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[29] Long-chain fatty acyl-CoA thioesterase from Mycobacterium smegmatis
242
FATTY ACID SYNTHESIS
[29]
the enzyme protein after ammonium sulfate fractionation gives the same value, it is likely that the 2.1 S form is structurally stable and is not a result of proteolysis during purification. The purified enzyme is composed of one polypeptide with a molecular weight of 19,000. The Stokes' radius of the enzyme is 19/~.3 Substrate Specificity. At saturating levels of substrates and in the presence of serum albumin, maximal rate of hydrolysis is found with palmitoyl-CoA, the activity decreasing upon either increasing or decreasing the length of the saturated fatty acyl-CoA chains.ll Lauroyl-CoA, myristoyl-CoA, and stearoyl-CoA are hydrolyzed at about 20, 55, and 30% the rate, respectively, of that of palmitoyl-CoA, a The purified enzyme also hydrolyzes unsaturated acyl-CoA esters. The rate of oleoylCoA hydrolysis is only 15% of the rate of palmitoyl-CoA, a No hydrolysis is detectable with decanoyl-CoA or acyl-CoA of shorter chain length. No hydrolysis of palmitoyl-L-carnitine, cholesteryl oleate, tripalmitoyl glycerol, and dipalmitoylphosphatidylcholine is detectable with the purified enzyme. 3 The enzyme has neither palmitoyl-CoA synthase activity, nor carnitine palmitoyltransferase activity, nor acid and alkaline phosphatase activity. 3 The enzyme thus seems to be specific for longchain fatty acyl-CoA esters.
[29] L o n g - C h a i n F a t t y A c y l - C o A T h i o e s t e r a s e f r o m
Mycobacteriurn smegmatis By KENICHI K. YABUSAKI and CLINTON E. BALLOU A thioesterase, with a specificity for long-chain (C12-Cls) acyl-CoA derivatives is present in extracts of Mycobacterium smegmatis (ATCC 356). The action of this enzyme on palmitoyl-CoA is inhibited by the polymethylpolysaccharides found in mycobacteria. ~
Assay Procedure
Principle. The assay is based on the liberation of the thiol group of CoA, reaction (I), as measured by the increase in absorbance at 412 n m due to the reaction of free C o A with 5,5'-dithiobis(2-nitrobenzoicacid) (DTNB). 2
K. K. Y a b u s a k i a n d C. E. Ballou, J. Biol. Chem. 254, 12,314 (1979). z G. L. E l l m a n , Arch. Biochem. Biophys. 82, 70 (1959).
METHODS IN ENZYMOLOGY, VOL. 71
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181971-X
[29]
LONG-CHAIN FATTY ACYL-CoA THIOESTERASE
243
AcyI-SCoA + H~O ~ fatty acid + CoASH
(1)
Reagents Tris-HC1 buffer, 0.1 M, pH 8.0 Palmitoyl-CoA, 5/aM Ovalbumin, 7.0 mg/ml in 0.1 M Tris-HC1 buffer, pH 8.0 DTNB, 0.01 M in 0.1 M potassium phosphate buffer, pH 7.0 Procedure. The complete assay mixture contains 10/~mol of Tris-HC1 buffer, pH 8.0, 0.05 /zmol of DTNB, 0.01 /xmol of palmitoyl-CoA, and 1-1.5 mU of the thioesterase in a final volume of 2.0 ml. To observe initial rates of hydrolysis of palmitoyl-CoA, it was necessary to dilute the enzyme (final step of purification) severalfold. Typically, a 5-/zl aliquot of the stock thioesterase solution containing 0.125 mg of protein per milliliter was diluted to 0.2 ml with a solution of ovalbumin at 7.0 mg/ml in 0.1 M Tris-HC1 buffer, pH 8.0. After temperature equilibration and recording a base line, the sample cuvette was removed, diluted enzyme was added from a 4-/~1 microcapillary pipette, and a piece of parafilm was placed over the top of the cuvette, which was mixed by several inversions and then replaced in the sample holder. This total operation could be performed within 30 sec; the initial rates of hydrolysis were linear and were recorded over a period of 5 min or longer. A split-beam recording spectrophotometer with 0.05 or 0.1 full scale absorbance slide wire capability is convenient for the above measurements. A molar extinction coefficient of 13,600 is used to quantitate the DTNB reduction/ Since DTNB also reacts slowly with the thiol groups of proteins, the use of a split beam spectrophotometer is advantageous in that an aliquot of the protein solution can be added to the reference cuvette, which contains all assay components except the substrate, palmitoyl-CoA. Otherwise, it is necessary to correct the initial rates of reduction of DTNB by proteins by subtraction of controls in which palmitoyl-CoA is omitted. Units. An enzyme unit is defined as the amount of enzyme activity necessary to catalyze the hydrolysis of 1 /zmol of palmitoyl-CoA per minute under the above conditions. Specific activity is expressed as units per milligram of protein. Purification Procedure The following procedure, summarized in the table, yields an 8000-fold purification of the acyl-CoA thioesterase. 1 All operations were performed at 4° unless otherwise stated. Step I. Preparation of Crude Extract. Frozen M. smegmatis wet cells (200 g) were thawed, suspended in 800 ml of 0.1 M potassium phosphate buffer, pH 7.0, and broken by two passes through a Manton-Gaulin
244
[29]
FATTY ACID SYNTHESIS PURIFICATION OF Mycobacterium smegmatis THIOESTERASE
Step
Total activitya (units)
Specific activity (units/mgprotein)
Purification (fold)
Extract, 37,000gb Protamine sulfate extract Dialyzed (NH4)2SO4pellet DEAE-cellulose chromatography Hexylagarose chromatography BioGel A-0.5m chromatography
536 421 228 80 39 27
0.03 0.05 0.08 0.6 86 230
! 1.7 3 21 3007 8040
a Expressed as micromoles of CoA released per minute with 5 ~ substrate. b From 200 g of wet cell paste.
palmitoyl-CoA as
pressure cell operating at 10,000 psi. The broken cell suspension was centrifuged at 37,000 g for 1 hr. Step 2. Protamine Sulfate Extract. To the supernatant extract was added, slowly and with stirring, a 1% solution ofprotamine sulfate in 0.01 M potassium phosphate buffer, pH 7.0, to give a final ratio of 0.12 mg of protamine sulfate per milligram of protein. The solution was stirred for 30 min then centrifuged at 37,000 g for 15 min. The supernatant liquid containing the enzyme activity was removed, the pellet was washed by resuspension in 0.01 M potassium phosphate buffer, pH 7.0, and the suspension was centrifuged at 37,000 g for 15 min. This second supernatant was combined with the first. Step 3. Ammonium Sulfate Precipitation. The enzyme was precipitated from the above extract by adding ammonium sulfate to 55% of saturation. The mixture was stirred for 3 hr and then centrifuged at 16,000 g. The pellet was suspended in 0.01 M Tris-HC1 buffer, pH 8.0, and dialyzed for 24 hr against 7 liters of 0.01 M Tris-HC1 buffer, pH 8.0, with two changes of the buffer during that time. The protein solution was concentrated to 50 ml with an Amicon Diaflo apparatus fitted with a PM-10 membrane. Step 4. DEAE-Cellulose Chromatography. The concentrated protein solution from step 3 was applied to a DEAE-cellulose column (2.8 × 18 cm) equilibrated with 0.01 M Tris-HC1 buffer, pH 8.0, and fractions were collected by elution with the same buffer. After the absorbance at 280 nm dropped to a low value, a gradient of 0.01 to 0.5 M Tris-HC1, pH 8.0, was applied and fractions were collected. The major peak of thioesterase activity was eluted at a conductivity of 3-5 mmho. The active fractions were combined and concentrated to 5 ml, and glycerol was added to give a concentration of 10%. This solution was dialyzed against 4 liters of 0.01 M Tris-HCl, pH 8.0, containing 10% glycerol.
[29]
LONG-CHAIN FATTY A C Y L - C o A THIOESTERASE
245
Step 5. Hexylagarose Chromatography. The protein solution from step 4 was applied to a hexylagarose column (1.8 x 33 cm) equilibrated with 0.01 M Tris-HC1, pH 8.0, containing 10% glycerol, and the column was washed with the same buffer. After the absorbance at 280 nm dropped to a low value, the column was eluted with a gradient of 0.01 to 0.4 M TrisHC1, pH 8.0, containing 10% glycerol. The fractions that contained thioesterase activity were combined and concentrated to 1.5 ml. Step 6. BioGei A-O.5m Chromatography. The concentrated protein solution from step 5 was applied to a BioGel A-0.5m column (1.8 x 114 cm) equilibrated with 0.1 M Tris-HC1, pH 8.0, containing 10% glycerol. Fractions of 2.5 ml were collected by elution with the same buffer, and the active fractions were combined, concentrated, and stored at 4° in the final buffer. Properties
Purity. Preparations of thioesterase with a specific activity of 230 units per milligram of protein reveal two major components of about 22,000 and 20,000 MW and a few minor components by acrylamide gel electrophoresis under denaturing conditions. 1 Molecular Weight. The enzyme has an apparent molecular weight by gel permeation chromatography on Sephadex G-100 of about 40,000' and is probably composed of two subunits of about MW 20,000 from its properties during acrylamide gel electrophoresis under denaturing conditions. Requirements and Extent of Hydrolysis. Because of the high activity at the final stage of purification, in order to observe the initial rates of hydrolysis of palmitoyl-CoA, the enzyme has to be assayed in very dilute protein solutions. Dilution of the final stage purified enzyme with buffer alone causes a substantial loss in activity. Therefore, to stabilize the enzyme, a solution of ovalbumin I is used to dilute the enzyme instead of serum albumin, which is noted for its ability to bind palmitoyl-CoA. 3 The inactivation of enzyme activity by dilution with buffer alone is not observed at stages of purification prior to step 6. Thioesterase preparations from step 4, or at the final stage of purification in the presence of ovalbumin, will catalyze the complete conversion of palmitoyl-CoA to stoichiometric amounts of CoA and palmitate. Kinetic Parameters and pH Optimum. The thioesterase has an apparent K m and Vma x for monomeric palmitoyl-CoA of 9 # M and 107/anol of CoA released per minute per milligram of protein, respectively. With micellar palmitoyl-CoA as substrate, the thioesterase has an apparent K m 3 H. Knoche, T. W. Esders, and K. Bloch, J. Biol. Chem. 248, 2317 (1973).
246
FATTY ACID SYNTHESIS
[29]
and Vmax of 5/xM and 77/xmol of CoA released per minute per milligram of protein, respectively. It should be noted that the above Vma x data were determined on the purified enzyme after about 10 months of storage at 4° in the presence of 10% glycerol. These results indicate that the enzyme has good stability under these conditions. The optimal pH for thioesterase activity is 8.0. Specificity. At saturating levels of substrate, maximal initial rates of hydrolysis are found for palmitoyl-CoA and stearoyl-CoA. Activity decreases with decreasing length of the saturated fatty acyl chain. Approximately 5 times lower activity is observed with lauroyl-CoA over palmitoyl-CoA at equal concentrations. Very low rates of hydrolysis are observed for acyl-CoA thioesters of Ci0 chain length, and no detectable activity is observed for acyl chain lengths of C8 or less. Inhibitors. The mycobacterial polymethylpolysaccharides, 4 6-O-methylglucose polysaccharide (MGP) and 3-O-methylmannose polysaccharide, which form very stable complexes with palmitoyl-CoA, 5'6 are potent inhibitors of the thioesterase-catalyzed hydrolysis of both monomeric and micellar palmitoyl-CoA. The polysaccharides appear to have no effect on the Vma x but appear to increase the apparent K m with both monomeric and micellar palmitoyl-CoA by altering the free substrate concentration.' The effect of the polymethylpolysaccharides on the thioesterase-catalyzed hydrolysis of palmitoyl-CoA appears to result solely from the binding of the substrate, not from an interaction with the enzyme itself. This is suggested from the fact that MGP had no effect on the initial rate of hydrolysis of lauroyl-CoA a substrate that, because of its short lipid chain, forms only weak complexes with polymethylpolysaccharides. 1 The same conclusion is supported by the observation that the modified MGP, 1 which binds palmitoyl-CoA poorly, m also fails to inhibit. However, the possibility that the acyl-CoA-polymethylpolysaccharide complex itself is a very poor substrate has not been ruled out.
+ G. R. Gray and C. E. Ballou, this series, Vol. 35 [11]. K. Bloch, Adv. Enzymol. 45, 1 (1977). 6 K. K. Yabusaki and C. E. Ballou, Proc. Natl. Acad. Sci. U.S.A. 75, 691 (1978).
FATTY ACID SYNTHESIS
[29]
the enzyme protein after ammonium sulfate fractionation gives the same value, it is likely that the 2.1 S form is structurally stable and is not a result of proteolysis during purification. The purified enzyme is composed of one polypeptide with a molecular weight of 19,000. The Stokes' radius of the enzyme is 19/~.3 Substrate Specificity. At saturating levels of substrates and in the presence of serum albumin, maximal rate of hydrolysis is found with palmitoyl-CoA, the activity decreasing upon either increasing or decreasing the length of the saturated fatty acyl-CoA chains.ll Lauroyl-CoA, myristoyl-CoA, and stearoyl-CoA are hydrolyzed at about 20, 55, and 30% the rate, respectively, of that of palmitoyl-CoA, a The purified enzyme also hydrolyzes unsaturated acyl-CoA esters. The rate of oleoylCoA hydrolysis is only 15% of the rate of palmitoyl-CoA, a No hydrolysis is detectable with decanoyl-CoA or acyl-CoA of shorter chain length. No hydrolysis of palmitoyl-L-carnitine, cholesteryl oleate, tripalmitoyl glycerol, and dipalmitoylphosphatidylcholine is detectable with the purified enzyme. 3 The enzyme has neither palmitoyl-CoA synthase activity, nor carnitine palmitoyltransferase activity, nor acid and alkaline phosphatase activity. 3 The enzyme thus seems to be specific for longchain fatty acyl-CoA esters.
[29] L o n g - C h a i n F a t t y A c y l - C o A T h i o e s t e r a s e f r o m
Mycobacteriurn smegmatis By KENICHI K. YABUSAKI and CLINTON E. BALLOU A thioesterase, with a specificity for long-chain (C12-Cls) acyl-CoA derivatives is present in extracts of Mycobacterium smegmatis (ATCC 356). The action of this enzyme on palmitoyl-CoA is inhibited by the polymethylpolysaccharides found in mycobacteria. ~
Assay Procedure
Principle. The assay is based on the liberation of the thiol group of CoA, reaction (I), as measured by the increase in absorbance at 412 n m due to the reaction of free C o A with 5,5'-dithiobis(2-nitrobenzoicacid) (DTNB). 2
K. K. Y a b u s a k i a n d C. E. Ballou, J. Biol. Chem. 254, 12,314 (1979). z G. L. E l l m a n , Arch. Biochem. Biophys. 82, 70 (1959).
METHODS IN ENZYMOLOGY, VOL. 71
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181971-X
[29]
LONG-CHAIN FATTY ACYL-CoA THIOESTERASE
243
AcyI-SCoA + H~O ~ fatty acid + CoASH
(1)
Reagents Tris-HC1 buffer, 0.1 M, pH 8.0 Palmitoyl-CoA, 5/aM Ovalbumin, 7.0 mg/ml in 0.1 M Tris-HC1 buffer, pH 8.0 DTNB, 0.01 M in 0.1 M potassium phosphate buffer, pH 7.0 Procedure. The complete assay mixture contains 10/~mol of Tris-HC1 buffer, pH 8.0, 0.05 /zmol of DTNB, 0.01 /xmol of palmitoyl-CoA, and 1-1.5 mU of the thioesterase in a final volume of 2.0 ml. To observe initial rates of hydrolysis of palmitoyl-CoA, it was necessary to dilute the enzyme (final step of purification) severalfold. Typically, a 5-/zl aliquot of the stock thioesterase solution containing 0.125 mg of protein per milliliter was diluted to 0.2 ml with a solution of ovalbumin at 7.0 mg/ml in 0.1 M Tris-HC1 buffer, pH 8.0. After temperature equilibration and recording a base line, the sample cuvette was removed, diluted enzyme was added from a 4-/~1 microcapillary pipette, and a piece of parafilm was placed over the top of the cuvette, which was mixed by several inversions and then replaced in the sample holder. This total operation could be performed within 30 sec; the initial rates of hydrolysis were linear and were recorded over a period of 5 min or longer. A split-beam recording spectrophotometer with 0.05 or 0.1 full scale absorbance slide wire capability is convenient for the above measurements. A molar extinction coefficient of 13,600 is used to quantitate the DTNB reduction/ Since DTNB also reacts slowly with the thiol groups of proteins, the use of a split beam spectrophotometer is advantageous in that an aliquot of the protein solution can be added to the reference cuvette, which contains all assay components except the substrate, palmitoyl-CoA. Otherwise, it is necessary to correct the initial rates of reduction of DTNB by proteins by subtraction of controls in which palmitoyl-CoA is omitted. Units. An enzyme unit is defined as the amount of enzyme activity necessary to catalyze the hydrolysis of 1 /zmol of palmitoyl-CoA per minute under the above conditions. Specific activity is expressed as units per milligram of protein. Purification Procedure The following procedure, summarized in the table, yields an 8000-fold purification of the acyl-CoA thioesterase. 1 All operations were performed at 4° unless otherwise stated. Step I. Preparation of Crude Extract. Frozen M. smegmatis wet cells (200 g) were thawed, suspended in 800 ml of 0.1 M potassium phosphate buffer, pH 7.0, and broken by two passes through a Manton-Gaulin
244
[29]
FATTY ACID SYNTHESIS PURIFICATION OF Mycobacterium smegmatis THIOESTERASE
Step
Total activitya (units)
Specific activity (units/mgprotein)
Purification (fold)
Extract, 37,000gb Protamine sulfate extract Dialyzed (NH4)2SO4pellet DEAE-cellulose chromatography Hexylagarose chromatography BioGel A-0.5m chromatography
536 421 228 80 39 27
0.03 0.05 0.08 0.6 86 230
! 1.7 3 21 3007 8040
a Expressed as micromoles of CoA released per minute with 5 ~ substrate. b From 200 g of wet cell paste.
palmitoyl-CoA as
pressure cell operating at 10,000 psi. The broken cell suspension was centrifuged at 37,000 g for 1 hr. Step 2. Protamine Sulfate Extract. To the supernatant extract was added, slowly and with stirring, a 1% solution ofprotamine sulfate in 0.01 M potassium phosphate buffer, pH 7.0, to give a final ratio of 0.12 mg of protamine sulfate per milligram of protein. The solution was stirred for 30 min then centrifuged at 37,000 g for 15 min. The supernatant liquid containing the enzyme activity was removed, the pellet was washed by resuspension in 0.01 M potassium phosphate buffer, pH 7.0, and the suspension was centrifuged at 37,000 g for 15 min. This second supernatant was combined with the first. Step 3. Ammonium Sulfate Precipitation. The enzyme was precipitated from the above extract by adding ammonium sulfate to 55% of saturation. The mixture was stirred for 3 hr and then centrifuged at 16,000 g. The pellet was suspended in 0.01 M Tris-HC1 buffer, pH 8.0, and dialyzed for 24 hr against 7 liters of 0.01 M Tris-HC1 buffer, pH 8.0, with two changes of the buffer during that time. The protein solution was concentrated to 50 ml with an Amicon Diaflo apparatus fitted with a PM-10 membrane. Step 4. DEAE-Cellulose Chromatography. The concentrated protein solution from step 3 was applied to a DEAE-cellulose column (2.8 × 18 cm) equilibrated with 0.01 M Tris-HC1 buffer, pH 8.0, and fractions were collected by elution with the same buffer. After the absorbance at 280 nm dropped to a low value, a gradient of 0.01 to 0.5 M Tris-HC1, pH 8.0, was applied and fractions were collected. The major peak of thioesterase activity was eluted at a conductivity of 3-5 mmho. The active fractions were combined and concentrated to 5 ml, and glycerol was added to give a concentration of 10%. This solution was dialyzed against 4 liters of 0.01 M Tris-HCl, pH 8.0, containing 10% glycerol.
[29]
LONG-CHAIN FATTY A C Y L - C o A THIOESTERASE
245
Step 5. Hexylagarose Chromatography. The protein solution from step 4 was applied to a hexylagarose column (1.8 x 33 cm) equilibrated with 0.01 M Tris-HC1, pH 8.0, containing 10% glycerol, and the column was washed with the same buffer. After the absorbance at 280 nm dropped to a low value, the column was eluted with a gradient of 0.01 to 0.4 M TrisHC1, pH 8.0, containing 10% glycerol. The fractions that contained thioesterase activity were combined and concentrated to 1.5 ml. Step 6. BioGei A-O.5m Chromatography. The concentrated protein solution from step 5 was applied to a BioGel A-0.5m column (1.8 x 114 cm) equilibrated with 0.1 M Tris-HC1, pH 8.0, containing 10% glycerol. Fractions of 2.5 ml were collected by elution with the same buffer, and the active fractions were combined, concentrated, and stored at 4° in the final buffer. Properties
Purity. Preparations of thioesterase with a specific activity of 230 units per milligram of protein reveal two major components of about 22,000 and 20,000 MW and a few minor components by acrylamide gel electrophoresis under denaturing conditions. 1 Molecular Weight. The enzyme has an apparent molecular weight by gel permeation chromatography on Sephadex G-100 of about 40,000' and is probably composed of two subunits of about MW 20,000 from its properties during acrylamide gel electrophoresis under denaturing conditions. Requirements and Extent of Hydrolysis. Because of the high activity at the final stage of purification, in order to observe the initial rates of hydrolysis of palmitoyl-CoA, the enzyme has to be assayed in very dilute protein solutions. Dilution of the final stage purified enzyme with buffer alone causes a substantial loss in activity. Therefore, to stabilize the enzyme, a solution of ovalbumin I is used to dilute the enzyme instead of serum albumin, which is noted for its ability to bind palmitoyl-CoA. 3 The inactivation of enzyme activity by dilution with buffer alone is not observed at stages of purification prior to step 6. Thioesterase preparations from step 4, or at the final stage of purification in the presence of ovalbumin, will catalyze the complete conversion of palmitoyl-CoA to stoichiometric amounts of CoA and palmitate. Kinetic Parameters and pH Optimum. The thioesterase has an apparent K m and Vma x for monomeric palmitoyl-CoA of 9 # M and 107/anol of CoA released per minute per milligram of protein, respectively. With micellar palmitoyl-CoA as substrate, the thioesterase has an apparent K m 3 H. Knoche, T. W. Esders, and K. Bloch, J. Biol. Chem. 248, 2317 (1973).
246
FATTY ACID SYNTHESIS
[29]
and Vmax of 5/xM and 77/xmol of CoA released per minute per milligram of protein, respectively. It should be noted that the above Vma x data were determined on the purified enzyme after about 10 months of storage at 4° in the presence of 10% glycerol. These results indicate that the enzyme has good stability under these conditions. The optimal pH for thioesterase activity is 8.0. Specificity. At saturating levels of substrate, maximal initial rates of hydrolysis are found for palmitoyl-CoA and stearoyl-CoA. Activity decreases with decreasing length of the saturated fatty acyl chain. Approximately 5 times lower activity is observed with lauroyl-CoA over palmitoyl-CoA at equal concentrations. Very low rates of hydrolysis are observed for acyl-CoA thioesters of Ci0 chain length, and no detectable activity is observed for acyl chain lengths of C8 or less. Inhibitors. The mycobacterial polymethylpolysaccharides, 4 6-O-methylglucose polysaccharide (MGP) and 3-O-methylmannose polysaccharide, which form very stable complexes with palmitoyl-CoA, 5'6 are potent inhibitors of the thioesterase-catalyzed hydrolysis of both monomeric and micellar palmitoyl-CoA. The polysaccharides appear to have no effect on the Vma x but appear to increase the apparent K m with both monomeric and micellar palmitoyl-CoA by altering the free substrate concentration.' The effect of the polymethylpolysaccharides on the thioesterase-catalyzed hydrolysis of palmitoyl-CoA appears to result solely from the binding of the substrate, not from an interaction with the enzyme itself. This is suggested from the fact that MGP had no effect on the initial rate of hydrolysis of lauroyl-CoA a substrate that, because of its short lipid chain, forms only weak complexes with polymethylpolysaccharides. 1 The same conclusion is supported by the observation that the modified MGP, 1 which binds palmitoyl-CoA poorly, m also fails to inhibit. However, the possibility that the acyl-CoA-polymethylpolysaccharide complex itself is a very poor substrate has not been ruled out.
+ G. R. Gray and C. E. Ballou, this series, Vol. 35 [11]. K. Bloch, Adv. Enzymol. 45, 1 (1977). 6 K. K. Yabusaki and C. E. Ballou, Proc. Natl. Acad. Sci. U.S.A. 75, 691 (1978).