Choline acetyltransferase and carnitine acetyltransferase in the placenta of the mouse

('omp Biot/;em t>hl~ml, 1977. I.I, 5{~(', lip 163 to 1(19 Pcrqltollm Ptl'3~ Printed m Gr~'ar Britai,l

CHOLINE ACETYLTRANSFERASE AND CARNITINE A C E T Y L T R A N S F E R A S E IN THE P L A C E N T A O F THE M O U S E FRANK WI!LSCH AND St:SAN K. McCARTIIY Department of Pharmacology, Michigan State University. East Lansing, MI 48824. U.S.A. (Receired 30 June 1976)

Abstract--l. The major l-~'~C-acetyl labelled metabolite as identified by high voltage electrophorcsis and thin layer chromatograph) was l-~'~C-acetylcarnitinc when crude or dialyzed mouse placenta homogenates were incubated in the absence or presence of exogenous I-carnitine respectively and with 1-~4C-acetylcoenzyme A. 2. Extensive dialysis removed >90",, of endogenous substrates and should be performed prior to an.,, measurements of acctylating cnz) mc activity. 3. Dial),zcd placenta homogenatc s)nthesized only small quantities of ~4C-acctylcholine, and the relationship of this product to choline acetyltransfcrase was equivocal. 4. Some kinetic characteristic.'s of carnitine acetyltransferase obtained by partial purification with ammonium sulfate were determined

INTRODUCTION

Acetylcholinc (A('h) is most intensely investigated as a transmitter substance in nervous tissue, but this ester also occurs in remarkable concentrations in the noninnervated human placenta (Rama Sastry et al., 19761 It has been proposed that ACh may play a role in controlling the permeability of those membranes which separate maternal from fetal blood stream (Bull et al., 1961; Koelle, 1967; Harbison et al.. 1975. 1976). For studies designed to elucidate the physiological signiticance of placental ACh an animal model would bc desirable. However, previous reports indicated that the strikingly high concentrations of ACh were unique for the human placenta. Correspondingly, activity of choline acetyltransferase (acetyl-CoA : choline O-acetyltransferase, ChAc, E.C.2.3.1.6), the enzyme responsible for the biosynthesis of ACh. correlated well with the high tissue content of ACh in human placenta but was absent in all species investigated except for some higher primates [Hebb & Ratkovic, 1962). Subsequently it was shown with a sensitive radioisotopic assay that placental homogenates from a variet~ of domestic and laboratory animals synthesized exceedingly small quantities of a product which behaved like authentic ~'*C-ACh (Wclsch. 19741. Elaborate isolation proccdurcs were necessary which precluded a simple quantitative determination of ChAt. Results obtained by White & Wu 11973) in the heart demonstrated that high activity of carnitinc acctyltransfcrasc (acetylCoA : carnitinc O-acctyltransferase. CarAc, E.C.2.3.1.7) could cause serious errors in the quantitative estimation of ChAt. and our previous observations from the guinea pig placenta suggested one m~uor cationic metabolitc unrelated to A C h (Wclsch, 1974) which might be acctylcarnitinc [AcCar). An existing microassay for ('hAc has recently bccn modilicd to overcome the drawbacks of nonspccificity of other radiochcmical methods which arc based on l-~'ff_'-acctylcocnzymc A (AcCoA) as an acetyl donor

for I-t4C-ACh synthesis (Fonnum. 1975). This method can distinguish ~4C-ACh from ~'LC-acctylcarnitinc (AcCar) because it uses a highly specitic liquid cation exchange reaction (Fonnum, 1969) to isohtte ~'~C-ACh as an ion pair with tctraphenylboron (TPB). The present communication reports the application of this new ChAc assay to the placenta of the mouse and evaluates its suitability to determine the enzyme activity quantitatively. In addition it was the purpose of the experiments to obtain more information about the nature of the major ~4C-labcllcd metabolitc whose synthesis was previously recognized (Wclsch, 1974).

MATERIALS

ANt)

METHODS

Animals

Placentas from Swiss Webster mice maintained in a breeding colony in our department were obtained through the courtesy of Dr. James E. Gibson. The mice were sacrificed during the last week of pregnancy. Preparation of placentas.lor analysis Placentas obtained were pooled from a single animal or on 2 occasions from 3-8 animals if subsequent partial purification of the enzyme was planned. The tissue was thoroughly washed in ice-cold 0.9~'~, Na(l. blotted and weighed. In a few cases the material was then homogenized (Polytron homogenizer, Brinkmann Instruments. Westbury, NY: two periods of 15 sec each at a setting of 5) in 10 mM ED-IA (pH 7.4) containing 0.5",, Triton X-It.X) (Fonnum. 1975) to yield homogenatcs of l:2 to 1:5 (w v). When dial)sis was to be performed placentas were homogcnized in 5 mM sodium phosphate buffer (p}t 7.4) which contained 0.1 mM EDTA and 1'~, butanol (Roskoski et al., 1974). A small Ix)rtion of such homogenates was retained ~hile most of the material was dialyzed for 24 hr at 4 'C against 3 changes of 200 volumes each of the dialysis buffer used by White & Wu (1973). Analytical procedures (at Determination of ChAt. The assay of ChAc was performed on extensively dialyzed homogenatcs with the

163

164

. FRANK WELSCH AND SUSAN K. M C C A R r t l Y

method described by Fonnum (1975) and on some occasions with the differential assay of Hamprecht & Amano (1974). The final conccntrations of thc components of the incubation mixture in a total volume of 1('/,) /A were: 300 mM NaCL 50 mM sodium phosphate buffer (pH 7.4), 20 mM E D T A (pH 7.4), 8 mM choline iodide, 0.1 mM physostigmine and 0.2 mM AcCoA. The latter was made up from 0. I ,uCi (220,000 dis/min) of I-~'~C-AcCoA (New England Nuclear, Boston, MA) and carrier AcCoA (Cat. No. 6200 P.L. Biochemicals, Milwaukee, WI). The purity of carrier AcCoA was checked by u.v. spectrophotometry in potassium phosphate buffer (pH 7.0). Absorption was measured at 232, 250. 260, 280 and 290 nm. The ratios referred to the 260 nm absorption were calculated and found to agree with the values stated by P.L. and New England Nuclear. The radiochemical purity of ~'~C-AcCoA was examined by high voltage ch:ctrophoresis (HVE. CAMAG, New Berlin, WI) in 0.015 M sodium citrateS3.04",] EDTA buffer (pH 4.0), conditions under which there was a single peak of radioactivity 11 cm anodal when 100 V/cm were applied for 30 min. The solutions required for the ChAc assay were made up such that a suitable amount of enzyme was present in 50 I~1, while 40 ]tl contained all other ingredients except for AcCoA. Ten ~1 of the latter were added to start the enzyme reaction in prewarmed 6 x 50 mm disposable culture tubes at 37'C for incubation times ranging from 2 to 120 min. Following the flushing-out of the microtube contents and addition of extraction and scintillation tluids (for details see Fonnum, 1975), the scintillation vials were put into a horizontal position and gently shaken for 3{.)sec on a reciprocating shaker to accomplish liquid cation exchange of ~4C-ACh synthesized into the upper organic layer (with or without TPB). This mode of shaking had no deleterious effects on the blanks containing boiled tissue (about 800 dis,.' min = 0.3'~,i). All samples were assayed in duplicate. Radioactivity was determined in a model 3380 liquid scintillation spectrometer (Packard Instruments} equipped with a model 544 absolute activity analyzer for automatic external standardization. (b) Determination of CarAc. This enzyme was assayed in a final volume of 100 l~1 with minor modifications of the anion exchange procedure (Schrier & Shuster, 19671 applying the precautions previously found to be necessary (White & Wu, 1973: Welsch, 1974). Undialyzed and dialyzed homogenatcs and a CarAt which was partially purified according to White & Wu (1973) were examined with the method described in that report. Boiled enzyme was used for blank values. During the course of these experiments it became apparent that the concentrations of l-carnitine and AcCoA had to bc raised in order to achieve saturation of CarAc from mouse placenta. Protein was determined with the Folin phenol reagent (Lowry et al., 1951). (c) ldentication cq" radiolabelled metabolites. Radioactive products isolated with either the liquid cation exchange method or by anion exchange column chromatography were examined by HVE on 2.5 cm wide strips of Whatman No. 1 paper with formic acid-acctic acid buffer (Potter & Murphy, 1967). Authentic ACh and d.l-AcCar (Sigma, St. Louis, MOI were electrophoresed on adjacent tracks and migrated 26 27 cm and 19 20 cm respectively when 100 V/cm were applied for 30 min. Authentic markers (20 ug) were localized by iodine vapor staining, while strips containing radioactivity wcrc passed through a radiochromatogram scanner (Packard Instruments) or cut into 1 cm sections for liquid scintillation counting in toluene scintillator, lodinc staining consistently produced much narrower bands than determination of radioactivity revealed. This is a phenomenon common to paper chromatography and HVE (McCarty ct al., 1973). Descending paper chromatography was performed according to Markwcll et aL (1973) and thin layer chromatography (TLC) on silica gel

plates (Silica Gel 60. No. 5763, Brinkmann, Westbury. NYI with methanol:acetone: HCI = 90: 10: ltl v,v) as the developing solvent (Hosein ct aL. 19701. Radioactivity, on "I-LC plates was localized by scraping one cm bands of plate coating into counting vials to which 2 ml of water and 10 ml of tritosol scintillation fluid {Fricke. 19751 were added RESt,LTS 1. Determination o[" C h A c As in the case of heart muscle (White & Wu, 1973; Roskoski et al.. 1974) dialysis of mouse placenta homogenates proved to be quite etticicnt in removing endogenous substrates. The dialyzable tissue constituents wcrc probably responsible for the large background reactions which occurred when only I~'C-AcCoA was provided (Wclsch, 1974). In the current experiments dialysis removed more than 9()'~., of the endogenous factors responsible for the formation of l-a'*C-acetyl labelled cationic products in the absence of a second substrate (Table 1). This obserwttion revealed that extensive dialysis was necessary as a first step in any attempt to determine ChAc in thc mouse placenta. In addition, dialysis appeared to remove some inhibitory factor from the homogcnate. because C a r A t determined on dialyzed tissue was higher than the enzyme activity of undialyzcd preparations (scc below). Because only small amounts of I-~'~C'-ACh were expcctcd to be synthesized, the concentration of dialyzed homogenate was raised to 25 mg,100 itl of incubation volume. Tubes wcrc incubated for time intervals of up to 1 hr and analyzed with the liquid cation exchange method (Tablc 2). Only radioactivity extractable when TPB was present was attributed to the formation of a specific ion pair between ~4C-ACh and TPB. Thcse samples reached barely 1.5 2 times the disintegrations of the blank values. Although there was a modest rise with increasing incubation time, this gain was not linearly related to time. In order to assure the reliability of Fotmum's assay method, total rat brain, rat brain corpus striatum and human term placenta homogcnatcs were examined as representative tissues with high ChAc. Placentas from dog, rabbit, rat or human chorion and amnion were assayed as examples of tissues with low ChAc activity. Table 1. Effects of dialysis on the incorporation of l-~'~C-acetyl groups into cationic products Experiment No. 1 No. 2 Undialyzed Dialyzed

10786 1446 (7.3".)

18695 773 (4. I '~i,}

Mouse placenta homogenates were kept in a coldroom either undialyzed or dial)zed as describ~ in Methods. Both preparations were then incubated (No. I = 6 mg tissue, 15 min; No. 2 = 10 mg, 10 rain) in an incubation medium containing all components including 2 mM I-~'*('-AcCoA (-~4tX).00(I dis.rain) except for choline or l-carnitinc. Duplicate samples were analyzed by anion exchange chromatography, and values shov, n arc averages of disintegrations:rain.

165

Choline acetyltransferase and carnitine acetyltransfcrase Table 2. Incorporation of l-a4C-acetyl groups into tetraphcnylboron extractable material Incubation Time (min) Blank 15 30 ~)

Extraction Method Acetonitrile Acetonitrile + TPB 771 2042 2322 2259

Net ACh dis:min

nmolcs ACh synthesized per g xh-

1(134 1350 1535

16.32 9.80 5.62

850 3176 3672 3794

* Radioactivity believed to be attributable to t'~C-ACh after deduction of the acctonitrile contribt,tions. Dialyzed mouse placenta homogenatc (25 mg) was incubated with the method of Fonnum (1975) except for that 0.1 pCi (~- 220.000 dis/min) ~'~C-AcCoA was provided in each tubc. Samples wcrc extracted with acetonitrile only or with acctonitrile containing 5 mg tctraphen31boron (TPB) per ml. Blanks consisted of boiled tissue. Values are expressed as disintegrations per min Idis/min) and represent mean valu~.~ of duplicate determinations from a representative experiment. G o o d agreement was found with reported values for the high ChAc tissues [/~moles Ach synthesized/g fresh tissue x h ~ : total brain = 8.1; corpus striatum 33.0; human placenta = 7.0). In regard to rat, rabbit and dog placenta the same reservations concerning a quantitation of the data as expressed in Table 2 for the mouse placenta were indicated. To further ensure the reliability of thc liquid cation exchange assay other samples were examined with the differential assay recommended by Hamprccht & Amano (1974). In this procedure two parallel sets of tubes were incubated either with the complete ChAc assay medium or with a medium were physostigminc was omitted and replaced by acetylcholinesterase (acetylcholine acetylhydrolase, ACHE, E.C. 3.1.1.7)). Any ACh synthesized would be immediately hydrolyzed by ACHE, and the differences in radioactivity eluted from an anion exchange column may be considered to be due to laC-ACh formation. This approach was not more successful for quantitative determinations in placentas with low rates of ACh synthesis than the ion pair extraction. Qualitative verification of the presence of ~'~C-ACh in the TPB extractable radioactivity from mouse placenta was achieved by displacing ~4C-ACh from TPB by means of dilute HCI (Fonnum, 1969). Following lyophilization of the acid the residue was dissolved in a small volume of elcctrophoresis buffer and analyzed by HVE. A single peak corresponding in its electrophoretic mobility to authentic I-'4C-ACh was found when the paper strip was scanned for radioactivity. Considering the well established high specificity of TPB for ion pair formation with ACh (Fonnum, 1969: Goldberg and McCaman, 1973) and our previous differentiation and identification (Welsch, 1974) this was bclicvcd to provide sufficient evidence for the conclusion that some ~4C-ACh had been synthesized by mouse placentas.

2. Determination oJ CarAc As little as 250 ug of crude or dialyzed mouse placenta homogenate in the presence of both AcCoA and 1-carnitine catalyzed the synthesis of easily measurable amounts of ~4C-acetyl labelled material which did not bind to the anion exchange resin. The values obtained from dialyzed samples were higher than when the crude homogenate was used (Fig. 1) suggesting that dialysis had removed an unidentified inhibitory factor. Omission of 1-carnitine from the medium

resulted in values equal to boiled blanks which wcrc carried through the entire procedure. Althot,gh the rate of product formation was linearly related to tissue concentration and to incubation time up to 10 min as judged from graphical plots of disintegrations vs incubation time. the slope of the line did not pass through the zero point (Fig. I). A reaction which appeared to be non-enzymatic may have contributed to this phenomenon. When all components were mixed and the tube immediately chilled and diluted with 500 ILl ice-cold water, an amount of radioactivity could nevertheless be eluted from the ion exchange columns which was about equal to the y-intercept determined by graphical means. Therelbre. it appeared that it was justified to deduct this value from each time point prior to calculating the amount of product synthesized. During the linear phase the rate of product formation ranged from 26.0 to 65.0 umoles of ~'*C-metabolite synthesized/g of dial2,zcd 4

3

% ,IN

E 2

D, 'll

1

,

,

,

,

|

,

,

8 Incubation

timel

,

,

|

,

10 rain

Fig. I. Formation of 14C-acetylated material by dialyzed ( e - - - e ) and undialyzed ( & - - & ) mouse placcnta homo-

genatc. Homogenate (0.25 mg) was incubated with 2 mM 14C-AcCoA ((I.2 ,uCi) and 10 mM l-carnitine for the times indicated in the abscissa. The ordinate shows the net disintegrations per min after deduction of the column blank ( -~600 dis.:min).

166

FRANK WELSCH AND SUSAN K . M C C A R T H Y

.I

""-'*°"liiiil 20 pg

100-

~

-I

20 pg

.

0

~

50-

0

&

o.~'~ Start

5

,;)-

;s

2;

cm

.

3o

Fig. 2. Localization of radioactivity on an eleetropherogram by paper strip scanning. The material analyzed derived from an acid ethanol extract of pooled anion exchange column effluents which was subjected to high voltage electrophoresis. Full scale pen deflection in the vertical direction represents 1000 cpm with about 20'.,/ocounting efficiency for ~'*C. The upper part of the figure shows the localization of the authentic standards d,l-AcCar and ACh as revealed by iodine vapor staining. mouse placenta xh -~ (three determinations performed on different homogenates of pooled placentas; 288--722 nmoles/mg protein xh-~). The identification of the ~'*C-labelled material which appeared to be a product of enzymatic synthesis and quite likely AcCar was pursued by pooling the anion exchange column eluates from several columns. The effluent was lyophilized and extracted with a small volume of 95~o ethanol containing 0.2°J~, glacial acetic acid. The soluble material was first analyzed by high voltage electrophoresis and three radioactive areas were found on the electropherogram (Fig. 2). Authentic standards were spotted with the addition of reconstituted column effluent because initial experience indicated that unidentified constituents of this material could retard the eleetrophoretic mobility when compared to standards alone. Two radioactive regions on the electropherogram, 1 2 cm cathodal and 25 26 cm cathodal, were minor while the overriding peak of radioactivity had mobility identical to authentic d,l-AcCar at 19.5 cm. The migration of the smallest amount of radioactivity at 26 cm corresponded to ACh. The region believed to represent ~4C-AcCar was eluted fi'om the paper and analyzed by three different procedures, i.e. descending paper chromatography, reelectrophoresis under similar conditions for 20 rain and TLC. The least ,satisfactory separation was obtained with paper chromatography because radioactivity, although peaking with an R r = 0.4 identical to authentic AcCar, was spread out over 9 cm. The electrophoretic mobility of a single area of radioactivity corresponded to authentic AcCar (Fig. 3A). Comparable results were obtained on the TEC plate were the R r = 0.45 of AcCar coincided with the highest content of radioactivity synthesized by the mouse placenta (Fig. 3B). All these observations strengthened the conclusion that the major acetylated ~4C-labelled metabolite was indeed ~4C-AcCar. Some prvperties o] partially purified CarAe. The substratc concentrations required for saturation of the placental enzyme arc indicatcd in Table 3. The enzyme appeared to be saturated by 1-carnitine conccntrations of about 10 mM, and higher concen-

trations resulted in substrate inhibition of AcCar synthesis. The apparent average K,,, value of the two enzyme batches with respect to l-carnitine determined graphically from Lineweaver Burk plots was 0.78 mM at a fixed concentration of 2 mM I-AcCoA and variable amounts of l-carnitine. The apparent Km value with regard to the former substrate was 0.6 mM. This value has to be considered as only approximate since no attempt was made to correct for the possibility of AcCoA being subjcct to other reactions such as deacvlation. Because of cost considerations concerning ~gC-AcCoA all determinations were rouA

B .

"v'i!iiiil

v.;.,°°.,

d,I-AcCar

-"

,o4.

--0

TLC

-~

E

----

--

2

,o: - . , , .

15

I--

18

cfft

. . . .

21

--,,: . . . . . 6

cnl

8

7

10

Fig. 3. Analysis of radioactivit~ in prepurified extract. The major peak of radioactive material corresponding in electrophoretic mobility to authentic AcCar as localized by paper strip scanning was eluted from the paper. Panel A: Repeat of high voltage electrophoresis (HVE) and distribution of ~4C as revealed by liquid scintillation counting of I cm paper sections. Panel B: Localization of 14C on thin layer chromatography (TLCI plates in one cm wide scrapings of plate coating. The upper part of the figure in both panels shows the appearance of an iodine vapor stained paper strip (HVE) or plate area (TL(') showing the localization of the authentic standard d,l-AcCar.

Choline acetyltransferase and carnitine acetyltransferase Table 3. Effect of substrate concentrations on mouse placenta camitine acetyltransferase activity ~4C-Acetyl CoA mM Activity 0.025 0.05 0.1 0.125 0.2 0.5 1.0 2.0

250.1 400.5 687.5 883.9 1089.9 2223.3 3322.3 4248.9

mM

I-Carnitine Activity

0.5 1.0 2.0 5.0 10.0 20.0

2383.0 3409.2 4487.5 5245.0 5475.0 4600.0

Enzyme activities (nmolcs/mg protein xh -t) are the average of duplicatc determinations performed in two separate assays. Tubes containing partially purified CarAc (6.5 pg protein) were incubated for 10 min at fixed l-carnitine (10 mM)or s'*C-AcCoA (2 mMI concentrations while the second substrate was varicd as indicated, t4C-AcCar was isolated by anion exchange chromatography. tinely performed with 2 mM AcCoA (2000 dis/min/ nmolcl, a concentration 3.33 times the apparent K,,,. Rates of AcCar synthesis (Vm,0 as determined from double reciprocal plots differed among the two batches of partially purified enzyme. Batch No. 1 had a value of 74.5 _+ 9.7 nmoles/mg protein min -t (2 + S.D. 4 separate determinations) while batch No. 2 synthesized 18.1 _+ 2.4 (2 + S.D., 6 determinations), although the apparent K,, values agreed closely. The difference may be due to the method of enzyme preparation. For the second purification placentas from mice were obtained on successive days over a period of 2--3 weeks. Pooled placentas from each animal were homogenized and stored in frozen condition ( - 2 0 " C ) until the day when all homogenates were combined for dialysis treatment and purification. Although CarAc appeared to be stable in frozen crude homogenates for the period under consideration, it could not be ruled out that the storage might have affected the yield of enzyme in the ammonium sulfate precipitation procedure. Batch No. 1 was prepared immediately after collecting the placentas. Some of the kinetic properties of the mouse placenta enzyme were quite comparable to a similar preparation from rabbit heart where the specific activity of CarAc was 68 nmoles/mg protein × m i n (Whitc & Wu, 1973). The K,, value with respect to carnitine was 2.6 mM in brain homogenates (d,l-carnitine; McCarman et al., 1966) and 0.31 mM in partially purified heart extract (l-earnitine, Fritz & Schultz, 1965). Half saturation of the mouse placenta enzyme occurred at a concentration of l-carnitine of 0.78 raM. For comparison with a better defined enzyme preparation, the K,, for l-carnitine was determined using commercially distributed highly purified CarAc from pigeon breast muscle for which a K,, value of 0.4 mM was reported (White & Wu, 1973). In our hands the value was found to be 0.8 mM. The discrepancy may be due in part to the fact that the l-carnitine was not anhydrous yet was used without recrystallization. With respect to AcCoA the K,, = 0.6 mM was higher than that of rather crude brain (McCaman et al., 1965) and liver or kidney enzyme (Markwell et al., 1973). Quality control of both the carrier and the labelled AcCoA revealed con-

167

formity with established criteria (see Methods) but deacylases were not ruled out which may have reduced the available AcCoA concentration. In concentrations up to 10 mM authentic dA-AcCar had no inhibitory effect on CarAc. Another point of interest was to examine the substrate specificity of the partially purified CarAc from mouse placenta. This was of concern in view of the observations that choline ean serve as a low attinity substrate for CarAc leading to the formation of ~'*C-ACh by CarAc from choline and ~4C-AcCoA (White & Wu, 1973; Roskoski et al., 1974). Therefore. 10 mM choline iodide was added simultaneously with variable concentrations of l-carnitine to tubes which contained the complete incubation medium with 2 mM AcCoA. Choline was a weak competitive inhibitor of carnitine acetylation to AcCar (Fig. 4), as revealed by the consistently lower disintegrations in the samples containing choline. This implied that choline competed with l-carnitine for access sites, to CarAc. In order to obtain more specific information about the possibility that CarAc caused the acetylation of choline when dialyzed homogenate was incubated with choline, bromoacetylcholine (100 pM) or naphthylvinylpyridine (50 itM) was added to the incubation mixture. In the concentrations which were examined both compounds arc well known for their ability to inhibit ChAc activity (White & Wu, 1973; Welsch. 1974b; Roskoski et al., 1974). There was no significant effect on the radioactivity which was extracted into the organic phase containing TPB. DISCUSSION

The results of the present study revealed remarkable differences of mouse placenta homogenates in the ability to acetylate either choline or carnitine. Thus, prior to any attempt to measure either ChAc or CarAc activity, it would be essential to dialyze the homogenates extensively before using them for biochemical analyses of acctylating enzyme activity. ,112

111

1 [S'~ • 1°-4

Fig. 4. Effects of choline on acetylation of l-carnitine by I-t4C-AcCoA and CarAc from mouse placenta. Tubes containing partially purified CarAc (17.25 #g protein) were incubated for 10 min with t4C-AcCoA (2 mM), choline (10 mM) and variable concentrations of l-carnitine. ~4C-AcCar was isolated by anion exchange chromatography (0-----@: control; A---- A: 10 mM choline present).

168

FRANK WELSCtt AND SUSAN K. MCCARTHY

These findings confirm for the placenta the observations reported in heart muscle (White & Wu, 1973; Roskoski et al.. 1974). l-rom thc analysis of the labclled products formed by dialyzed placenta homogenate it appeared that small quantities of ~'*C-ACh were present suggesting ChAc activity. However, it would be difficult to quantify the data tTablc 1) because of lack of linearity of thc ~'~C-ACh synthcsizing reaction with respect to incubation timc. This poor correlation was probably not duc to an inadequacy of the extraction method. The ion pair extraction with TPB could extract ACh in the low picomolc range (Goldberg & McCaman, 1973) and appeared reliablc over a wide range of ACh concentrations (Fonnum. 1969). If one would use the valuc of 16 nmolcs ~4C-ACh synthesized/g tissue xh-~ (Tablc 1) and compared this to the quantity of ~'~C-AcCar synthesized (26 72 pmoles,"g xh-~), the molar ratio was at least 1:1(1/t3, a figure which is in the same ordcr of magnitude as that in the guinea pig heart (Roskoski et al.. 1974). This widc disparity explained the need for dialysis, without which thc overriding CarAt reaction, supplied with substrate by the presence of enough endogcnous carnitine, will deplctc the AcCoA by synthcsis of AcCar. Dialysis prior to ('arAc assays appcared to removc some unidentified inhibitory factor (Fig. 1), making this pretreatmcnt equally desirable. Also for any kinctic analyses dialysis would bc rccommendable in order to remove the apparcntly htrge quantities of endogenous carnitint. It could not be dccided with certainty whether the ~*C-ACh formed in vitro was synthesized by CarAc or ChAc bccause the effects of classical ChAc inhibiting drugs on ~4C-acetyl transfer ,,,,'ere not significant. However, in rivo determinations of ACh itself have qualitatively shown thc presence of small amounts of a conapound which eluted during pyrolysis gas chromatography with the same retention time as demethylatcd authentic ACh (Welsch, unpublishcd observations: Stevens et al., 1976; Harbison et al., 1976). The latter group of investigators also reported a gestational period depcndcnt activity of ChAc in the mouse placenta which synthesized bctwccn 50 and 70 nmolcs of ACh.g tissue xh-~, but details of the method wcrc not provided. Therefore, it is difficult to compare our results with those observations publishcd in abstract form and to decide whcthcr the product dcscribcd was indeed ACE In the present study the mousc placenta appeared to be quitc different as regards the cholinergic system because of the very low ChAc activity, which appeared to correlate well with the equally low endogcnous ACh content. This was in contrast to the placenta of highcr primates such as the rhesus monkey where the placental cholincrgic system is almost as abundant (Welsch, manuscript in prcparation) as in human placcnta (Rama Sastry et al.. 1976; Welsch, 1974b). It is difficult to explain thc functional significance of these striking species diffcrcnces in the concentrations of the cholincrgic system because the physiological role of ACh in the placcnta is poorly understood. CarAc is an enzyme which has bccn implicated in mitochondrial transport processes related to fatty acid metabolism. This enzymc may have a special role in thc transfer of acetyl groups across mitochondrial mem-

brane barriers (McCaman et al.. 1965; Markwcll et al., 1973).

In a pilot experiment we examined a dialyzed human term placenta homogcnate and found CarAc to bc about the .same as ChAc activity (5 10 ltmoles of either product synthesizcd/g fresh tissuc xh depending on which of the two substrates was supplied). This result might explain why thc ion exchange method (Schrier & Shuster, 1967) could bc applied to crude human term placenta homogenates to measure ChAc yet failed with other plaecntas, including the mouse placenta, where large discrepancies existed between an ovcrriding activity of CarAc and low or questionable ChAc activity. In the absence of choline human term placenta synthesized only about 5')(, of the cationic materials produced in the presence of cholinc while the mouse values were indistinguishable (Welsch, 1974). The present rcsults underlinc the difficulties encountered in the use of a common laboratory animal placenta for studies of thc cholinergic system and its role in placental function. Acknowledgements -We thank Dr. Loran Bieber, Department of Biochemistry at Michigan State University. for his generous supply of I-carnitinc and his gift of pigeon breast muscle CarAc (Sigma). This study was supported by the U.S. Public Health Service, NIH grant HD-07091. and in part by grant 1-444 from The National Foundation-March of Dimes.

REFERENCES Bt:LI. G., HEaB C. O. & R ATKOVt("D. (1961) Choline acctylase in the human placenta at different stages of development. Nature, Lond. 190, 1202. FOb,'~L'M F. (1975) A rapid radiochemical method for the determination of choline acetyltransfcrasc. J. Neurochem. 24, 407-409. FON~t;M F. (1969) Isolation of choline esters from aqueous solutions by extraction with sodium tetraphenyllx~ron in organic solvents. Biochem. d. 113, 291-298. FRICKE U. (1975) Tritosol: A new scintillation cocktail based on Triton X-I(KI. Anal. Biochcm. 63, 555--558. Fatrz I. B. & SCHL'L'rZS. (1965) Carnitine acetyltransfcrase. II. Inhibition by carnitine analogues and by sulfhydryl reagents. J. biol. Chem. 240, 2188-2192. GOLDBERGA. M. & MCCAMANR. E. (1973) The determination of picomole amounts of acctylcholinc in mammalian brain. J. Neurochem. 20, 1 8. HAMPnF!c.r B. & AMA~O T. (1974) Diffcrential assay for choline acetyltransferasc. Anal Bioehem. 57. 162-172. HJ:BB C. O. & RA'I'KOVlCD. (1962) Choline acctylasc in the placenta of man and other species..I. Physiol.. L,md. 163, 307 313. HARBISON R. D.. OLUI~ADEWOT., DWIVt~DI C. & RAMA SAS'rRV B. V. (1975) Proposed role of the placcntal cholinergic system in the regulation of fetal growth and development. In Basic and Thcrapeutic Aspects qf Perinatal Pharmacology (Edited by MORSELLI P. L.. GARA'r-I'X~IS. & SI-:RENIF.) pp. 107-120. Raven Prcss, Ncw York. HARatSON R. D.. SrEw~s M. W., DwlvEtn C. & F'A~T M. (1976) Regulation of fetal growth by a placcntal cholinergic system. Fedn Proc. I"cdn Am. Soes exp. Biol. 35, 611, Abstr. 2234. Hos~!tn E. A., KA'rO A., VL~: E. & HU.L A. H. (1970) The identification of acetyl-l-carnitylcholine in rat brain extracts and the comparison of its cholinomimctic properties with acetylcholine. Can. J. physiol. PharmacoL 48, 709-722. KOrLLt~ G. B. (1967) Physiological functions of acctylcholine. Neurosei. Res. Prof4r. Bull. 5, 44.

Choline acetyltransfcrase and carnitine acetyltransferase LOWRY O. H., ROSliBROUGI! N. J., FARR A. L. & RANDALL R. J. (1951)Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MCCAMAN R. E.. MCCAMAN M. W. & STAFFORD M. L. (1966) Carnitine acetyltransferase in nervous tissue. J. biol. Chem. 241,930-934. MCCART¥ L. P.. K>qGHr A. S. & ('HENOWt~TH M. B. (1973) Incorporation of t'tC-choline into phospholipids in the isolated phrenic nerve diaphragm preparation of the rat. J. Neurochem. 20, 487-494. MARKWI:LL M. A.. McGROARrV E. J., BH!m!R L. & TOEmiRr N. E (1973)The subeellular distribution of carnitine acetyltransfcrase in mammalian liver and kidney. J. biol. ('hem. 248, 3426 3432. Pox [ER 1.. T. & Mt'RPW,' W. (1967) Electrophoresis of acetylcholinc, choline and related compot, nds. Biochem. Pharmacol. 16, 1386 1388. RAMA SASI'RY B. V., OLt;I~tADI!WO J., HARBISON R. D. & SCHMH)I D. E. [1976} Human placental cholinergic system: Occurrence, distribution and variation with gestational age of accctylcholine in human placenta. Biochem. Pharmacol. 25, 425-2,31.

169

ROSKOSKI R., JR., MAYER H. F.. & SCttMID P. G. (1974) choline acetyltransferase activity in guinea-pig heart #1 vitro. J. Neurochem. 23. 1197-1200. SCHRIeR B. K. & SHt:SIeR L. (1967) A simplified radiochemical assay for choline acetyltransferase. J. Neurochem. 14, 977-985. STEVENS M. W.. DWlVEDJ C. & HARBISON R. D. (1976) Studies on the placental cholinergic system in rodents. Ahstr. 15th AmL Meet..4m. Soc. To x'icol, p. 69, Abstr. 86. WELSCH F. (1974a)Choline acetyltransferase in aneural tissue: evidence for the presence of the enzyme in the placenta of the guinea pig and other species. Am. J. Obstet. Gynecol. 118, 849-856. WI-:t,SCH F. (1974b) Choline acetyltransferase of human placenta during the first trimester of pregnancy. Experientia 30, 162 163. WrnrE H. L. & Wu J. C. (1973) Choline and carnitine acetyltransferase of heart. Biochem. 12, 841--846.