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A simple radioassay of acetate thiokinase
ANALYTICAL
BIOCHEMISTRY
A Simple
38, 469474
Radioassay
M. SLADEK, Biochemisches
Institut
CHR.
der Universitit,
(1970)
of Acetate BARTH,
AND
Hermann
Received April
Thiokinase K. DECKER
Herokrstr.
7, 78 Freiburg,
Germany
22, 1970
Several methods are available for the determination of acetate thiokinase (acetyl-CoA synthetase, EC 6.2.1.1) activity. During studies of its subcellular distribution they were found to be of limited applicability. With the optical assay using citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) (1) no acetate thiokinase activity could be detected in crude mitochondrial extracts (2). An isotope method coupled to citrate synthase (3) is likely to give too low results with mitochondrial extracts because further conversion of labeled citrate cannot be excluded. The radiochromatographic assay with carnitine acetyltransferase (EC 2.3.1.7) was reported to yield varying recoveries of total cellular activity (4). The calorimetric hydroxamate method (5) is too insensitive for detecting minor activities of some cell compartments and is also of low specificity because the formation of any active acyl compound is measured. In this paper a rapid and generally applicable isotope assay of acetate thiokinase is described having high sensitivity and specificity. It applies the principle of “isoionic-exchange chromatography” (6) to the separat,ion of the reaction substrate “C-acetate, and the product, 14Cacetohydroxamate. MATERIALS
AND
METHODS
Sodium 1-14C-acetate was purchased from Amersham, Great Britain (sp.act. 57 mCi/mmole, batch 122). CoASH,l citrate lyase (EC 4.1.3.6), citrate synthase (EC 4.1.3.7), ATP, glutathione, and acetate kinase (EC 2.7.2.1), were obtained from Boehringer Mannheim GmbH, Germany, ion-exchange resins from SERVA, Heidelberg, Germany. Before use Dowex 2X-8, 200400 mesh, was cycled twice with 2 N NaOH ,and 10% HCl and converted to the acetate form with a mixture of 9 N acetic acid and 0.9 ib! sodium acetate. ’ Abbreviations glutathione.
CoASH = coenzyme A ; ATP = adenosine 469
triphosphate;
GSH =
470
SLADEK,
BARTH,
AND
DECKER
Other materials were of reagent grade and purchased from Merck, D,armstadt, Germany. Acetohydroxamie acid was prepared from acetic acid anhydride and hydroxylamine and recrystallized from ethyl acetate (m.p. 86°C). 1-14CAcetohydroxamic acid was prepared enzymically with acetate kinase and purified by ion-exchange thin-layer chromatography on polyethylene iminocellulose (MN Polygram Cel PEI, Macherey & Nagel, Diiren, Germany) with butanol saturated with 0.05 M LiCl as solvent. RI values for acetic acid were 0.02, for acetohydroxamic acid 0.15. Free hydroxylamine solutions were prepared by batchwise neutralizing about 25 mmoles of NH,OH*HCl with Dowex 2X-8, OH- form, 2W 400 mesh (glass electrode). The resin was filtered off and the solution was concentrated to approximately 5 ml in UUCUO. The recovery of free hydroxylamine was about 55%; it fell drastically if the volume was reduced to less than 5 ml during solvent evaporation. Hydroxylamine was titrated manganometrically (7). Potassium ATP was prepared ‘by passing sodium ATP through ma column of Amberlite IR-120 in the K+ form. PROCEDURES
Enzyme Assay. Triethanolamine hydrochloride 200 mM, pH 8.0; CoASH 0.5 mM; GSH 10 mM; MgCl, 10 mM; potassium ATP 5 mM; alkali ion free hydroxylamine 400 mM; potassium acetate 10 mM (2.4 X 10B dpm lJ4C-‘acetate) ; enzyme preparation. Total volume 1.0 ml. GoASH and GSH were dissolved in buffer immediately before use. The reaction was carried out at 25°C for 20 min and stopped by addition of 1.0 ml of 3 M acetic acid followed by boiling for 5 min. For optimal separation of acetohydroxamate and acetic acid the incubation mixture must be adjusted to pH 4 by addition of 3 M acetic acid bbefore chromatography. Stan&~-d Artulyticat Procedure. 0.5 ml of the centrifuged assay mixture was applied to a column (149 X 5 mm) of Dowex 2X-8, 200-490 mesh, acetate form. The column was rinsed twice with 0.5 ml portions of water and the eluate discarded. Acetohydroxamic acid was then eluted with 3 ml of 0.8 M acetic acid. This fraction was collected in a scintillation vial and 10 ml of Bray scintillator (11) was added. It was not necessary to regulate the flow rate of the column. Recovery of authentic 1-14C-acetohydroxamic acid after the standard analytical procedure was better than 92%. Columns were regenerated with 25 ml of 3 M sodium acetate followed by 5 ml of 0.8 N acetic acid. Radioactivity was measured in a Packard scintillation spectrometer model 3380. The measurements were corrected by internal standardization. Standard
RADIOASSAY
OF
ACETATE
THIOKINASE
471
This standard assay was compared to the radioassay based on citrate synthase (3). In the latter method the recovery of isolated citrate (citrate lyase method (8)) was found to average 80%. This value was accounted for in calculating reaction rates. Homogenates Livers from male Wistar rats (200-230 gm) were freed from blood by in situ perfusion with 0.3 M sucrose, buffered with 10 mM triethanolamine hydrochloride, pH 7.2, and homogenized according to PotterElvehjem in the same medium; cell fractions were subsequently sonicated (Branson Sonifier, model J17A, 4 periods of 10 set, setting 2.5). The whole liver homogenate was usually taken for assay ; sometimes a particle-free supernatant was used. Protein was determined by the biuret method (12). REHJLTS
A simple and rapid assay of acetate thiokinase activity using labeled acetate as substrate and hydroxlamine as acyl acceptor affords an easy and quantitative separation of small amounts of acetohydroxamate from an excess of acetate. This was accomplished by isoionic-exchange chromatography, which allows collection of acetate-free acetohydroxamate in a volume of only 3 ml (Fig. 1). Thus, the entire labeled hydroxamate formed in an enzymic assay can be transferred to one counting vial and its radioactivity so determined quantitatively. “C-Acetohydroxamic acid formation catalyzed by acetate thiokinase activity of liver homogenates was found to be linear with time and proportional to protein concentration (Fig. 2). Linear relationship indicates that the specific radioactivity of the substrate was not altered by endogenous acetate of liver homogenate. Assays were run in duplicate. The difference between parallels averaged 2.5%. With the standard analytical procedure propionic acid and propionohydroxamic acid follow the same elution pattern as acetic and acetohydroxamic acid. Therefore propionate activation can be assayed in the same manner. The method does not, however, allow quantitative separation of butyric acid and butyrohydroxamic acid (2). ,4 comparison of the assay described here with the radioactive method coupled to citrate synthase (3) gave identical results when carried out with the same cytoplasmic extract of rat liver (1.01 pmole acetohydroxamic acid formed/min X gm wet liver tissue, 25°C). This argues against an inhibitory effect of hydroxylamine on the enzyme. In fact, no change in acetate thiokinase activity was seen when the concentration of hydroxylamine was varied from 200 to 400 nnVf (2).
472
SLADEK, .
.
BARTH,
.
AND
DECKER
.
-b-
lO&*
l 0.8 M
40
t
CHg
COOH
3M
CH, COO-No+
:1$-
\1 :1d-
f
10
12
14
t6
18
ml
FIG. 1. Separation of acetic acid and acetohydroxamic procedure.” For details see “Methods.”
acid by “standard
analytical
P 150 -
100 .
56 .
L 0.40 Min
o.so
1.2 m g Protein
1.6
2.0
2, 1
FIQ. 2. Acetohydroxamic acid formation by rat liver acetate thiokinase with varying time of incubation (a) and changing amounts of homogenate protein (b). For details of incubation and analytical procedure see Standard Enzyme Assay and Standard Analytical Procedure.
RADIOASSAY
OF
ACETATE
473
THIOKINASE
Table 1 indicates that an impurity of the labeled acetate appears in the acetohydroxamic acid fraction. This was confirmed by ion-exchange paper chromatography of an assay mixture. Radioactivity amounting to the blank value was found on a spot different from both acetic and acetohydroxamic acid. Unless the labeled substrate (acetate) is purified extensively, the sensitivity of this method is limited by this contaminant. With the batch of l-14C-acetate used here and under the standard assay conditions the radioactivity of this impurity corresponded to 15 nmoles of product formation (Table 1). TABLE 1 Sensitivity of ‘Standard Enzyme Assay”0 Acetohydroxamic dpm/assay
acid,
Corresponding to nmole acetohydroxamic acid formed 170.0 21.5 14.8 15.0 14.1
Standard assay CoASH omit,ted With heat-inactivated ensyme Incubation at 0°C Incubation of substrate and buffer only
40,752 5,160 3,556 3,600 3,334
n For assay conditions see “Methods.” to 10 mg tissue was used.
0.2 ml of a whole liver homogenate equivalent
DISCUSSION
The method described here fulfills necessary criteria of a reliable enzyme assay. It measures the catalytic conversion of the specific substrate only; this is achieved by use of a labeled substrate which, in addition, renders the method very sensitive. Also, by forming a stable hydroxamic acid derivative it excludes spontaneous or enzymic conversions of the reaction product. Using different kinds of tissue fractionation methods in rat liver and kidney, the sum of thiokinase activities was always better than 87% for acetate and propionate activation (2). The separation of product from substrate is accomplished by the application of “isoionic-exchange chromatography” developed in our laboratory (6). By using acetic acid as eluent it allows retardation of the reaction substrate acetate (isoion) which can be calculated from the total ion-exchange capacity of the resin. The reaction product appears with the column effluent (void volume) in agreement with its pK value of 9.32 (10).
474
2. 4. 5. 6.
7. 8. 9. 10.
11. 12.
BARTH,
AND
DECKER
REFERENCES R. W., AND BALLARD, F. J., Biochem. J. 105, 527 (1968). SLADEK, M., Thesis, Freiburg, 1970. HEPP, D., PR%SE, E., WEISS, H., AND WIELAND, O., Biochem. 2. 344, 87 (1966). AAS, M., AND BREMER, J., Biochim. Biophys. Acta 164, 157 (1968). EISENBERQ, M. A., Biochim. Biophys. Acta 16, 58 (1965). THAUER, R. K., RUPPRECHT, E., AND JUNGERMANN, K., Anal. Biochem. 38, 461 (1970). p. 63. de Gruyter, Berlin 1961. JANDER, G., AND JAHR, K. F., “Massanalyse,” MOELLERING, M., AND GRUBER, W., Anal. Biochem. 17, 369 (1966). WEBSTER, 5. T., .I. Biol. Chem. 240, 5504 (1966). FISHBEIN, W. N., DALY, Y., AND STREETER, C. L., And. Biochem. 28, 13 (1969). BRAY, G. A., Ad Biochem. 1, 279 (1960). BEISENHERZ, G., BOLTZE, H. J., BUTCHER, TH., CZOK, R., GARBADE, K. M., MEYERARENDT, E., AND PFLEIDERER, G., Z. Naturforsch. Sb, 555 (1953).
1. HANSON, 3.
SLADEK,
BIOCHEMISTRY
A Simple
38, 469474
Radioassay
M. SLADEK, Biochemisches
Institut
CHR.
der Universitit,
(1970)
of Acetate BARTH,
AND
Hermann
Received April
Thiokinase K. DECKER
Herokrstr.
7, 78 Freiburg,
Germany
22, 1970
Several methods are available for the determination of acetate thiokinase (acetyl-CoA synthetase, EC 6.2.1.1) activity. During studies of its subcellular distribution they were found to be of limited applicability. With the optical assay using citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) (1) no acetate thiokinase activity could be detected in crude mitochondrial extracts (2). An isotope method coupled to citrate synthase (3) is likely to give too low results with mitochondrial extracts because further conversion of labeled citrate cannot be excluded. The radiochromatographic assay with carnitine acetyltransferase (EC 2.3.1.7) was reported to yield varying recoveries of total cellular activity (4). The calorimetric hydroxamate method (5) is too insensitive for detecting minor activities of some cell compartments and is also of low specificity because the formation of any active acyl compound is measured. In this paper a rapid and generally applicable isotope assay of acetate thiokinase is described having high sensitivity and specificity. It applies the principle of “isoionic-exchange chromatography” (6) to the separat,ion of the reaction substrate “C-acetate, and the product, 14Cacetohydroxamate. MATERIALS
AND
METHODS
Sodium 1-14C-acetate was purchased from Amersham, Great Britain (sp.act. 57 mCi/mmole, batch 122). CoASH,l citrate lyase (EC 4.1.3.6), citrate synthase (EC 4.1.3.7), ATP, glutathione, and acetate kinase (EC 2.7.2.1), were obtained from Boehringer Mannheim GmbH, Germany, ion-exchange resins from SERVA, Heidelberg, Germany. Before use Dowex 2X-8, 200400 mesh, was cycled twice with 2 N NaOH ,and 10% HCl and converted to the acetate form with a mixture of 9 N acetic acid and 0.9 ib! sodium acetate. ’ Abbreviations glutathione.
CoASH = coenzyme A ; ATP = adenosine 469
triphosphate;
GSH =
470
SLADEK,
BARTH,
AND
DECKER
Other materials were of reagent grade and purchased from Merck, D,armstadt, Germany. Acetohydroxamie acid was prepared from acetic acid anhydride and hydroxylamine and recrystallized from ethyl acetate (m.p. 86°C). 1-14CAcetohydroxamic acid was prepared enzymically with acetate kinase and purified by ion-exchange thin-layer chromatography on polyethylene iminocellulose (MN Polygram Cel PEI, Macherey & Nagel, Diiren, Germany) with butanol saturated with 0.05 M LiCl as solvent. RI values for acetic acid were 0.02, for acetohydroxamic acid 0.15. Free hydroxylamine solutions were prepared by batchwise neutralizing about 25 mmoles of NH,OH*HCl with Dowex 2X-8, OH- form, 2W 400 mesh (glass electrode). The resin was filtered off and the solution was concentrated to approximately 5 ml in UUCUO. The recovery of free hydroxylamine was about 55%; it fell drastically if the volume was reduced to less than 5 ml during solvent evaporation. Hydroxylamine was titrated manganometrically (7). Potassium ATP was prepared ‘by passing sodium ATP through ma column of Amberlite IR-120 in the K+ form. PROCEDURES
Enzyme Assay. Triethanolamine hydrochloride 200 mM, pH 8.0; CoASH 0.5 mM; GSH 10 mM; MgCl, 10 mM; potassium ATP 5 mM; alkali ion free hydroxylamine 400 mM; potassium acetate 10 mM (2.4 X 10B dpm lJ4C-‘acetate) ; enzyme preparation. Total volume 1.0 ml. GoASH and GSH were dissolved in buffer immediately before use. The reaction was carried out at 25°C for 20 min and stopped by addition of 1.0 ml of 3 M acetic acid followed by boiling for 5 min. For optimal separation of acetohydroxamate and acetic acid the incubation mixture must be adjusted to pH 4 by addition of 3 M acetic acid bbefore chromatography. Stan&~-d Artulyticat Procedure. 0.5 ml of the centrifuged assay mixture was applied to a column (149 X 5 mm) of Dowex 2X-8, 200-490 mesh, acetate form. The column was rinsed twice with 0.5 ml portions of water and the eluate discarded. Acetohydroxamic acid was then eluted with 3 ml of 0.8 M acetic acid. This fraction was collected in a scintillation vial and 10 ml of Bray scintillator (11) was added. It was not necessary to regulate the flow rate of the column. Recovery of authentic 1-14C-acetohydroxamic acid after the standard analytical procedure was better than 92%. Columns were regenerated with 25 ml of 3 M sodium acetate followed by 5 ml of 0.8 N acetic acid. Radioactivity was measured in a Packard scintillation spectrometer model 3380. The measurements were corrected by internal standardization. Standard
RADIOASSAY
OF
ACETATE
THIOKINASE
471
This standard assay was compared to the radioassay based on citrate synthase (3). In the latter method the recovery of isolated citrate (citrate lyase method (8)) was found to average 80%. This value was accounted for in calculating reaction rates. Homogenates Livers from male Wistar rats (200-230 gm) were freed from blood by in situ perfusion with 0.3 M sucrose, buffered with 10 mM triethanolamine hydrochloride, pH 7.2, and homogenized according to PotterElvehjem in the same medium; cell fractions were subsequently sonicated (Branson Sonifier, model J17A, 4 periods of 10 set, setting 2.5). The whole liver homogenate was usually taken for assay ; sometimes a particle-free supernatant was used. Protein was determined by the biuret method (12). REHJLTS
A simple and rapid assay of acetate thiokinase activity using labeled acetate as substrate and hydroxlamine as acyl acceptor affords an easy and quantitative separation of small amounts of acetohydroxamate from an excess of acetate. This was accomplished by isoionic-exchange chromatography, which allows collection of acetate-free acetohydroxamate in a volume of only 3 ml (Fig. 1). Thus, the entire labeled hydroxamate formed in an enzymic assay can be transferred to one counting vial and its radioactivity so determined quantitatively. “C-Acetohydroxamic acid formation catalyzed by acetate thiokinase activity of liver homogenates was found to be linear with time and proportional to protein concentration (Fig. 2). Linear relationship indicates that the specific radioactivity of the substrate was not altered by endogenous acetate of liver homogenate. Assays were run in duplicate. The difference between parallels averaged 2.5%. With the standard analytical procedure propionic acid and propionohydroxamic acid follow the same elution pattern as acetic and acetohydroxamic acid. Therefore propionate activation can be assayed in the same manner. The method does not, however, allow quantitative separation of butyric acid and butyrohydroxamic acid (2). ,4 comparison of the assay described here with the radioactive method coupled to citrate synthase (3) gave identical results when carried out with the same cytoplasmic extract of rat liver (1.01 pmole acetohydroxamic acid formed/min X gm wet liver tissue, 25°C). This argues against an inhibitory effect of hydroxylamine on the enzyme. In fact, no change in acetate thiokinase activity was seen when the concentration of hydroxylamine was varied from 200 to 400 nnVf (2).
472
SLADEK, .
.
BARTH,
.
AND
DECKER
.
-b-
lO&*
l 0.8 M
40
t
CHg
COOH
3M
CH, COO-No+
:1$-
\1 :1d-
f
10
12
14
t6
18
ml
FIG. 1. Separation of acetic acid and acetohydroxamic procedure.” For details see “Methods.”
acid by “standard
analytical
P 150 -
100 .
56 .
L 0.40 Min
o.so
1.2 m g Protein
1.6
2.0
2, 1
FIQ. 2. Acetohydroxamic acid formation by rat liver acetate thiokinase with varying time of incubation (a) and changing amounts of homogenate protein (b). For details of incubation and analytical procedure see Standard Enzyme Assay and Standard Analytical Procedure.
RADIOASSAY
OF
ACETATE
473
THIOKINASE
Table 1 indicates that an impurity of the labeled acetate appears in the acetohydroxamic acid fraction. This was confirmed by ion-exchange paper chromatography of an assay mixture. Radioactivity amounting to the blank value was found on a spot different from both acetic and acetohydroxamic acid. Unless the labeled substrate (acetate) is purified extensively, the sensitivity of this method is limited by this contaminant. With the batch of l-14C-acetate used here and under the standard assay conditions the radioactivity of this impurity corresponded to 15 nmoles of product formation (Table 1). TABLE 1 Sensitivity of ‘Standard Enzyme Assay”0 Acetohydroxamic dpm/assay
acid,
Corresponding to nmole acetohydroxamic acid formed 170.0 21.5 14.8 15.0 14.1
Standard assay CoASH omit,ted With heat-inactivated ensyme Incubation at 0°C Incubation of substrate and buffer only
40,752 5,160 3,556 3,600 3,334
n For assay conditions see “Methods.” to 10 mg tissue was used.
0.2 ml of a whole liver homogenate equivalent
DISCUSSION
The method described here fulfills necessary criteria of a reliable enzyme assay. It measures the catalytic conversion of the specific substrate only; this is achieved by use of a labeled substrate which, in addition, renders the method very sensitive. Also, by forming a stable hydroxamic acid derivative it excludes spontaneous or enzymic conversions of the reaction product. Using different kinds of tissue fractionation methods in rat liver and kidney, the sum of thiokinase activities was always better than 87% for acetate and propionate activation (2). The separation of product from substrate is accomplished by the application of “isoionic-exchange chromatography” developed in our laboratory (6). By using acetic acid as eluent it allows retardation of the reaction substrate acetate (isoion) which can be calculated from the total ion-exchange capacity of the resin. The reaction product appears with the column effluent (void volume) in agreement with its pK value of 9.32 (10).
474
2. 4. 5. 6.
7. 8. 9. 10.
11. 12.
BARTH,
AND
DECKER
REFERENCES R. W., AND BALLARD, F. J., Biochem. J. 105, 527 (1968). SLADEK, M., Thesis, Freiburg, 1970. HEPP, D., PR%SE, E., WEISS, H., AND WIELAND, O., Biochem. 2. 344, 87 (1966). AAS, M., AND BREMER, J., Biochim. Biophys. Acta 164, 157 (1968). EISENBERQ, M. A., Biochim. Biophys. Acta 16, 58 (1965). THAUER, R. K., RUPPRECHT, E., AND JUNGERMANN, K., Anal. Biochem. 38, 461 (1970). p. 63. de Gruyter, Berlin 1961. JANDER, G., AND JAHR, K. F., “Massanalyse,” MOELLERING, M., AND GRUBER, W., Anal. Biochem. 17, 369 (1966). WEBSTER, 5. T., .I. Biol. Chem. 240, 5504 (1966). FISHBEIN, W. N., DALY, Y., AND STREETER, C. L., And. Biochem. 28, 13 (1969). BRAY, G. A., Ad Biochem. 1, 279 (1960). BEISENHERZ, G., BOLTZE, H. J., BUTCHER, TH., CZOK, R., GARBADE, K. M., MEYERARENDT, E., AND PFLEIDERER, G., Z. Naturforsch. Sb, 555 (1953).
1. HANSON, 3.
SLADEK,