Radioassay of the stereospecificity of 2-monoacylglycerol acyltransferase

ANALYTICAL

BIOCHEMISTRY

119, 4-11 (1982)

Radioassay of the Stereospecificity 2-Monoacylglycerol Acyltransferase F. MANGANARO, Department

A. KUKSIS,’

of Biochemistry, and Banting and Best Department University of Toronto, Toronto, Ontario

of

AND J. J. MYHER of Medical MSG IL6,

Research Canada

(112 ColIege

Street),

Received May 13, 1981 The 2-monoacylglycerol acyltransferase (EC 2.3.1.22, acylglycerol palmitoyl transferase) catalyzes the synthesis of X-l,2-diacylglycerols from 2-monoacylglycerol and acyl CoA with an apparently variable stereochemical specificity. A microassay for determining the ratio of m-1,2- and sn-2,3-diacylglycerols formed by the acylation of radioactive 2-monoacylglycerol in intact cells or in cell-free systems in the presence of free fatty acids and cofactors has been developed. The diacylglycerols are isolated by thin-layer chromatography using nonradioactive racemic diacylglycerols as carriers. The enantiomer content is determined following a chemical synthesis of X- 1,2-diacyiphosphatidylcholines and a stereospecific stepwise release of the sn1,2- and sn-2,3-diacylglycerols by phospholipase C. By using thin-layer chromatography for the isolation of the hydrolysis products, known samples ranging in enantiomer ratios from 0.05 to 20 and containing 5000 to 200,000 cpm can be assayed to within 1% of the major and within 10% of the minor enatiomer content. The method is applicable to the determination of the enantiomer content of X-1,2-diacylglycerols generated via other acyltransferases and via lipolysis of triacylglycerols and diacylglycerolphospholipids in other biological systems.

The stereospecific assay of X- 1,2-diacylglycerols via the corresponding phosphatidylphenols and phospholipase A2 is a wellestablished experimental routine ( l-4), and it has been extensively utilized in the determination of the positional distribution of fatty acids in natural triacylglycerols (4). Likewise, this method has been widely applied in the analysis of natural mixtures of diacylglycerols (5,6), including those arising specifically from the action of lipoprotein lipase (7,8), lingual lipase (9,10), and he-

ture is based on a preferential destruction of the sn-1,2- and a complete enzymatic inertness of the sn-2,3- enantiomers. This difficulty is overcome in the stereospecific analysis proposed by Myher and Kuksis ( 14). This technique is based on an intermediate synthesis of X-l ,2-phosphatidylcholines, which are then stepwise degraded with phospholipase C. This enzyme initially releases the sn1,2-diacylglycerols and subsequently the sn2,3-diacylglycerols, which can be separately isolated and assessed for the composition and molecular association of the fatty acids by a variety of methods. The latter technique has thus far been applied only to stereospecific analyses of triacylglycerol structure (14-16). In the present communication, we describe a further utilization of the above principle in a stereochemical radioassay of the diacylglycerol products of sn-2-monoacylglycerol acyltransferase (EC 2.3.1.22, acyl-

patic lipase ( 11) on natural triacylglycerols, and from that of the monoacylglycerol acyl-

transferase on 2-monoacylglycerol ( 12,13). A major drawback to this technique is the impossibility of recovering the pure enantiomeric diacylglycerols for an analysis of the fatty-acid composition or molecular association, because the resolution of the mix’ To whom correspondence should be addressed. 0003-2697/82/010004-08$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

4

2-MONOACYLGLYCEROL

glycerol palmitoyl transferase) in intact mucosal cells and in cell-free fractions prepared from them, An abstract has appeared (17). MATERIALS

AND METHODS

All chemicals and solvents were of reagent grade or better quality and were supplied by Fisher Scientific Company, Toronto, Canada. Phospholipase C, phospholipase AZ, and trioleoylglycerol (99%) were supplied by Sigma Chemical Company, St. Louis, Missouri. [ 2-3H]Glycerol trioleate ( 1-2 Ci/ mmol) was obtained from ICN Chemicals and Radioisotopes (Montreal, Quebec Canada), while dipalmitoylphosphatidyl-[N-Me“C]choline (50-200 mCi/mmol) was purchased from NEN Canada (Lachine, Quebec, Canada). Nonradioactive and radioactive sn-glycerol-2-oleate and rac-glycerol1,Zdioleate were prepared from the corresponding glycerol trioleates by digestion with pancreatic lipase, as previously described ( 1). The sn-2-glycerol monooleate and the sn- 1,2(2,3)-glycerol dioleates were isolated by thin-layer chromatography using boric acid-impregnated silica gel (18). The acylglycerols were recovered by elution with chloroform:methanol (2: 1). The sn- 1,2-diacylglycerol component was removed by brief hydrolysis with phospholipase C after conversion of the rat- 1,2-diacylglycerols to the corresponding phosphatidylcholines, as described by Myher and Kuksis (14). Mixed acid sn- 1,2-diacylglycerol-3-phosphorylcholine was isolated from egg yolk (19). Alternatively, rat- 1,Zdioleoylglycerols were generated from the trioleoylglycerol by Grignard degradation (2) and the enantiomers were recovered and purified as above. Source of acyitransferase. Both intact villus cells and villus cell homogenates served as sources of the monoacylglycerol acyltransferase activity. The villus cells were prepared by the method of Hoffman and Kuksis (20) from the jejunal mucosa scrapings of male Wistar rats (250-350 g) by digestion with hyaluronidase. The cell ho-

ACYLTRANSFERASE

ASSAY

5

mogenates and sonicates were prepared as described by Manganaro and Kuksis (21). The purified enzyme was obtained by sucrose-gradient centrifugation (22). Biosynthesis of X-1,2-diacylglycerols. Radioactive X- 1,2-diacylglycerols were biosynthesized by incubating sn-Zoleoyl[3H]glycerol and free fatty-acid micelles with either intact villus cells or appropriate subcellular fractions fortified with ATP and CoA, as previously described (21). For optimum analysis, the incubations were conducted with substrates of 100,000 to 200,000 counts. The reaction products were isolated by preparing a total lipid extract with chloroform:methanol(2:1) (23). Along with the chloroform phase were added 0.25 mg of rat-1,2-dioleoylglycerol per lo6 cells or an equivalent amount of homogenate or sonicate. The X- 1,2-diacylglycerols were isolated by thin-layer chromatography with boric acid-impregnated plates as described above. Outline of stereospecific radioassay. An aliquot of the radioactive diacylglycerols along with the nonradioactive rat- 1,2-diacylglycerol carrier (0.75 mg) was converted into rat-1,2-diacylphosphatidylcholine as described by Myher and Kuksis (14). The phosphatidylcholines were isolated and purified by thin-layer chromatography using silica gel H and choloroform:methanol:acetic acid:water (7545: 12:6) as the developing solvent (24). The phosphatidylcholines were recovered by extraction of the gel with chloroform:methanol:water:acetic acid (50:39:10:1) (25). Th e extracts were partitioned with 4 M ammonia and dried as previously described (14). The recovered radioactive X- 1,2-diacylphosphatidylcholines were then hydrolyzed at 32°C in two steps with phospholipase C. The sn- 1,2-diacylglyceroh released by a 2-min digestion were isolated by thin-layer chromatography on the 5% borate-treated silica gel using chloroform: acetone (94:6) as the solvent (18). The residual sn-2,3-diacylphosphatidylcholines were recovered from the origin of the plate and were subjected to an additional 4-h

6

MANGANARO,

KUKSIS,

digestion with phospholipase C. The released sn-2,3-diacylglycerols were isolated as above. At each isolation step, an aliquot of the solution was used for counting and the rest of the material processed further. The stereospecific analysis of the acylation products of the sn-2-oleoylglycerol was completed by a preparation of the tert-butyl dimethylsilyl ethers, which were resolved according to the degree of unsaturation of AgNOj-thin-layer chromatography using 1% methanol in chloroform as the developing solvent ( 16). This routine allowed the identification of all major diacylglycerol species of both enantiomers and a more precise assessment of the products of the acyltransferase activity. A parallel stereospecific assay of the X- 1,2-diacylphosphatidylcholines with phospholipase A2 was carried out as described by Myher and Kuksis ( 14). Calculations. Molar proportions of the sn1,2- and sn-2,3-diacylglycerols were calculated from the ratio of the radioactivity in the glycerol moiety of the appropriate reaction products. Where necessary, corrections were made for the incompleteness of digestion with phospholipase C on the basis of the extent of hydrolysis of the radioactive internal standard (sn-1,2-dipalmitoylphosphatidyl[Me-14C]choline). The corrected sn1,2-/sn-2,3-enantiomer ratio was calculated to be equal to ( 1OOa + bX):( 1OOb - bX), where a and b are the uncorrected relative values of the sn-1,2- and sn-2,3-enantiomers and X is the percentage of the undigested standard. RESULTS Generation

of X-l, 2- Diacylglycerols

In Table 1 are given the yields of radioactive X- 1,2-diacylglycerols obtained by incubating 2-monooleoyl [3H]glycerol (0.25 mg; 4,440,OOO dpm), along with stoichiometric amounts of nonradioactive palmitic acid (0.31 mg), with the isolated villus cells, cell homogenates, and sonicates, and with

AND

MYHER TABLE

1

YIELDS OF RADIOACTIVE X-1,2-DIACYLGLYCEROLS FOLLOWING INCUBATION OF 2-MONOOLEOYL[‘HIGLYCEROL AND PALMITIC ACID WITH VARIOUS PREPARATIONSOFMONOACYLGLYCEROLACYLTRANSFERASE” Source of monoacylglycerol acyltransferase Isolated

villus

Amount protein bxd cells

of

Yield of X-1,2diacylglycerolb (4-m)

1.37 1.21 1.13 1.29

8,630 9,580 10,710 9,951

Cell homogenates

2.20 2.21 2.35 2.12

518,990 532,220 550,500 523,321

Cell

1.21 1.29 1.22 1.25

186,874 195,091 191,431 189,762

0.08 0.06 0.05 0.08

9,323 5,470 7,513 10,521

sonicates

Purified

enzyme

“The incubation medium contained 0.25 mg monoacylglycerol(4,44O,OOO dpm) and 0.3 1 mg palmitic acid along with the cofactors (homogenates, sonicates, and purified enzyme) and were incubated for 30 min at 37°C. *The results are single determinations.

purified 2-monoacylglycerol acyltransferase of rat intestinal mucosa. Characteristically, lO,OOO- 100,000 dpm/mg diacylglycerol were recovered from each preparation, depending upon the exact radioactivity of the substrate and the extent of endogenous dilution. In those instances where an insufficient mass of diacylglycerol was present for thin-layer chromatographic isolation of the products (less than 0.1 mg), 0.25 mg of nonradioactive rat- 1,2-diacylglycerols were added as carriers. In all instances, the diacylglycerols were extracted and purified by thin-layer chromatography under conditions designed to minimize the isomerization of the acylglycerols.

2-MONOACYLGLYCEROL

Synthesis of Radioactive X-1,2Phosphatidylcholine

In Table 2 are given the yields of radioactive X- 1,2-diacylphosphatidylcholines as a percentage of the radioactivity in the X1,2-diacylglycerol starting materials. The yields averaged 50-70s and were adequate for processing radioactivities as low as 2000 dpm. We completed the reaction without isolation of intermediates. A thin-layer chromatographic examination of the products of the initial phosphorylation indicated that all the X- 1,2-diacylglycerol had reacted, but that, in addition to the monoester, the corresponding chlorohydrin had also been formed and was apparently responsible for the nonquantitative yield of the overall transformation. The radioactive X- 1,2-diaTABLE

2

YIELDS OF RADIOACTIVEX-~JDIACYLPHOSPHATIDYLCHOLINES FOLLOWING INTRODUCTION PHATEANDCHOLINEGROUPSINTOTHERADIOACTIVE X-I,%DIACYLGLYCEROLSa

OF PHOS-

Radioactivity of X- 1,2-d& acylglycerols

Radioactivity of X- 1,2-diacylphosphatidylcholine@

Yield @J)

3,119 28,130 30,560 393,521 22,482 62,325 28,005 2,367 25,732 20,176

1,875 15,329 16,895 194,750 14,007 32,176 17,995 1,559 15,220 15,772

60 54 55 49 62 52 64 66 61 78

a The diacylglycerols (1.0 mg) were converted into the phosphatides by treatment with a chilled solution of chloroform:pyridine:phosphorus oxychloride 1.9:1.9:0.1 (by volume) for 1 h at 0°C followed by 1 h at 25”C, when the mixture was poured onto 200 mg of dry powdered choline chloride. The mixture was stirred at 30°C for 15 h, diluted with 20 pl of water, and stirred for 30 min more. The reaction mixture was then evaporated under a stream of nitrogen and the phosphatidylcholines extracted (14). ’ The results are single determinations.

ACYLTRANSFERASE

ASSAY

7

cylphosphatidylcholines were isolated by thin-layer chromatography as described above and were subjected to a stereospecific digestion with phospholipase C as detailed below. Standardization

of Product Assay

The product assay was standardized by verifying that the amounts of enzyme used and the incubation conditions selected were adequate for a complete hydrolysis of the total mass of sn- 1,2-diacylphosphatidylcholines potentially present in the X-1,2-diacylphosphatidylcholine mixture. In Table 3 it is shown that a minimum of 0.5 units of the enzyme are required to effect a complete release of the sn- 1,2-diacylglycerols from 0.5 mg of rat- 1,2-diacylphosphatidylcholines within 2 min. The released sn-1,2-diacylglycerols were not contaminated by sn2,3-diacylglycerols even at higher enzyme concentrations (2 units) as the enantiomer ratio remained stable. The extent of the hydrolysis of the sn- 1,2-diacylglycerols was independently monitored by means of a radioactive marker, sn- 1,2-diacylphosphatidyl[Me14C]choline, which also served to correct the enantiomer ratio when complete hydrolysis of the sn-1,2-enantiomers was not obtained. The results of a stereospecific assay of known mixtures of radioactive diacylglycerol enantiomers are given in Table 4. It is seen that samples ranging in enantiomer ratios from 0.05 to 20 and containing 5000-200,000 cpm may be assayed t,o within +l% of the major and to within *lo% of the minor enantiomer content by means of phospholipase C digestion in the absence of corrections by internal markers. By using radioactive sn- 1,2-diacylphosphatidyl[ Me“C]choline as an internal marker, the accuracy of even a substandard enzymatic assay of the minor enantiomer could be increased by a factor of 25 (Table 3). A parallel assay carried out with phospholipase A2 (results not shown) gave comparable es-

8

MANGANARO,

KUKSIS, TABLE

AND

MYHER

3

EFFECTOFCONCENTRATIONOFPHOSPHOLIPASEC IJPONRELEASEOFSII-I,%DIACYLGLYCEROLS FROM raC-1,2-DIACYLPHOSPHATIDYLCHOLINES

Determined ratio Enzyme” (units) 0.005 0.010 0.020 0.050 0.075 0.100

0.250 0.500 0.750 1.oo 1.50 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00

Phosphatidylcholine (mg) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.25 0.50 0.75 1.00 1.50 2.0 2.5 3.0 4.0 5.0

Hydrolysis* (WI 4 4 IO 22 36 42 84 96 98 96 96 98 100 100 100

100 100 100 loo 100 100 100

sn-1,22 2 5 11

18 21 42 48 49 48 48 49 49 50 50 41 48 49 50 48 41 48

Corrected ratio’

sn-2,3-

sn-1,2-

sn-2,3

98 98 95 89 82 79 58 52 51 52 52 51 51 50 50 53 52 51 50 52 53 52

50 51 52 50 48 49 49 50 49 49 48 50 50 50 50 49 48 49 50 49 49 48

50 49 48 50 52 51 51 50 51 51 52 50 50 50 50 51 52 51 50 51 51 52

a One unit of enzyme is defined as the amount of enzyme that liberates 1.0 rmol of water-soluble organic phosphorus from sn-1,2-diacylphosphatidylcholine, pH 7.3, at 37°C. The added enzyme concentration was 1 unit/ ml. The digestions were carried out for 2 min. * The extent of hydrolysis was determined by counting the radioactivity recovered by tic in the unhydrolyzed phosphatidylcholine fraction of the added sn-l,2-dipalmitoylphosphatidyl[Me-’4C]choline internal standard. ’ Corrected ratios were obtained using sn- 1,2-dipalmitoylphosphatidyl [Me-‘4C]choline as internal standard, as discussed in text.

timates for the relative ratio of the enantiomers. On the basis of the above experiments, optimum hydrolyses of the X-l ,2-diacylphosphatidylcholines were carried out by employing 1 unit of enzyme/O.5 mg of substrate and completing the initial digestion in 2 min and the final digestion in 4 h. The release of the sn-2,3-diacylglycerols could be speeded up by increasing the amount of enzyme in relation to the residual substrate. Assay of Total Diacylglycerol Products The enantiomer content of the total X-1,2diacylglycerols following radiolabeling from

sn-2-monooleoylglycerol during incubation with either intact cells or cell-free preparations was assayed by comparing the radioactivites recovered in the sn- 1,2-diacylglycer01s and in the residual sn-2,3-diacylphosphatidylcholines. In Table 5 is shown the reproducibility of repeat analyses of selected samples of X- 1,2-diacylglycerols recovered from incubations of the radioactive monoacylglycerol and palmitic acid with intact cells and with the cell homogenates and sonicates. Some 66-69s of the total radioactivity was associated with the sn-1,2-enantiomers from the cells and from the cell

2-MONOACYLGLYCEROL

ACYLTRANSFERASE TABLE

9

ASSAY

4

EFFECT OF VARIATIONS IN ENANTIOMERIC DIACYLPHOSPHATIDYLCHOLINE RATIO UPON QUANTITATIVE RELEASE OF Sn- 1,2-DIACYLGLYCEROLS BY PHOSPHOLIPASE C ’ Known ratios of enantiomeric phosphatidylcholine?

Estimated ratios of enantiomeric phosphatidylcholines

sn- I ,2-

sn-2,3-

Hydrolysis (%)

50 95 5 30 70 5’ 95’

50 5 95 70 30 95 5

100 100 100 loo 100 100 100

sn- 1,249.8 93.5 5.9 29.2 69.3 4.9 94.1

sn-2,3-

+ 0.7 + 0.8 k 0.8 + 0.6 f 0.7 IL 0.7 +- 0.6

50.2 6.5 94.1 70.7 30.7 95.1 5.9

iz f f + + * +

0.7 0.8 0.8 0.6 0.7 0.7 0.6

’ The reaction medium contained a total of 0.5 mg of X-1,2-diacylphosphatidylcholine and 2 units of enzyme along with the buffer, as described in text. The incubations were done at 32°C for 2 min. and the extent of hydrolysis was determined as described in Table 3. b Samples contained lOO,OOO-200,000 cpm; means -+ SD of three experiments, except where indicated. ’ Samples contained 5000-6000 cpm; means f SD of three experiments.

sonicates, while the cell homogenates gave essentially equal proportions of the two enantiomers. The variability of the estimates for the enantiomer content could be reduced by running out the residual phosphatidylcholines from the origin of the thin-layer chromatographic plate by following the neutral solvent development with the development in the phospholipid solvent system. A

parallel analysis of aliquots of the same sample with phospholipase A2 confirmed the results obtained with phospholipase C within 6%. Assay of Molecular Species of m-1,2- and sn-2,3-Diacylglycerols

The stereospecific assay of the molecular species of diacylglycerols produced by the

TABLE

5

REPRODUCIBILITY OF ESTIMATES OF ENANTIOMERIC DIACYLGLYCEROL RATIOS IN PRODUCTS OF MONOACYLGLYCEROL ACYLTRANSFERASE” Enantiomer ratios* Phospholipase C

Phospholipase Al

Enzyme source

sn- 1,2-

sn-2,3-

sn- 1,2-

sn-2,3-

Villus cells Cell homogenates Cell sonicates

67 f 3 51+5 68 -t 5

33 + 3 49 f 5 32 k 5

69 f 3 50 + 2 66 + 3

31 k 3 50 k 2 34 + 3

’ In these experiments, the proportion of the sn-2,3-diacylglycerols in the phospholipase C digests was estimated from the radioactivity of the residual sn-2,3-diacylphosphatidylcholines. The proportion of the sn-1,2- and sn-2,3diacylglycerols in the phospholipase A2 digests was estimated from the radioactivity in the lyso (sn-1) phosphatidylcholines and in the residual sn-2,3-diacylphosphatidylcholines, respectively. b Means +- SD of 10 (phospholipase C) and 3 (phospholipase AZ) experiments.

IO

MANGANARO,

KUKSIS, AND MYHER

monoacylglycerol acyltransferase is based on an argentation-chromatographic resolution of the diacylglycerols according to degree of unsaturation, within each enantiomer type. Separate bands were obtained for the saturates, monoenes, dienes, trienes, and tetraenes for each of the sn-1,2- and sn-2,3diacylglycerol enantiomers recovered from the incubation of radioactive sn-2-monooleoylglycerols and palmitic acid with the various enzyme preparations. A comparison to reference standards revealed that the bulk of the radioactivity in the diacylglycerols was recovered in the monoene fraction of both sn- 1,2- and sn-2,3-diacylglycerols, as would be expected if the 2-monooleoylglycerol was esterified mainly with the added free palmitic acid. Smaller amounts of radioactivity, however, were also present in the diene and triene bands. The radioactivity in the saturates is attributed to the presence of saturated species in the sn-2-monooleoyl[ ‘HIglycerol. The finding of comparable amounts of radioactivity in the various unsaturation classes of the sn-1,2- and sn2,3-diacylglycerols indicates that a common pool of free fatty acids was utilized for the acylation of both sn-1 and sn-3- positions of the 2-monoacylglycerol molecule. It does not follow, however, that both sn-1,2-and sn-2,3diacylglycerols were necessarily produced at equal rates. DISCUSSION

The proposed procedure for assaying the diacylglycerol products of the sn-2-monoacylglycerol acyltransferase allows a determination of the molecular association and fatty-acid specificity of the reaction in addition to the positional distribution of the acids also provided by the Brockerhoff analysis. The new procedure requires a reasonable standardization of the reaction conditions, because an incomplete hydrolysis of the sn-1,2-diacylphosphatidylcholines will result in an overestimation of the sn-2,3enantiomers, while an overdigestion with

phospholipase C will result in an overestimation of the sn-1,2- enantiomers. This is due to the incomplete specificity of the phospholipase C for the enantiomeric diacylphosphatidylcholines ( 14). To guard against possible nonrepresentative stereospecific hydrolysis of small amounts of one of the enantiomers in the presence of excessive amounts of the other, the digestions with phospholipase C were performed in the presence of a large excess of racemic carriers made up of the anticipated molecular species of phosphatidylcholine. The rat- 1,2-diacylglycerols generated from randomized corn oil triacylglycerols were well suited for the synthesis of the desired rat-phosphatidylcholines. In the presence of such carriers, the phospholipase C digestion was always carried out under essentially constant conditions of substrate loading (0.5 mg) and enantiomer content (5050). The most accurate estimates of the enantiomer ratios were obtained by comparing the radioactivities in the sn-1,2- and the sn-2,3-diacylglycerols. However, adequate estimates of the enantiomer proportions could also be derived by comparing the radioactivities in the initially released sn1,2-diacylglycerols and in the residual sn2,3-diacylphosphatidylcholines. The latter approach was comparable to that used in the Brockerhoff analysis, where the enantiomer content is derived from comparisons of radioactivity in the lyso and in the residual phosphatidylcholines. For both approaches, the addition of trace amounts of sn- 1,2-diacylphosphatidyl[ Me“C]cho1ine provided extra confidence, as it indicated the extent of hydrolysis of the sn1,2- enantiomers obtained in each digestion, as well as provided a basis for correcting results from incomplete digestions. The corrections can be conveniently performed by a differential counting of 14C and ‘H, provided the substrate and the internal standard have been appropriately labeled. Morley et al. (26) bad previously used deuterium-la-

2-MONOACYLGLYCEROL

beled enantiomeric diacylglycerols as substrates in an assay of the stereospecific hydrolysis of rat- 1,2-diacylglycerols by lipoprotein lipase. Christie (3) had employed rat-diacylglycerols isotopically labeled in fatty acids as a means of correcting for losses during chromatographic separations and isolation of the intermediates required for the stereospecific analysis of triacylglycerols via the phosphatidylphenols. The main advantages of the present radioassay of the stereospecificity of the 2monoacylglycerol acyltransferase are twofold. First, the procedure allows the physical resolution of the diacylglycerol enantiomers, the composition of which can be determined and thus a complete identification of the products obtained. Second, the possibility of incomplete lipolysis is guarded against by means of an added internal standard, which also serves to correct the analytical results if necessary. The disadvantage of the procedure is the need for time-consuming preparation and purification of derivatives. The assay system is applicable to determination of the stereochemical composition of molecular species in diacylglycerol mixtures arising from the activity of other acyltransferases and of lipases. It is hoped that the development of a sensitive stereospecific microassay for the products of acylglycerol acyltransferase will lead to a better understanding of the action of both synthetic and degradative enzymes of acylglycerol metabolism than is possible at present. ACKNOWLEDGMENT These studies were supported by funds from the Ontario Heart Foundation and the Medical Research Council of Canada.

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Rex Rex

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ACYLTRANSFERASE

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II

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