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[60] Carnitine acetyltransferase from pigeon breast muscle
[60]
CARNITINEACETYLTRANSFERASEFROM PIGEON BREAST
387
[60] C a r n i t i n e A c e t y l t r a n s f e r a s e f r o m P i g e o n B r e a s t M u s c l e [EC 2.3.1.7
Acetyl-CoA:carnitine O-aeetyltransferase] By J. F. A. CIJASE
O-Acetyl-(--)-carnitine + CoASH ~ (-)-carnitine + acetyl-Cok Assay Methods Method A: The Direct Assay Principle. Enzyme preparations of specific activity in excess of 1 unit per milligram of protein per milliliter (see below) are assayed most satisfactorily by direct spectrophotometric observation of the reacting substrafes. At 232 m~, there is an increase in molar extinction of 4.5 )< 103 cm-1 on the acetylation of CoASH,1 whereas carnitine and acylcarnitine solutions do not absorb at this wavelength.2 The catalyzed reaction may be followed in either the forward or reverse direction, as it is readily reversible (Keq = 0.6).s Reagents Tris-HC1 buffer, 1.0 M, pH 7.8 Neutral EDTA, 0.1 M CoASH, 10 mg/ml. The solid coenzyme is dissolved freshly in water Acetyl-DL-carnitine hydroehloride, 0.1 M, prepared according to Fraenkel and Friedmann' and dissolved in water Enzyme: 0.02-0.1 unit of carnitine acetyltransferase is a suitable amount for assay. This corresponds to about 0.2--1.0 /~g of the crystalline enzyme from pigeon breast muscle,~ and gives an increase in extinction at 232 m~ of 0.05-0.25 per minute in the system described below Procedure. The assay system contains 0.2 ml of Tris-HC1, 0.005 ml of EDTA, 0.05 ml of CoASH, enzyme, and water in a final volume of 1.95 ml. This mixture, in a cell of 10 mm light path, is equilibrated at 25 ° in a spectrophotometer with a temperature-controlled cell housing. On the addition of 0.05 ml of acetylcarnitine, the extinction of the solution at 1E. R. Stadtman, Vol. I [137]. 2D. J. Pearson, Biochem. J. 95, 23c (1965). 3I. B. Fritz, S. K. Schultz, and P. A. Stere, J. Biol. Chem. 238, 2509 (1963). G. Fraenkel and S. Friedmann, Vitamins Hormones 15, 73 (1957). 6j. F. A. Chase, D. J. Pearson, and P. K. Tubbs, Biochim. Biophys. Acta 96, 162 (1965).
388
REACTIONS YIELDING ACETYL-COA
[60]
232 m~ increases linearly with time for 1-2 minutes. Bovine serum albumin was included in another description" but is now omitted as it has no effect on the activity or stability of the enzyme. Units. One unit of enzyme is t h a t amount which catalyzes the acetylation of 1 micromole of CoASH per minute in the above system. Protein is determined spcctrophotometrically at 260 and 280 m~ according to Layne. 6 Specific activity is expressed as units per milligram of protein.
Method B: Carnitine Acetyltrans]erase in Crude Tissue Extracts Crude extracts contain too much 232 m~ absorbing material for the direct assay to be applicable. Carnitine acetyltransferase activity may be detected in such preparations in a coupled system2, 7 Principle. Acetyl-CoA, formed from acetylcarnitine and CoASH, is removed to form citrate in the presence of malate, NAD, and excess citrate synthase and malate dehydrogenase, the reduction of NAD being followed at 340 m~. Acetylcarnitine q- CoASH ~ carnitine -b acetyl-CoA Acetyl-CoA -~ oxaloacetate -~ citrate ~ CoASH Malate -t- NAD ~ oxaloacetate T NADH~ Acetylcarnitine -[- malate -~ N A D --~ carnitine ~ citrate -{- NADH~
Reagents L-Malate, 1.0 M, p H 8.0 NAD, 10 raM, p H 6.0 NaCN, l0 m M Citrate synthase: a crystalline suspension of the enzyme from pig heart, s ca. 5 mg of protein/ml (or see footnote 8a) Malate dehydrogenase: an ammonium sulfate suspension of the pig heart enzyme,' ca. 5 mg of protein/ml 6E. Layne, Vol. III [73]. N. R. Marquis and I. B. Fritz, J. Biol. Chem. 240, 2193 (1965). 'P. A. Srere and G. W. Kosicki, J. Biol. Chem. 236, 2557 (1961). ,a It may be noted that a substantial copurification of carn/tine acetyltransferase and citrate synthase occurs during steps 1--4 of the purification procedure. Most of the citrate synthase, which is present in an amount comparable to that of the carnitine enzyme in the eluate from step 4 is, however, left in solution after the calcium phosphate gel treatment. If desired, it may be adsorbed by the further addition of 5 ml of gel per I00 ml of eluate. Citrate synthase may then be eluted and crystallized as described for the pig heart enzyme,' when it is suitable for use in assay method B. ' Commercial preparations are available, or see this volume [18-21].
[50]
CARNITINE ACETYLTRANSFERASE FROM PIGEON BREAST
389
Tris-HC1, pH 7.8, EDTA, CoASH, acetyl-DL-carnitine and carnitine acetyltransferase solutions as for assay method A Procedure. Combine Tris-HC1, 0.2 ml; EDTA, 0.05 ml; L-malate, 0.05 ml; NAD, 0.05 ml; NaCN, 0.2 ml (if the preparation shows NADH.~ oxidase activity); CoASH, 0.25 ml; malate dehydrogenase, 0.005 ml; citrate synthase, 0.01 ml; carnitine acetyltransferase, 0.04-0.20 unit; and water to a volume of 1.95 ml. Equilibrate the mixture in a spectrophotometer at 25 ° as described in method A. On the addition of 0.05 ml of acetylcarnitine, an increase in extinction at 340 m~ is observed. For the calculation of specific activity, it is assumed that the extinction coefficient for NAD reduction at this wavelength is 6.22 X 103 cm-1. Specific activities may then be expressed in a manner analogous to that given for method A. This procedure gives erroneously low estimates of enzymatic activity because less than 1 equivalent of NADH~ is produced per equivalent of acetyl-CoA formed,2,1° but it is adequate for comparative purposes. Other Methods
Assays that have been devised to study the reverse reaction between acetyl-CoA and (--)-carnitine include a relatively insensitive hydroxamate procedure to follow acetylcarnitine formation,~1 and the use of the thiol reagent 5,5"-dithiobis-(2-nitrobenzoic acid) (DTNB) to follow CoASH release2,12 The latter technique has the disadvantage that DTNB slowly inactivates carnitine acetyltransferase2 A very sensitive isotopeexchange assay has also been described TM with its application to detection of the enzyme in nervous tissue. Purification Procedure This procedure is essentially the same as published elsewhere2 All operations were conducted in a cold-room at 4 ° unless otherwise indicated. Pigeon breast muscle, excised as soon as possible after death, m a y be stored frozen for several months without loss of extractable activity. Step 1. Extraction. Stored muscle is thawed, sliced, and homogenized for I minute in a Waring blendor with 3 volumes of cold (--5 °) 2 0 % ethanol containing 0 . 4 M KCI. The homogenate is centrifuged for 10 minutes at 23,000 g, filtered through muslin to remove fat, and dialyzed IoW. Buckel and If. Eggerer, Biochem. Z. 343, 29 (1965). "S. Friedmann and G. Fraenkel, Arch. Biochem. Biophys. 59, 491 (1955). 12I. B. Fritz and S. K. Sehultz, J. Biol. Chem. 240, 2188 (1965). ~R. E. McCaman, M. W. McCaman, and M. L. Stafford, J. Biol. Chem. o,41, 930 (1966).
390
REACTIONS YIELDING ACETYL-COA
[60]
against two changes of 2 m M potassium phosphate buffer, pH 7.5, containing 0.5 mM EDTA. Step ~. Ammonium Sul]ate Fractionation. Any precipitate formed on dialysis is discarded, and solid ammonium sulfate is added to give 50~ saturation (312 g/liter). After 30 minutes, the precipitate is collected by centrifugation and discarded. Further ammonium sulfate (82 g/liter) is added to the supernatant to give 62.5% saturation. The protein precipitated contains over 90% of the enzyme activity; it is dissolved in 0.1 M phosphate, pH 7.5, and dialyzed as before. Step 3. Acetone Fractionation. The dialyzate is cooled to 0 ° and 0.47 of its volume of acetone is added slowly, with stirring; the temperature of the mixture is lowered progressively to --7% After 30 minutes, the precipitate is centrifuged, dissolved in 0.1 M phosphate, pit 7.5, and dialyzed as in step 1. It has been found repeatedly that attempts to redissolve the acetone pellet in dilute buffer (e.g., 10 raM) result in almost complete loss of enzyme activity, and this should be avoided. Step ~. Fractionation on DBAE-Cellulose. The dialyzed preparation is applied to a column of DEAE-cellulose (35 g of DEAE per gram of protein) equilibrated with 2 mM phosphate, pH 7.5. The column is washed with 2 column volumes of 10 mM phosphate, pH 7.5, followed by 3 volumes of 15 mM buffer. This removes about half of the protein; no transferase activity should be present in the 10 mM eluate, and only a trace in the 15 mM fraction. The enzyme is eluted with 4 column volumes of 25 mM phosphate, pH 7.5. Step 5. Calcium Phosphate Gel. One milliliter of calcium phosphate gel14 (34 mg/ml, dry weight) is added per 100 ml of 25 mM phosphate eluate; this absorbs all the enzyme. The gel is washed 3 times with 0.1 M phosphate, pH 7.5, and the enzyme is eluted in 0.4 M phosphate, containing 10% ammonium sulfate2 ~ Step 6. Fractionation on Sephadex G-IO0. Ammonium sulfate (55 g) is added to each 100 ml of gel eluate, and the precipitate is dissolved in a minimal volume of 0.1 M phosphate, pH 7.5. Fractions (2-4 ml) of this solution are applied to a column of Sephadex G-100 (4 cm X 21 cm) connected to a reservoir of the same buffer. The first 45 ml of eluate contains no protein and is discarded; thereafter, 5 ml fractions are taken and assayed for protein (absorption at 280 m~) and enzymatic activity. Two, slightly overlapping, protein peaks emerge. The first, after 65 ml of eluate have been collected, contains enzymatically inactive colored material; the second, at ll0 ml, corresponds with the enzyme activity. No further protein emerges after 150 ml, and a pool is made of fractions cont4D. Keilin and E. F. Hartree, Proc. Roy. Soc. Lo~zdon B124, 397 (1938); see al.~o Vol. I [11].
[60]
CARNITINE ACETYLTBANSFERASE FROM PIGEON BREAST
391
raining 90-95% of the total activity. I m p u r e material appearing at low elution volumes is discarded. Step 7. Crystallization. The pooled fractions from step 6 are concentrated b y adding a m m o n i u m sulfate to about 90% saturation (65 g/100 ml) and the precipitate is dissolved in 0.1 M phosphate, p H 7.5, to give a solution containing about 10 mg of protein per milliliter. This is cooled to 0 ° and solid a m m o n i u m sulfate is added until a faint turbidity appears. Any traces of denatured brown material t h a t precipitate are discarded, and the solution is then stored at 4 °. Crystals of carnitinc acetyltransferase appear overnight and continue to grow for several days in the form of fine needles? Recrystallization is cffectcd by a repetition of this process and is encouraged b y "seeding" the incipiently turbid solution with preformed crystals. The specific activity of the recrystallized enzyme is 118-119 units/rag, a value which is unaltered by further recrystallizations. The purification procedure is summarized in the table for a preparation starting from 900 g of pigeon muscle and yielding 15 mg of crystalline enzyme. No difficulties have been encountered in reproducing these results so long as the warning given in step 3 is heeded. The method m a y be scaled-up at least 4-fold with a proportionate increase in yield. PURIFICATION PROCEDURE FOR CARNITINE ACETYLTRANSFERASE FROM PIGEON BREAST ~IUSCLE
Step and fraction
Volume (ml)
Specific Total activity Units/ activity Protein (units/rag Yield mla (units b) (mg/ml) protein) (%)
1. Centrifuged extract 2660 3.1 2. 50--62.5% Ammonium 170 43.7 sulfate fraction 3. 0-32% Acetone fraction 140 36.5 4. DEAE-cellulose 1970 1.08 25 mM eluate 5. Calcium phosphate 102 17.0 gel eluate 6. Sephadex G-100 eluate 50 32.8 7. Crystallization First crystals 1.7 717 First recrystallization 1.35 438 Second recrystallization 1.75 334
8250 7430
39 0 64.0
0.18 0.68
100 90
5130 2130
24.2 0.11
1.51 9.8
62 26
1734
0.68
25.0
21
1640
0.73
45.0
20
1219 592 584
6.6 3.7 2.8
108 118 119
15 ---
" Activity measurements for steps 1 and 2 were made using the coupled assay (method B). Thereafter the direct assay (method A) was employed. b One unit of enzyme is the amount that catalyzes the acetylation of 1 micromole of CoASH per minute in the assay conditions.
392
REACTIONS YIELDING ACETYL-COA
[50]
Properties Stability. The crystalline enzyme is stored conveniently as a suspension in 60-70% saturated ammonium sulfate, when it is completely stable at 4 °. Dilute solutions (10-100 ~g/ml) in 0.1 M phosphate, pH 7.5, also may be stored in the refrigerator; they lose only 10-12% of their activity in 2 months. Effects o] pH. Carnitine acetyltransferase shows a broad pH optimum between 7.0 and 8.0 for both forward and reverse reactions2 ,15 It is stable overnight at pH 5.5, but becomes rapidly and irreversibly inactivated above pH 8.6.8 Purity. Recrystallized carnitine acetyltransfcrasc is colorless in solution and is homogeneous in the ultracentrifuge, on chromatography on Sephadex G-100, and on electrophoresis on cellulose acetate paper. 1~ It is also free of acetyl-CoA hydrolase, citrate synthase, malate dehydrogenase, and palmitoylcarnitine transferase activities. Molecular Weight. A value of 58,000 ± 3000 is indicated by sedimentation equilibrium 1' and gel filtration18 methods. Inhibitors. The enzyme is inhibited by a range of reagents (iodoacetamide, p-chloromercuribenzoate, DTNB, N-ethylmaleimidc) which are somewhat specific for protein thiol groups2,12,1~ Divalent cations are also inhibitory. Specificity. The enzyme is highly specific for (--)-carnitine and CoA, (--)-norearnitine and 3'-dephospho-CoA being the only known analogs which are also substrates2 ,1~,15 (-k)-Carnitine is a competitive inhibitor (K~--173 ~ / ) 1 ~ for (--)-carnitine and its derivatives. 12'1~ Group transfer between CoASH and (--)-carnitine is catalyzed with n-acyl groups containing up to 10 carbon atoms, and Vm~ for the reaction falls off with increasing chain length2 ,~° Palmitoyl-CoA is not a substrate, 3 but acts as a potent competitive inhibitor with respect to both (--)-carnitine and acetyl-CoA (K~ -~ 0.43 p.M).2° Affinity for Substrates. Michaelis constants for substrates of pigeon breast muscle carnitine acetyltransferase at pH 7.8 and 30 ° have been found to be as follows: K,,, CoASH ~-- 37 ~M; K~, acetyl-CoA : 34 p.M; K~, (--)-carnitine----120 pit/; Kin, acetyl-(--)-carnitine-----350 ~M. ~9 Similar results have been reported for the enzyme from other sources, ~,~3 and it ~ppears that these K,~ values represent dissociation constants (K~) for the enzyme-substrate complexes involved. 19,'~° ~J. F. A. Chase, Biochem. J. 1{}4, 503 (1967). '~J. Kohn, Nature 181, 839 (1958). ~TD. A. Yphantis, Ann. N.Y. Acad. Sci. 88, 586 (1960). ~sp. Andrews, Biochem. J. 91, 222 (1964). ~gj. F. A. Chase and P. K. Tubbs, Biochem. J. 99, 32 (1966). ~J. F. A. Chase, Biochem. J. 104, 510 (1967).
[60]
CARNITINE ACETYLTRANSFERASE FROM PIGEON BREAST
393
Turnover Number. Taking the molecular weight of pigeon breast muscle carnitine as 58,000, it may be calculated from the experimentally determined value of V~z [the maximum velocity at infinite concentration of both CoASH and acetyl-(--)-carnitine] that the turnover number of the enzyme at pH 7.8 and 30 ° is 29,000 moles of substrate transformed per mole of enzyme per minute. The corresponding value for the back reaction between acetyl-CoA and (--)-carnitine is 23,000. Distribution. Carnitine acetyltransferase is distributed widely in animal tissues 7,2~,22 and is also present in the yeast Saccharomyces carlsbergensis. 23 In general, it is found in highest concentration in tissues (e.g., heart and skeletal muscle) which show a high rate of fat oxidation, and in sperm.
,1A. M. Th. Beenakkers and M. Klingenberg, Biochim. Biophys. Aeta 84, 207 (1964). ~N. R. Marquis and I. B. Fritz, J. Biol. Chem. 240, 2197 (1965). U Personal communication from Miss Ann Light, University of Bristol, Bristol, England.
CARNITINEACETYLTRANSFERASEFROM PIGEON BREAST
387
[60] C a r n i t i n e A c e t y l t r a n s f e r a s e f r o m P i g e o n B r e a s t M u s c l e [EC 2.3.1.7
Acetyl-CoA:carnitine O-aeetyltransferase] By J. F. A. CIJASE
O-Acetyl-(--)-carnitine + CoASH ~ (-)-carnitine + acetyl-Cok Assay Methods Method A: The Direct Assay Principle. Enzyme preparations of specific activity in excess of 1 unit per milligram of protein per milliliter (see below) are assayed most satisfactorily by direct spectrophotometric observation of the reacting substrafes. At 232 m~, there is an increase in molar extinction of 4.5 )< 103 cm-1 on the acetylation of CoASH,1 whereas carnitine and acylcarnitine solutions do not absorb at this wavelength.2 The catalyzed reaction may be followed in either the forward or reverse direction, as it is readily reversible (Keq = 0.6).s Reagents Tris-HC1 buffer, 1.0 M, pH 7.8 Neutral EDTA, 0.1 M CoASH, 10 mg/ml. The solid coenzyme is dissolved freshly in water Acetyl-DL-carnitine hydroehloride, 0.1 M, prepared according to Fraenkel and Friedmann' and dissolved in water Enzyme: 0.02-0.1 unit of carnitine acetyltransferase is a suitable amount for assay. This corresponds to about 0.2--1.0 /~g of the crystalline enzyme from pigeon breast muscle,~ and gives an increase in extinction at 232 m~ of 0.05-0.25 per minute in the system described below Procedure. The assay system contains 0.2 ml of Tris-HC1, 0.005 ml of EDTA, 0.05 ml of CoASH, enzyme, and water in a final volume of 1.95 ml. This mixture, in a cell of 10 mm light path, is equilibrated at 25 ° in a spectrophotometer with a temperature-controlled cell housing. On the addition of 0.05 ml of acetylcarnitine, the extinction of the solution at 1E. R. Stadtman, Vol. I [137]. 2D. J. Pearson, Biochem. J. 95, 23c (1965). 3I. B. Fritz, S. K. Schultz, and P. A. Stere, J. Biol. Chem. 238, 2509 (1963). G. Fraenkel and S. Friedmann, Vitamins Hormones 15, 73 (1957). 6j. F. A. Chase, D. J. Pearson, and P. K. Tubbs, Biochim. Biophys. Acta 96, 162 (1965).
388
REACTIONS YIELDING ACETYL-COA
[60]
232 m~ increases linearly with time for 1-2 minutes. Bovine serum albumin was included in another description" but is now omitted as it has no effect on the activity or stability of the enzyme. Units. One unit of enzyme is t h a t amount which catalyzes the acetylation of 1 micromole of CoASH per minute in the above system. Protein is determined spcctrophotometrically at 260 and 280 m~ according to Layne. 6 Specific activity is expressed as units per milligram of protein.
Method B: Carnitine Acetyltrans]erase in Crude Tissue Extracts Crude extracts contain too much 232 m~ absorbing material for the direct assay to be applicable. Carnitine acetyltransferase activity may be detected in such preparations in a coupled system2, 7 Principle. Acetyl-CoA, formed from acetylcarnitine and CoASH, is removed to form citrate in the presence of malate, NAD, and excess citrate synthase and malate dehydrogenase, the reduction of NAD being followed at 340 m~. Acetylcarnitine q- CoASH ~ carnitine -b acetyl-CoA Acetyl-CoA -~ oxaloacetate -~ citrate ~ CoASH Malate -t- NAD ~ oxaloacetate T NADH~ Acetylcarnitine -[- malate -~ N A D --~ carnitine ~ citrate -{- NADH~
Reagents L-Malate, 1.0 M, p H 8.0 NAD, 10 raM, p H 6.0 NaCN, l0 m M Citrate synthase: a crystalline suspension of the enzyme from pig heart, s ca. 5 mg of protein/ml (or see footnote 8a) Malate dehydrogenase: an ammonium sulfate suspension of the pig heart enzyme,' ca. 5 mg of protein/ml 6E. Layne, Vol. III [73]. N. R. Marquis and I. B. Fritz, J. Biol. Chem. 240, 2193 (1965). 'P. A. Srere and G. W. Kosicki, J. Biol. Chem. 236, 2557 (1961). ,a It may be noted that a substantial copurification of carn/tine acetyltransferase and citrate synthase occurs during steps 1--4 of the purification procedure. Most of the citrate synthase, which is present in an amount comparable to that of the carnitine enzyme in the eluate from step 4 is, however, left in solution after the calcium phosphate gel treatment. If desired, it may be adsorbed by the further addition of 5 ml of gel per I00 ml of eluate. Citrate synthase may then be eluted and crystallized as described for the pig heart enzyme,' when it is suitable for use in assay method B. ' Commercial preparations are available, or see this volume [18-21].
[50]
CARNITINE ACETYLTRANSFERASE FROM PIGEON BREAST
389
Tris-HC1, pH 7.8, EDTA, CoASH, acetyl-DL-carnitine and carnitine acetyltransferase solutions as for assay method A Procedure. Combine Tris-HC1, 0.2 ml; EDTA, 0.05 ml; L-malate, 0.05 ml; NAD, 0.05 ml; NaCN, 0.2 ml (if the preparation shows NADH.~ oxidase activity); CoASH, 0.25 ml; malate dehydrogenase, 0.005 ml; citrate synthase, 0.01 ml; carnitine acetyltransferase, 0.04-0.20 unit; and water to a volume of 1.95 ml. Equilibrate the mixture in a spectrophotometer at 25 ° as described in method A. On the addition of 0.05 ml of acetylcarnitine, an increase in extinction at 340 m~ is observed. For the calculation of specific activity, it is assumed that the extinction coefficient for NAD reduction at this wavelength is 6.22 X 103 cm-1. Specific activities may then be expressed in a manner analogous to that given for method A. This procedure gives erroneously low estimates of enzymatic activity because less than 1 equivalent of NADH~ is produced per equivalent of acetyl-CoA formed,2,1° but it is adequate for comparative purposes. Other Methods
Assays that have been devised to study the reverse reaction between acetyl-CoA and (--)-carnitine include a relatively insensitive hydroxamate procedure to follow acetylcarnitine formation,~1 and the use of the thiol reagent 5,5"-dithiobis-(2-nitrobenzoic acid) (DTNB) to follow CoASH release2,12 The latter technique has the disadvantage that DTNB slowly inactivates carnitine acetyltransferase2 A very sensitive isotopeexchange assay has also been described TM with its application to detection of the enzyme in nervous tissue. Purification Procedure This procedure is essentially the same as published elsewhere2 All operations were conducted in a cold-room at 4 ° unless otherwise indicated. Pigeon breast muscle, excised as soon as possible after death, m a y be stored frozen for several months without loss of extractable activity. Step 1. Extraction. Stored muscle is thawed, sliced, and homogenized for I minute in a Waring blendor with 3 volumes of cold (--5 °) 2 0 % ethanol containing 0 . 4 M KCI. The homogenate is centrifuged for 10 minutes at 23,000 g, filtered through muslin to remove fat, and dialyzed IoW. Buckel and If. Eggerer, Biochem. Z. 343, 29 (1965). "S. Friedmann and G. Fraenkel, Arch. Biochem. Biophys. 59, 491 (1955). 12I. B. Fritz and S. K. Sehultz, J. Biol. Chem. 240, 2188 (1965). ~R. E. McCaman, M. W. McCaman, and M. L. Stafford, J. Biol. Chem. o,41, 930 (1966).
390
REACTIONS YIELDING ACETYL-COA
[60]
against two changes of 2 m M potassium phosphate buffer, pH 7.5, containing 0.5 mM EDTA. Step ~. Ammonium Sul]ate Fractionation. Any precipitate formed on dialysis is discarded, and solid ammonium sulfate is added to give 50~ saturation (312 g/liter). After 30 minutes, the precipitate is collected by centrifugation and discarded. Further ammonium sulfate (82 g/liter) is added to the supernatant to give 62.5% saturation. The protein precipitated contains over 90% of the enzyme activity; it is dissolved in 0.1 M phosphate, pH 7.5, and dialyzed as before. Step 3. Acetone Fractionation. The dialyzate is cooled to 0 ° and 0.47 of its volume of acetone is added slowly, with stirring; the temperature of the mixture is lowered progressively to --7% After 30 minutes, the precipitate is centrifuged, dissolved in 0.1 M phosphate, pit 7.5, and dialyzed as in step 1. It has been found repeatedly that attempts to redissolve the acetone pellet in dilute buffer (e.g., 10 raM) result in almost complete loss of enzyme activity, and this should be avoided. Step ~. Fractionation on DBAE-Cellulose. The dialyzed preparation is applied to a column of DEAE-cellulose (35 g of DEAE per gram of protein) equilibrated with 2 mM phosphate, pH 7.5. The column is washed with 2 column volumes of 10 mM phosphate, pH 7.5, followed by 3 volumes of 15 mM buffer. This removes about half of the protein; no transferase activity should be present in the 10 mM eluate, and only a trace in the 15 mM fraction. The enzyme is eluted with 4 column volumes of 25 mM phosphate, pH 7.5. Step 5. Calcium Phosphate Gel. One milliliter of calcium phosphate gel14 (34 mg/ml, dry weight) is added per 100 ml of 25 mM phosphate eluate; this absorbs all the enzyme. The gel is washed 3 times with 0.1 M phosphate, pH 7.5, and the enzyme is eluted in 0.4 M phosphate, containing 10% ammonium sulfate2 ~ Step 6. Fractionation on Sephadex G-IO0. Ammonium sulfate (55 g) is added to each 100 ml of gel eluate, and the precipitate is dissolved in a minimal volume of 0.1 M phosphate, pH 7.5. Fractions (2-4 ml) of this solution are applied to a column of Sephadex G-100 (4 cm X 21 cm) connected to a reservoir of the same buffer. The first 45 ml of eluate contains no protein and is discarded; thereafter, 5 ml fractions are taken and assayed for protein (absorption at 280 m~) and enzymatic activity. Two, slightly overlapping, protein peaks emerge. The first, after 65 ml of eluate have been collected, contains enzymatically inactive colored material; the second, at ll0 ml, corresponds with the enzyme activity. No further protein emerges after 150 ml, and a pool is made of fractions cont4D. Keilin and E. F. Hartree, Proc. Roy. Soc. Lo~zdon B124, 397 (1938); see al.~o Vol. I [11].
[60]
CARNITINE ACETYLTBANSFERASE FROM PIGEON BREAST
391
raining 90-95% of the total activity. I m p u r e material appearing at low elution volumes is discarded. Step 7. Crystallization. The pooled fractions from step 6 are concentrated b y adding a m m o n i u m sulfate to about 90% saturation (65 g/100 ml) and the precipitate is dissolved in 0.1 M phosphate, p H 7.5, to give a solution containing about 10 mg of protein per milliliter. This is cooled to 0 ° and solid a m m o n i u m sulfate is added until a faint turbidity appears. Any traces of denatured brown material t h a t precipitate are discarded, and the solution is then stored at 4 °. Crystals of carnitinc acetyltransferase appear overnight and continue to grow for several days in the form of fine needles? Recrystallization is cffectcd by a repetition of this process and is encouraged b y "seeding" the incipiently turbid solution with preformed crystals. The specific activity of the recrystallized enzyme is 118-119 units/rag, a value which is unaltered by further recrystallizations. The purification procedure is summarized in the table for a preparation starting from 900 g of pigeon muscle and yielding 15 mg of crystalline enzyme. No difficulties have been encountered in reproducing these results so long as the warning given in step 3 is heeded. The method m a y be scaled-up at least 4-fold with a proportionate increase in yield. PURIFICATION PROCEDURE FOR CARNITINE ACETYLTRANSFERASE FROM PIGEON BREAST ~IUSCLE
Step and fraction
Volume (ml)
Specific Total activity Units/ activity Protein (units/rag Yield mla (units b) (mg/ml) protein) (%)
1. Centrifuged extract 2660 3.1 2. 50--62.5% Ammonium 170 43.7 sulfate fraction 3. 0-32% Acetone fraction 140 36.5 4. DEAE-cellulose 1970 1.08 25 mM eluate 5. Calcium phosphate 102 17.0 gel eluate 6. Sephadex G-100 eluate 50 32.8 7. Crystallization First crystals 1.7 717 First recrystallization 1.35 438 Second recrystallization 1.75 334
8250 7430
39 0 64.0
0.18 0.68
100 90
5130 2130
24.2 0.11
1.51 9.8
62 26
1734
0.68
25.0
21
1640
0.73
45.0
20
1219 592 584
6.6 3.7 2.8
108 118 119
15 ---
" Activity measurements for steps 1 and 2 were made using the coupled assay (method B). Thereafter the direct assay (method A) was employed. b One unit of enzyme is the amount that catalyzes the acetylation of 1 micromole of CoASH per minute in the assay conditions.
392
REACTIONS YIELDING ACETYL-COA
[50]
Properties Stability. The crystalline enzyme is stored conveniently as a suspension in 60-70% saturated ammonium sulfate, when it is completely stable at 4 °. Dilute solutions (10-100 ~g/ml) in 0.1 M phosphate, pH 7.5, also may be stored in the refrigerator; they lose only 10-12% of their activity in 2 months. Effects o] pH. Carnitine acetyltransferase shows a broad pH optimum between 7.0 and 8.0 for both forward and reverse reactions2 ,15 It is stable overnight at pH 5.5, but becomes rapidly and irreversibly inactivated above pH 8.6.8 Purity. Recrystallized carnitine acetyltransfcrasc is colorless in solution and is homogeneous in the ultracentrifuge, on chromatography on Sephadex G-100, and on electrophoresis on cellulose acetate paper. 1~ It is also free of acetyl-CoA hydrolase, citrate synthase, malate dehydrogenase, and palmitoylcarnitine transferase activities. Molecular Weight. A value of 58,000 ± 3000 is indicated by sedimentation equilibrium 1' and gel filtration18 methods. Inhibitors. The enzyme is inhibited by a range of reagents (iodoacetamide, p-chloromercuribenzoate, DTNB, N-ethylmaleimidc) which are somewhat specific for protein thiol groups2,12,1~ Divalent cations are also inhibitory. Specificity. The enzyme is highly specific for (--)-carnitine and CoA, (--)-norearnitine and 3'-dephospho-CoA being the only known analogs which are also substrates2 ,1~,15 (-k)-Carnitine is a competitive inhibitor (K~--173 ~ / ) 1 ~ for (--)-carnitine and its derivatives. 12'1~ Group transfer between CoASH and (--)-carnitine is catalyzed with n-acyl groups containing up to 10 carbon atoms, and Vm~ for the reaction falls off with increasing chain length2 ,~° Palmitoyl-CoA is not a substrate, 3 but acts as a potent competitive inhibitor with respect to both (--)-carnitine and acetyl-CoA (K~ -~ 0.43 p.M).2° Affinity for Substrates. Michaelis constants for substrates of pigeon breast muscle carnitine acetyltransferase at pH 7.8 and 30 ° have been found to be as follows: K,,, CoASH ~-- 37 ~M; K~, acetyl-CoA : 34 p.M; K~, (--)-carnitine----120 pit/; Kin, acetyl-(--)-carnitine-----350 ~M. ~9 Similar results have been reported for the enzyme from other sources, ~,~3 and it ~ppears that these K,~ values represent dissociation constants (K~) for the enzyme-substrate complexes involved. 19,'~° ~J. F. A. Chase, Biochem. J. 1{}4, 503 (1967). '~J. Kohn, Nature 181, 839 (1958). ~TD. A. Yphantis, Ann. N.Y. Acad. Sci. 88, 586 (1960). ~sp. Andrews, Biochem. J. 91, 222 (1964). ~gj. F. A. Chase and P. K. Tubbs, Biochem. J. 99, 32 (1966). ~J. F. A. Chase, Biochem. J. 104, 510 (1967).
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CARNITINE ACETYLTRANSFERASE FROM PIGEON BREAST
393
Turnover Number. Taking the molecular weight of pigeon breast muscle carnitine as 58,000, it may be calculated from the experimentally determined value of V~z [the maximum velocity at infinite concentration of both CoASH and acetyl-(--)-carnitine] that the turnover number of the enzyme at pH 7.8 and 30 ° is 29,000 moles of substrate transformed per mole of enzyme per minute. The corresponding value for the back reaction between acetyl-CoA and (--)-carnitine is 23,000. Distribution. Carnitine acetyltransferase is distributed widely in animal tissues 7,2~,22 and is also present in the yeast Saccharomyces carlsbergensis. 23 In general, it is found in highest concentration in tissues (e.g., heart and skeletal muscle) which show a high rate of fat oxidation, and in sperm.
,1A. M. Th. Beenakkers and M. Klingenberg, Biochim. Biophys. Aeta 84, 207 (1964). ~N. R. Marquis and I. B. Fritz, J. Biol. Chem. 240, 2197 (1965). U Personal communication from Miss Ann Light, University of Bristol, Bristol, England.