The Isolation and General Properties of Escherichia coli Malonyl Coenzyme A-Acyl Carrier Protein TransacyIase*

SUMMARY Malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli was purified 4800-fold by procedures which included chromatography on DEAE-cellulose, Sephadex G-100, Sephadex G-75, DEAE-Sephadex, and preparative polyacrylamide gel electrophoresis. The purified enzyme was shown to be homogeneous by electrophoresis on polyacrylamide gels, by sedimentation equilibrium centrifugation, and by amino acid analysis. A molecular weight of 36,660, obtained with carboxymethylated enzyme by sedimentation equilibrium measurements, agreed with deter-minations on the native enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (36,500) and by Sephadex G-100 column chromatography (37,000). An szo,W = 2.31 S was determined for the enzyme by sedimentation velocity measurements. Amino acid analysis and determination of the isoelectric point (pH 4.65) both showed the enzyme to be acidic. The purified enzyme was inhibited by N-ethyl-maleimide and to a lesser extent by iodoacetamide, but inhibition by


SUMMARY
Malonyl coenzyme A-acyl carrier protein transacylase of Escherichia coli was purified 4800-fold by procedures which included chromatography on DEAE-cellulose, Sephadex G-100, Sephadex G-75, DEAE-Sephadex, and preparative polyacrylamide gel electrophoresis. The purified enzyme was shown to be homogeneous by electrophoresis on polyacrylamide gels, by sedimentation equilibrium centrifugation, and by amino acid analysis.
A molecular weight of 36,660, obtained with carboxymethylated enzyme by sedimentation equilibrium measurements, agreed with determinations on the native enzyme by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (36,500) and by Sephadex G-100 column chromatography (37,000). An szo,W = 2.31 S was determined for the enzyme by sedimentation velocity measurements.
Amino acid analysis and determination of the isoelectric point (pH 4.65) both showed the enzyme to be acidic.
The purified enzyme was inhibited by N-ethylmaleimide and to a lesser extent by iodoacetamide, but inhibition by both reagents was significantly increased at pH values above 7.3. Preincubation of the enzyme with malonyl-CoA protected against inactivation by both sulfhydryl reagents.
Reduction of aged enzyme preparations by incubation with dithiothreitol was shown to stimulate enzyme activity approximately S-fold. These experiments suggest that a reduced sulfhydryl group(s) on the enzyme is required for maximal catalytic activity.
The de novo synthesis of fatty acids in Escherichia coli has been shown to require the initial transacylation of acetyl (Reaction 1) * This work was supported in part by the National Science Foundation (GB-38676X) and the National Institutes of Health (l-ItOl-HL-10406).
For the preceding paper of this series see Reference 1.
$ Junior Fellow of the Center for the Biology of Natural Systems, Washington University, St. Louis, Missouri.
These studies are taken from a thesis submitted bv F. E. Ruth in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Chemistrv. Washineton Universitv. St. Louis. Missouri. Present addre&,'Depar&ent of Microbidlogy and Golecular Genetics, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115. and malonyl groups (Reaction 3) from coenzyme A to acyl carrier protein (2)(3)(4).
The transfer of acetate and malonate to ACP' converts these acyl groups to thioester forms which are characteristic of acyl intermediates in E. coli fatty acid synthesis and which are strictly required for the condensation reactions catalyzed by @ketoacyl-ACP synthetase (Reactions 2 and 4). The formation of acetyl-ACP in this scheme is followed by transfer of the acetyl group to a sulfhydryl site on the condensing enzyme (Econd) with the formation of an acetyl-E,,,d intermediate and the release of ACP (Reaction 2). Transacylation of malonate from CoA to ACP (Reaction 3) provides the substrate for the condensation of acetyl-E,,,d and malonyl-ACP (Reaction 4).
The enzymes, acetyl-CoA-ACP transacylase and malonyl-CoA-ACP transacylase, which catalyze Reactions 1 and 3, respectively, have been partially purified and characterized from E. coli cell extracts (2,5). In a recent report on the purification of E. coli malonyl transacylase an apparently homogeneous protein was isolated and found to have a molecular weight of 35,500 to 40,000 (6). The enzyme was shown to catalyze the reversible transfer of malonate between CoA, ACP, pantetheine, cysteamine or N-acetylcysteamine, and kinetic evidence indicated a Ping Pong Ui Ui reaction mechanism.
Inhibition studies with the isolated enzyme, furthermore, indicated that malonyl transacylase was insensitive to the sulfhydryl reagents, N-ethylmaleimide and iodoacetamide, but was inactivated by phenylmethylsulfonylfluoride, a potent serine protease inhibitor.
The sensitivity of the enzyme to phenylmethylsulfonylfluoride, together with the observation that the linkage in the malonyl-enzyme intermediate was stable to performic acid oxidation, led to the proposal of a non-thiol mechanism of malonyl transfer by the E. coli enzyme.
The mechanism proposed by Joshi and Wakil (6) was analogous nmoles of DPNH, 0.4 ~g of fl-hydroxyacyl-CoA dehydrogenase to that first suggested for malonyl transacylase observed in yeast (195 pmoles per min per mg of protein at 37"), 10 nmoles of refatty acid synthetase (7,8) and similar also to the mechanism duced ACP, 10 nmoles of acetyl-ACP, 15 nmoles of malonyllater extended to the same enzyme in the pigeon liver fatty acid CoA, 5 pg of P-ketoacyl-ACP synthetase (3 pmoles per min per synthetase (9,10). The transfer of malonate to ACP by the mg of protein at 25"), and 0.08 to 0.8 milliunits of malonyl trans-E. coli enzyme was suggested to proceed through the intermediate acylase in a total volume of 0.1 ml. Reactions were started by esterification of malonate to a hydroxyamino acid residue, pre-the addition of malonyl transacylase, and the oxidation of DPNH sumably serine, in the active site of the enzyme.
was measured spectrophotometrically at 340 nm in a Gilford Since the reported insensitivity of purified E. coli malonyl recording spectrophotometer at 25". Under the above conditransacylase to sulfhydryl reagents contradicted earlier observa-tions the change in absorbance was linear for a minimum period tions with impure enzyme (2, 5) as well as a previous report from of 4 min. A unit of enzyme activity is defined as an amount of this laboratory using purified enzyme (II), a more detailed ex-enzyme catalyzing the formation of 1.0 pmole of DPN per min amination of the enzyme's sensitivity to these agents was under-under the assay conditions. taken.
A report is made here of the isolation of E. coli malonyl Alternatively, a precipitation assay was used in which malonyl transacylase by a procedure yielding enzyme with a specific ac-transfer from [i4C]malonyl-CoA was followed directly by measurtivity 2.8 times that previously reported for pure enzyme (6). ing

Materials
Malonyl-CoA and CoA were obtained from P-L Biochemicals Inc. N-Ethylmaleimide, iodoacetamide, iodoacetic acid, Dpantetheine, DPNH as well as ,&hydroxyacyl-CoA dehydrogenase (pig heart, 195 units per mg) were purchased from Sigma Chemical Co. The molecular weight standards, pepsin and was added to assure complete precipitation of the [14C]malonyl-ACP, the insoluble material was separated by centrifugation, and the pellet was washed twice with 10% trichloroacetic acid (0.4 ml) and once with diethyl ether (0.4 ml).
[14C]Malonyl groups were released from protein by incubation of the washed pellet in 0.2 ml of 1 ~\i NaOH at 37" for 15 min. The alkaline solutions were transferred to 20 ml of Bray's solution (16) and radioactivity was measured in a Packard Tri-Carb model 3380 liquid scintillation spectrometer equipped with an absolute activity analyzer (AAA-model 544). The nonspecific precipitation of ovalbumin (twice crystallized) were obtained from Worthington radioactivity was less than 1% of experimental values in this Biochemical Corp., myoglobin (horse heart twice crystallized) procedure. Protein was determined by the microbiuret method and cytochrome c (horse heart) from Mann Biochemicals Inc., of Munkres and Richards (17) or by the method of Lowry et and bovine serum albumin (Fraction V, fatty acid poor) from al. (18). Pentex Biochemicals.
Urea (ultra pure) and guanidine HCl Analytical Polyacrylamide Gel Electrophoresis-The method of (ultra pure) were products of Schwara-Mann Biochemicals Inc. Davis (19) was used in the preparation of polyacrylamide disc Whatman DEAE-cellulose ion exchange resins were obtained gels for analysis of the enzyme. Tubes containing 1.0 ml of 15oj, through H. Reeve Angel and Co., Sephadex gels were purchased polyacrylamide separating gel (5 x 50 mm), 0.2 ml of stacking from Pharmacia Fine Chemicals Inc., and hydroxylapatite (Bio-gel, and sample containing 10 to 100 pg of protein in a volume Gel HTP) was from Bio-Rad Laboratories. Radioactive of 0.1 ml were subjected to electrophoresis at a current of 1 ma [1,3-%2]malonyl-CoA (18.5 and 19.8 mCi per mmole) and [l-per tube at room temperature using a Tris-glycylglycine (pH i4C]iodoacetic acid (13.9 mCi per mmole) were obtained from 8.5) electrode buffer system. Samples were applied as a 5% New England Nuclear Inc. ACP was prepared by the method glycerol solution layered beneath the upper electrode buffer which of Majerus et al. (12), and acetyl-ACP by the method of Alberts contained bromphenol blue tracking dye. At the end of elecet al. (13). The enzyme P-ketoacyl-ACP synthetase was purified trophoresis the gels were cooled in an ice bath and then stained according to the method of Prescott and Vagelos (14).
overnight in a solution of 0.01% Coomassie blue in 12.5% trichloroacetic acid.

Methods
Analysis of gels for radioactivity or enzyme activity was performed on l-to 2-mm slices of unstained gels cut Malonyl Transacylase Assays-Two assay procedures were used for the measurement of malonyl transacylase activity.
A coupled spectrophotometric assay was used during purification procedures and inhibitor studies.
The formation of malonyl-ACP by malonyl transacylase was coupled to the condensation of malonyl-ACP and acetyl-ACP by P-ketoacyl-ACP synthetase and the reduction of acetoacetyl-ACP by &hydroxyacyl-CoA dehydrogenase in the presence of DPNH. The routine assay mixture contained 10 pmoles of potassium phosphate, pH 7.0, 2.5 pmoles of dithiothreitol, 0.1 pmole of EDTA (pH 7.0), 30 with a razor blade. Elution of enzyme from the gels was performed by mincing the gel slice in a volume of 0.1 M potassium phosphate, pH 6.9, containing 0.01 M dithiothreitol and 0.001 M EDTA and storing overnight at 4".

Purijication of Malonyl Transacylase
Previously published procedures (2, 5, 6, 15) for the purification of malonyl transacylase from extracts of E. coli were modi-fied in these experiments to permit both an increased yield of pure enzyme of higher specific activity and the simultaneous isolation of /3-ketoacyl-ACP synthetase.
The sequence of purification steps used in this scheme is described below and the results are reported in Table I. Ammonium sulfate Fractionation-All of the following purif? cation procedures, except where indicated, were conducted at 4". Five pounds of frozen E. coli B (full log, enriched medium, from Grain Processing Corp., Muscatine, Iowa), were thawed in 5 liters of buffer containing 0.02 M potassium phosphate, pH 7.0, 0.05 M 2-mercaptoethanol, and 0.001 M EDTA (Buffer A). The suspended cells were broken by passage through a Manton-Gaulin submicron disperser at 9,000 p.s.& and the ruptured cells were brought to 25% saturation with ammonium sulfate.
The precipitate was removed by centrifugation in a refrigerated Sorvall RCB-B at 15,000 x g for 30 min, and the pellet was resuspended in Buffer A to a protein concentration of 20 mg per ml. The resulting suspension, which contained 5% ammonium sulfate, was again ruptured by passage through the disperser and brought to 25y0 saturation by the addition of solid ammonium sulfate. The precipitate was removed by centrifugation, and the supernatant was combined with the original 257, supernatant.
After dilution to a protein concentration of 20 mg per ml the pooled supernatants were brought to 55% ammonium sulfate saturation, and the precipitate was removed by centrifugation.
The bulk of the enzyme was recovered from the 55% supernatant by the addition of solid ammonium sulfate to 95% saturation (Table I). The precipitate which was removed by centrifugation was dissolved in a minimal volume of Buffer A and dialyzed overnight against 40 volumes of the same buffer.
A streptomycin sulfate step for removal of nucleic acids was sometimes employed at this stage. When used, streptomycin was added as a concentrated solution in the proportion of 2 g per g of protein to a protein solution of conductivity 1.5 mmhos. The precipitate was allowed to form for 30 min and was removed by centrifugation.
DEAE-cellulose-23 Batching-The dialyzed or streptomycintreated extract was applied to DEAE-cellulose-23 equilibrated with Buffer A (15 mg of protein per ml of gravity settled resin) by direct mixing in a fritted bottom funnel (Bel-Art Products). The resin containing the adsorbed enzyme was washed twice with 1.5 volumes of Buffer A followed by two washes with 1.5 volumes of Buffer A containing 0.125 M LiCl (conductivity, 5.3 mmhos).
Malonyl transacylase was eluted with two washes of 1. After washing with 1 volume of the equilibrating buffer, the column was eluted with a lovolume linear gradient of 0.075 to 0.20 M LiCl in Buffer A (conductivity, 3.5 to 8.5 mmhos).
Peak malonyl transacylase activity was found at a conductivity of 5.8 mmhos.
The pooled fractions containing the enzyme were dialyzed overnight against 20 volumes of Buffer A. Hydrozylapatite Batching-The dialyzed eluate from the previous step (conductivity, 1.5 mmhos) was mixed with hydroxylapatite (50 g dry weight adsorbent per g of protein) which had been washed with several volumes of Buffer A. This step was performed in a fritted glass funnel under vacuum.
Under these conditions the enzyme was not retained by the hydroxylapatite and was recovered quantitatively in the filtrate. The filtrate was concentrated by pressure filtration to a volume of 40 ml.
Xephadex G-100 Chromatography-Molecular sizing of the purified extract was first performed on a 1.35-liter column (4.5 x 85.0 cm) of Sephadex G-100 (40 to 120 mesh) equilibrated and eluted with Buffer A containing 0.01 M dithiothreitol in place of 2-mercaptoethanol (Buffer A-dithiothreitol). 2-Mercaptoethanol was replaced by dithiothreitol as the reducing agent in all subsequent purification steps. The 40-ml sample was applied by layering as a 5% glycerol solution under a solution of Buffer Adithiothreitol and the column was eluted at a rate of 0.3 ml per min. Fractions were collected every 8 min and assayed for protein by measuring absorbance at 280 nm and for malonyl transacylase activity.
Tubes containing enzyme were pooled and concentrated by pressure dialysis to a volume of 9.0 ml.
Sephadex G-75 Chromatography-A more restrictive sizing of the extract was next accomplished by chromatography on Sephadex G-75. The sample, which had been concentrated and made 5% in glycerol, was applied to a 320 ml (2.5 x 90.0 cm) Sephadex G-75 (10 to 40 mesh) column equilibrated with Buffer A-dithiothreitol.
The column was eluted with the same buffer at a flow rate of 0.15 ml per min, and fractions were collected every 10 min. Fractions containing enzyme were pooled.
The pH of the solution was adjusted to 8.5 by addition of NaOH, and the dithio- The fractions containing enzyme were subjected to analysis by standard 15% polyacrylamide disc gel electrophoresis (19). This analysis aided in selecting, on the basis of the relative positions of contaminating proteins, the fractions to be pooled for subsequent preparative polyacrylamide gel electrophoresis.
A typical elution profile for this step, together with the range of fractions pooled after electrophoretic analysis (Fractions 140 to 151) is shown in Fig. 1. Tail fractions of the peak area were pooled and rechromatographed to generate additional suitable enzyme.
The pooled material was concentrated by pressure dialysis to a protein concentration of 4 to 7 mg per ml.

Preparative Polyacrylamide
Gel Electrophoresis-The highest purity of the enzyme was obtained by a final electrophoresis procedure on 15yo polyacrylamide at pH 8.5. A Canalco PD-1 preparative disc gel apparatus with a large upper column (PD2/ 320) was employed.
The separating and stacking gels used were the same as those of the standard pH 8.5 analytical acrylamide system with the exception that the cross-linking of the separating r 800 a e ;.
. Fifteen milliliters of separating gel (15% acrylamide-0.4 y0 bisacrylamide), polymerized with ammonium persulfate, and 5 ml of stacking gel, polymerized with riboflavin and ultraviolet light, were used for the electrophoresis of 10 mg of protein.
Polymerization, electrophoresis, and elution of the gels were carried out at 4". The sample, 2 to 3 ml containing 10 mg of protein, 5% glycerol, and bromphenol blue tracking dye, was applied to the top of the stacking gel, and Tris-glycylglycine buffer, pH 8.5, was layered above. Electrophoresis was begun at 8 ma and continued until the tracking dye entered the separating gel; the amperage was then increased to 12. Eluting buffer (0.2 M imidazole-HCl, pH 7.0, 0.01 M dithiothreitol, 0.001 M EDTA) was initially pumped at a flow rate of 0.4 ml per min until the tracking dye was eluted; the flow rate was increased to 0.8 ml per min during protein collection.
A profile of enzyme activity eluted from the gel by this procedure is shown in Fig. 2. Fractions were examined by standard analytical polyacrylamide disc gel electrophoresis.
Those fractions which showed a single band upon staining with Coomassie blue (Fructions 335 to 555) were pooled for further characterization.
The yield of pure enzyme by this procedure represented 25 to 30% of the material applied to the gel. The impure enzyme of the tail fractions was subjected to repeat electrophoresis to improve the recovery. The total yield of enzyme activity from the initial electrophoresis ranged from 80 to 90'%, but this yield decreased when the procedure was repeated.
Comments on Purijication Procedure-The purification of malonyl transacylase by this procedure provided enzyme in good yield which appeared homogeneous by several criteria.
Electrophoresis of 60 Hg of pure enzyme on standard polyacrylamide disc gels at pH 8.5 revealed a single band that stained with Coomassie blue (Fig. 3). A second gel, that was not stained, was sliced and the slices were eluted with Buffer A-dithiothreitol. Enzyme assays of the eluted material indicated that malonyl transacylase was present at the same RF as the single protein band. The use of 15y0 polyacrylamide gels in this analysis was found necessary for the discrimination of contaminating proteins which were observed to have RF characteristics similar to that of the enzyme.
Electrophoresis on 10% polyacrylamide gels containing 0.1% sodium dodecyl sulfate at pH 7.0 also revealed a single protein band which was stained with a solution of Coomas-  sie blue in 12% trichloroacetic acid and 5% methanol. Electrophoretically purified enzyme was also found to be homogeneous from its sedimentation properties and from amino acid analyses described below.
The maximum specific activity of pure enzyme (1850 units per mg) obtained by this procedure is 2.8 times greater than that previously reported (6). Although the coupled spectrophotometric assay used in obtaining this value differs from the assay procedures previously employed for this enzyme, a comparison with the acid precipitation assay used by others showed that the two methods gave similar results.   25". An aliquot of malonyl transacylase was alkylated by incubation with iodoacetamide at a final concentration of 50 mM for 30 min at 25". The alkylation was stopped by the addition of a 5-fold excess of 2-mercaptoethanol. llioth the reduced native enzyme and the alkylated enzyme were dialyzed overnight against buffer containing 0.1 M sodium phosphate, pH 7.2, 1% 2-mercaptoethanol, and 1% sodium dodecyl sulfate and subjected to electrophoresis on 10% polyacrylamide gels containing 0.1% sodium dodecyl sulfate. The proteins were located in the gels by staining for 16 hours with a solution of 0.1% Coomassie blue in 12'% trichloroacetic acid and 5% methanol.
Migration distances of both the native and alkylated malonyl transacylase were similar, indicating molecular weights of 36,500 and 35,500, respectively, by comparison with standards (Fig. 4). Electrophoresis of the enzyme on 15Cr, polyacrylamide gels containing 0.1% sodium dodecyl sulfate gave similar results.
Sephadex G-100 Gel Filtration-An approximation of the molecular weight of malonyl transacylase from its elution properties in gel filtration was made using Sephadex G-100 equilibrated and eluted with 0.02 M potassium phosphate, pH 7.0, 0.01 M dithiothreitol, and 0.001 M EDTA.
The elution volumes, V,, of protein standards varying in molecular weights from 12,400 to 67,000 were used in the calibration.
The proteins were applied to the column in 3.0 ml containing 5y0 glycerol and eluted at a rate of 0.32 ml per min. Fraction volumes were determined for each 5-min collection period.
The elution volumes observed in this manner were used in molecular weight estimates as described by Andrews (21).

Sedimentation Equilibrium
Centrifugation-An estimate of the molecular weight of reduced and carboxymethylated malonyl transacylase by equilibrium centrifugation was made using the high speed meniscus depletion method of Yphantis (22). Malonyl transacylase which had been purified by preparative electrophoresis was reduced and alkylated as described above. Centrifugation of the samples was conducted under the identical conditions and the protein concentration across each cell was recorded photographically at 19 and 23 hours with the use of Rayleigh interference optics (Fig. 6). The molecular weight of carboxymethylated enzyme determined by this method is 36,660. A similar value was obtained for all three protein concentrations tested.
Sedimentation Velocity-Sedimentation rate measurements of malonyl transacylase, which had been reduced by prior treatment with dithiothreitol as described above, were conducted using a synthetic boundary cell and a Spinco model E analytical ultracentrifuge equipped with schlieren optics. Fig. 7 illustrates the boundary peak observed and its rate of migration.
The presence of a single absorbance peak, whose symmetry was maintained throughout the centrifugation, confirms the relative homogeneity of the protein sample. An SZO+, of 2.31 S was c-alculated from this experiment.
The partial specific volume, V, used in this calculation (0.7389 cm3 per g) was estimated from the observed amino acid composition by the method of Cohn and Edsall (23).
Amino Acid Composition-The results of amino acid analyses of electrophoretically pure malonyl transacylase after 24 and 72 hours of acid hydrolysis in 6 N HCl at 110" are shown in Table II. The presence of 6 half-cystine residues was indicated by the performic acid oxidation method of Moore (25) and confirmed by carboxymethylation using iodo[l-Wlacetic acid as described by Hirs (26). When the enzyme was reduced and then alkylated in 0.1 M Tris-HCl, pH 8.6, containing 6 M guanidine-HCl and labeled iodoacetic acid in loo-fold excess over the enzyme concentration for 10 min at 25", there was good agreement between the number of carboxymethylcysteine residues detected by amino acid analysis and the amount of label in an aliquot of protein precipitated by addition of trichloroacetic was made to quantitate the number of glutamine or asparagine residues present; however, an isoelectric point of pH 4.65 for the native enzyme, as determined by gel isoelectric focusing (Fig. 8)) suggests the presence of a large number of free acidic residues. pH Optimum-The effect of pH on the catalytic activity of malonyl transacylase was determined by direct measurement of the rate of malonyl-ACP formation. Fig. 9   Inhibition by Alkylating Agents-Malonyl transacylase purified by the procedure described in this report was inhibited by Nethylmaleimide, but the degree of inhibition was dependent on the pH of the reaction (Table III).
Whereas the enzyme was inhibited by only 37.5% when incubated with 11.5 mM N-ethylmaleimide for 15 min (25") at pH 7.3, total inhibition occurred under the same conditions at pH 8.6. A similar pH effect was observed when iodoacetamide and iodoacetic acid were tested as alkylating agents; however, maximal inhibition by 25 mM concentrations of these reagents at pH 8.6 was only 74% and 52oj,, respectively.
Malonyl-CoA at a concentration of 25 pM completely protected the enzyme against 25 mM concentrations (pH 8.6) of the alkylating agents, as measured both by effects on enzyme activity (not shown) and the binding of malonate to the enzyme (27 tially observed in the Sephadex G-100 step of enzyme purification when dithiothreitol replaced 2-mercaptoethanol as the reducing agent (Table I), was readily demonstrated with aged enzyme preparations.
Enzyme which had been stored at 4" for several weeks showed a 5-fold stimulation of catalytic activity when exposed to 0.05 M dithiothreitol at pH 8.6 (Table IV). Although the thiol reducing properties of dithiothreitol have been reported to be quite rapid at pH 7.0 (28)) it appeared that an enhancement of this stimulation occurred when the enzyme was reduced at pH 8.6. The more rapid reduction of the enzyme at pH 8.6 is consistent with the greater inactivation of the enzyme by the alkylating agents, N-ethylmaleimide and iodoacetamide, observed at higher pH values. DISCUSSION Purification of malonyl transacylase by the method described in this report resulted in a maximum specific activity of the homogeneous enzyme (1850 units per mg) which exceeded by 2.8-fold that previously reported (653 units per mg) for the presumably pure enzyme (6). Although this discrepancy might be attributable in part to impurities, a more likely explanation would appear to reside in the relative reduction states of the enzyme in the different preparations.
As seen in Table I, the use of dithiothreitol in place of 2-mercaptoethanol in the Sephadex G-100 and subsequent purification steps resulted in an increase in the total enzyme activity of the preparation. Evidence that this stimulation resulted from a reduction of the enzyme was derived from experiments in which dithiothreitol was added  to a preparation of enzyme which was stored at 4" for several weeks (Table IV).
The effect of dithiothreitol in restoring activity to the enzyme would suggest that a sulfhydryl group(s) sensitive to oxidation and reduction has an important structural or functional role in the catalytic process.