Fatty-Acid Synthesis in Lactating-Goat Mammary Gland

Transcription

1 Eur. J. Biochem. 95, (1979) FattyAcid Synthesis in LactatingGoat Mammary Gland 2. MediumChain Fatty Acid Synthesis Inger GRUNNET and Jens KNUDSEN Institute of Biochemistry, Odense University (Received November 22, 1978) Addition of goat, rat, rabbit and cow mammary microsomal fraction to fatty acid synthetase of goat mammary gland induced CIO fatty acid synthesis. When the microsomal fractions from rat, rabbit and cow mammary gland were incubated with their respective fatty acid synthetases only the cow enzyme synthesized significant amounts of Clo compared to the purified fatty acid synthetases alone. The goat mammary microsomal fraction was not able to induce CIO synthesis by rat and rabbit mammarygland fatty acid synthetase, but only by the goat and cow enzyme. Both goat and cow mammarygland fatty acid synthetase have in contrast to the rabbit and rat enzymes the ability to hydrolyse mediumchain acylthioesters. We therefore suggest a novel mechanism for mediumchain fatty acid synthesis in lactatinggoat mammary gland. This mechanism involves an activation of the mediumchain acylthioester hydrolase within the fatty acid synthetase of goat mammary gland by an unknown microsomal factor. In the preceding paper dealing with mediumchain fatty acid synthesis in goat mammary gland (C8:o and Clo,o) we reported that tissue slices and particlefree supernatant incubated with microsomal fraction synthesized both short, medium and longchain fatty acids [l]. In contrast the purified fatty acid synthetase and particlefree supernatant alone could not synthesize significant amounts of mediumchain fatty acids. Furthermore it was not possible to show the presence of a cytosolic mediumchainterminating acylthioester hydrolase, like the one found in rabbit and rat mammary gland [2,3]. In the present paper the effect of the microsomal fraction on mediumchain fatty acid synthesis is further examined in experiments with purified fatty acid synthetase. MATERIALS AND METHODS Materials used were as described in [l]; [114C] acylcoa esters were synthesized according to 121. Purgication of Fatty Acid Synthetases Mammary gland fatty acid synthetase was purified from lactating goat, cow, rat and rabbit as previously described for cow [4]. Preparation of Microsomal Fractions Microsomal fractions from lactating goat, cow, rat and rabbit mammary gland were prepared as previously described [5]. Incubation with Purified Fatty Acid Synthetase Incubation systems b or c as in [l] were used. Microsomal fraction was added as indicated in the tables. All incubations were stopped by adding 5 M NaOH and the samples were analysed for total fatty acids [5]. Measurement of the Thioesterase Specificity of Cow MammaryGland Fatty Acid Synthetase The thioesterase specificity of cow mammarygland fatty acid synthetase towards acylcoa model substrates was measured as described [6]. To avoid the problems of critical micellar concentrations, the specificity was measured at low substrate concentrations i.e. 3 pm. Measurement of Protein Proteins were precipitated by 15 % (w/v) trichloroacetic acid and analysed by the method of Lowry et al. [7] with bovine serum albumin as standard. RESULTS Fatty Acid Synthesis by Purgied LactatingGoat MammaryGland Fatty Acid Synthetase in the Presence of Goat Mammary Microsomal Fraction Incubation of microsomal fraction and purified fatty acid synthetase from lactatinggoat mammary

2 ~ ~ ~~~ _ ~~ 504 MediumChain FattyAcid Synthesis in Goat Mammary Gland. 2 Table 1. The effect of microsomal fraction on the chain length of fatty acids synthesized by purified goat mammarygland fatty acid synthetase Incubations (15 min) were as described in Methods. The incubations contained 207 pg fatty acid synthetase (specific activity 850 nmol NADPH oxidized x min' x mg protein'), 35 pg acetylcoa carboxylase (specific activity 260 nmol malonylcoa formed x min' x mg protein') and 100 pm [l'4c]acetylcoa (5.52 Ci/mol) in a total volume of 1 ml. The values for the proportions of fatty acid synthesized are means of two determinations. For total fatty acid synthesized mean f S.D. is shown Experiment Microsomal Total fatty acid Fatty acid synthesized fraction synthesized ~ ~ ~ ~ ~ ~ (acetate incorporated c40 CSO c80 ClOO c120 c140 c160 c180 from [l14c]acetylcoa) mg protein f ' I f f Table 2. Fatty acid synthesis by the microsomal fraction and heat sensitivity of the microsomal induction of mediumchain fatty acid synthesis by purified goat mammarygland fatty acid synthetase Incubations (15 min) were as described in Methods. The incubation mixture contained fatty acid synthetase (specific activity 1100 nmol NADPH oxidized x mini x mg protein') and microsomal fraction as indicated in the table and 40 pm [114C]acetylCoA (5.95 Ci/mol) in a total volume of 1 ml. MalonylCoA was infused in a rate of 5 nmol/min. The values for the properties of fatty acids synthesized are means of two determinations. For total fatty acid synthesized mean f S.D. is shown Experi Fatty acid Microsomal Total fatty acid Fatty acid synthesized ment synthetase fraction synthesized ~ ~ ~ ~. ~~ ~~ (acetate incorporated c4:o cs:o c8.0 cl0:o c1z:o c14:o c16:o c18: from [l14c]acetylcoa) mg protein ~ f boiled f gland in a buffer containing the substrates needed for fatty acid and triglyceride synthesis and with ratelimiting malonylcoa concentration, resulted in induction of mediumchain fatty acid synthesis. The total and relative amount of Cl0 synthesized was increased, whereas that of longerchain fatty acids was decreased compared to incubations with fatty acid synthetase alone (Table 1). Increasing the amount of added microsomal protein decreased total fatty acid synthesis and shifted the chain length towards shortchain fatty acids. The microsomal fraction itself synthesized only small amounts of shortchain and longchain fatty acids and no mediumchain fatty acids (Table 2). Boiling of the microsomal fraction destroyed the ability to induce mediumchain fatty acid synthesis (Table 2). The results indicate that the microsomal fraction is involved in the control of fatty acid chain termination in goat mammary gland, and that the controlling factor is heatlabile and therefore probably a protein factor. The above described induction of CIO fattyacid synthesis by the microsomal fraction could be due to either the presence in goat mammary microsomal fraction of a mediumchainterminating acylthio ester hydrolase like the one found in rabbit mammary gland cytosol [2] or to activation of the mediumchain acylthioester hydrolase present in the goat mammarygland fatty acid synthetase complex [6]. To test these two different possibilities the following experiments were carried out. Fatty Acid Synthesis by Goat MammaryGland Fatty Acid Sjwthetase in the Presence of Dgjrrerit Mammary Microsomal Fractions Fatty acid synthetase from goat mammary gland was incubated with the microsomal fractions from goat, rat, cow and rabbit mammary gland. Both cow, rat and rabbit mammary microsomal fraction induced, like the goat mammary microsomal fraction, mediumchain fatty acid synthesis by goat mammarygland fatty acid synthetase (Table 3). However, rat and rabbit mammary microsomal fractions could not induce mediumchain fatty acid synthesis when incubated with their corresponding fatty acid synthetases (Table 4). The pattern of fatty acids synthesized in these experiments were similar to that synthesized by the purified fatty acid synthetases alone (Table 5).

3 ~ ~ I. Grunnet and J. Knudsen 505 Table 3. Fatty acid synthesis by goat mammarygland fatty acid synthetase incubated with mammarygland microsomal fractions of different species Incubations were made as described in Methods. The incubation mixtures contained 100 pg fatty acid synthetase (specific activity: 980 nmol NADPH oxidized x min' x mg protein'), 200 pg microsomal protein, and 40 pm [l'4c]acetylcoa (specific activity 3.8 Ci/mol) in a total volume of 0.5 ml. MalonylCoA was infused at a rate of 2.1 nmol/min. The values for the properties of fatty acids synthesized are means of two determinations. For total fatty acid synthesized mean 2 S.D. is shown ~ of mammary gland of mammary synthesized fatty acid microsomal (acetate incorporated c40 c60 CS0 clo0 cl20 c140 c160 c180 synthetase fraction from [l'4c]acetylcoa) ~.~ Goat 75 f Goat goat 85f Goat cow 71 f Goat rabbit 72i Goat rat 83 I Table 4. Fatty acid synthesis by different species of mammarygland fatty acid synthetases and their respective microsomal fr&tions Incubations were as described in Table 3. The specific activities of the fatty acid synthetases were: cow 260, rabbit 740 and rat 1340 nmol NADPH oxidized x min' x mg protein' of mammary gland of mammary synthesized fatty acid microsomal (acetate incorporated c4:o c6:o c8:o clo0 c12:0 C14:O c16:o c18:o synthetase fraction from [1'4C]acetylCoA) ~. _ ~. ~ ~ cow Rabbit Rat cow 5.2 f 0.3 rabbit rat 8.0 f Table 5. Fatty acid synthesis by mammarygland fatty acid synthetases of different species Incubations were as described in Tables 3 and 4 except that no microsomal protein was added Source Total fatty acid Fatty acid synthesized of mammary gland synthesized fatty acid synthetase (acetate incorporated c4:o c6:0 c8:o ClO.0 c12:o c14:o c16:o c18:o from [l'4c]acetylcoa).~ Goat cow Rabbit Rat ~ ~ f f f Therefore the effect of rabbit and rat mammary microsoma1 fraction on C10 synthesis by goat mammarygland fatty acid synthetase could not be explained by a contamination of the microsomal fraction with mediumchain acylthioester hydrolase. The results suggest that the induction of mediumchain fatty acid synthesis by goat mammarygland fatty acid synthetase with mammary microsomal fractions is unspecific and not due to the presence of mediumchainterminating acylthioester hydrolase in the goat mammary microsomal fraction. Like goat, cow mammarygland fatty acid synthetase is also able to synthesize Cl0 fatty acids, when incubated with cow mammary microsomal fraction (Tables 4 and 5). This indicates that ruminant, cow and goat, mammarygland fatty acid synthetases may differ from the rat and rabbit mammary enzymes with respect to the ability to terminate fatty acid synthesis at mediumchain length. Further indication for this is obtained in the following experiment. Fatty Acid Synthesis by Goat, Cow, Rut and Rabbit MammaryGland Fatty Acid Synthetases in the Presence of Gout Mammary Microsomal Fraction Fatty acid synthetases from goat, cow, rat and rabbit mammary gland were incubated with substrates necessary for fatty acid and triglyceride synthesis in

4 ~~ ~ 506 MediumChain FattyAcid Synthesis in Goat Mammary Gland. 2 Table 6. Fatty acid synthesis by different species of mammaryglandfatty acid synthetases incubated with goat microsomal fraction Incubations were made as described in Tables 3 and 4 of mammary gland of mammary synthesized fatty acid microsomal (acetate incorporated c4:o c6:o c8:o cl0:o C12:O c14:o c16:o c18:o synthetase fraction from [l'4c]acetylcoa) Goat goat 8.5 i 0.4 cow goat 8.8 I 0.3 Rabbit goat 6.1 & 1.8 Rat goat I I I the presence of goat mammary microsomal fraction. As in the previous experiments, malonylcoa was infused continuously at low rate. Goat mammary microsomal fraction was able to induce mediumchain fatty acid I synthesis by both goat and cow mammarygland fatty acid synthetases, but not with the rat and rabbit mammarygland enzymes (Table 6). Control experiments showed that none of the purified fatty acid synthetases under these conditions synthesized significant amounts of mediumchain fatty acids themselves (Table 5). This strongly suggests that the ruminant, cow and goat, mammary gland fatty acid synthetase differs from rat and rabbit mammarygland fatty acid synthetases with regard to the ability to terminate fatty acid synthesis at mediumchain length. Specijiicity of the Terminating Thioesterase in Cow MammaryGland Fatty Acid Synthetase We have previously shown that goat mammarygland fatty acid synthetase in contrast to the rabbit enzyme can hydrolyse mediumchain acylcoa model substrates [6]. This indicates that the goat mammarygland fatty acid synthetase has an inherent ability to terminate fatty acids at mediumchain length. The results in Fig.1 show that also cow mammarygland fatty acid synthetase can hydrolyse mediumchain length fatty acids with 18% of the activity found for palmitoylcoa. This further support the hypothesis that goat and cow mammarygland fatty acid synthetase differs from the rat and rabbit mammary enzyme in the ability to terminate fatty acid synthesis at mediumchain length. DISCUSSION The present results strongly suggest that goat and cow mammarygland fatty acid synthetases in contrast to the rat and rabbit enzymes have the ability to terminate fatty acid synthesis at mediumchain length. However, interaction with an unknown heatlabile I. " c4:o ' f6:o c8:o cl0:o clz:o c14:o c160 c180 AcylCoA chain length Fig. 1. Acylthioesterase specificity of' cow mammarygland fatty acid synthetase. The enzyme (1 pg) was incubated for 10 min in experiments with C4:0 and c6:o acylcoa esters, 2 min with c8:oc12:0 acylcoa esters, and 0.5 min with c14:0c18:0 acylcoa esters in a total volume of 0.5 ml Table I. Mediumchain fatty acid synthesis by goat, cow, rabbit and rat mammarygland fatty acid synthetases Summary of Tables 3, 4, 5 and 6. n.d., not determined Microsomal fraction Mediumchain fatty acid synthesis with fatty acid synthetase from goat cow rabbit rat Goat cow + n.d. n.d. Rabbit n.d. n.d. Rat + n.d. n.d. factor in the microsomal fraction is required to ensure mediumchain fatty acid synthesis by the two fatty acid synthetases. It is unlikely that this heatlabile factor could be a mediumchainterminating acylthioester hydrolase, like the one found in rabbit and rat mammary gland [2,3]. This conclusion is based upon the experiments with rat, rabbit and goat microsoma1 fractions and purified fatty acid synthetases, the results of which are summarized in Table 7. All these microsomal fractions were able to induce mediumchain fatty acid synthesis by goat mammary

5 I. Grunnet and J. Knudsen 507 gland fatty acid synthetase, but not with the enzymes from rat and rabbit mammary gland. Both goat and cow mammary gland fatty acid synthetases can hydrolyse mediumchain acylcoa esters (Fig. 1, [6]). In addition it has not been possible to show the presence of a mediumchainterminating acylthioester hydrolase in the cytosol of lactatinggoat mammary gland [l]. We therefore suggest that mediumchain fatty acid synthesis in lactatinggoat mammary gland is accomplished through an activation of the mediumchain acylthioester hydrolase within the fatty acid synthetase by an unknown microsoma1 factor. It is interesting that rabbit and rat mammarygland microsomal fraction also contain this factor. Both these microsomal fractions synthesize triglycerides with a high content of mediumchain fatty acids in vivo. The factor might therefore be a general component in the triglyceridesynthesizing systems that are involved in synthesis of triglycerides containing high proportions of mediumchainlength fatty acids. This is a novel mechanism for mediumchain fatty acid termination, different from the one found in rat and rabbit mammary gland. As mentioned earlier, mediumchain fatty acids are terminated in these tissues by a separate low molecular cytosolic acylthioester hydrolase. The fact that both goat and cow mammarygland fatty acid synthetase can terminate fatty acids at mediumchain length indicates that this might be a general phenomenon for ruminant fatty acid synthetases. Unpublished results with sheep mammarygland fatty acid synthetase further support this. We thank Annelise Wendelboe Andersen, Inge Fabricius and Conny Henneberg for excellent technical assistance. REFERENCES 1. Grunnet, I. & Knudsen, J. (1979) Eur. J. Biochem. 95, Knudsen, J., Clark, S. & Dils, R. (1977) Biochem. J. 160, Libertini, L. & Smith, S. (1978) J. Biol. Chem. 253, Knudsen, J. (1972) Biochim. Biophys. Acla, 280, Knudsen, J. (1976) Comp. Biochem. Physiol. 53B, Grunnet, I. & Knudsen, J. (1978) Biochem. Biophys. Res. Commun. 80, Lowry, 0. H., Roseborough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, I. Grunnet and J. Knudsen, Biokemisk Institut, Odense Universitet, Campusvej 55, DK5230 Odense M, Denmark