in italics.
EXTRACTION
ANALYSIS
OF
lyzer (Cobas Bio) greatly facilitated this analysis: 24 samples could be analyzed for 20 metabolites in 1 day. A standard error of less than 10% between different extracts is obtainable for most metabolites. When the same experiments are performed on different batches of yeast cells, occasionally a larger variation is observed. This stresses the time- andcondition-dependent character of metabolite levels in living cells. The method described in this paper presents the opportunity to study changes in yeast metabolism on a very short time scale. In Fig. 1 intracellular accumulation of glucose and G6P within 1 s is clear, whereas other metabolites do not change on this time scale. Measurements of changes in metabolite levels within seconds after glucose addition can be used to elucidate the way in which the glycolytic flux in yeast is regulated by its metabolite concentrations. A paper about such an investigation is in preparation. A further study of the mechanism of glucose uptake is in progress. It may be noted that the method of quenching in cold methanol might be used also for other organisms (other yeast species and bacteria, plant, and animal cell cultures), leaving their membranes intact, although we did not test this. It might even be a useful method in related
GLYCOLYTIC
123
METABOLITES
fields like the study of rapid protein modifications example, phosphorylation by protein kinases).
(for
ACKNOWLEDGMENTS The work was supported in part by a grant from Gist-brocades, The Netherlands. The technical support of Frits Mol and Karel Jaspers is greatly appreciated. We are indebted Jan Berden, Bert Oehlen, and Pieter Postma for valuable discussions and careful reading of the manuscript.
REFERENCES 1. Bray,
2.
B. (7. t 1964) in Rapid Mixing and Sampling Techniques in Biochemistry (Chance, B., Eisenhardt, R. H., Gibson, Q. H., and Louberg-Holm, K. K., Eds.), pp 195-203, Academic Press, New York. Ballou, D. P., and Palmer, G. A. (1974) Anal. Chem. 46, 124% 12<53.
3< Lowry,
Randall,
R. J.
4.
Analysis,
Vol.
0. H., Rosebrough, N. J., Farr, A. L., and (1951) J. Biol. Chem. 193, 265-275. Bergmeyer, H. U. (1985a) Methods of Enzymatic VI, VCH Publishers, Deerfield Beach, FL.
5I Bergmeyer,
H. U. (1985b) Methods of Enzymatic Analysis, Vol. VII, VCH Publishers, Deerfield Beach, FL. Saez, M. J., and Lagunas, R. (1976) Mol. Cell. Biochem. 13,73-78.
6. 7. Bencini,
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D. A., Wild,
132,254-X8.
J. R., and
O’Donovan,
G. A. (1983)
Anal.