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Reversible enzymatic acetylation of carnitine
.ARCHIVES
OF
Reversible
BIOCHEMISTRY
AND
BIOPHYSICS
Enzymatic Acetylation
69, 491-501 (19%)
of Carnitinel~ 2
S. Friedman and G. Fraenkel From the Department of Entomology, University of Illinois,
Urbana, Illinois
Received June 15, 1955
The isolation of carnitine from mammalian muscle in 1905 (l), and its subsequent identification as -r-trimethylamino-fl-hydroxybutyrate (2) led to a few attempts to assess its significance in metabolism. The discovery that acetylcholine was the mediator of the nerve impulse was the signal for a flurry of investigation into whether acetylcarnitine was another compound which perhaps had the same function. None of this work succeeded in even hinting at a possible role for this compound
(3). In 1948, Fraenkel (4) discovered a new growth factor for the larva of the beetle, Tenebrio molitor. Since it was required in exceedingly small amounts and could be replaced by a soluble fraction of yeast, it was na,med vitamin B, (T = Tenebrio). In 1952, Carter et al. (5) succeeded in isolating this vitamin, and upon subjecting it to analysis, found that it was identical with carnitine. As a result of these findings new investigations were instituted on the role of carnitine in the metabolism of higher animals. In the course of these studies, it was found that carnitine appeared to inhibit enzymatic acetylations performed by soluble preparations from pigeon liver acetone powders. This was later found to be due to an enzyme in these crude extracts which is active in the acetylation of carnitine to form acetylcarnitine. It is in support of these results that the present paper is written. 1Someof the information presented in this paper was discussedin the Biological Chemistry section of the annual meeting of the Federation of American Societies for Experimental Biology in Atlantic City, N. J., April 12-16, 1954. f This investigation
was supported in part by a grant from The Office of the
Surgeon General, Dept. of the Army (MD-308). 491
492
S. FRIEDMAN AND G. FRAENKEL MATERIALS
AND METHODS
Acetone powders of pigeon liver were prepared according to Kaplan and Lipmann (6). Extracts of these powders were made by rubbing them up with 10 vol. of 0.02 M NaHCOI in an ice-cold mortar and centrifuging the resulting suspension at 25,000 X g for 10 min. at 0°C. The extracts so obtained were fractionated immediately with solid (NH&SOI at 0°C. and dialyzed against 100 vol. of 0.02 M NaHC03 at 3°C. for 12 hr. The dialyzed extracts could be kept for 3 months at -2O’C. without appreciable loss in activity. Sheep liver extracts were prepared and fractionated in the same manner. 0-Acetylcarnitine was prepared by treating dl-carnitine.HCl with 10 vol. of acetyl chloride at room temperature in a stoppered flask for 2448 hr. with intermittent shaking to break up the cake which formed after a short time. The excess acetyl chloride was then decanted off and the solid dried in uucuo. O-Acetylcarnitine was separated from residual carnitine by a fractional crystallization procedure which involved dissolving the solid in the least amount of glacial acetic acid and adding increasing amounts of anhydrous acetone. The compound was identified as the hydrochloride, which melted at 136-163” with bubbling, and by formation of the gold chloride derivative which had a melting point of 160-162”. Chromatographic identification as described below showed the compound to be entirely free of carnitine. Acetyl-p-aminobenzoic acid (PAB) was measured by the method of Bratton and Marshall (7) using a Klett-Summerson calorimeter with a No. 54 filter. Hydroxamic acid formation was determined by a slight modification of the method of tipmann and Tuttle (8). Acethydroxamic acid was demonstrated chromatographically by the method of Stadtman and Barker (9). 0-Acetylcarnitine was measured quantitatively by a modification of the method of Hestrin (10). A chromatographic separation of carnitine and acetylcarnitine was effected by the method of Friedman et al. (ll), using the acid solvent system described in that paper. When a separation of these two compounds was necessary in the presence of protein, the protein was removed by precipitation from the reaction mixture with 20 vol. of 95oJ,ethanol. After centrifugation, the supernatant was taken down to dryness and the solids resolubilized in a small volume of absolute ethanol. This was applied directly to the chromatographic paper. Separation of acetylcholine and acetylcarnitine was attained using descending chromatography on strips of Whatman No, 1 paper at 37°C. The solvent system was butanol saturated with water, and this mixture was permitted to drip off the end of the paper for a few hours after complete development. Protein and salt were removed from the reaction mixtures, prior to this chromatographic separation, by the method of Banister, Whittaker, and Wijesundera (12). The spots were made visible through the method of Whittaker and Wijesundera (13). Choline determinations were carried out using the method of Horowitz and Beadle (14). The cholineless Neumspora crassa was obtained through the kindness of Dr. B. Connor Johnson of the University of Illinois. Clostridium kluyveri cells were obtained through the courtesy of Dr. I. C. Gunsalus of the University of Illinois. Extracts were made from frozen cells in the following manner: The frozen cells were ground with 2.5 times their weight of Alumina A-301 in an ice-cold mortar until a gummy paste was formed. Phosphate buffer (0.02 M, pH 7.2) was added in a weight-volume ratio of 1:3.3, and the
ACETYLATION
493
OF CARNITINE
TABLE I Inhibition of Enzymatically 6atalyzed PAB Acetylation by dl-Carnitine Complete reaction mixture: Na acetate 20 pM, ATP 5 PM, CoA 10 units, cysteine.HCl 10 a, Na bicarbonate 200 @, Nit citrate 40 NM, MgClr 4 pM, PAB 54 pg., pigeon liver extract (404% (NH&SOI fraction) 0.3 ml., in 1.03ml. Incu-
bated at 37°C. for 1.5 hr. 1. Pigeon liver extracts PAB acetykted Pg.
System
Complete Complete Complete Complete Complete
+ + + -
dl-carnitine (10 PM) betaine (10 pM) p-hydroxybutyrate (10 PM) acetate
suspension was centrifuged. The supernatant
36 0 36 36 0
was used for all of the determina-
tions involving bacteria. Lithium acetylphosphate was prepared according to Stadtman and Lipmann (15). Dipotasaium adenosine tripkiosphate (ATP) and coenzyme A (CoA) were
purchased from Pabst Laboratories: Acetylcholine chloride was obtained from Merck and.Co., and dl-carnitine.HCl and Chemicals Co.
was obtained from International
Minerals
RESULTS
The initial observations concerned with carnitine inhibition of PAB acetylation were made on crude pigeon liver extracts. Under these conditions, inhibition was very variable, but with further frationation of the extracts using solid (NH&S04, a much more reproducible inhibition could be demonstrated. Table I expresses the result of a sample experiment using an enzyme preparation which was partially purified (ca. 5X) by the above method. The specificity of this inhibition was somewhat delimited by substituting other compounds for carnitine. Neither glycine betaine nor j3-hydroxybutyrate, when tested at the same concentrations as carnitine, were active in producing any suppression of PAB acetylation. To determine the site of inhibition, experiments were done using a system other than the acetate-activating system for generating acetyl CoA. This could then be coupled with the PAB-acetylating enzyme from pigeon liver (found in the same ammonium sulfate fraction as the acetate-activating enzyme) fog analysis of inhibition. The phosphotransacetylase system from CZ. kluyveti was chosen as a means of generating acetyl CoA from acetyl phosphate (16). When carnitine was
494
S. FRIEDMAN
AND
G.
FRAENKEL
TABLE II Inhibition of Enzymatically Catalyzed PAB Acetylation by dl-Carnitine Complete reaction mixture: Lithium acetylphosphate 10 rM, Na bicarbonate 80 pM, cysteine.HCl 10 p&l, CoA 10 units, PAB 54 pg., pigeon liver extract (407OoJc(NH&SO4 fraction) 0.25 ml., Cl. kluyveri extract 0.2 ml., in 1.13 ml. Incubated 2 hr. at 28°C. 2. Clostridium kluyveri extracts system
PAB acetylated a.
12 3 0
Complete Complete f dl-carnitine (10 PM) Complete - acetylphosphate
added to a reaction mixture containing acetylphosphate, CE. kluyveri extract, CoA, pigeon liver extract, and PAB, a strong inhibition of acetyl PAB formation occurred. Table II illustrates a typical experiment. Since both methods of producing acetyl CoA were inhibited by addition of carnitine, the possibility of formation of an acetylated carnitine was next taken into consideration. The rationale for this was as TABLE Reversal of dl-Car&tine
I. Cl. kluyveri Complete Complete Complete Complete II. Pigeon liver Complete Complete Complete Complete
III
Inhibition of Enzymatically by O-Acetylcarnitine System
Catalyzed PAB
+ dl-carnitine (10 pM) + dl-carnitine (10 pM) + 0-acetylcarnitine - acetylphosphate -I- dl-carnitine + dl-carnitine - acetate
(10 pM) (10 PM) + 0-acetylcarnitine
Acetylation PAB acetylated trg.
(10 PM)
(10 PM)
49 14 31 0 30 4 17 0
Complete reaction mixture : Cl. kluyveri: Lithium acetylphosphate 10 NM, Na bicarbonate 80 a, cysteine. HCl 10 GM, CoA 10 units, PAB 54 pg., pigeon liver extract (4&70% (NH&SO4 fraction) 0.25 ml., CZ. kluyveri extract 0.2 ml., in 1.13 ml. Incubated 2 hr. at 28°C. Pigeon liver: Na acetate 20 pM, ATP 5 pM, CoA 10 units, cysteine.HCl 10 &, Na bicarbonate 200 PM, Na citrate 40 PM, MgClz 4 pM, PAB 54 pg., pigeon liver
extract (40-70% (NH&SO, 1.5 hr.
fraction)
0.3 ml., in 1.03 ml. Incubated at 37°C. for
ACETYLATION
OF
CARNITINE
495
follows: If carnitine were competing with PAB for active acetate, its presence would effectively inhibit the formation of acetyl PAB. Acetylcarnitine was therefore prepared and the hypothesis tested. Acetylcarnitine added to a reaction mixture containing quantities of carnitine high enough to inhibit the reaction was found to reverse the inhibition. The results of this are shown in Table III. An equivalent amount of free acetate did not produce the same effect. Enzymatic formation of acetylcarnitine could be shown in a reaction mixture containing acetate, ATP, Mg++, CoA, carnitine, and the pigeon liver preparation. The methods whereby this was demonstrated were both chemical and chromatographic. The first method utilized the fact that 0-acetylcarnitine is an ester and, as such, should respond to the Hestrin test. The linearity of this response is shown in Fig. 1. In this experiment, various concentrations of synthetic 0-acetylcarnitine were placed in cuvettes, 0.1 ml. of 28 % NHzOH.HCl was added, followed by 0.5 ml. of 14 % NaOH. The tubes were shaken, and 1.0 ml. of 3 N HCl was added followed by 1.0 ml. of 5% FeC13 in 0.1 N HCl. Under these conditions, acetylcarnitine gives approximately the same density readings as acetylcholine of equal concentrations. When the
pM
FIG. 1. Response of scetylcarnitine
ACETYL
CARNITINE
to modified He&in test. To each cuvette was added a given concentration of 0-acetylcarnitine, and then in order 0.1 ml. of 28% NH*OH.HCl, and 0.5 ml. of 14% NaOH. After shaking, 1.0 ml. of 3 N HCl was added, followed by 1.0 ml. of 5% FeCla in 0.1 N HCl.
496
S. FRIEDMAN
AND
G.
FRAENKEL
TABLE IV Enzymatic Formation of Acetylcarnitine as Measured by Modified Hestrin Test Complete reaction mixture: Na acetate 20 pM, ATP 5 pM, CoA 20 unite, cysteine.HCl 10 pM, Na bicarbonate 200 &f, MgC12 4 pM, Na citrate 40 PM, dl-carnitine 20 PM, pigeon liver extract (40-70% (NH&SO, fraction) 0.6 ml. Incubated 30 min. at 37°C. .4cetylcarnitine, rM formed hydroxamic acid
System
Complete Complete - dl-carnitine Complete - CoA Complete - enzyme
as
1.0 0.2 0.2 0.2
enzymatic test was made, a He&in-positive compound was formed, as can be seen by Table IV. As is noted in the table, &he reaction is dependent upon the presence of CoA. To prove that this was acetylcarnitine, the reaction mixture was deproteinized, spotted on Whatman No. 1 paper, chromatographed using the acid solvent system of Friedman et al. (II), and developed by the method described in the dame paper. The results were as shown in Fig. 2. A spot corresponding to that of acetylcarnitine, which moves at an RI of 0.70 in this sytem, was quite easily seen. The reversal of this reaction, which entails the donation of acetyl groups from 0-acetylcarnitine fo CoA was investigated, using as ac-
CARN
AC CARN
EXPT
CON-I
FIQ. 2. Chromatographic separation of carnitine and acetylcarnitipe on Whatman No. 1 paper at room temperature in the acid solvent system of Friedman et al.
(11).
ACETYLATION
OF
TABLE Enzymatic
Acetylations
V
Using Acetylcarnitine
as Substrate PAB formed Pg.
system
Acetate, complete Acetate, complete - ATP-Mg Acetate, complete - ATP-Mg + dl-carnitine Acetylcarnitine, complete Acetylcholine, complete
497
CARNITINE
(20 &f)
50 0 0 34 0
Hydroxamic acid formed r‘+f
1.33 0 1.0
Complete reaction mixture : Acetate : Na acetate 20 pM, ATP 5 pM, CoA 20 units, cysteine .HCl 10 pM, Na bicarbonate 200 pM, MgCll4 PM, Na citrate 40 pM, pigeon liver extract (40-70yo (NH&!0 fraction) 0.3 ml. Incubated 30 min. at 37°C. Acetylcarnitine: acetylcarnitine 20 pM, CoA 20 units, cysteine .HCl 10 &f, Na bicarbonate 200 pM, pigeon liver extract (40-700Jo(NH&SOI fraction) 0.3 ml. Acetylcholine : same as acetylcarnitine, except acetylcholine. Cl (20 &f) present instead of acetylcarnitine. For PAB determinations, 54 rg. PAB added. For h:ydroxamic acid determinations, 100 pM of hydroxylamine .HCl (neutralized to pH 6.8 nith 14% NaOH) added.
ceptors both PAB and hydroxylamine. Table V illustrates the formaof these two compounds. In the absence of Mg++ and ATP, acetylcarnitine reacts to form the derivatives mentioned above. Acetate and carnitine added as cosubstrates do not react in the a,bsence of Mg++ and ATP. Acetylcholine is also inactive in these experiments. The same reaction of acetylcarnitine takes place using a sheep liver extract instead of the pigeon liver. The dependence of this reaction upon CoA is expressed in Fig. 3. Since it is well known that esters react chemically with hydroxylamine in alkaline solution, the spontaneous reaction of acetylcarnitine with hydroxyla,mine was measured and compared with the enzymatic one. The results of this are shown in Fig. 4. The proof of acetyl donation by acetylcarnitine rests in the formation of acethydroxamic acid, which can be identified by the chromatographic method of Stadtman and Barker. When acetylcarnitine is incubated with CoA and hydroxylamine in the presence of the pigeon liver preparation for a 1-hr. period, treated to remove protein and salts, and then chromatographed, a spot with an Rf value of 0.50, corresponding to acethydroxamic acid is produced. Controls in which no enzyme or CoA are added form spots of very much smaller dimensions. This tion of acetyl derivatives
498
S. FRIEDMAN
AND
G.
FRAENKEL
reaction is due to the spontaneous formation of acethydroxamic acid, as mentioned above. When acetylcarnitine is split, two possibilities exist as to end products of the reaction. If the split were made at the ester linkage, the resulting products would be “acetyl” and carnitine, whereas if the split occurred at the a-P carbon linkage, the products would be “acetyl” and acetylcholine. In order to definitely eliminate the latter possiblity from consideration, although all of the evidence was against it, two methods were used. Since the chromatographic separation of acetylcholine and acetylcarnitine is feasible, reaction mixtures of the type heretofore used were incubated for 2 hr. at 37”C., and after removal of the protein and salt, were spotted on Whatman No. 1 paper. The chromatographic separation offered no rewards, in that no acetylcholine could be identified. In spite of the fact that the chromatograms were not completely satisfactory due to slight streaking, the only identifiable spot was acetylcarnitine. The second method used in the search for acetylcholine was the Neurospora choline assay based on the fact that a mutant strain of Neurospora m-ma requires for normal growth a small amount of choline in addition to its basal medium. If acetylcholine were formed
FIG. 3. Coenzyme A dependence of enzymatic from acetylcarnitine. Reation mixture contained: specified, cyzteine.HCl 10 &f, Na bicarbonate (neutralized to pH 6.8 with 14% NaOH) 100 PM, (NH&SOI fraction) 0.3 ml.
formation of hydroxamic acid acetylcarnitine 20 PM, CoA as 200 ai hydroxylamine.HCl pigeon liver extract (46-70%
ACETYLATION
0 TINE-
499
OF CARNITINE
60 MINUTES
I20
FIG. 4. Comparison of spontaneous and enzymatic formation of hydroxamic acid from acetylcarnitine. Reaction mixture contains: acetylcarnitine 20 a, cysteine.HCl 10 PM, CoA 20 units, hydroxylamine.HCl (neutralized to pH 6.8 with 14% NaOH) 100 pM, Na bicarbonate 200 &I. Pigeon liver extract (40-7095 (NH&S01 fraction) added for enzymatic formation (boiled, and added for measurement of spontaneous formation) 0.3 ml.
as a reaction product of the split of acetylcarnitine, treatment of the reaction mixture after incubation with a small amount of strong base would split the acetylcholine to choline and acetate, and the choline could then be determined. Since the Neurospora assay is sensitive to less than 5 pg., anyacetylcholineformed would be recognized. The results of these experiments were completely negative. DISCUSSION
The role of acetyl CoA as an intermediate in biological systems is so well documented that it is not necessary to mention it here. The number of compounds which are active in donating acetyl groups to coenzyme A are limited, however, by the fact that the energy level of this compound is too high to permit the usual type of low-energy group-transfer reactions. Acetylcarnitine is able to make this transfer, but the properties which put it into an energy state permitting it to do this have not yet been accounted for. One may assume that they are involved with bhe
500
S. FRIEDMAN AND G. FRAENKEL
nature of the ester linkage (a secondary ester) and its neighboring quaternary ammonium group. Up to this time, the enzyme which catalyzes the reaction between acetylcarnitine and coenzyme A has not been purified enough to determine a true equilibrium constant for the reaction. Once this is done, however, it may be possible to include a new class of compounds among those which are now known to play such an important role in group-transfer reactions. Acetylcarnitine has not yet been found in animals, but there are a few indirect supports for believing that it may have some function in metabolism. The first is the effectiveness of deoxycarnitine as a competitive inhibitor of carnitine activity in the mealworm, Tenebrio molitor (17). This compound, lacking a hydroxyl group on the P-carbon, is the only one of more than twenty analogs and derivatives tested which is known to have any effect on carnitine utilization. These results establish the importance of a substitutable group on the P-carbon. Acetylcarnitine is also known to be active in replacing carnitine in the diet of Tenebrio (17). The limited sensitivity of the assay method does not permit a definite statement as to its activity, but it is known to be just as active and possibly more so. It is, of course, possible that a deacylase in the gut of the insect may split the acetylated compound to a nonacetylated one. Investigations are at present being carried on in an effort to further characterize this acetylating system and elucidate its function. SUMMARY
of enzymatic acetylation of PAB by 1. An apparent inhibition carnitine has been found to be due to an enzyme which is active in the acetylation of carnitine. 2. This enzyme, which is found in both pigeon and sheep liver extracts, seems to catalyze the following reaction: 0-acetylcarnitine + CoA ti acetyl CoA + carnitine 3. The possible importance of secondary esters in group-transfer reactions is discussed. REFERENCES 1. GULEWITSCH,W., AND KRIMBERG, R., 2. physiol. Chem. 46, 326 (1905). 2. TOMITA, M., 2. physiol. Chem. 168, 42 (1926). 3. STRACK, E., AND FOSTERLINQ, K., Arch. exptl. Path& Pharmakol. 186, 612 (1937). 4. FRAENKEL, G., BLEWETT, M., AND COLES, M., Nature 181, 981 (1948). 5. CARTER, H.E., BHATTACBARYYA, P.K., WEIDMAN, K.R., ANDFRAENKEL,G., Arch Biochem. and Biophys. 38, 405 (1952).
ACETYLATION
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
OF
CARNITINE
501
KAPLAN, N. O., AND LIPMANN, F., .I. Biol. Chem. 174, 37 (1948). BRATTON, A. C., AND MABSHALL, E. K., JR., J. Biol. Chem. 129, 537 (1939). LIPMANN, F., AND TUTTLE, L. C., J. Biol. Chem. 161, 415 (1945). STADTMAN, E. R., AND BARKER, H. A., J. Biol. Chem. 184,769 (1950). HESTRIN, S., J. Biol. Chem. 180, 249 (1949). FRIEDMAN, S., MCFARLANE, J. E., BHATTACHARYYA, P. K., AND FRAENKEL, G., Arch. Biochem. and Biophys. 69, 484 (1955). BANISTER, J., WHITTAKER, V. P., AND WIJESUNDERA, S., J. PhysioZ. (London) 121, 55 (1953). WHITTAKER, V. P., AND WIJESUNDERA, S., Biochem. J. 61, 348 (1952). HOROWITZ, N. H., AND BEADLE, q. W., J. BioZ. Chem. 160, 325 (1943). STADTMAN, E. R., AND LIPMANN, F., J. BioZ. Chem. 186,549 (1950). STADTMAN, E. R., NOVELLI, G. D., AND LIPMANN, F., J. BioZ. Chem. 191, 365 (1951). BHATTACJURYYA, P. K., FRIEDMAN, S., AND FRAENKEL, G., Arch. Biochem. and Biophys. 64, 424 (1955).
OF
Reversible
BIOCHEMISTRY
AND
BIOPHYSICS
Enzymatic Acetylation
69, 491-501 (19%)
of Carnitinel~ 2
S. Friedman and G. Fraenkel From the Department of Entomology, University of Illinois,
Urbana, Illinois
Received June 15, 1955
The isolation of carnitine from mammalian muscle in 1905 (l), and its subsequent identification as -r-trimethylamino-fl-hydroxybutyrate (2) led to a few attempts to assess its significance in metabolism. The discovery that acetylcholine was the mediator of the nerve impulse was the signal for a flurry of investigation into whether acetylcarnitine was another compound which perhaps had the same function. None of this work succeeded in even hinting at a possible role for this compound
(3). In 1948, Fraenkel (4) discovered a new growth factor for the larva of the beetle, Tenebrio molitor. Since it was required in exceedingly small amounts and could be replaced by a soluble fraction of yeast, it was na,med vitamin B, (T = Tenebrio). In 1952, Carter et al. (5) succeeded in isolating this vitamin, and upon subjecting it to analysis, found that it was identical with carnitine. As a result of these findings new investigations were instituted on the role of carnitine in the metabolism of higher animals. In the course of these studies, it was found that carnitine appeared to inhibit enzymatic acetylations performed by soluble preparations from pigeon liver acetone powders. This was later found to be due to an enzyme in these crude extracts which is active in the acetylation of carnitine to form acetylcarnitine. It is in support of these results that the present paper is written. 1Someof the information presented in this paper was discussedin the Biological Chemistry section of the annual meeting of the Federation of American Societies for Experimental Biology in Atlantic City, N. J., April 12-16, 1954. f This investigation
was supported in part by a grant from The Office of the
Surgeon General, Dept. of the Army (MD-308). 491
492
S. FRIEDMAN AND G. FRAENKEL MATERIALS
AND METHODS
Acetone powders of pigeon liver were prepared according to Kaplan and Lipmann (6). Extracts of these powders were made by rubbing them up with 10 vol. of 0.02 M NaHCOI in an ice-cold mortar and centrifuging the resulting suspension at 25,000 X g for 10 min. at 0°C. The extracts so obtained were fractionated immediately with solid (NH&SOI at 0°C. and dialyzed against 100 vol. of 0.02 M NaHC03 at 3°C. for 12 hr. The dialyzed extracts could be kept for 3 months at -2O’C. without appreciable loss in activity. Sheep liver extracts were prepared and fractionated in the same manner. 0-Acetylcarnitine was prepared by treating dl-carnitine.HCl with 10 vol. of acetyl chloride at room temperature in a stoppered flask for 2448 hr. with intermittent shaking to break up the cake which formed after a short time. The excess acetyl chloride was then decanted off and the solid dried in uucuo. O-Acetylcarnitine was separated from residual carnitine by a fractional crystallization procedure which involved dissolving the solid in the least amount of glacial acetic acid and adding increasing amounts of anhydrous acetone. The compound was identified as the hydrochloride, which melted at 136-163” with bubbling, and by formation of the gold chloride derivative which had a melting point of 160-162”. Chromatographic identification as described below showed the compound to be entirely free of carnitine. Acetyl-p-aminobenzoic acid (PAB) was measured by the method of Bratton and Marshall (7) using a Klett-Summerson calorimeter with a No. 54 filter. Hydroxamic acid formation was determined by a slight modification of the method of tipmann and Tuttle (8). Acethydroxamic acid was demonstrated chromatographically by the method of Stadtman and Barker (9). 0-Acetylcarnitine was measured quantitatively by a modification of the method of Hestrin (10). A chromatographic separation of carnitine and acetylcarnitine was effected by the method of Friedman et al. (ll), using the acid solvent system described in that paper. When a separation of these two compounds was necessary in the presence of protein, the protein was removed by precipitation from the reaction mixture with 20 vol. of 95oJ,ethanol. After centrifugation, the supernatant was taken down to dryness and the solids resolubilized in a small volume of absolute ethanol. This was applied directly to the chromatographic paper. Separation of acetylcholine and acetylcarnitine was attained using descending chromatography on strips of Whatman No, 1 paper at 37°C. The solvent system was butanol saturated with water, and this mixture was permitted to drip off the end of the paper for a few hours after complete development. Protein and salt were removed from the reaction mixtures, prior to this chromatographic separation, by the method of Banister, Whittaker, and Wijesundera (12). The spots were made visible through the method of Whittaker and Wijesundera (13). Choline determinations were carried out using the method of Horowitz and Beadle (14). The cholineless Neumspora crassa was obtained through the kindness of Dr. B. Connor Johnson of the University of Illinois. Clostridium kluyveri cells were obtained through the courtesy of Dr. I. C. Gunsalus of the University of Illinois. Extracts were made from frozen cells in the following manner: The frozen cells were ground with 2.5 times their weight of Alumina A-301 in an ice-cold mortar until a gummy paste was formed. Phosphate buffer (0.02 M, pH 7.2) was added in a weight-volume ratio of 1:3.3, and the
ACETYLATION
493
OF CARNITINE
TABLE I Inhibition of Enzymatically 6atalyzed PAB Acetylation by dl-Carnitine Complete reaction mixture: Na acetate 20 pM, ATP 5 PM, CoA 10 units, cysteine.HCl 10 a, Na bicarbonate 200 @, Nit citrate 40 NM, MgClr 4 pM, PAB 54 pg., pigeon liver extract (404% (NH&SOI fraction) 0.3 ml., in 1.03ml. Incu-
bated at 37°C. for 1.5 hr. 1. Pigeon liver extracts PAB acetykted Pg.
System
Complete Complete Complete Complete Complete
+ + + -
dl-carnitine (10 PM) betaine (10 pM) p-hydroxybutyrate (10 PM) acetate
suspension was centrifuged. The supernatant
36 0 36 36 0
was used for all of the determina-
tions involving bacteria. Lithium acetylphosphate was prepared according to Stadtman and Lipmann (15). Dipotasaium adenosine tripkiosphate (ATP) and coenzyme A (CoA) were
purchased from Pabst Laboratories: Acetylcholine chloride was obtained from Merck and.Co., and dl-carnitine.HCl and Chemicals Co.
was obtained from International
Minerals
RESULTS
The initial observations concerned with carnitine inhibition of PAB acetylation were made on crude pigeon liver extracts. Under these conditions, inhibition was very variable, but with further frationation of the extracts using solid (NH&S04, a much more reproducible inhibition could be demonstrated. Table I expresses the result of a sample experiment using an enzyme preparation which was partially purified (ca. 5X) by the above method. The specificity of this inhibition was somewhat delimited by substituting other compounds for carnitine. Neither glycine betaine nor j3-hydroxybutyrate, when tested at the same concentrations as carnitine, were active in producing any suppression of PAB acetylation. To determine the site of inhibition, experiments were done using a system other than the acetate-activating system for generating acetyl CoA. This could then be coupled with the PAB-acetylating enzyme from pigeon liver (found in the same ammonium sulfate fraction as the acetate-activating enzyme) fog analysis of inhibition. The phosphotransacetylase system from CZ. kluyveti was chosen as a means of generating acetyl CoA from acetyl phosphate (16). When carnitine was
494
S. FRIEDMAN
AND
G.
FRAENKEL
TABLE II Inhibition of Enzymatically Catalyzed PAB Acetylation by dl-Carnitine Complete reaction mixture: Lithium acetylphosphate 10 rM, Na bicarbonate 80 pM, cysteine.HCl 10 p&l, CoA 10 units, PAB 54 pg., pigeon liver extract (407OoJc(NH&SO4 fraction) 0.25 ml., Cl. kluyveri extract 0.2 ml., in 1.13 ml. Incubated 2 hr. at 28°C. 2. Clostridium kluyveri extracts system
PAB acetylated a.
12 3 0
Complete Complete f dl-carnitine (10 PM) Complete - acetylphosphate
added to a reaction mixture containing acetylphosphate, CE. kluyveri extract, CoA, pigeon liver extract, and PAB, a strong inhibition of acetyl PAB formation occurred. Table II illustrates a typical experiment. Since both methods of producing acetyl CoA were inhibited by addition of carnitine, the possibility of formation of an acetylated carnitine was next taken into consideration. The rationale for this was as TABLE Reversal of dl-Car&tine
I. Cl. kluyveri Complete Complete Complete Complete II. Pigeon liver Complete Complete Complete Complete
III
Inhibition of Enzymatically by O-Acetylcarnitine System
Catalyzed PAB
+ dl-carnitine (10 pM) + dl-carnitine (10 pM) + 0-acetylcarnitine - acetylphosphate -I- dl-carnitine + dl-carnitine - acetate
(10 pM) (10 PM) + 0-acetylcarnitine
Acetylation PAB acetylated trg.
(10 PM)
(10 PM)
49 14 31 0 30 4 17 0
Complete reaction mixture : Cl. kluyveri: Lithium acetylphosphate 10 NM, Na bicarbonate 80 a, cysteine. HCl 10 GM, CoA 10 units, PAB 54 pg., pigeon liver extract (4&70% (NH&SO4 fraction) 0.25 ml., CZ. kluyveri extract 0.2 ml., in 1.13 ml. Incubated 2 hr. at 28°C. Pigeon liver: Na acetate 20 pM, ATP 5 pM, CoA 10 units, cysteine.HCl 10 &, Na bicarbonate 200 PM, Na citrate 40 PM, MgClz 4 pM, PAB 54 pg., pigeon liver
extract (40-70% (NH&SO, 1.5 hr.
fraction)
0.3 ml., in 1.03 ml. Incubated at 37°C. for
ACETYLATION
OF
CARNITINE
495
follows: If carnitine were competing with PAB for active acetate, its presence would effectively inhibit the formation of acetyl PAB. Acetylcarnitine was therefore prepared and the hypothesis tested. Acetylcarnitine added to a reaction mixture containing quantities of carnitine high enough to inhibit the reaction was found to reverse the inhibition. The results of this are shown in Table III. An equivalent amount of free acetate did not produce the same effect. Enzymatic formation of acetylcarnitine could be shown in a reaction mixture containing acetate, ATP, Mg++, CoA, carnitine, and the pigeon liver preparation. The methods whereby this was demonstrated were both chemical and chromatographic. The first method utilized the fact that 0-acetylcarnitine is an ester and, as such, should respond to the Hestrin test. The linearity of this response is shown in Fig. 1. In this experiment, various concentrations of synthetic 0-acetylcarnitine were placed in cuvettes, 0.1 ml. of 28 % NHzOH.HCl was added, followed by 0.5 ml. of 14 % NaOH. The tubes were shaken, and 1.0 ml. of 3 N HCl was added followed by 1.0 ml. of 5% FeC13 in 0.1 N HCl. Under these conditions, acetylcarnitine gives approximately the same density readings as acetylcholine of equal concentrations. When the
pM
FIG. 1. Response of scetylcarnitine
ACETYL
CARNITINE
to modified He&in test. To each cuvette was added a given concentration of 0-acetylcarnitine, and then in order 0.1 ml. of 28% NH*OH.HCl, and 0.5 ml. of 14% NaOH. After shaking, 1.0 ml. of 3 N HCl was added, followed by 1.0 ml. of 5% FeCla in 0.1 N HCl.
496
S. FRIEDMAN
AND
G.
FRAENKEL
TABLE IV Enzymatic Formation of Acetylcarnitine as Measured by Modified Hestrin Test Complete reaction mixture: Na acetate 20 pM, ATP 5 pM, CoA 20 unite, cysteine.HCl 10 pM, Na bicarbonate 200 &f, MgC12 4 pM, Na citrate 40 PM, dl-carnitine 20 PM, pigeon liver extract (40-70% (NH&SO, fraction) 0.6 ml. Incubated 30 min. at 37°C. .4cetylcarnitine, rM formed hydroxamic acid
System
Complete Complete - dl-carnitine Complete - CoA Complete - enzyme
as
1.0 0.2 0.2 0.2
enzymatic test was made, a He&in-positive compound was formed, as can be seen by Table IV. As is noted in the table, &he reaction is dependent upon the presence of CoA. To prove that this was acetylcarnitine, the reaction mixture was deproteinized, spotted on Whatman No. 1 paper, chromatographed using the acid solvent system of Friedman et al. (II), and developed by the method described in the dame paper. The results were as shown in Fig. 2. A spot corresponding to that of acetylcarnitine, which moves at an RI of 0.70 in this sytem, was quite easily seen. The reversal of this reaction, which entails the donation of acetyl groups from 0-acetylcarnitine fo CoA was investigated, using as ac-
CARN
AC CARN
EXPT
CON-I
FIQ. 2. Chromatographic separation of carnitine and acetylcarnitipe on Whatman No. 1 paper at room temperature in the acid solvent system of Friedman et al.
(11).
ACETYLATION
OF
TABLE Enzymatic
Acetylations
V
Using Acetylcarnitine
as Substrate PAB formed Pg.
system
Acetate, complete Acetate, complete - ATP-Mg Acetate, complete - ATP-Mg + dl-carnitine Acetylcarnitine, complete Acetylcholine, complete
497
CARNITINE
(20 &f)
50 0 0 34 0
Hydroxamic acid formed r‘+f
1.33 0 1.0
Complete reaction mixture : Acetate : Na acetate 20 pM, ATP 5 pM, CoA 20 units, cysteine .HCl 10 pM, Na bicarbonate 200 pM, MgCll4 PM, Na citrate 40 pM, pigeon liver extract (40-70yo (NH&!0 fraction) 0.3 ml. Incubated 30 min. at 37°C. Acetylcarnitine: acetylcarnitine 20 pM, CoA 20 units, cysteine .HCl 10 &f, Na bicarbonate 200 pM, pigeon liver extract (40-700Jo(NH&SOI fraction) 0.3 ml. Acetylcholine : same as acetylcarnitine, except acetylcholine. Cl (20 &f) present instead of acetylcarnitine. For PAB determinations, 54 rg. PAB added. For h:ydroxamic acid determinations, 100 pM of hydroxylamine .HCl (neutralized to pH 6.8 nith 14% NaOH) added.
ceptors both PAB and hydroxylamine. Table V illustrates the formaof these two compounds. In the absence of Mg++ and ATP, acetylcarnitine reacts to form the derivatives mentioned above. Acetate and carnitine added as cosubstrates do not react in the a,bsence of Mg++ and ATP. Acetylcholine is also inactive in these experiments. The same reaction of acetylcarnitine takes place using a sheep liver extract instead of the pigeon liver. The dependence of this reaction upon CoA is expressed in Fig. 3. Since it is well known that esters react chemically with hydroxylamine in alkaline solution, the spontaneous reaction of acetylcarnitine with hydroxyla,mine was measured and compared with the enzymatic one. The results of this are shown in Fig. 4. The proof of acetyl donation by acetylcarnitine rests in the formation of acethydroxamic acid, which can be identified by the chromatographic method of Stadtman and Barker. When acetylcarnitine is incubated with CoA and hydroxylamine in the presence of the pigeon liver preparation for a 1-hr. period, treated to remove protein and salts, and then chromatographed, a spot with an Rf value of 0.50, corresponding to acethydroxamic acid is produced. Controls in which no enzyme or CoA are added form spots of very much smaller dimensions. This tion of acetyl derivatives
498
S. FRIEDMAN
AND
G.
FRAENKEL
reaction is due to the spontaneous formation of acethydroxamic acid, as mentioned above. When acetylcarnitine is split, two possibilities exist as to end products of the reaction. If the split were made at the ester linkage, the resulting products would be “acetyl” and carnitine, whereas if the split occurred at the a-P carbon linkage, the products would be “acetyl” and acetylcholine. In order to definitely eliminate the latter possiblity from consideration, although all of the evidence was against it, two methods were used. Since the chromatographic separation of acetylcholine and acetylcarnitine is feasible, reaction mixtures of the type heretofore used were incubated for 2 hr. at 37”C., and after removal of the protein and salt, were spotted on Whatman No. 1 paper. The chromatographic separation offered no rewards, in that no acetylcholine could be identified. In spite of the fact that the chromatograms were not completely satisfactory due to slight streaking, the only identifiable spot was acetylcarnitine. The second method used in the search for acetylcholine was the Neurospora choline assay based on the fact that a mutant strain of Neurospora m-ma requires for normal growth a small amount of choline in addition to its basal medium. If acetylcholine were formed
FIG. 3. Coenzyme A dependence of enzymatic from acetylcarnitine. Reation mixture contained: specified, cyzteine.HCl 10 &f, Na bicarbonate (neutralized to pH 6.8 with 14% NaOH) 100 PM, (NH&SOI fraction) 0.3 ml.
formation of hydroxamic acid acetylcarnitine 20 PM, CoA as 200 ai hydroxylamine.HCl pigeon liver extract (46-70%
ACETYLATION
0 TINE-
499
OF CARNITINE
60 MINUTES
I20
FIG. 4. Comparison of spontaneous and enzymatic formation of hydroxamic acid from acetylcarnitine. Reaction mixture contains: acetylcarnitine 20 a, cysteine.HCl 10 PM, CoA 20 units, hydroxylamine.HCl (neutralized to pH 6.8 with 14% NaOH) 100 pM, Na bicarbonate 200 &I. Pigeon liver extract (40-7095 (NH&S01 fraction) added for enzymatic formation (boiled, and added for measurement of spontaneous formation) 0.3 ml.
as a reaction product of the split of acetylcarnitine, treatment of the reaction mixture after incubation with a small amount of strong base would split the acetylcholine to choline and acetate, and the choline could then be determined. Since the Neurospora assay is sensitive to less than 5 pg., anyacetylcholineformed would be recognized. The results of these experiments were completely negative. DISCUSSION
The role of acetyl CoA as an intermediate in biological systems is so well documented that it is not necessary to mention it here. The number of compounds which are active in donating acetyl groups to coenzyme A are limited, however, by the fact that the energy level of this compound is too high to permit the usual type of low-energy group-transfer reactions. Acetylcarnitine is able to make this transfer, but the properties which put it into an energy state permitting it to do this have not yet been accounted for. One may assume that they are involved with bhe
500
S. FRIEDMAN AND G. FRAENKEL
nature of the ester linkage (a secondary ester) and its neighboring quaternary ammonium group. Up to this time, the enzyme which catalyzes the reaction between acetylcarnitine and coenzyme A has not been purified enough to determine a true equilibrium constant for the reaction. Once this is done, however, it may be possible to include a new class of compounds among those which are now known to play such an important role in group-transfer reactions. Acetylcarnitine has not yet been found in animals, but there are a few indirect supports for believing that it may have some function in metabolism. The first is the effectiveness of deoxycarnitine as a competitive inhibitor of carnitine activity in the mealworm, Tenebrio molitor (17). This compound, lacking a hydroxyl group on the P-carbon, is the only one of more than twenty analogs and derivatives tested which is known to have any effect on carnitine utilization. These results establish the importance of a substitutable group on the P-carbon. Acetylcarnitine is also known to be active in replacing carnitine in the diet of Tenebrio (17). The limited sensitivity of the assay method does not permit a definite statement as to its activity, but it is known to be just as active and possibly more so. It is, of course, possible that a deacylase in the gut of the insect may split the acetylated compound to a nonacetylated one. Investigations are at present being carried on in an effort to further characterize this acetylating system and elucidate its function. SUMMARY
of enzymatic acetylation of PAB by 1. An apparent inhibition carnitine has been found to be due to an enzyme which is active in the acetylation of carnitine. 2. This enzyme, which is found in both pigeon and sheep liver extracts, seems to catalyze the following reaction: 0-acetylcarnitine + CoA ti acetyl CoA + carnitine 3. The possible importance of secondary esters in group-transfer reactions is discussed. REFERENCES 1. GULEWITSCH,W., AND KRIMBERG, R., 2. physiol. Chem. 46, 326 (1905). 2. TOMITA, M., 2. physiol. Chem. 168, 42 (1926). 3. STRACK, E., AND FOSTERLINQ, K., Arch. exptl. Path& Pharmakol. 186, 612 (1937). 4. FRAENKEL, G., BLEWETT, M., AND COLES, M., Nature 181, 981 (1948). 5. CARTER, H.E., BHATTACBARYYA, P.K., WEIDMAN, K.R., ANDFRAENKEL,G., Arch Biochem. and Biophys. 38, 405 (1952).
ACETYLATION
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
OF
CARNITINE
501
KAPLAN, N. O., AND LIPMANN, F., .I. Biol. Chem. 174, 37 (1948). BRATTON, A. C., AND MABSHALL, E. K., JR., J. Biol. Chem. 129, 537 (1939). LIPMANN, F., AND TUTTLE, L. C., J. Biol. Chem. 161, 415 (1945). STADTMAN, E. R., AND BARKER, H. A., J. Biol. Chem. 184,769 (1950). HESTRIN, S., J. Biol. Chem. 180, 249 (1949). FRIEDMAN, S., MCFARLANE, J. E., BHATTACHARYYA, P. K., AND FRAENKEL, G., Arch. Biochem. and Biophys. 69, 484 (1955). BANISTER, J., WHITTAKER, V. P., AND WIJESUNDERA, S., J. PhysioZ. (London) 121, 55 (1953). WHITTAKER, V. P., AND WIJESUNDERA, S., Biochem. J. 61, 348 (1952). HOROWITZ, N. H., AND BEADLE, q. W., J. BioZ. Chem. 160, 325 (1943). STADTMAN, E. R., AND LIPMANN, F., J. BioZ. Chem. 186,549 (1950). STADTMAN, E. R., NOVELLI, G. D., AND LIPMANN, F., J. BioZ. Chem. 191, 365 (1951). BHATTACJURYYA, P. K., FRIEDMAN, S., AND FRAENKEL, G., Arch. Biochem. and Biophys. 64, 424 (1955).