- Email: [email protected]
Acyl-CoA synthetases in guinea-pig liver mitochondria
Biochimica et Biophysics Acta, 441 (1976) 260-267 @ Elsevier Scientific Publishing Company, Amsterdam
BBA
-
Printed
in The Netherlands
56837
ACYL-CoA
SYNTHETASES
PURIFICATION PROPIONYL-CoA
P.H.E.
IN GUINEA-PIG
AND CHARACTERIZATION SYNTHETASE
LIVER
MITOCHONDRIA
OF A DISTINCT
GROOT
With the technical
assistance
of L.M. SCHEEK
Department of Biochemistry I. Faculty of Medicine, Box 1738, Rotterdam (The Netherlands) (Received
March
Ist,
Erasmus
IJniLlersity Rotterdam,
P.O.
1976)
Summary Guinea-pig liver mitochondria contain three soluble ATP-dependent acylCoA synthetases: (a) a medium-chain acyl-CoA synthetase, (b) a salicylate activating enzyme, and (c) a propionyl-CoA synthetase. A complete separation of these enzymes has been accomplished and the resulting preparation of propionyl-CoA synthetase (Spec. act. 4 units/mg protein) accepts acetate, propionate and butyrate as substrates with a high preference for propionate.
Introduction Recently, indications were found that cow and guinea-pig liver mitochondria contain a short-chain acyl-CoA synthetase showing high preference for propionate as substrate [ 1,2]. Acetyl-CoA synthetase (EC 6.2.1.1) and butyryl-CoA synthetase, both purified from beef heart mitochondria by Webster and coworkers [ 3,4], are absent in guinea-pig liver mitochondria, but are present in most other tissues, including heart [ 5,6]. Inside the mitochondria, short-chain acyl-CoA synthetases are localized in the matrix compartment [ 7-91. The present work describes a purification of guinea-pig liver propionyl-CoA synthetase and includes studies on the substrate specificity of this enzyme. During purification, two other ATP-dependent acyl-CoA synthetases could be separated. One enzyme exhibited the fatty acid substrate specificity of medium-chain acyl-CoA synthetase [lo] *, purified by Mahler et al. from beef* Medium-chain awl-CoA synthetase (EC 6.2.1.2; highest V with heptanoate) has obtained the recommended name butyryl-CoA synthetase [ 111 which in our opinion is confusing. We will use the name butyryl-CoA synthetase only for the type of enzyme identical in substrate specificity to the beef heart enzyme [4] (highest V with butyrate).
261
liver particles, and another was similar to the salicylate activating enzyme, purified by Killenberg et al. [12] from beef liver particles, an enzyme which also activates medium-chain fatty acids. Materials and Methods Reagents. [ 1-14C Jfatty acids and [carboxyl-‘4C]salicylic acid and benzoic acid were supplied by the Radiochemical Centre (Amersham, England), mixed with the free acids and neutralized with KOH. The final specific activities were 0.2-0.5 Ciimol. Phosphocellulose P-11 and DEAE-Sephadex A-50 were supplied by Whatman Biochemicals (Maidstone, England) and Pharmacia (Uppsala, Sweden) respectively, and pretreated as recommended by the manufacturers. Yeast inorganic pyrophosphatase was a product of Sigma Chemicals (St. Louis, U.S.A.). All other reagents were obtained as described earlier [ 2,13,14], Purification procedures. Lysosome-poor mitochondria were isolated from four guinea-pig livers (about 90 g tissue) as described before [2,14,15], suspended in about 60 ml ATP-containing salt medium (80 mM Tris . HCl, 40 mM KCI, 1 mM Mg&, 1 mM ATP, 0.2 mM dithiothreitol, pH 7.5) and disrupted by ultrasound. Membranes were sedimented (60 min at 300 000 X g,,,) and the supernatant (700-900 mg protein) was used for the purification of propionylCoA synthetase (Table I, stage 1). This fraction was adjusted to pH 8 and a saturated solution of (NH.+)$04 of pH 8, was added dropwise to a final concentration of 0.25 g/ml (0°C). The precipitate was removed (10 mm at 30 000 X g,,,) and discarded. The supe~at~t was brought to a (NH4)$04 concentration of 0.32 g/ml and the precipitate collected by centrifugation and dissolved in about 20 ml ATP-containing salt medium (Table I, stage 2). 50% of the propionyl-CoA synthetase activity and 30-40% of the octanoyl-CoA and salicylylCoA synthetase activities are recovered in this fraction. The 0.25-6.32 g/ml (NH4)#04 fraction was dialyzed overnight against 10 mM potassium phosphate buffer (pH 6.5), containing 0.2 mM dithiothreitol and 0.1 mM EDTA. TABLE I PURIFICATION
OF PROPIONYL-CoA
SYNTHETASE
For details consult the text. Stage of purification
Volume (ml)
“rotai protein
Propionate activation
(me)
Spec. act. (munits/
Tot. act. (units)
Recovery
92
100
(%)
m&G Soluble mitochondrial fraction 0.25-0.32 g (NH&SO,qf ml 1st dialysis Phosphocellulose 2nd dialysis DEAE-Sephadex
63
707
20 22 44 6.1 50
153 120 9.9 6.7 0.7
130 300 292 1480 1030 4140
46 35.3 14.6 6.9 2.9
50 38 16 7.5 3.2
262
After centrifugation (Table I, stage 3) and dilution of the cIe= solution with the same buffer to 100 ml, it was applied to a phosphocellulose column (12 X 2.5 cm), previously equilibrated with the buffer used (flow rate 60 ml/h, temperature 4°C). The absorption at 280 nm of the effluent was monitored continuously. The column was washed with phosphate buffer until no more protein was eluted (after about 100 ml). Neither propionyl-CoA nor salicylyl-CoA synthetase activities and only a low octanoyl-CoA synthetase activity (less than 10% of the applied activity) could be detected in the effluent, Then a gradient elution was carried out, employing 500 g phosphate buffer in the mixing chamber and an equal weight of phosphate buffer containing 1 M KC1 in the reservoir at the start of the elution. Fractions of 11 ml were collected and tested for propionyl-, octanoyl- and sakylyl-CoA synthetase activities (Fig. 1). OctanoylCoA synthetase activity was eluted in the first protein peak (at 100 mM KCl). The most active fractions were pooled, protein precipitated with (NH4)ZSU4 (0,383 g/ml) and the precipitate preserved at 4°C for later studies (mediumchain acyl-CoA synthetase, see Table 111). Propionyl- and salicylyl-CoA synthetase activities were eluted in a shoulder after a second protein peak (at 190 mM KCl) had emerged. The most active fractions were pooled (Table 1, stage 4) and protein precipitated with ( NH&SO4 (0.38 g/ml). The precipitate was collected by centrifugation, dissolved in 6 ml Tris - HCl buffer (100 mM Tris . HQ, 0.2 mM dithiothreitol, 0.1 mM EDTA, pW 8.0), dialyzed overnight against the same buffer and cleared by centrifugation (Table I, stage 5). The resulting solution was applied to a D~A~~ephad~x column (35 x 2.5 cm), previously equilibrated with the same Tris =HCl buffer (flow rate 20 ml/h, temperature 4°C). The column was eluted under starting conditions and the fractions (5.5 ml each} were tested for C,-CoA, C,-CoA and salicylyl-CoA syn-
L 100
i-
04
0
_l 0
600 ml
EFFLUENT
Fig. I. The separation of medium-chain atyf-CoA synthetase from propionyl-CoA synthetase illus the &i&ate activating mz~rne by pbosphoceliulose ~~ro~a~~~a~~y. Conditions of the phosphocellufose chromatography arc given in the text. ~------I, propianyECoA synthetase activities: A- - - - - -A, salicylyl-COA synthetase activities; *- - - - - -*, octanoyl-CoA synthebase activities: ------, E28Qnm;------, KCI. C3-, Cg- and salicylate activations were determined at carbosvlic acid cancentrations of 2.0.5 and 1 mM respectivety.
263
mU mU ?iiTKl5
CL
01
10 -I-
O-LO ml
EFFLUENT
Fig. 2. The separation of propionyl-CoA synthetase from the salicylate activating enzyme by DEAESephadex chromatography. Conditions of the DEAE-Sephadex chromatography are given in the text. m---m, propionyl-CoA synthetase activities: l -. -. -. - 0, butyryl-CoA synthetase activities: A - - - - - - A salicvlyl-CoA synthetase activities; -, protein profile. C3 and C4 activations were measrued at 2 mM fatty acid, salicylate activation at 1 mM salicylate. Not all salicylyl-CoA synthetase had left the column in this experiment. In this figure, it can be seen that butvrate is activated by both enzymes.
thetase activities, and for protein content (Fig. 2). A complete separation of the propionyl-CoA synthetase and the salicylate activating enzyme was accomplished. Fractions which catalyzed the highest rate of propionate activation were pooled (Table I, stage 6). Salicylyl-CoA synthetase was treated similarly and used in the study shown in Table III (salicylate activating enzyme). Purified propionyl-CoA synthetase was found to be unstable. Freezing leads to a complete loss of activity. We kept this enzyme, as well as salicylyl-CoA synthetase, at 4” C after precipitation with ammonium sulphate. Acyl-CoA synthetase assays. The principles of our short- and medium-chain acyl-CoA synthetase activity determinations have been described earlier [ 2,13, 141. The incubation mixture (final volume, 0.25 ml) consisted of: 40 mM tritine . KOH, 10 mM MgC12, 8 mM ATP, 1.2 mM EDTA, 5 mM phosphoenolpyruvate, 1.35 mM coenzyme A, 8 units/ml of both pyruvate kinase and adenylate kinase, 1.5 units/ml inorganic pyrophosphatase, [l-14C] fatty acid, 5 mM L-carnitine and 4 units/ml carnitine acetyltransferase. The pH of the final mixture was 8.0 at 37°C. The reaction was started after 2 min preincubation at 37°C with the addition of the enzyme source and stopped after 10 min with 1.2 ml ethanol. The reaction product [ l-14C] acylcarnitine was separated from [ 1-‘4C] fatty acids on Dowex 50W (H’) 200-500 mesh (and not 50-100 mesh [ 131) as described before. Salicylyl-CoA and benzoyl-CoA formation were measured according to Killenberg et al. [12] in a medium (final volume, 0.25 ml) similar to that used in our fatty acid activation assay, except that carnitine and carnitine acetyltransferase were omitted. Radioactive salicylate or benzoate were present at 1 mM. The reaction was started after 2 min preincubation at 37°C with the enzyme source and stopped after 10 or 20 min with 0.05 ml 2 M HC104 and 0.6
261
ml water. The enzyme fraction to be tested was omitted from the blanks. The unreacted radioactive salicylate or benzoate was removed in 5 extractions with 5 ml diethyl ether; the complete waterphase, containing the radioactive CoAderivative, was transferred to a counting vial containing 10 ml In&a-gel. Units of enzyme activity are given in pmol of product formed per min at 37°C. Protein was measured by a micro-adaptation of the method of Lowry et al. [ 161. Because dithiothreitol interferes with these measurements, protein was first precipitated in 5% trichloroacetic acid and spun down. Bovine serum albumin standards were treated in a similar way. Results and Discussion During our first attempts to purify propionyl-Coil synthetase from guineapig liver mitochondria, it became evident that two other ATP-dependent acylCoA synthetases were contaminants: medium-chain acyl-CoA synthetase and the salicylate activating enzyme. Both enzymes show activities with short-chain fatty acids (compare Table III). During the purification of the propionyl-CoA synthetase, octanoyl- and salicylyl-CoA synthetase activities were measured as well. The specific activities of acyl-CoA formation, measured in soluble fractions of guinea-pig liver mitochondria, were 114 i 15 (4), 72 * 4 (4) and 2.3 + 0.3 (3) munits/mg protein with propionate (2 mM), octanoate (0.5 mM) and salicylate (1 mM) respectively (mean f S.E.M. and number of experiments in parenthesis}. ~opionyl-CoA synthetase was freed from medium”chain acyl-CoA synthetase and the salicylate activating enzyme on phosphocellulose and DEAESephadex respectively (Figs. 1 and 2). A 30-times purification was accomplished (Table I). Purified propionyl-CoA synthetase was used in kinetic studies. Lineweaver-Burk plots of the kinetics of acetate, propionate and butyrate activation by this enzyme are given in Fig. 3. The calculated kinetic parameters are shown in Table II. The high preference of this enzyme for propionate is evident from these data. Propionate is a strong competitive inhibitor of acetate and butyrate activation (Figs. 3A and 3C). The pi values for propionate, calTABLE
II
KINETIC
PARAMETERS
OF PURIFIED
E’ROPIONYL-CoA
SYNTHETASE
A preparation of purified propionyl-CoA synthetase (Table I. stage 6) was used in these experiments. Short-chain fatty acid activation was determined as shown under Methods. When shown to be present. 1 mM unlabeled propionate was used. K,, Kh and V values were calculated from the data given in Fig. 3, using the least square method. 1’ values are given as percentage of the V found for propionate activation. Km values for ATP. M&l2 and coenzyme A of this enzyme with its substrate propionate were found to be 3.2, 1.5 and 0.45 mM. respectively (EDTA was omitted from the assay: the concentration of one reactant was varied while all other reactants were present at nearly saturating concentrations). Activation tested (‘4C-labeled)
In the presence (unlabeled)
Acetate Acetate Propionate Butyrate Butyrate I
1 mM propionate
of
K’ GW
Km (mM)
Ki (mM)
121
0.35
17
0.38
32 0.43 4.6
1 mM propionate -.
V (%) 72 71 100 44 46
265
Fig. 3. Lineweaver-Burk plots of acetate, propionate svnthetase. For details consult Table II and the text.
and butyrate
activation
by purified
propionyl-CoA
culated from these plots (0.35 mM and 0.38 mM respectively) agree well with the K, value for propionate (0.43 mM) which indicates that our preparation of propionyl-Coil synthetase is free of other short-chain fatty acid activating enzymes. During the purification of propionyl-CoA synthetase, fractions containing two other acyl-CoA synthetases could be separated. The substrate specificities of these enzymes were studied (Table III) and shown to be similar to beef-liver medium-chain acyl-CoA synthetase (activating C4-C12; highest V with C, ; activating also benzoate, but not salicylate [ 10,12]), and the beef-liver salicylate
266 TABLE
III
SUBSTRATE SPECIFICITIES OF THE THREE ATP-DEPENDENT ACYL-CoA ENT IN THE SOLUBLE FRACTION OF GUINEA-PIG LIVER MITOC~ONDRIA
SYNTHETASES
PRES-
Preparations of medium-chain awl-CoA synthetase (spec. act. with hexanoate 1000 munitsiml! protein), propionyl-CoA synthetase (spec. act. with propionate 4000 munits/mg) and salicylyl-CoA synthetase (spec. act. with hexanoate 720 munits/mg) were obtained as described in Methods. Velocities of acyl-CoA formation are expressed as percentages of the velocities found with the favoured fatty acids. The following carboxylic acid concentrations were used: C2, Cj and Cq, 5 mM: C,j, 2 mM (medium-chain awl-CoA synthetase) or 5 mM; Cg, 1 mM (medium-chain acyl-CoA synthetase) or 5 mM: Cl*, C~J and C16.1 mM complexed to 0.143 mM bovine serum albumin (according to method 1 of Groat and Hiilsmann [131); satieylate and benzoate 1 mM. Activation
tested
Observed acyl-CoA Medium-chain
Acetate Propionate Butyrate Hexanoate Octanoate Laurate Myristate PaImitate Benzoate salicy1ate
0.2 3 35 100 58 14 5 1 14 0.02
synthetase activity
acyl-CoA
(“a)
Propionyl-CoA thetase
syn-
Salicylate
II 100 19 0 0 -_ -
0 16 74 100 6 -
0 0
122 8
activating
activating enzyme (highest V with C6 and benzoate, but also accepting salicylate as the substrate 1121). These two enzymes present in ~inea-pig liver mitochondria may be involved in the initiation of medium-chain fatty acid oxidation and may also have a role in the excretion of aromatic carboxylic acids of vegetable sources by activation followed by conjugation with glycin [ 12,171. The distinct propionyl-CoA synthetase in livers of herbivores is likely to be involved in the activation of propionate produced by microbial fermentation of cellulose in the rumen (~minants) or caecum (guinea-pig). In vivo studies in sheep have shown that propionate is mainly metabolized by the liver, while acetate is mainly metabolized in extrahepatic tissues [IS]. The presence of the distinct propionyl-CoA synthetase in liver mitochondria would guarantee a rapid activation of propionate, to be used in synthetic reactions including gluconeogenesis, even in the presence of acetate, which is the main product of the cellulose fermentation. A tissue distribution study of Scholte and Groot [5] in guinea-pig and rat has shown that the presence of a propionyl-CoA synthetase is rather unique for liver (only adipose tissue seems to be equipped with this enzyme) which fits in with the concept of the special role of the liver in propionate metabolism.
Acknowledgement The author is greatly indebted to Professor and his interest in this investigation.
Dr. W.C. Hiilsmann
for his advice
References 1 Ash, R., and Baird G.D. (1973) 2 Grout, 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
P.H.E.
(197.5)
Biochim.
Biochem. Biophys.
J. 136. Acta 380,
311-319 12-20.
Campagnari. F., and Webster. L.T. (1963) J. Biol. Chem. 238, 1628-1633 Webster, L.T.. Grrowin, L.D., and Takita, L. (19fi5) J. Biol. Chem. 240, 29-34 Scholte, H.R.. and Groat. P.H.E. (1975) Biochim. Biophvs. Acta 409, 283-296 Groat, P.H.E.. Scholtc. H.R., and Hiilsmann. W.C. (1976) Advances in Lipid Research (Paoletti. R. and Kritchevskv, D.. eds.). Vol. XIV, pp. 75-126. Academic Press, New York Aas, M., and Bremer, J. (1968) Biochim. Biophys. Acta 164, 157-166 Aas, M. (1971) Biochim. Biophys. Acta 231, 32-47 Scholtr, H.R., Wit-Peters. E.M.. and Bakker J.C. (1971) Biochim. Biophys. Acta 231, 479486 Mahler. H.R., Wakil. S.J.. and Bock, R.M. (1953) J. Biol. Chem. 204, 453468 Flctrkin. M., and StotF. E.H. (1973) Comprehensive Biochemistry, Vol. XIII, Enzyme Nomenclature, Elsevier, Amsterdam. Kilfenberg, P.G., Davidson. E.D., and Webster, L.T. (1971) Mol. Pharmacof. 7, 260-268 Groat, P.H.E.. and Hiilsmann, W.C. (1973) Biochim. Biophys. Aeta 316. 124-135 Groat, P.H.E., Van Loon. C.M.I., and Hiilsmann. W.C. (1974) Biochim. Biophys. Acta 337, I-12 Loewfmstein. J., Scholte. H.R., and Wit-Pccters. E.M. (1970) Biochim. Biophys. Acta 223, 432436 Lowry. O.H.. Rosebrough. N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Forman. W.B., Davidson. E.D., and Webster, L.T. (1971) Mol. Phamacol. 7. 247-259 Bergman. EN., and Wolff. J.E. (1971) Am. J. Physiol. 221. 586-592
BBA
-
Printed
in The Netherlands
56837
ACYL-CoA
SYNTHETASES
PURIFICATION PROPIONYL-CoA
P.H.E.
IN GUINEA-PIG
AND CHARACTERIZATION SYNTHETASE
LIVER
MITOCHONDRIA
OF A DISTINCT
GROOT
With the technical
assistance
of L.M. SCHEEK
Department of Biochemistry I. Faculty of Medicine, Box 1738, Rotterdam (The Netherlands) (Received
March
Ist,
Erasmus
IJniLlersity Rotterdam,
P.O.
1976)
Summary Guinea-pig liver mitochondria contain three soluble ATP-dependent acylCoA synthetases: (a) a medium-chain acyl-CoA synthetase, (b) a salicylate activating enzyme, and (c) a propionyl-CoA synthetase. A complete separation of these enzymes has been accomplished and the resulting preparation of propionyl-CoA synthetase (Spec. act. 4 units/mg protein) accepts acetate, propionate and butyrate as substrates with a high preference for propionate.
Introduction Recently, indications were found that cow and guinea-pig liver mitochondria contain a short-chain acyl-CoA synthetase showing high preference for propionate as substrate [ 1,2]. Acetyl-CoA synthetase (EC 6.2.1.1) and butyryl-CoA synthetase, both purified from beef heart mitochondria by Webster and coworkers [ 3,4], are absent in guinea-pig liver mitochondria, but are present in most other tissues, including heart [ 5,6]. Inside the mitochondria, short-chain acyl-CoA synthetases are localized in the matrix compartment [ 7-91. The present work describes a purification of guinea-pig liver propionyl-CoA synthetase and includes studies on the substrate specificity of this enzyme. During purification, two other ATP-dependent acyl-CoA synthetases could be separated. One enzyme exhibited the fatty acid substrate specificity of medium-chain acyl-CoA synthetase [lo] *, purified by Mahler et al. from beef* Medium-chain awl-CoA synthetase (EC 6.2.1.2; highest V with heptanoate) has obtained the recommended name butyryl-CoA synthetase [ 111 which in our opinion is confusing. We will use the name butyryl-CoA synthetase only for the type of enzyme identical in substrate specificity to the beef heart enzyme [4] (highest V with butyrate).
261
liver particles, and another was similar to the salicylate activating enzyme, purified by Killenberg et al. [12] from beef liver particles, an enzyme which also activates medium-chain fatty acids. Materials and Methods Reagents. [ 1-14C Jfatty acids and [carboxyl-‘4C]salicylic acid and benzoic acid were supplied by the Radiochemical Centre (Amersham, England), mixed with the free acids and neutralized with KOH. The final specific activities were 0.2-0.5 Ciimol. Phosphocellulose P-11 and DEAE-Sephadex A-50 were supplied by Whatman Biochemicals (Maidstone, England) and Pharmacia (Uppsala, Sweden) respectively, and pretreated as recommended by the manufacturers. Yeast inorganic pyrophosphatase was a product of Sigma Chemicals (St. Louis, U.S.A.). All other reagents were obtained as described earlier [ 2,13,14], Purification procedures. Lysosome-poor mitochondria were isolated from four guinea-pig livers (about 90 g tissue) as described before [2,14,15], suspended in about 60 ml ATP-containing salt medium (80 mM Tris . HCl, 40 mM KCI, 1 mM Mg&, 1 mM ATP, 0.2 mM dithiothreitol, pH 7.5) and disrupted by ultrasound. Membranes were sedimented (60 min at 300 000 X g,,,) and the supernatant (700-900 mg protein) was used for the purification of propionylCoA synthetase (Table I, stage 1). This fraction was adjusted to pH 8 and a saturated solution of (NH.+)$04 of pH 8, was added dropwise to a final concentration of 0.25 g/ml (0°C). The precipitate was removed (10 mm at 30 000 X g,,,) and discarded. The supe~at~t was brought to a (NH4)$04 concentration of 0.32 g/ml and the precipitate collected by centrifugation and dissolved in about 20 ml ATP-containing salt medium (Table I, stage 2). 50% of the propionyl-CoA synthetase activity and 30-40% of the octanoyl-CoA and salicylylCoA synthetase activities are recovered in this fraction. The 0.25-6.32 g/ml (NH4)#04 fraction was dialyzed overnight against 10 mM potassium phosphate buffer (pH 6.5), containing 0.2 mM dithiothreitol and 0.1 mM EDTA. TABLE I PURIFICATION
OF PROPIONYL-CoA
SYNTHETASE
For details consult the text. Stage of purification
Volume (ml)
“rotai protein
Propionate activation
(me)
Spec. act. (munits/
Tot. act. (units)
Recovery
92
100
(%)
m&G Soluble mitochondrial fraction 0.25-0.32 g (NH&SO,qf ml 1st dialysis Phosphocellulose 2nd dialysis DEAE-Sephadex
63
707
20 22 44 6.1 50
153 120 9.9 6.7 0.7
130 300 292 1480 1030 4140
46 35.3 14.6 6.9 2.9
50 38 16 7.5 3.2
262
After centrifugation (Table I, stage 3) and dilution of the cIe= solution with the same buffer to 100 ml, it was applied to a phosphocellulose column (12 X 2.5 cm), previously equilibrated with the buffer used (flow rate 60 ml/h, temperature 4°C). The absorption at 280 nm of the effluent was monitored continuously. The column was washed with phosphate buffer until no more protein was eluted (after about 100 ml). Neither propionyl-CoA nor salicylyl-CoA synthetase activities and only a low octanoyl-CoA synthetase activity (less than 10% of the applied activity) could be detected in the effluent, Then a gradient elution was carried out, employing 500 g phosphate buffer in the mixing chamber and an equal weight of phosphate buffer containing 1 M KC1 in the reservoir at the start of the elution. Fractions of 11 ml were collected and tested for propionyl-, octanoyl- and sakylyl-CoA synthetase activities (Fig. 1). OctanoylCoA synthetase activity was eluted in the first protein peak (at 100 mM KCl). The most active fractions were pooled, protein precipitated with (NH4)ZSU4 (0,383 g/ml) and the precipitate preserved at 4°C for later studies (mediumchain acyl-CoA synthetase, see Table 111). Propionyl- and salicylyl-CoA synthetase activities were eluted in a shoulder after a second protein peak (at 190 mM KCl) had emerged. The most active fractions were pooled (Table 1, stage 4) and protein precipitated with ( NH&SO4 (0.38 g/ml). The precipitate was collected by centrifugation, dissolved in 6 ml Tris - HCl buffer (100 mM Tris . HQ, 0.2 mM dithiothreitol, 0.1 mM EDTA, pW 8.0), dialyzed overnight against the same buffer and cleared by centrifugation (Table I, stage 5). The resulting solution was applied to a D~A~~ephad~x column (35 x 2.5 cm), previously equilibrated with the same Tris =HCl buffer (flow rate 20 ml/h, temperature 4°C). The column was eluted under starting conditions and the fractions (5.5 ml each} were tested for C,-CoA, C,-CoA and salicylyl-CoA syn-
L 100
i-
04
0
_l 0
600 ml
EFFLUENT
Fig. I. The separation of medium-chain atyf-CoA synthetase from propionyl-CoA synthetase illus the &i&ate activating mz~rne by pbosphoceliulose ~~ro~a~~~a~~y. Conditions of the phosphocellufose chromatography arc given in the text. ~------I, propianyECoA synthetase activities: A- - - - - -A, salicylyl-COA synthetase activities; *- - - - - -*, octanoyl-CoA synthebase activities: ------, E28Qnm;------, KCI. C3-, Cg- and salicylate activations were determined at carbosvlic acid cancentrations of 2.0.5 and 1 mM respectivety.
263
mU mU ?iiTKl5
CL
01
10 -I-
O-LO ml
EFFLUENT
Fig. 2. The separation of propionyl-CoA synthetase from the salicylate activating enzyme by DEAESephadex chromatography. Conditions of the DEAE-Sephadex chromatography are given in the text. m---m, propionyl-CoA synthetase activities: l -. -. -. - 0, butyryl-CoA synthetase activities: A - - - - - - A salicvlyl-CoA synthetase activities; -, protein profile. C3 and C4 activations were measrued at 2 mM fatty acid, salicylate activation at 1 mM salicylate. Not all salicylyl-CoA synthetase had left the column in this experiment. In this figure, it can be seen that butvrate is activated by both enzymes.
thetase activities, and for protein content (Fig. 2). A complete separation of the propionyl-CoA synthetase and the salicylate activating enzyme was accomplished. Fractions which catalyzed the highest rate of propionate activation were pooled (Table I, stage 6). Salicylyl-CoA synthetase was treated similarly and used in the study shown in Table III (salicylate activating enzyme). Purified propionyl-CoA synthetase was found to be unstable. Freezing leads to a complete loss of activity. We kept this enzyme, as well as salicylyl-CoA synthetase, at 4” C after precipitation with ammonium sulphate. Acyl-CoA synthetase assays. The principles of our short- and medium-chain acyl-CoA synthetase activity determinations have been described earlier [ 2,13, 141. The incubation mixture (final volume, 0.25 ml) consisted of: 40 mM tritine . KOH, 10 mM MgC12, 8 mM ATP, 1.2 mM EDTA, 5 mM phosphoenolpyruvate, 1.35 mM coenzyme A, 8 units/ml of both pyruvate kinase and adenylate kinase, 1.5 units/ml inorganic pyrophosphatase, [l-14C] fatty acid, 5 mM L-carnitine and 4 units/ml carnitine acetyltransferase. The pH of the final mixture was 8.0 at 37°C. The reaction was started after 2 min preincubation at 37°C with the addition of the enzyme source and stopped after 10 min with 1.2 ml ethanol. The reaction product [ l-14C] acylcarnitine was separated from [ 1-‘4C] fatty acids on Dowex 50W (H’) 200-500 mesh (and not 50-100 mesh [ 131) as described before. Salicylyl-CoA and benzoyl-CoA formation were measured according to Killenberg et al. [12] in a medium (final volume, 0.25 ml) similar to that used in our fatty acid activation assay, except that carnitine and carnitine acetyltransferase were omitted. Radioactive salicylate or benzoate were present at 1 mM. The reaction was started after 2 min preincubation at 37°C with the enzyme source and stopped after 10 or 20 min with 0.05 ml 2 M HC104 and 0.6
261
ml water. The enzyme fraction to be tested was omitted from the blanks. The unreacted radioactive salicylate or benzoate was removed in 5 extractions with 5 ml diethyl ether; the complete waterphase, containing the radioactive CoAderivative, was transferred to a counting vial containing 10 ml In&a-gel. Units of enzyme activity are given in pmol of product formed per min at 37°C. Protein was measured by a micro-adaptation of the method of Lowry et al. [ 161. Because dithiothreitol interferes with these measurements, protein was first precipitated in 5% trichloroacetic acid and spun down. Bovine serum albumin standards were treated in a similar way. Results and Discussion During our first attempts to purify propionyl-Coil synthetase from guineapig liver mitochondria, it became evident that two other ATP-dependent acylCoA synthetases were contaminants: medium-chain acyl-CoA synthetase and the salicylate activating enzyme. Both enzymes show activities with short-chain fatty acids (compare Table III). During the purification of the propionyl-CoA synthetase, octanoyl- and salicylyl-CoA synthetase activities were measured as well. The specific activities of acyl-CoA formation, measured in soluble fractions of guinea-pig liver mitochondria, were 114 i 15 (4), 72 * 4 (4) and 2.3 + 0.3 (3) munits/mg protein with propionate (2 mM), octanoate (0.5 mM) and salicylate (1 mM) respectively (mean f S.E.M. and number of experiments in parenthesis}. ~opionyl-CoA synthetase was freed from medium”chain acyl-CoA synthetase and the salicylate activating enzyme on phosphocellulose and DEAESephadex respectively (Figs. 1 and 2). A 30-times purification was accomplished (Table I). Purified propionyl-CoA synthetase was used in kinetic studies. Lineweaver-Burk plots of the kinetics of acetate, propionate and butyrate activation by this enzyme are given in Fig. 3. The calculated kinetic parameters are shown in Table II. The high preference of this enzyme for propionate is evident from these data. Propionate is a strong competitive inhibitor of acetate and butyrate activation (Figs. 3A and 3C). The pi values for propionate, calTABLE
II
KINETIC
PARAMETERS
OF PURIFIED
E’ROPIONYL-CoA
SYNTHETASE
A preparation of purified propionyl-CoA synthetase (Table I. stage 6) was used in these experiments. Short-chain fatty acid activation was determined as shown under Methods. When shown to be present. 1 mM unlabeled propionate was used. K,, Kh and V values were calculated from the data given in Fig. 3, using the least square method. 1’ values are given as percentage of the V found for propionate activation. Km values for ATP. M&l2 and coenzyme A of this enzyme with its substrate propionate were found to be 3.2, 1.5 and 0.45 mM. respectively (EDTA was omitted from the assay: the concentration of one reactant was varied while all other reactants were present at nearly saturating concentrations). Activation tested (‘4C-labeled)
In the presence (unlabeled)
Acetate Acetate Propionate Butyrate Butyrate I
1 mM propionate
of
K’ GW
Km (mM)
Ki (mM)
121
0.35
17
0.38
32 0.43 4.6
1 mM propionate -.
V (%) 72 71 100 44 46
265
Fig. 3. Lineweaver-Burk plots of acetate, propionate svnthetase. For details consult Table II and the text.
and butyrate
activation
by purified
propionyl-CoA
culated from these plots (0.35 mM and 0.38 mM respectively) agree well with the K, value for propionate (0.43 mM) which indicates that our preparation of propionyl-Coil synthetase is free of other short-chain fatty acid activating enzymes. During the purification of propionyl-CoA synthetase, fractions containing two other acyl-CoA synthetases could be separated. The substrate specificities of these enzymes were studied (Table III) and shown to be similar to beef-liver medium-chain acyl-CoA synthetase (activating C4-C12; highest V with C, ; activating also benzoate, but not salicylate [ 10,12]), and the beef-liver salicylate
266 TABLE
III
SUBSTRATE SPECIFICITIES OF THE THREE ATP-DEPENDENT ACYL-CoA ENT IN THE SOLUBLE FRACTION OF GUINEA-PIG LIVER MITOC~ONDRIA
SYNTHETASES
PRES-
Preparations of medium-chain awl-CoA synthetase (spec. act. with hexanoate 1000 munitsiml! protein), propionyl-CoA synthetase (spec. act. with propionate 4000 munits/mg) and salicylyl-CoA synthetase (spec. act. with hexanoate 720 munits/mg) were obtained as described in Methods. Velocities of acyl-CoA formation are expressed as percentages of the velocities found with the favoured fatty acids. The following carboxylic acid concentrations were used: C2, Cj and Cq, 5 mM: C,j, 2 mM (medium-chain awl-CoA synthetase) or 5 mM; Cg, 1 mM (medium-chain acyl-CoA synthetase) or 5 mM: Cl*, C~J and C16.1 mM complexed to 0.143 mM bovine serum albumin (according to method 1 of Groat and Hiilsmann [131); satieylate and benzoate 1 mM. Activation
tested
Observed acyl-CoA Medium-chain
Acetate Propionate Butyrate Hexanoate Octanoate Laurate Myristate PaImitate Benzoate salicy1ate
0.2 3 35 100 58 14 5 1 14 0.02
synthetase activity
acyl-CoA
(“a)
Propionyl-CoA thetase
syn-
Salicylate
II 100 19 0 0 -_ -
0 16 74 100 6 -
0 0
122 8
activating
activating enzyme (highest V with C6 and benzoate, but also accepting salicylate as the substrate 1121). These two enzymes present in ~inea-pig liver mitochondria may be involved in the initiation of medium-chain fatty acid oxidation and may also have a role in the excretion of aromatic carboxylic acids of vegetable sources by activation followed by conjugation with glycin [ 12,171. The distinct propionyl-CoA synthetase in livers of herbivores is likely to be involved in the activation of propionate produced by microbial fermentation of cellulose in the rumen (~minants) or caecum (guinea-pig). In vivo studies in sheep have shown that propionate is mainly metabolized by the liver, while acetate is mainly metabolized in extrahepatic tissues [IS]. The presence of the distinct propionyl-CoA synthetase in liver mitochondria would guarantee a rapid activation of propionate, to be used in synthetic reactions including gluconeogenesis, even in the presence of acetate, which is the main product of the cellulose fermentation. A tissue distribution study of Scholte and Groot [5] in guinea-pig and rat has shown that the presence of a propionyl-CoA synthetase is rather unique for liver (only adipose tissue seems to be equipped with this enzyme) which fits in with the concept of the special role of the liver in propionate metabolism.
Acknowledgement The author is greatly indebted to Professor and his interest in this investigation.
Dr. W.C. Hiilsmann
for his advice
References 1 Ash, R., and Baird G.D. (1973) 2 Grout, 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
P.H.E.
(197.5)
Biochim.
Biochem. Biophys.
J. 136. Acta 380,
311-319 12-20.
Campagnari. F., and Webster. L.T. (1963) J. Biol. Chem. 238, 1628-1633 Webster, L.T.. Grrowin, L.D., and Takita, L. (19fi5) J. Biol. Chem. 240, 29-34 Scholte, H.R.. and Groat. P.H.E. (1975) Biochim. Biophvs. Acta 409, 283-296 Groat, P.H.E.. Scholtc. H.R., and Hiilsmann. W.C. (1976) Advances in Lipid Research (Paoletti. R. and Kritchevskv, D.. eds.). Vol. XIV, pp. 75-126. Academic Press, New York Aas, M., and Bremer, J. (1968) Biochim. Biophys. Acta 164, 157-166 Aas, M. (1971) Biochim. Biophys. Acta 231, 32-47 Scholtr, H.R., Wit-Peters. E.M.. and Bakker J.C. (1971) Biochim. Biophys. Acta 231, 479486 Mahler. H.R., Wakil. S.J.. and Bock, R.M. (1953) J. Biol. Chem. 204, 453468 Flctrkin. M., and StotF. E.H. (1973) Comprehensive Biochemistry, Vol. XIII, Enzyme Nomenclature, Elsevier, Amsterdam. Kilfenberg, P.G., Davidson. E.D., and Webster, L.T. (1971) Mol. Pharmacof. 7, 260-268 Groat, P.H.E.. and Hiilsmann, W.C. (1973) Biochim. Biophys. Aeta 316. 124-135 Groat, P.H.E., Van Loon. C.M.I., and Hiilsmann. W.C. (1974) Biochim. Biophys. Acta 337, I-12 Loewfmstein. J., Scholte. H.R., and Wit-Pccters. E.M. (1970) Biochim. Biophys. Acta 223, 432436 Lowry. O.H.. Rosebrough. N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Forman. W.B., Davidson. E.D., and Webster, L.T. (1971) Mol. Phamacol. 7. 247-259 Bergman. EN., and Wolff. J.E. (1971) Am. J. Physiol. 221. 586-592