566th MEETING, CAMBRIDGE
287
The activity of diacylglycerol acyltransferase towards [l-14C]butyryl-CoA was tested first by using exogenous dipalmitoylglycerol or dioleoylglycerol, which were the substrates used by Pynadeth & Kumar (1964). N o incorporation of [l-14Clbutyryl-CoA into triacylglycerol was observed. However, incorporation of [l -14C]butyryl-CoA into triacylglycerol was successfully demonstrated when membrane-bound 1,2-dipalmitoyl-~n-[2-~H]glycerol(formed from membrane-bound [3H]phosphatidic acid by the microsomal Mgz+-dependent phosphatidate phosphohydrolase (EC 3.1.3.4) was used as substrate (Table 2). The ratio of [l-14C]butyric acid to [2-3H]glycerol in triacylglycerol that contained short-chain fatty acids was 0.95, and the amount of [2-3H]glycerol incorporated equalled the decrease in the incorporation into dipalmitoylglycerol. This clearly established that the membrane-bound dipalmitoylglycerol was the butyryl acceptor. With [l-14C]palmitoyl-CoA and membrane-bound dipalmitoylglycerol, [l-14C]palmitic acid was incorporated into triacylglycerol that contained long-chain fatty acids, and at a rate three times that of butyrate incorporation into triacylglycerolthat contained short-chain fattyacids. The synthesis of triacylglycerols that contained short-chain fatty acids was also observed when [l-"V]butyryl-CoA was incubated in the presence of MgZf and microsomal fractions which contained membrane-bound [3H]phosphatidic acid. Thus, in this experiment, butyryl-CoA was present during the formation of [3H]dipalmitoylglycerol.Under these conditions the synthesis of triacylglycerol was three times greater than that obtained above using pre-formed membrane-bound diacylglycerol. The high rate of incorporation of butyryl-CoA into the sn-3 position of triacylglycerol that contained short-chain fatty acids compared with its negligible rate of incorporation into phosphatidic acid, shows that triacylglycerols that contain short-chain fatty acids are synthesized by the microsomal fraction of cow mammary gland by the acylation of long-chain 1,2-diacyl-sn-glyceroIswith short-chain acyl-CoA esters. This biosynthetic route is consistent with the distribution of short-chain fatty acids in the triacylglycerols of ruminant milk. We thank Jette Berhrk for excellent technical assistance. Askew, E. W., Emery, R. S. & Thomas, J. W. (1971) Lipids 6, 326-331 Bligh, E. G. & Dyer, W . J. (1959) Can. J. Biochem. Physiol. 37, 911-917 Kennedy, E. P. (1961) Fed. Proc. Fed. Am. SOC.Exp. Biol. 20,934-940 Kuksis, A. (1972) Prog. Chem. Furs Other Lipids 12, 1-163 Marai, L., Breckenridge, W. C. & Kuksis, A. (1969) Lipids 4, 562-570 Pitas, R. E., Sampugna, J. & Jensen, R. G. (1967) J. Dairy Sci. 50, 1332-1335 Pynadeth, T. I. & Kumar, S. (1964) Biochim. Biophys. Actu 84,251-263
Fatty Acid Elongation by Preparations from Pisum sufivum JOHN L. HARWOOD and PAUL BOLTON Department of Biochemistry, University College, P.O. Box 78, Cardiff CF1 IXL, Wales, U.K.
Fatty acid synthesis had been studied in a number of subcellular preparations from higher plants (c.f. Harwood, 1975). Evidence has been presented that at least three enzymes are involved in the production of saturated fatty acids. These are, respectively, fatty acid synthetase, which forms palmitic acid; palmitate elongase, which produces stearate; and at least one further elongase, which synthesizes the very-long-chain fatty acids, Cz0,Cz2etc.. Palmitate elongase apparently originates from the chloroplast (plastid) stroma (Harwood, 1974; Weaire & Kekwick, 1975) and utilizes palmitoyl acyl-carrier protein as substrate with malonyl-CoA as the unit of addition (Harwood, 1974; Jaworski et al., 1974). The enzyme has thus far been studied in two maturing seed tissues, namely those of avocado and safflower. Formation of very-long-chain fatty acids takes place in microsomal preparations where malonyl-CoA is again the Cz donor (Harwood & Stumpf, 1971; Kolattukudy & Buckner, 1972; Bolton &
Vol. 5
BIOCHEMICAL SOCIETY TRANSACTIONS Hanvood, 1976). Rapid production of these acids occurs in conjunction with cutin or suberin synthesis, for which the compounds are important precursors. Subcellular fractions from germinating pea (Pisum sativum), which had been shown previously to be active in elongating fatty acids (Harwood & Stumpf, 1971 ; Harwood, 1975), were used to study the reactions further. The ‘105000gsupernatant’ synthesized palmitic acid de novo and stearic acid by elongation. The microsomal fraction, by contrast, also formed very-Iong-chain fatty acids (arachidic acid etc.). The cofactor requirements for both fractions were similar, with the exception that acylcarrier protein was absolutely required for the supernatant, whereas its omission from the microsomal incubation medium still allowed considerable fatty acid synthesis from malonyl-CoA. These results are similar to those obtained in other experiments with subcellular fractions from plants, and, presumably, reflect the considerable endogenous concentrations of acyl-carrier protein in membrane preparations. Since a-oxidation of stearate and the very-long-chain fatty acids showed that radioactivity from [14C]malonyl-CoA was only present at the carboxyl end of the molecules, an endogenous primer acyl chain must have been used. Analysis of the endogenous lipids in the microsomal and supernatant fractions showed that phosphatidylcholine was the major component in the microsomal fraction, and triacylglycerol in the supernatant. Unesterified fatty acids represented less than 3 % of the total lipid of either fraction. The products of fatty acid synthesis from [14C]malonyl-CoA by microsomal fractions were examined and it was found that the majority of the 14C-labeIledfatty acids formed were localized in the complex lipid fractions as 0-esters rather than as acyl-carrier-protein esters or CoA esters. Together with the fact that plant tissues are apparently unable to form palmitoyl or stearoyl acyl-carrier proteins from
Table 1. Eflect of exogtxous lipids on fatty acid synthesis by pea microsomal fractions Meansf s.D. are given, with the number of experiments in parentheses. Incubations were carried out in the presence of 0.5% Triton X-100. Addition None Dipalmitoylphosphatidylethanolamine Dipalmitoylphosphatidylcholine Stearic acid Distearoylphosphat idylcholine
Fatty acid synthesis Elongation (%of total (% of control) fatty acid synthesis) lOOf 3 53
176225 (2) 161 f 7 (6) 91f 7 (1) 1023-12 (2)
63 62 54 53
Table 2. Efect of various reagents on microsomal fatty acid synthesis Results are expressed as a percentage of the total fatty acid synthesis in the control. M e a n s k s . ~are . given. Fatty acid synthesis (% of control) (meanSfS.D.) Addition c (final concn.) Nature of addition ... Preincubation Added to medium None 100f4 100f6 Sodium arsenite (1 nm) 57t6 59f 10 N a F (3 m ~ ) 76Ifr8 60f9 EDTA (1 m) 89+5 EGTA (1 mM) look5 5,5’-Dithiobis-(2-nitrobenzoic acid) (1mM) 16 k 4 56f2 Iodoacetamide ( 5 m ) 5k2 4+1
-
1977
289
566th MEETING, CAMBRIDGE
the respective free fatty acids (cf. Harwood, 1975), these results indicated the possibility that pea microsomal fractions could utilize complexlipids as the source of, fist, palmitate and then stearate for elongation. To study the elongation enzymes of the microsomal fraction more thoroughly, sohbilization was attempted with a variety of detergents. Of these, only concentrations of Triton X-100(below 1% final concn.) were non-inhibitory. At this concentration of detergent it was found that activity was still present in very-large-molecular-weight particles eluted with the void volume of Sephadex G-200. Moreover, the ability of microsomal fractions to synthesize very-Iong-chainfatty acids was lost by Triton X-100 treatment. Palmitate and stearate synthesis was unaffected or sIightly increased. Addition of dipalmitoylphosphatidylcholine was made to microsomal fractions in an attempt to increase elongation rates. The phospholipid consistently increased total incorporation of [14C]malonyl-CoA into fatty acids, and the percentage elongation observed also occurred at higher rates. However, [1-'4C]palmitoylphosphatidylcholine,when added exogenously in the presence of Triton X-100,was not elongated, in spite ofthefact that the phospholipid was incorporated into the microsomal lipid by substitution. Moreover, further experiments showed that the stimulation of [14C]malonyl-CoA incorporation into microsomal fatty acids by exogenous lipids containing palmitate was relatively unspecific. Therefore in contrast with soluble palmitate elongase, the exact nature of the substrate used by the particular elongase remains undefined. However, it should be noted that addition of stearic acid or distearoylphosphatidylcholinefailed to increase fatty acid synthesis (Table 1)in the presence of Triton X-100. The effect of some inhibitors, chelating agents and thiols on microsomal fatty acid synthesis is shown in Table 2. Thiol-group reagents caused inhibition of fatty acid formation. Arsenite was found, as in other plant systems, to specifically prevent palmitate elongation without decreasing fatty acid synthetase activity. The inhibition caused by fluoride was not, apparently, due to chelation of essential divalent cations, since EDTA and EGTA failed to significantly alter fatty acid synthesis. Some dramatic differences were seen in the extent of inhibition of thiols when they were added to the assay system rather than preincubated with the microsomal fraction. These probably reflect the relative affinities of thiol groups on microsomal proteins, coenzyme-A and acyl-carrier protein. These studies were supported by a grant from the Science Research Council. Bolton, P. & Harwood, J. L. (1976) Phytochemistry in the press Harwood, J. L. (1974) Lipids 9, 850-854 Harwood, J. L. (1975) in Recent Advances in the Chemistry and Biochemistry of Plant Lipids (Galliard, T. & Mercer, E. I., eds.), pp. 43-93, Academic Press, London and New York Harwood, J. L. & Stumpf, P.K. (1971) Arch. Biochem. Biophys. 142, 281-291 Jaworski, J. G., Goldschmidt,E. E. & Stumpf, P. K. (1974) Arch. Eiochem. Eiophys. 163,769-776 Kolattukudy, P.E. & Buckner, J. S. (1972) Eiochern. Eiophys. Res. Commun. 46,801-807 Weiare, P. J. & Kekwick, R. G. 0. (1975) Eiochem. J. 146,439445
VOl. 5
10
287
The activity of diacylglycerol acyltransferase towards [l-14C]butyryl-CoA was tested first by using exogenous dipalmitoylglycerol or dioleoylglycerol, which were the substrates used by Pynadeth & Kumar (1964). N o incorporation of [l-14Clbutyryl-CoA into triacylglycerol was observed. However, incorporation of [l -14C]butyryl-CoA into triacylglycerol was successfully demonstrated when membrane-bound 1,2-dipalmitoyl-~n-[2-~H]glycerol(formed from membrane-bound [3H]phosphatidic acid by the microsomal Mgz+-dependent phosphatidate phosphohydrolase (EC 3.1.3.4) was used as substrate (Table 2). The ratio of [l-14C]butyric acid to [2-3H]glycerol in triacylglycerol that contained short-chain fatty acids was 0.95, and the amount of [2-3H]glycerol incorporated equalled the decrease in the incorporation into dipalmitoylglycerol. This clearly established that the membrane-bound dipalmitoylglycerol was the butyryl acceptor. With [l-14C]palmitoyl-CoA and membrane-bound dipalmitoylglycerol, [l-14C]palmitic acid was incorporated into triacylglycerol that contained long-chain fatty acids, and at a rate three times that of butyrate incorporation into triacylglycerolthat contained short-chain fattyacids. The synthesis of triacylglycerols that contained short-chain fatty acids was also observed when [l-"V]butyryl-CoA was incubated in the presence of MgZf and microsomal fractions which contained membrane-bound [3H]phosphatidic acid. Thus, in this experiment, butyryl-CoA was present during the formation of [3H]dipalmitoylglycerol.Under these conditions the synthesis of triacylglycerol was three times greater than that obtained above using pre-formed membrane-bound diacylglycerol. The high rate of incorporation of butyryl-CoA into the sn-3 position of triacylglycerol that contained short-chain fatty acids compared with its negligible rate of incorporation into phosphatidic acid, shows that triacylglycerols that contain short-chain fatty acids are synthesized by the microsomal fraction of cow mammary gland by the acylation of long-chain 1,2-diacyl-sn-glyceroIswith short-chain acyl-CoA esters. This biosynthetic route is consistent with the distribution of short-chain fatty acids in the triacylglycerols of ruminant milk. We thank Jette Berhrk for excellent technical assistance. Askew, E. W., Emery, R. S. & Thomas, J. W. (1971) Lipids 6, 326-331 Bligh, E. G. & Dyer, W . J. (1959) Can. J. Biochem. Physiol. 37, 911-917 Kennedy, E. P. (1961) Fed. Proc. Fed. Am. SOC.Exp. Biol. 20,934-940 Kuksis, A. (1972) Prog. Chem. Furs Other Lipids 12, 1-163 Marai, L., Breckenridge, W. C. & Kuksis, A. (1969) Lipids 4, 562-570 Pitas, R. E., Sampugna, J. & Jensen, R. G. (1967) J. Dairy Sci. 50, 1332-1335 Pynadeth, T. I. & Kumar, S. (1964) Biochim. Biophys. Actu 84,251-263
Fatty Acid Elongation by Preparations from Pisum sufivum JOHN L. HARWOOD and PAUL BOLTON Department of Biochemistry, University College, P.O. Box 78, Cardiff CF1 IXL, Wales, U.K.
Fatty acid synthesis had been studied in a number of subcellular preparations from higher plants (c.f. Harwood, 1975). Evidence has been presented that at least three enzymes are involved in the production of saturated fatty acids. These are, respectively, fatty acid synthetase, which forms palmitic acid; palmitate elongase, which produces stearate; and at least one further elongase, which synthesizes the very-long-chain fatty acids, Cz0,Cz2etc.. Palmitate elongase apparently originates from the chloroplast (plastid) stroma (Harwood, 1974; Weaire & Kekwick, 1975) and utilizes palmitoyl acyl-carrier protein as substrate with malonyl-CoA as the unit of addition (Harwood, 1974; Jaworski et al., 1974). The enzyme has thus far been studied in two maturing seed tissues, namely those of avocado and safflower. Formation of very-long-chain fatty acids takes place in microsomal preparations where malonyl-CoA is again the Cz donor (Harwood & Stumpf, 1971; Kolattukudy & Buckner, 1972; Bolton &
Vol. 5
BIOCHEMICAL SOCIETY TRANSACTIONS Hanvood, 1976). Rapid production of these acids occurs in conjunction with cutin or suberin synthesis, for which the compounds are important precursors. Subcellular fractions from germinating pea (Pisum sativum), which had been shown previously to be active in elongating fatty acids (Harwood & Stumpf, 1971 ; Harwood, 1975), were used to study the reactions further. The ‘105000gsupernatant’ synthesized palmitic acid de novo and stearic acid by elongation. The microsomal fraction, by contrast, also formed very-Iong-chain fatty acids (arachidic acid etc.). The cofactor requirements for both fractions were similar, with the exception that acylcarrier protein was absolutely required for the supernatant, whereas its omission from the microsomal incubation medium still allowed considerable fatty acid synthesis from malonyl-CoA. These results are similar to those obtained in other experiments with subcellular fractions from plants, and, presumably, reflect the considerable endogenous concentrations of acyl-carrier protein in membrane preparations. Since a-oxidation of stearate and the very-long-chain fatty acids showed that radioactivity from [14C]malonyl-CoA was only present at the carboxyl end of the molecules, an endogenous primer acyl chain must have been used. Analysis of the endogenous lipids in the microsomal and supernatant fractions showed that phosphatidylcholine was the major component in the microsomal fraction, and triacylglycerol in the supernatant. Unesterified fatty acids represented less than 3 % of the total lipid of either fraction. The products of fatty acid synthesis from [14C]malonyl-CoA by microsomal fractions were examined and it was found that the majority of the 14C-labeIledfatty acids formed were localized in the complex lipid fractions as 0-esters rather than as acyl-carrier-protein esters or CoA esters. Together with the fact that plant tissues are apparently unable to form palmitoyl or stearoyl acyl-carrier proteins from
Table 1. Eflect of exogtxous lipids on fatty acid synthesis by pea microsomal fractions Meansf s.D. are given, with the number of experiments in parentheses. Incubations were carried out in the presence of 0.5% Triton X-100. Addition None Dipalmitoylphosphatidylethanolamine Dipalmitoylphosphatidylcholine Stearic acid Distearoylphosphat idylcholine
Fatty acid synthesis Elongation (%of total (% of control) fatty acid synthesis) lOOf 3 53
176225 (2) 161 f 7 (6) 91f 7 (1) 1023-12 (2)
63 62 54 53
Table 2. Efect of various reagents on microsomal fatty acid synthesis Results are expressed as a percentage of the total fatty acid synthesis in the control. M e a n s k s . ~are . given. Fatty acid synthesis (% of control) (meanSfS.D.) Addition c (final concn.) Nature of addition ... Preincubation Added to medium None 100f4 100f6 Sodium arsenite (1 nm) 57t6 59f 10 N a F (3 m ~ ) 76Ifr8 60f9 EDTA (1 m) 89+5 EGTA (1 mM) look5 5,5’-Dithiobis-(2-nitrobenzoic acid) (1mM) 16 k 4 56f2 Iodoacetamide ( 5 m ) 5k2 4+1
-
1977
289
566th MEETING, CAMBRIDGE
the respective free fatty acids (cf. Harwood, 1975), these results indicated the possibility that pea microsomal fractions could utilize complexlipids as the source of, fist, palmitate and then stearate for elongation. To study the elongation enzymes of the microsomal fraction more thoroughly, sohbilization was attempted with a variety of detergents. Of these, only concentrations of Triton X-100(below 1% final concn.) were non-inhibitory. At this concentration of detergent it was found that activity was still present in very-large-molecular-weight particles eluted with the void volume of Sephadex G-200. Moreover, the ability of microsomal fractions to synthesize very-Iong-chainfatty acids was lost by Triton X-100 treatment. Palmitate and stearate synthesis was unaffected or sIightly increased. Addition of dipalmitoylphosphatidylcholine was made to microsomal fractions in an attempt to increase elongation rates. The phospholipid consistently increased total incorporation of [14C]malonyl-CoA into fatty acids, and the percentage elongation observed also occurred at higher rates. However, [1-'4C]palmitoylphosphatidylcholine,when added exogenously in the presence of Triton X-100,was not elongated, in spite ofthefact that the phospholipid was incorporated into the microsomal lipid by substitution. Moreover, further experiments showed that the stimulation of [14C]malonyl-CoA incorporation into microsomal fatty acids by exogenous lipids containing palmitate was relatively unspecific. Therefore in contrast with soluble palmitate elongase, the exact nature of the substrate used by the particular elongase remains undefined. However, it should be noted that addition of stearic acid or distearoylphosphatidylcholinefailed to increase fatty acid synthesis (Table 1)in the presence of Triton X-100. The effect of some inhibitors, chelating agents and thiols on microsomal fatty acid synthesis is shown in Table 2. Thiol-group reagents caused inhibition of fatty acid formation. Arsenite was found, as in other plant systems, to specifically prevent palmitate elongation without decreasing fatty acid synthetase activity. The inhibition caused by fluoride was not, apparently, due to chelation of essential divalent cations, since EDTA and EGTA failed to significantly alter fatty acid synthesis. Some dramatic differences were seen in the extent of inhibition of thiols when they were added to the assay system rather than preincubated with the microsomal fraction. These probably reflect the relative affinities of thiol groups on microsomal proteins, coenzyme-A and acyl-carrier protein. These studies were supported by a grant from the Science Research Council. Bolton, P. & Harwood, J. L. (1976) Phytochemistry in the press Harwood, J. L. (1974) Lipids 9, 850-854 Harwood, J. L. (1975) in Recent Advances in the Chemistry and Biochemistry of Plant Lipids (Galliard, T. & Mercer, E. I., eds.), pp. 43-93, Academic Press, London and New York Harwood, J. L. & Stumpf, P.K. (1971) Arch. Biochem. Biophys. 142, 281-291 Jaworski, J. G., Goldschmidt,E. E. & Stumpf, P. K. (1974) Arch. Eiochem. Eiophys. 163,769-776 Kolattukudy, P.E. & Buckner, J. S. (1972) Eiochern. Eiophys. Res. Commun. 46,801-807 Weiare, P. J. & Kekwick, R. G. 0. (1975) Eiochem. J. 146,439445
VOl. 5
10
