Cocoa butter biosynthesis. Purification and characterization of a soluble sn-glycerol-3-phosphate acyltransferase from cocoa seeds.

Glycerol-3-phosphate acyltransferase has been purified from the post-microsomal supernatant of cocoa seeds using differential ammonium sulfate solubility along with anion exchange and gel filtration chromatography. Chromatofocusing and isoelectric focusing revealed a series of proteins with acyltransferase activity having isoelectric points close to 5.2. Gel filtration on Sephacryl S-300 in 500 mM NaCl, along with polyacrylamide gel electrophoresis (denaturing and non-denaturing) and immunochemical analysis, gave evidence that the native enzyme has a molecular weight of 2 X 10(5) and consists of an aggregate of 10 Mr 20,000 subunits. The highly purified enzyme carries an acyl donor, probably acyl-CoA, although this is not firmly established. The hydrophobic nature of the purified enzyme was demonstrated by its firm binding to octyl-Sepharose. Mass spectrometric analysis of reaction products revealed the presence of both palmitic and stearic acids. Considering that 1) the fatty acids were derived from the purified enzyme; 2) they were found exclusively in the 1-position of glycerol 3-phosphate; 3) the fatty acid positioning and composition is consistent with that found in cocoa butter, the major storage product of cocoa seeds; and 4) the enzyme is found in the post-microsomal supernatant, it seems reasonable to conclude that the first step in cocoa butter biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase in the cytoplasm of cocoa cotyledon cells.

Glycerol-3-phosphate acyltransferase has been purified from the post-microsomal supernatant of cocoa seeds using differential ammonium sulfate solubility along with anion exchange and gel filtration chromatography. Chromatofocusing and isoelectric focusing revealed a series of proteins with acyltransferase activity having isoelectric points close to 5.2.
Gel filtration on Sephacryl S-300 in 500 mM NaCl, along with polyacrylamide gel electrophoresis (denaturing and non-denaturing) and immunochemical analysis, gave evidence that the native enzyme has a molecular weight of 2 x 10' and consists of an aggregate of 10 M. 20,000 subunits.
The highly purified enzyme carries an acyl donor, probably acyl-CoA, although this is not firmly established. The hydrophobic nature of the purified enzyme was demonstrated by its firm binding to octyl-Sepharose.
Mass spectrometric analysis of reaction products revealed the presence of both palmitic and stearic acids. Considering that 1) the fatty acids were derived from the purified enzyme; 2) they were found exclusively in the 1-position of glycerol 3-phosphate; 3) the fatty acid positioning and composition is consistent with that found in cocoa butter, the major storage product of cocoa seeds; and 4) the enzyme is found in the postmicrosomal supernatant, it seems reasonable to conclude that the first step in cocoa butter biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase in the cytoplasm of cocoa cotyledon cells.
The unique physical properties of cocoa butter, the major storage product of cocoa seeds, stem from the fact that it is a mixture of triacylglycerols composed of 85% oleic acid in the 2-position, whereas the 1-and 3-positions are occupied by palmitic and stearic acids (1,2). Lipids make up over 50% of the dry weight of mature cocoa seeds, whereas triacylglycerols make up about 96% of the lipids (3).
Plant triacylglycerol biosynthesis proceeds through the complete glycerol 3-phosphate pathway from l-monoacylglycerol 3-phosphate to 1,2-diacylglycerol 3-phosphate, to 1,2diacylglycerol, to triacylglycerol (4, 5). This laboratory is engaged in a systematic study of the enzymes responsible for the biosynthesis of cocoa butter, and the present study is concerned with the purification and characterization from * This work was supported in part by awards from the Hershey Foods Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cocoa seeds of glycerol-P acyltransferase, the enzyme catalyzing the first step in the pathway. A preliminary report of this work has been published (6).

Preliminary Purification
Although several studies concern the occurrence and biosynthesis of triacylglycerol in seeds, information on plant glycerol-3-phosphate acyltransferase is limited to reports on the enzyme from spinach and pea chloroplasts (7)(8)(9). In early experiments on the enzymes of cocoa butter biosynthesis, we discovered the majority of glycerophosphate acyltransferase' activity in the post-microsomal supernatant fraction of cocoa seed extracts. The enzyme has now been extensively purified following very closely the procedures described by Bertrams and Heinz (8). Summary of a typical purification is given in Table I. In some preparations, where initial specific activities were low due to uncertainties of protein measurements caused by the presence of large amounts of pigments, enrichments exceeding 1000-fold were achieved. The specific activities of our pure preparations are strikingly similar to those reported for the pea chloroplast enzyme (8). The enzyme can be prepared either from fresh beans, or from beans kept at -80 "C for several months, though yields and specific activities are lower for the latter. Similar results were obtained using mature (180 days after pollination) or immature (120 days after pollination) seeds.

Chromatofocusing and Isoelectric Focusing
Following preliminary purification, the enzyme preparation was passed through a chromatofocusing column. The results of this experiment are shown in Fig. 1. It is seen that a small amount of glycerophosphate acyltransferase activity elutes at an isoelectric point of 6.2 but the majority of the enzyme Portions of this paper (including "Experimental Procedures" and Figs. 1s-5s) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-3424, cite the authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
The abbreviations used are: glycerophosphate acyltransferase, glycerol-3-phosphate acyltransferase; ACP, acyl carrier protein.  Isoelectric focusing using vertical tube polyacrylamide gels confirmed the results of the chromatofocusing experiment (Fig. 2). Thus, we conclude that cocoa seed glycerophosphate acyltransferase exists in several molecular forms distinguishable by small charge differences.
Isoelectric focusing of purified pea chloroplast acyltransferase activity demonstrated the existence of two forms of the enzyme with apparent isoelectric points of 6.3 and 6.6 (8).
The two forms co-purified through all other purification steps. In contrast, spinach chloroplast acyltransferase was found to exist in a single molecular form with isoelectric point 5.2 (8).

Hydrophobic Chromatography
Detection of enzyme activity in all preliminary purification steps, as well as in the chromatofocusing and isoelectric focusing experiments, was achieved without addition of a fatty acid donor to reaction mixtures. In addition, it was determined that, even with highly purified glycerophosphate acyltransferase preparations, addition of either acyl-CoA or acyl-ACP had no effect on enzyme activity. We reasoned that if the enzyme was the source of the fatty acid donor, it should be possible to demonstrate this by hydrophobic chromatography. Post-S-300 glycerophosphate acyltransferase was subjected to hydrophobic chromatography on an octyl-Sepharose column as described by Rock and Garwin (11). The procedure resulted in considerable loss of enzyme activity but the major recoverable activity was found in the fraction (peak C) eluted from the column with 25% isopropanol (Fig. 3). Peak C was 10 times more active than peak B and, in common with all prior samples in the purification, did not require addition of an exogenous fatty acid donor to the reaction mixture. That is to say, the enzyme preparation itself was the source of the fatty acid transferred to glycerol 3-phosphate. Protein present in peak A , on the other hand, had essentially no enzyme activity when palmitoyl-CoA was not added to the reaction mixture. However, a small amount of activity was seen when palmitoyl-CoA was added.
The hydrophobic chromatography experiment clearly demonstrates that long chain fatty acids are associated with the purified enzyme. Rock and Garwin (11) used model compounds to show that ACP does not bind to octyl-Sepharose, whereas acyl-ACP binds strongly but can be eluted with solutions of 2-propanol. They showed that acyl-ACP binds to the hydrophobic gel via the acyl chain, strength of binding being proportional to fatty acid chain length. Although our experiment strongly suggests that purified cocoa glycerophosphate acyltransfera~ has long chain fatty acids associated, it does not prove that the fatty acid derives from acyl-ACP since it is known that acyl-CoA binds strongly to some proteins (12). The soluble glycerophosphate acyltransferase from pea and spinach chloroplasts could use either acyl-CoA or acyl-ACP as a fatty acid donor with preference for the latter (9). It is possible that both potential fatty acid donors are present in the purified cocoa seed glycerophosphate acyltransferase preparations.
Presence of CoA in purified glycerophosphate acyltransferase preparations is suggested from the results of experiments where post-S-300 glycerophosphate acyltransferase was incubated with ["Cjpalmitate and Aerobacter aerogenes acyl-CoA synthetase. Polyacrylamide gel electrophoresis of reaction mixtures followed by gel drying and fluorography revealed a band in the low molecular weight region where acyl-CoA migrates. The results of this experiment imply that free CoA as well as acyl-CoA is associated with the purified enzyme.

Kinetic Data
Typical Michaelis-Menten kinetics were obtained when a glycerol 3-phosphate concentration curve was run in the absence of added fatty acid donors. A K,,, of 4.2 mM was found, a value similar to that reported for the spinach chloroplast enzyme (8). Reaction velocity was linear for 10 min, then slowed, and finally stopped after 2-3 h.
Purified soluble cocoa seed glycerophosphate acyltransferase could be made responsive to added acyl-CoA by first incubating the enzyme with glycerol 3-phosphate, then passing the incubation mixture over a Sephadex G-50 column. The enzyme recovered after this treatment gives a typical Michaelis-Menten substrate concentration curve with a K , for stearoyl-CoA of about 4 p~, again similar to values for the spinach and pea chloroplast enzymes.

Analysis of Reaction Products
We conducted experiments to determine the chemical nature of the products formed when glycerol 3-phosphate was incubated with cocoa seed extracts. Peak C from the octyl-Sepharose column (Fig. 3) was the enzyme source used for these experiments. Detailed protocols for the analyses are given under "Experimental Procedures." First, it was determined by thin layer chromatography that a single fatty acid was transferred to glycerol 3-phosphate forming monoacylgIycero1 3-phosphate. An important property of acyltransferase is the positional specificity. To determine whether the fatty acid transferred was in position 1 or 2 of glycerol 3-phosphate, the reaction product, monoacylglycerol 3-phosphate, was isolated and dephosphorylated by alkaline phosphatase, and the resulting monoacylglycerol analyzed for isomeric composition by thin layer chromatography. The results clearly established that acylation occurred exclusively in the 1-position.
The pea and spinach chloroplast glycerophosphate acyltransferase also possess high positional specificity, both directing acyl groups into the C-1 position of glycerol with negligible acylation of C-2 (8).
Finally, the fatty acids transferred were shown by mass spectrometric analysis to be palmitic and stearic. All these observations are consistent with the conclusion that the enzyme purified from the soluble fraction of cocoa seed extracts is the first enzyme in the biosynthetic pathway leading from glycerol 3-phosphate to cocoa butter.

A n t i~~s to Cocoa Seed ~l y c e r~p~~p~t e A~~t r a n s f R~w i r n r n~~~s a~
to Demonstrate Presence of Antibodies-Antibodies to cocoa seed glycerophosphate acyltransferase were induced in a New Zealand White rabbit by injecting isoelectric focusing purified enzyme homogenized with polyacrylamide gel slices (see Fig. 2). Detailed protocols describing the immunological procedures are given under "Experimental Purified cocoa seed glycerophosphate acyltransferase after octyl-Sepharose chromatography ( l a n e A, 100 pg) or after Sephacryl-S-300 chromatography ( l o n e E, 10 pg) was denatured and reduced by heating for 3 min in 1.5-10-fold excess of loading buffer (63 mM Tris-HCI, pH 6.8; 12% glycerol; 1% sodium dodecyl sulfate, 180 mM 2-mercaptoethanol; 0.01% bromphenol blue). Standards of known molecular weight were treated in the same way. Electrophoresis was conducted in 15% polyacrylamide separating gel, using 6% stacking gels in the presence of 1% sodium dodecyl sulfate in a Tris-HCI buffer system (14). Duplicate gels were run; molecular weight standards and lones A and E on the left were stained with Coomassie Brilliant Blue, destained, and photographed. Lanes A and E on the right show the results of a Western blot to unmodified nitrocellulose followed by radiographic detection with anti-glycerophosphate acyltransferase and '251-labeled protein A.
Procedures." Presence of antibodies in rabbit serum was demonstrated by solid-phase radioimmunoassay (13). The amount of '9-protein A bound by purifie: glycerophosphate acyltransferase antigen-antibody complex was about 9 times greater when antiserum was added to the microtiter wells compared to the amount bound when preimmune serum was added (Fig. 4A).
Antibody Inhibition of Glycerophosphate Acyltransferase Activity to Demonstrate Specificity-Ability of the antibody to inhibit glycerophosphate acyltransferase activity was demonstrated by conducting the usual enzyme assay after incubation with varying amounts of antiserum or control (preimmune) serum. Fig. 4B shows the decreasing amount of enzyme activity observed after incubation with increasing amounts of antiserum protein.

Molecular Weight and Subunit Structure
The molecular weight of nondenatured glycerophosphate acyltransferase as determined by gel-filtration chromatography on Sephacryl S-300 columns in 500 mM NaCl is estimated to be about 200,000. Enzyme assay of gel slices obtained after polyacrylamide gel electrophoresis under nondenaturing conditions confirmed this estimate. However, when gel electrophoresis was performed in the presence of sodium dodecyl sulfate, the vast majority of protein, estimated by Coomassie Blue staining, representing all proteins present except for minor contaminants, was concentrated in a single area of the gel with electrophoretic mobility corresponding to a molecular weight of about 20,000. Antibodies to cocoa seed glycerophosphate acyltransferase formed immune complexes only with this polypeptide (Fig. 5 ) , confirming that the M , 20,000 protein was an enzyme subunit. Hence, we conclude that the native molecular weight of cocoa seed glycerophosphate acyltransferase is 2 X lo5 and that the enzyme is composed of 10 M, 20,000 peptides. Both molecular forms of pea chloroplast glycerophosphate actyltransferase had molecular weights of 42,000, whereas the spinach enzyme was slightly larger (8). No subunit determinations were reported for these enzymes. Lamb and Fallon (12) reported that 1 mg of rat microsomal protein could bind about 68 nmol of palmitoyl-CoA, all of which could be removed by incubation with high concentrations of albumin. If we assume an average molecular weight of 100,000 for the rat microsomal protein, it can be calculated that each nanomole of protein binds 6.8 nmol of palmitoyl-CoA. Incubation of highly purified cocoa seed glycerophosphate acyltransferase under assay conditions for up to 3 h, long enough to allow transfer of all bound fatty acid, resulted in formation of 100 nmol of product for each nanomole of enzyme, or 10 nmol of bound fatty acid donor/subunit. The amount of product formed was a linear function of protein concentration.