312
GENERAL ANALYTICAL METHODS
[39]
[39] M e a s u r e m e n t o f M a l o n y l C o e n z y m e A B y ROBERT W. GUYNN and RICHARD L. VEECH
Malonyl-CoA is a key intermediate in fatty acid synthesis. It is generated in the cytoplasm by acetyl-CoA carboxylase (EC 6.4.1.2)' and utilized both in fatty acid synthesis de novo (by fatty acid synthase multienzyme complex)~-4 and in chain lengthening processes (by the microsomal chain lengthening system)2 Therefore, since the steady state concentration of malonyl-CoA will be a function of the rate of production by acetyl-CoA carboxylase and the rate of utilization by the fat synthesizing systems, measurement of this metabolite is a useful tool in the study of the control of fatty acid synthesis in vivo. Measurement of malonyl-CoA has proved to be especially useful when combined with an estimate of the rate of fatty acid synthesis in vivo. `~
Principle
Malonyl-CoA is measured enzymatically in tissue extracts with purified rat liver fatty acid synthase which, using NADPH and acetyl-CoA or another suitable primer, converts malonyl-CoA to fatty acids. The oxidation of NADPH is followed spectrophotometrically at 340 nm, 2 molecules of NADPH being oxidized for each molecule of malonyl-CoA consumed. Acetyl-CoA -~ n malonyl-CoA- -~ 2n NADPH -~- 3n H + CH3(CH2CH2)~_ICH2CH2COOH ~- 2n NADP + +nCO2+nH20+ ( n + 1) CoA Tissue Preparation
The liver is freeze-clamped and powdered under liquid nitrogen. A 1:5 extract of the powdered liver is prepared in 0.85 M HCI04 and centrifuged at 35,000 g for 15 minutes at 0% The resulting supernatant is carefully neutralized with KOH, taking care not to exceed pH 7.0, and the ~S. J. Wakil, E. B. Titchener, and D. M. Gibson, Biochim. Biophys. Acta 29, 225 (1958). S. J. Wakil and J. Ganguly,J. Amer. Chem. Soc. 81, 2597 (1959). 3 R. O. Brady, R. M. Bradley, and E. G. Trams, J. Biol. Chem. 235, 3093 (1960). 4F. Lynen, Biochem. J. 102, 381 (1967). 5S. Abraham, I. L. Chaikoff, and W. M. Bortz, Nature (London) 192, 1287 (1961). R. W. Guynn, D. Veloso, and R. L. Veech, J. Biol. Chem. 247, 7325 (1972).
[39]
MALONYL-CoA ASSAY
313
samples are allowed to gtand in ice for half an hour. The KC104 precipitate is packed by centrifugation and the supernatant is used for the assay. Perchloric acid extracts of tissue, although very inhibitory of fatty acid synthase, are convenient to use and permit extraction of malonyl-CoA without significant hydrolysis. The recovery of malonyl-CoA added to tissue was found to be 96%.
Assay Reagents All prepared in deionized water 1 M potassium phosphate buffer, pH 7.0 0.125 M EDTA, pH 7.0 2 mM acetyl-CoA (free acid or trilithium salt) Activating solution: 1 M potassium phosphate buffer containing 20 mM dithiothreitol and 2 mM EDTA, pH 7.0
Enzyme Rat liver fatty acid synthase is prepared by the method of Nepokroeff et aU The dialyzed enzyme is usually satisfactory for the assay after the second ammonium sulfate fractionation. The preparation must be checked for NADP ÷ or NADPH-using enzymes. The enzyme was assayed spectrophotometrically at 25°. 7,~ Occasionally batches of the enzyme will contain significant glucose-6-phosphate dehydrogenase activity which interferes with the malonyl-CoA assay since it reduces NADP ÷ formed during the fatty acid synthase reaction. In spite of the different molecular weights of glucose-6-phosphate dehydrogenase and fatty acid synthase they are not separated by Sephadex chromatography. Glucose-6-phosphate dehydrogenase contamination may be minimized by following the procedure of Nepokroeff et al. exactly. Special attention should be given to the feeding schedule and size of rat. T Before use the enzyme is activated by incubating for 30 minutes at 38 ° with an equal volume of activating solution.
Assay Procedure For convenience a reagent cocktail is prepared. An amount sufficient for five assays consists of 5 mg of NADPH dissolved in 2.0 ml 1 M potassium phosphate buffer, pH 7.0; 2.0 ml 0.125 M EDTA, pH 7.0; and 0.2 ml 2 mM acetyl-CoA. Although useful in activating the enzyme, C. M. Nepokroeff,M. R. Lakshmann,and J. W. Porter, this volume [6]. 8A unit of enzyme is defined as that amount of protein necessary to catalyze the utilization of 1 t~moleof malonyl-CoAper minute.
314
G E N E R A L ANALYTICAL METHODS
[39]
dithiothreitol is not added to the cuvette since it has been found to accelerate a nonenzymatic oxidation of the N A D PH in the presence of the tissue extract. High concentrations of NADPH seem to be necessary to insure a stoichiometric assay. Lowering the NADPH concentration by more than half results in a less reliable or nonlinear assay. Duplicate cuvettes of 1 cm light path are prepared, each containing 2.0 ml of neutralized tissue extract and 400 ul of the reagent cocktail. The cuvettes are placed in a dual beam spectrophotometer and recorder of sufficient stability at high optical densities to measure relatively small changes (0.010-0.080 OD unit). With full-scale expansion to 0.1 OD units a base line recording is taken. Since the cuvettes should be identical this base line will be flat. A significant slope to the base line which cannot be related to drift in the recording instrument implies that the cuvettes are not identical. The only satisfactory course in such a situation is to reprepare the cuvettes. If the base line is suitable, 2-5 X 10-.2 unit s of fatty acid synthase is added to one cuvette and an equal volume of diluted (1:2) activating solution to the other cuvette. The optical density change is followed by continuous recording until completion (approximately 30 minutes). At the end of the reaction, second additions of enzyme and diluted activating solution are made to determine the correction for absorption by the enzyme. Standard malonyl-CoA can then be added to both cuvettes to check the stoichiometry of the assay. In all cases once the enzyme has been added mixing of the cuvettes should be done gently since the enzyme is readily inactivated at interfaces. Calculations
The reaction is stoichiometric; 2 N A D PH molecules are oxidized for each malonyl-CoA molecule incorporated. Therefore, the concentration of malonyl-CoA in the neutralized extract can be calculated directly from the change in optical density, the final volume in the cuvette, the extract sample volume, and the extinction coefficient of N A D PH (c = 6.22 X I0 ~' cm2/mole) o: umoles malonyl-CoA/ml neutral extract, (2.0 ml sample volume) 6.22 Sensitivity Under the conditions of the assay the reaction is linear with 2-40 nmoles of added malonyl-CoA per euvette. ~ B. L. I t o r e e k e r a n d A. K o r n b e r g , J. Biol. Chem. 175, 385 (1948).
[40]
FATTY ACID DISTRIBUTION IN GLYCEROLIPIDS
315
Standardization
The standard malonyl-CoA is assayed in pure solution by the same procedure. In the absence of KCI04 the reaction is much more rapid than in tissue extracts. The value obtained has been found to agree well with the amount of CoA released by complete hydrolysis of the standard under mildly aklaline conditions in the presence of dithiothreitol. Normal Values The concentration of malonyl-CoA in liver tissue varies considerably under different conditions. The following values have been found in rat liver: Rats starved 24 to 48 hours or fed a high fat diet have 0.004-0.006 ~mole/g wet weight, rats fed ad libitum on a diet containing 4% fat have 0.013-0.015 ~mole/g wet weight, and rats meal-fed 3 hours a day on the same diet have 0.020-0.038 ~mole/g wet weight immediately after eating. Discussion
The assay so described has proved very useful in liver, but coenzyme A derivatives in other tissues are present in lower concentrations; for example, the concentration of malonyl-CoA in brain is below the sensitivity of the assay as described.
[40] Determination of the Positional Distribution of Fatty Acids in Glycerolipids By H. BROCKERHOFF Introduction
Glycerol has one secondary and two primary hydroxyl groups. The two primary groups are stereochemically distinguishable in spite of the fact that there is a plane of symmetry in the glycerol molecule, because esterification of one or the other primary group, e.g., with phosphoric acid, leads to optically active derivatives, and so does the esterification of each hydroxyl with different fatty acids. In the recommended and generally used nomenclature, glycerol is viewed in a Fischer projection with its secondary hydroxyl to the left (in L configuration), and the three carbons are numbered from top to bottom. Glycerol so numbered is called sn-glycerol, for stereospecifically nmnbered.
GENERAL ANALYTICAL METHODS
[39]
[39] M e a s u r e m e n t o f M a l o n y l C o e n z y m e A B y ROBERT W. GUYNN and RICHARD L. VEECH
Malonyl-CoA is a key intermediate in fatty acid synthesis. It is generated in the cytoplasm by acetyl-CoA carboxylase (EC 6.4.1.2)' and utilized both in fatty acid synthesis de novo (by fatty acid synthase multienzyme complex)~-4 and in chain lengthening processes (by the microsomal chain lengthening system)2 Therefore, since the steady state concentration of malonyl-CoA will be a function of the rate of production by acetyl-CoA carboxylase and the rate of utilization by the fat synthesizing systems, measurement of this metabolite is a useful tool in the study of the control of fatty acid synthesis in vivo. Measurement of malonyl-CoA has proved to be especially useful when combined with an estimate of the rate of fatty acid synthesis in vivo. `~
Principle
Malonyl-CoA is measured enzymatically in tissue extracts with purified rat liver fatty acid synthase which, using NADPH and acetyl-CoA or another suitable primer, converts malonyl-CoA to fatty acids. The oxidation of NADPH is followed spectrophotometrically at 340 nm, 2 molecules of NADPH being oxidized for each molecule of malonyl-CoA consumed. Acetyl-CoA -~ n malonyl-CoA- -~ 2n NADPH -~- 3n H + CH3(CH2CH2)~_ICH2CH2COOH ~- 2n NADP + +nCO2+nH20+ ( n + 1) CoA Tissue Preparation
The liver is freeze-clamped and powdered under liquid nitrogen. A 1:5 extract of the powdered liver is prepared in 0.85 M HCI04 and centrifuged at 35,000 g for 15 minutes at 0% The resulting supernatant is carefully neutralized with KOH, taking care not to exceed pH 7.0, and the ~S. J. Wakil, E. B. Titchener, and D. M. Gibson, Biochim. Biophys. Acta 29, 225 (1958). S. J. Wakil and J. Ganguly,J. Amer. Chem. Soc. 81, 2597 (1959). 3 R. O. Brady, R. M. Bradley, and E. G. Trams, J. Biol. Chem. 235, 3093 (1960). 4F. Lynen, Biochem. J. 102, 381 (1967). 5S. Abraham, I. L. Chaikoff, and W. M. Bortz, Nature (London) 192, 1287 (1961). R. W. Guynn, D. Veloso, and R. L. Veech, J. Biol. Chem. 247, 7325 (1972).
[39]
MALONYL-CoA ASSAY
313
samples are allowed to gtand in ice for half an hour. The KC104 precipitate is packed by centrifugation and the supernatant is used for the assay. Perchloric acid extracts of tissue, although very inhibitory of fatty acid synthase, are convenient to use and permit extraction of malonyl-CoA without significant hydrolysis. The recovery of malonyl-CoA added to tissue was found to be 96%.
Assay Reagents All prepared in deionized water 1 M potassium phosphate buffer, pH 7.0 0.125 M EDTA, pH 7.0 2 mM acetyl-CoA (free acid or trilithium salt) Activating solution: 1 M potassium phosphate buffer containing 20 mM dithiothreitol and 2 mM EDTA, pH 7.0
Enzyme Rat liver fatty acid synthase is prepared by the method of Nepokroeff et aU The dialyzed enzyme is usually satisfactory for the assay after the second ammonium sulfate fractionation. The preparation must be checked for NADP ÷ or NADPH-using enzymes. The enzyme was assayed spectrophotometrically at 25°. 7,~ Occasionally batches of the enzyme will contain significant glucose-6-phosphate dehydrogenase activity which interferes with the malonyl-CoA assay since it reduces NADP ÷ formed during the fatty acid synthase reaction. In spite of the different molecular weights of glucose-6-phosphate dehydrogenase and fatty acid synthase they are not separated by Sephadex chromatography. Glucose-6-phosphate dehydrogenase contamination may be minimized by following the procedure of Nepokroeff et al. exactly. Special attention should be given to the feeding schedule and size of rat. T Before use the enzyme is activated by incubating for 30 minutes at 38 ° with an equal volume of activating solution.
Assay Procedure For convenience a reagent cocktail is prepared. An amount sufficient for five assays consists of 5 mg of NADPH dissolved in 2.0 ml 1 M potassium phosphate buffer, pH 7.0; 2.0 ml 0.125 M EDTA, pH 7.0; and 0.2 ml 2 mM acetyl-CoA. Although useful in activating the enzyme, C. M. Nepokroeff,M. R. Lakshmann,and J. W. Porter, this volume [6]. 8A unit of enzyme is defined as that amount of protein necessary to catalyze the utilization of 1 t~moleof malonyl-CoAper minute.
314
G E N E R A L ANALYTICAL METHODS
[39]
dithiothreitol is not added to the cuvette since it has been found to accelerate a nonenzymatic oxidation of the N A D PH in the presence of the tissue extract. High concentrations of NADPH seem to be necessary to insure a stoichiometric assay. Lowering the NADPH concentration by more than half results in a less reliable or nonlinear assay. Duplicate cuvettes of 1 cm light path are prepared, each containing 2.0 ml of neutralized tissue extract and 400 ul of the reagent cocktail. The cuvettes are placed in a dual beam spectrophotometer and recorder of sufficient stability at high optical densities to measure relatively small changes (0.010-0.080 OD unit). With full-scale expansion to 0.1 OD units a base line recording is taken. Since the cuvettes should be identical this base line will be flat. A significant slope to the base line which cannot be related to drift in the recording instrument implies that the cuvettes are not identical. The only satisfactory course in such a situation is to reprepare the cuvettes. If the base line is suitable, 2-5 X 10-.2 unit s of fatty acid synthase is added to one cuvette and an equal volume of diluted (1:2) activating solution to the other cuvette. The optical density change is followed by continuous recording until completion (approximately 30 minutes). At the end of the reaction, second additions of enzyme and diluted activating solution are made to determine the correction for absorption by the enzyme. Standard malonyl-CoA can then be added to both cuvettes to check the stoichiometry of the assay. In all cases once the enzyme has been added mixing of the cuvettes should be done gently since the enzyme is readily inactivated at interfaces. Calculations
The reaction is stoichiometric; 2 N A D PH molecules are oxidized for each malonyl-CoA molecule incorporated. Therefore, the concentration of malonyl-CoA in the neutralized extract can be calculated directly from the change in optical density, the final volume in the cuvette, the extract sample volume, and the extinction coefficient of N A D PH (c = 6.22 X I0 ~' cm2/mole) o: umoles malonyl-CoA/ml neutral extract, (2.0 ml sample volume) 6.22 Sensitivity Under the conditions of the assay the reaction is linear with 2-40 nmoles of added malonyl-CoA per euvette. ~ B. L. I t o r e e k e r a n d A. K o r n b e r g , J. Biol. Chem. 175, 385 (1948).
[40]
FATTY ACID DISTRIBUTION IN GLYCEROLIPIDS
315
Standardization
The standard malonyl-CoA is assayed in pure solution by the same procedure. In the absence of KCI04 the reaction is much more rapid than in tissue extracts. The value obtained has been found to agree well with the amount of CoA released by complete hydrolysis of the standard under mildly aklaline conditions in the presence of dithiothreitol. Normal Values The concentration of malonyl-CoA in liver tissue varies considerably under different conditions. The following values have been found in rat liver: Rats starved 24 to 48 hours or fed a high fat diet have 0.004-0.006 ~mole/g wet weight, rats fed ad libitum on a diet containing 4% fat have 0.013-0.015 ~mole/g wet weight, and rats meal-fed 3 hours a day on the same diet have 0.020-0.038 ~mole/g wet weight immediately after eating. Discussion
The assay so described has proved very useful in liver, but coenzyme A derivatives in other tissues are present in lower concentrations; for example, the concentration of malonyl-CoA in brain is below the sensitivity of the assay as described.
[40] Determination of the Positional Distribution of Fatty Acids in Glycerolipids By H. BROCKERHOFF Introduction
Glycerol has one secondary and two primary hydroxyl groups. The two primary groups are stereochemically distinguishable in spite of the fact that there is a plane of symmetry in the glycerol molecule, because esterification of one or the other primary group, e.g., with phosphoric acid, leads to optically active derivatives, and so does the esterification of each hydroxyl with different fatty acids. In the recommended and generally used nomenclature, glycerol is viewed in a Fischer projection with its secondary hydroxyl to the left (in L configuration), and the three carbons are numbered from top to bottom. Glycerol so numbered is called sn-glycerol, for stereospecifically nmnbered.
