- Email: [email protected]
Affinity purification of anti-pigeon liver fatty acid synthetase immunoglobulin and comparative immunoreactivity of the catalytic reactions
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 201, No. 1, April 15, pp. 199-206, 1980
Affinity Purification lmmunoglobulin
of Anti-Pigeon Liver Fatty Acid Synthetase and Comparative lmmunoreactivity of the Catalytic Reaction9
SARVAGYA S. KATIYAR,2
FRANK A. LORNITZO, JOHN W. PORTER
Lipid Metabolism Laboratory, William Physiological Chemistry,
RICHARD E. DUGAN,
AND
S. Middleton Memorial Veterans Hospital, and the Department University of Wisconsin, Madison, Wisconsin 53706
Received July 6, 1979; revised
of
December 20, 1979
Rabbit anti-pigeon liver fatty acid synthetase antibody was prepared by affinity chromatography on Sepharose-fatty acid synthetase to near monospecificity (98% or more) as shown by immunodiffusion plates and rocket immunoelectrophoresis. Immunotitrations of the highly purified monospecific antibody against the overall activity and partial activities of fatty acid synthetase were then carried out. Only 6 ma1 of antibody/ma1 of enzyme was required to inactivate overall fatty acid synthetase activity and the condensation reaction, while 12 to 18 mol were required to partially inactivate the P-ketoacyl reductase and the malonyl- and acetyl-CoA transferases. Palmitoyl-CoA thioesterase (deacylase) activity was not inhibited by the antibody. The degree of inactivation of the partial reactions by antibody was not affected by dissociation of the fatty acid synthetase. Immunoprecipitation of the enzyme indicated that there are approximately 35 immunoreactive sites on the fatty acid synthetase molecule. The possible implications of these results to an understanding of the structural organization of pigeon liver fatty acid synthetase and its antigenic determinants are discussed.
It has been shown that large proteins may possess many determinants for antibody formation (1). However, little is actually known about these antigenic sites or the heterogeneity of the population of antibodies generated. At the present time, the characterization of antibody recognition sites in small proteins by methods such as those employing peptide analogs as antigen is being developed (2). If a large protein is also a multienzyme complex, then the effects of antigen-antibody interaction on the component enzymes can be compared by immunotitrations of the catalytic activities. Kumar et al. (3) raised the question of comparative immunoreactivity of the component ’ This investigation was supported in part by Grants AM 01333 and 21148 from the National Institute of Arthritis, Metabolic and Digestive Diseases of the National Institutes of Health, United States Public Health Service, and the Medical Research Service of the Veterans Administration. ’ Present address: Department of Chemistry, Indian Institute of Technology, Kanpur 208016, India.
enzymes of chicken liver fatty acid synthetase. They found that only one component activity was very sensitive to inhibition from interaction with antibody. However, their antiserum was only partially purified by ammonium sulfate precipitation. It is now well known that supposedly homogeneous preparations of antigens sometimes give rise to antisera having contaminating antibodies. Therefore, the use of monospecific IgG3 for biochemical research has become increasingly important in recent years. In the present paper we report procedures for the purification of the IgG to pigeon liver fatty acid synthetase to near monospecificity. In this procedure, the purified antibody is bound to an affinity column of purified fatty acid synthetase and then eluted with 4.5 M MgCl,. This antibody was used in this study to determine the number of antigenie sites on the pigeon liver fatty acid synthetase complex. It was also used to deters Abbreviation
199
used: IgG, immunoglobulin
G.
0003-9861/80/050199-08$02.00/O Copyright 0 1980 by Academic press, Inc. All rights of reproduction in any form reserved.
200
KATIYAR
mine the effect of antibody-antigen interaction on each of the catalytic activities present in the fatty acid synthetase complex. EXPERIMENTAL
PROCEDURE
Chemicals. Acetyl-CoA and malonyl-CoA were obtained from P-L Biochemicals, and [l-‘4C]acetyl-CoA and [2-‘4C]malonyl-CoA were purchased from New England Nuclear. NADPH, S-acetoacetyl-N-acetylcysteamine, and fatty acid-free bovine serum albumin were obtained from Sigma. Agarose (Type I), used for crossed rocket immunoelectrophoresis, was a product of Sigma. Preparation of fatty acid synthetase. Pigeon liver fatty acid synthetase was purified by the standard procedure reported by Muesing and Porter (4). Preparation of antibody. Antibody was made in response to subcutaneous injections into a rabbit of purified pigeon liver fatty acid synthetase. Doses of 5 mg of enzyme protein mixed with Freund’s adjuvant were administered at lo-day intervals. The purification of the rabbit serum IgG fraction was carried out by a procedure similar to that of Livingston (5) and included a 50% ammonium sulfate fractionation and DEAE-cellulose chromatography. This IgG fraction was further purified by immunoaffinity chromatography. Immunouf$nity chromatography. Activation of the agarose by cyanogen bromide was carried out by the method of March et a2. (6). Meanwhile, 50 mg of purified pigeon liver fatty acid synthetase (4) was dialyzed twice for 2 h each in 20 times its volume of 0.2 M potassium phosphate buffer, pH 7.0, to remove mercaptans. The pH of this solution was increased to 8.6 by the addition of 2 M sodium carbonate and the activated gel was then added to the fatty acid synthetase solution and gently stirred overnight at room temperature. The gel was washed on a sintered glass funnel with water, followed by 0.9% NaCl containing 1 mM EDTA. Unbound fatty acid synthetase was measured by biuret analysis of the filtrate and the amount of bound enzyme was calculated indirectly. The gel was packed in a column, 0.2 cm I.D., to a height of 1.5 cm, and 1 mg of bovine serum albumin in 0.9% NaCl was passed through the column prior to passage of the rabbit serum IgG. Rabbit serum IgG, purified through DEAE-cellulose chromatography as described above, was placed on the column. The flow rate did not exceed 1.2 ml/h/O.03 cm2 cross se&ion of the column. The affinity gel was washed with 0.9% NaCl containing I mM EDTA and then eluted with 4.5 M magnesium chloride which had been neutralized to an apparent pH of 6.4 with 1 M Tris-acetate. The IgG fractions were pooled, concentrated, and dialyzed in a Schleicher and Schuell collodion bag against 0.9% sodium chloride and 1 mM EDTA. A
ET AL. final dialysis was carried out against 0.02 M potassium phosphate, pH 8.0. The antibody was stored frozen at -20°C at a concentration of 3.5 mg/ml. Ouchterlon,y double diffusion studies. Immunodiffusion studies were performed by the method of Ouchterlony. Gels on glass plates were prepared from 0.5% (w/v) agarose in 0.9% NaCl. Crossed immunoelectrophoresis. Crossed immunoelectrophoresis was carried out by the method described byAxelsoneta1. (7). Gels were prepared by pouring 1% agarose in barbital buffer, pH 8.6, at an ionic strength of 0.02, onto glass plates (10 x 8 cm). After the agarose hardened, antigen (DEAE-cellulose purified fatty acid synthetase) was applied to a well punched into one corner of the gel plate, and then electrophoresis in the first dimension was carried out. A 2 x 8-cm section of the gel containing the antigen which had been electrophoresed in one dimension was allowed to remain on the glass plate and the remainder of the gel was removed from the plate with a razor blade. On the part of the glass plate from which the gel was removed, 10 ml of agarose (1Yo)containing immunoaffinity-purified anti-fatty acid synthetase IgG, was now poured and electrophoresis in the second dimension was then performed. After electrophoresis the gel was pressed and dried. Staining of the proteins in the gel was carried out with Coomassie brilliant blue for 10 min. The plates were destained in ethanol:acetic acid:water (4.5:1.0:4.5) for three time periods of 10 min each. Quantitative imrnunoprecipitation. Five milligrams of DEAE-cellulose-purified antibody protein was mixed with varying amounts of DEAE-cellulose-purified fatty acid synthetase in each of a series of test tubes containing 0.9% sodium chloride, 1.0% Triton X-100, and 10 mM potassium phosphate buffer, pH 8.0, in a volume of 2 ml. The mixtures were maintained at 22°C for 2 h, then at 4°C for 20 h. After centrifugation, the precipitates were repeatedly washed and then assayed for protein. The equivalence point of antigen and antibody was determined by the standard procedure. lmmunotitrations of enzymatic activities. Small quantities of purified fatty acid synthetase and affinitypurified anti-fatty acid synthetase antibody were incubated together in 0.2 M potassium phosphate buffer for 45 min at 30°C. A series of incubations was performed with the amount of fatty acid synthetase constant and the amount of antibody varied. In other titrations, enzyme was varied and antibody was constant. The effect of antibody on the catalytic activity of the fatty acid synthetase complex for fatty acid synthesis and its component reactions was determined by enzyme assay for each of the individual component catalytic activities. The lines and intercepts obtained from titrations with varying amounts of fatty acid synthetase were determined by linear regression. Enzyme assays. The necessary substrates were
IMMUNOREACTIVITY
OF REACTIONS
added for the determination of activity in the presence of varying amounts of antibody and in the absence of antibody. All assays except deacylase contained 200 pgiml of bovine serum albumin. In the deacyiase assay, the albumin is omitted because it prevents quantitative extraction of the product palmitic acid. Fatty acid synthesis. The concentrations of substrates in the assay mixture were malonyl coenzyme A, 100 pM; acetyl coenzyme A, 33 PM; and NADPH, 100 FM. The reaction was followed spectrophotometrically at 340 nm. Ketoreductase. The concentrations of substrates in the assay mixture were N-acetyl-S-acetoacetylcysteamine, 4.4 mgiml, and NADPH, 100 pM. NADPH oxidation was measured as for fatty acid synthesis and reaction volumes for both assays were 1 ml. Deacylase. [‘%]Palmitoyl coenzyme A was diluted with nonradioactive palmitoyl coenzyme A to a specific activity of 1 x lo4 dpm/pg. Assay mixtures for deacylase activity contained 0.01 M potassium phosphate, approximately 5 pg of fatty acid synthetase, and up to 35 pg of protein of anti-fatty acid synthetase antibody in a l-ml volume. The reaction was started by the addition of 2.16 kg of [‘%]palmitoyl coenzyme A. Incubations were carried out for 3 min at 30°C and the reaction was stopped by the addition of 30 ~1 of 60% perchloric acid. After the addition of 1 ml of ethanol, the reaction mixture was extracted three times with 2-ml portions each of petroleum ether. Radioactivity was determined in the petroleum ether extracts in a liquid scintillation spectrometer. Mixtures with a constant amount of antibody contained 12 fig of anti-fatty acid synthetase antibody and 2.5 to 15.0 kg of fatty acid synthetase. Acetyland malonyl-CoA transacylases. Assays were carried out by measuring the exchange of ‘“Clabeled acyl groups between CoA and pantetheine (8). Immunotitration mixtures with a constant amount of antigen contained 10 pg of fatty acid synthetase, 10 to 75 pg antibody, and 20 Fg albumin, in 0.20 ml of a 0.2 M potassium phosphate: 1 tIIM EDTA buffer, pH 7.0. These mixtures were incubated for 45 min at 30°C before completion of the assay. Pantetheine, 330 nmol in 50 ~1 of 0.2 M potassium phosphate buffer, was then placed in each of a series of small test tubes in an ice-water bath. A 20-~1 aliquot of immunotitration mixture (0.25 to 2.0 pg fatty acid synthetase protein) was then added to each tube. The exchange reaction was started by the addition of 40 ~1 of l“‘C]acetyl-coenzyme A (15,000 dpm, 10 nmol) or [W]malonyl-coenzyme A (20,000 dpm, 10 nmol), and the reaction was stopped after 5 min by the addition of 20 ~1 of 2 N acetic acid. The mixture was then pipetted onto a BioRad AGl-X8 (Cl-) ion exchange column 4 x 20 mm. The product was washed from the column with 4 ml of 2 N acetic acid, and an aliquot was assayed for radioactivity.
OF FATTY
ACID
SYNTHESIS
201
Condensation reaction. The condensation of acetyland malonyl-CoA thioesters was assayed by following the formation of triacetic lactone, the product formed in the absence of NADP+ and NADPH, by light adsorption at 280 nm (9). Antigen-antibody mixtures (0.225 ml) for the forward immunotitration contained a constant amount of fatty acid synthetase (3 yg) and varying amounts of antibody in the range of 1 to 100 pg of protein, whereas in the reverse titration, antibody was held constant at 1.2 Fg and fatty acid synthetase was varied between 1 and 7 pg. Each mixture was incubated for 45 min at 30°C and then centrifuged at 25009 for 5 min. The supernatant solution was placed in a microcuvette at 30°C and the substrate mixture containing acetyl-coenzyme A, 25 nmol, and malonyl-coenzyme A, 51.5 nmol, in 25 ~1 was added. Assays were carried out with a pinhole slit in a Gilford spectrophotometer. Determination of molecular weight of fatty acid synthetase:lgG complex. Fully active pigeon liver fatty acid synthetase, 100 fig of protein, was diluted with buffer to a final concentration of 0.1 M potassium chloride, 0.02 M potassium phosphate, and 1 ITIM EDTA. Two milligrams of serum albumin was added and the final volume was adjusted to 5.0 ml. An amount of monospecific antipigeon liver fatty acid synthetase IgG to just inactivate the fatty acid synthetase was then added. The antibody-antigen mixture was incubated at 30°C for 30 min and then concentrated to 1 ml with a Schleicher and Schuell dialysis membrane at 4°C. This mixture was passed without further delay into a Sepharose 6B column (1.5 x 64 cm). Eluate fractions were assayed for light absorption at 280 nm and for palmitoyl-CoA deacylase activity. The average molecular weight of the fatty acid synthetase:IgG complex was determined by extrap olation of a plot of V, against the logs of the molecular weights for thyroglobulin and rat liver fatty acid synthetase. RESULTS
Putification
of Antibody
Rabbit serum protein (10 mg), purified by ammonium sulfate fractionation and DEAEcellulose chromatography, was placed on an affinity column containing DEAE-cellulosepurified pigeon liver fatty acid synthetase as the bound ligand. Most of the protein eluted without binding. The bound protein was then eluted with 4.5 M MgC&. A typical profile of binding and elution of anti-pigeon liver fatty acid synthetase antibody is shown in Fig. 1. The yield of IgG specific for fatty acid synthetase was found to be 21%, and this compared well with the 24%
202
KATIYAR a&b,,, 1.
0
P
c
IO ELUATE (ml)
,,,b -P
ET AL.
Crossed-rocked immunoelectrophoresis of the antiserum purified on the immunoaffinity column containing bound DEAEcellulose-purified fatty acid synthetase against DEAE-cellulose-purified fatty acid synthetase, showed the presence of one major and one trace rocket (Fig. 3). This indicates that the antibody reacted with a trace (approximately 2%) of another antigen which was present in the DEAE-cellulosepurified preparation of fatty acid synthetase. However, when sucrose density gradientpurified fatty acid synthetase was bound to the affinity gel, the crossed-rocket immunoelectrophoresis of the antibody to DEAEcellulose purified enzyme showed only one rocket. 20
FIG. 1. Immunoaffinity chromatography of DEAEcellulose-purified rabbit serum immunoglobulins. Ten milligrams of protein in 0.02 M potassium phosphate, pH 8.0, solution (a) were placed on a Sepharose 4B: fatty acid synthetase affinity gel column (0.2 g of gel); the column was then washed with (b) 0.9% sodium chloride containing 1 mM EDTA, pH 7.0; (c) 4.5 M magnesium chloride neutralized to an apparent pH of 6.4, and finally with the above sodium chloride solution.
yield determined by quantitative precipitation of the antibody with fatty acid synthetase.
Quantitative
Precipitation
At the equivalence point 1.2 mg of a 5 mg aliquot of DEAE-cellulose-purified rabbit serum IgG was precipitated by purified pigeon liver fatty acid synthetase. This indicates a 24% yield of anti-pigeon liver fatty acid synthetase antibody in the IgG fraction. From the highest ratio of antibody to enzyme, it is calculated that 35 to 40 molecules of anti-fatty acid synthetase antibody are bound to one molecule of fatty acid synthetase.
Ouchterlony Double Diffusion and Crossed-Rocket ImmunoeEectroiDhoresis
The precipitin patterns obtained in Ouchterlony immunodiffusion studies are shown in Fig. 2. Affinity-purified antiserum gave a single line with DEAE-cellulosepurified fatty acid synthetase (Fig. 2, wells 1 and 2), and with the 100,OOOg supernatant solution of pigeon liver homogenate (wells 3 and 4). This indicates that the antiserum is monospecific. Cross-precipitation reactions of the antiserum, which did not bind to the immunoaffinity column, with varying concentrations of the DEAEcellulose-purified fatty acid synthetase, showed no precipitin lines indicating, therefore, that all the antiserum specific to fatty acid synthetase was bound to the column.
FIG. 2. Ouchterlony double diffusion analysis of anti-pigeon liver fatty acid synthetase antibody. The central well contained 10 pg of immunoaffinitypurified antipigeon liver fatty acid synthetase antibody. The outer wells marked 1 and 2 contained 5 and 10 pg of fatty acid synthetase, respectively, and the outer wells marked 3 and 4 contained 50 and 100 Kg of the 100,OOOg supernatant solution of the pigeon liver homogenate.
IMMLINOREACTIVITY
Comparative Immwnoreactivity Component Activities
OF
REACTIONS
OF FATTY
ACID
SYNTHESIS
203
of
The effects of increasing amounts of affinity-purified anti-fatty acid synthetase antibody on the catalytic activities of the fatty acid synthetase for fatty acid synthesis and for individual component reactions of this process are shown in Fig. 4. These results were obtained by immunotitrating a constant amount of antigen with increasing amounts of affinity-purified antibody. Inactivation patterns were similar for fatty acid synthesis and the condensation reaction, whereas transacylase and reductase activity components were progressively less sensitive to inactivation. The catalytic activity of the deacylase component was not inhibited by anti-fatty acid synthetase
MOLES ANTI-FATTY ACID SYNTHETASE MOLES FATTY ACID SYNTHETASE
FIG. 4. Immunotitration of the catalytic activities of pigeon liver fatty acid synthetase for fatty acid synthesis and its component partial reactions. Fatty acid synthetase and affinity-purified antibody in the ratios shown on the figures were mixed and incubated 45 min at 30°C before assay. (0) Fatty acid synthesis; (A) condensation reaction; (m) acetyl transacylation; (0) malonyl transacylation; (0) ketoreduction; (x) deacylation. The molecular weights used were 456,000 for the fatty acid synthetase and 150,000 for the antibody. (The amounts of antigen protein used for each titration were as follows: fatty acid synthesis, 2.5 Kg; condensation reaction, 3 pg; acetyl transacylation, 10 pg; malonyl transacylation, 10 pg; ketoreduction, 5 pg; deacylation, 5 wg.)
FIG. 3. Crossed immunoelectrophoresis of immunoaffinity-purified anti-pigeon liver fatty acid synthetase antibody. Fifty micrograms of DEAE-cellulose-purified fatty acid synthetase was applied to the gel. The first dimensional electrophoresis was carried out at 10 V/cm for 2 h at 25°C in 1% agarose in barbital buffer, pH 8.6, of an ionic strength of 0.02. The anode is to the right. The second dimensional immunoelectrophoretic run was carried out at 2 V/cm at 25°C for 18 h. The anode is at the top. The immunogel contained 1.1 fig anti-pigeon liver fatty acid synthetase antibody per cm’ gel area.
antibody. In fact, at low ratios of antibody to antigen, there appeared to be a slight increase in enzyme activity. The results were the same when DEAE-cellulosepurified antibody, rather than affinitypurified antibody was used. The effect of antibody on fatty acid synthesis and its component partial activities were also determined by titration with constant amounts of antibody and increasing amounts of enzyme. This reverse method provides a much sharper end point than the forward method of titration. The intercept on the y-axis of the linear plot of enzyme activity vs amount of enzyme protein is the amount of enzyme activity suppressed by the amount of anti-fatty acid synthetase antibody in the incubation mixture (Fig. 5).
KATIYAR
ET AL.
acylase reaction is essentially unaffected by the binding of antibody under nonprecipitation conditions. Effect of Dissociation of Fatty Acid Synthetase on Antigen-Antibody Interaction
FIG. 5. Immunotitration of the catalytic activities of pigeon liver fatty acid synthetase for fatty acid synthesis and for ketoreduction. Enzyme activity, expressed as rate of oxidation of NADPH (measured by the decrease in light absorption at 340 nm), is plotted against pg of enzyme protein. Before the spectrometric assay, affinity-purified anti-fatty acid synthetase antibody, 4.66 pg of protein, was incubated for 45 min at 30°C with the quantities of sucrose density gradient-purified fatty acid synthetase shown on the figure. (0) Ketoreduction; (0) fatty acid synthesis.
Similarly, the intercept on the x-axis is the amount of enzyme protein inactivated by the given amount of antibody. It is evident (Fig. 5) that the ketoreductase reaction is less sensitive to inactivation of catalytic activity by antibody than the fatty acid synthetase overall reaction. Table 1 lists the moles of anti-fatty acid synthetase antibody required to inactivate 1 mol of fatty acid synthetase activities for fatty acid synthesis and for the component partial reactions. The data in this table were obtained by “reverse” immunotitrations similar to the ones described above. The condensation reaction is, the most sensitive of the component reactions, requiring approximately 6 mol of antibody to inactivate 1 mol of fatty acid synthetase. The overall reaction for fatty acid synthesis is similarly inactivated. The transacylase reactions require more binding of antibody, and the ketoreductase requires near saturation binding (see quantitative precipitation) in order to reach minimum activity. The de-
Dissociation of fatty acid synthetase into half-molecular weight species affects the catalytic activities of the enzyme. Fatty acid synthesis and the condensation reaction are abolished, whereas the other component reactions such as acetyl transacylase and ketoreductase are reduced to two-thirds of their activity in the undissociated complex. However, dissociation of the enzyme to half-molecular weight subunits did not significantly change the sensitivities of the partial activities of these subunits on reaction with antibody. Molecular Weight of Antigen-Antibody Complex
The antigen-antibody complex formed by adding to pigeon liver fatty acid synthetase an amount of IgG that would just eliminate enzyme activity for fatty acid synthesis yielded one peak of protein on molecular filtration through Sepharose 6B. The molecular weight of this complex, as determined by light absorption at 280 nm and by palmitoyl-CoA deacylase activity, was TABLE
I
IMMUNOINHIBITION OF THE CATALYTIC OF FATTY ACID SYNTHETASE
Catalytic activity Fatty acid synthesis Condensation reaction Malonyl transacylation Acetyl transacylation Ketoreduction Deacylation
ACTIVITIES
Molar ratio (Antibody/antigen) 6.6 6.0 12 18 30 m
o Moles of anti-fatty acid synthetase antibody required to inhibit the catalytic activity of 1 mol of fatty acid synthetase as determined by extrapolation of the linear rate of inhibition obtained under conditions of antigen excess.
IMMUNOREACTIVITY
OF
REACTIONS
1.2 x 106. This value corresponds reasonably well with a complex containing 6 mol of IgG bound to 1 mol of undissociated fatty acid synthetase (calculated molecular weight of 1.43 x 106). There was no 280-nm absorbing material or deacylase activity in those eluate fractions which corresponded to the molecular weight of a complex between antifatty acid synthetase IgG and halfmolecular weight subunits of pigeon liver fatty acid synthetase (about half of 1.4 x 106, or 600,000 to 800,000). DISCUSSION
Studies on the rates of enzyme synthesis and degradation, especially those for antigens which vary markedly in animals in different nutritional states, are carried out by quantitative immunoprecipitation or immunotitration. The purity of the antiserum is therefore of critical importance, particularly in those studies in which immunoprecipitation is carried out. The results presented in this paper describe a general procedure for the purification of antiserum to pigeon liver fatty acid synthetase by immunoaffinity chromatography. The efficiency of the immunoaffinity column approaches 90%, as shown by the correspondence between the percentages of anti-fatty acid synthetase antibody removed by the column from the DEAEcellulose-purified antibody and by the quantitative immunoprecipitation with the antigen. The nearly monospecific antibody purified by the method indicated above has been used to study the comparative immunoreactivities of the catalytic activities of pigeon liver fatty acid synthetase. The inhibition of several of the component catalytic activities of the antigen by the antibody appears to be one of the important effects of antigenantibody interaction. The extent of inhibition due to antigen-antibody interaction was measured by the immunotitrations shown in Figs. 4 and 5. The two types of immunotitrations reported here give different types of information. The forward immunotitration (fixed amount of antigen, varying amount of antibody, Fig. 4) indicates the upper limit of inhibition reached in the
OF FATTY
ACID
SYNTHESIS
205
presence of excess of antibody. These titration curves are not linear except for a relatively short range of antibody/antigen molar ratios, as they are essentially saturation curves. On the other hand, reverse immunotitration (fixed amount of antibody, varying amount of antigen, Fig. 5) gives an essentially linear plot, which, when extrapolated to the y-axis, gives a measure of the limit of sensitivity of the catalytic sites of fatty acid synthetase to relatively small amounts of antibody. The data of Figs. 4 and 5 and Table I show the loss of activities for the component reactions as a function of antibody concentration. It is of interest to note that on the molar basis the Condensation-CO, exchange reaction is inhibited by 6 mol of purified antibody/m01 of fatty acid synthetase, whereas a larger molar excess of antibody is required for the inactivation of other partial activities; s-acetoacetyl-N-acetylcysteamine reductase required a molar excess of 30. These results, along with the quantitative precipitation data, demonstrate that while 6 molecules of antibody are sufficient to inactivate 1 molecule of fatty acid synthetase for the overall reaction, a total of 35 to 40 antibody molecules can bind to one molecule of the enzyme. The inhibition of the component reactions can be interpreted as follows: The mechanism of inhibition of the overall and partial reactions of fatty acid synthesis may involve conformational change or steric hindrance, or both, of the enzyme complex. A conformational change induced by the binding of 6 antibody molecules per fatty acid synthetase molecule might be sufficient to disrupt the condensation reaction. In this reaction, a precise geometry between the two binding sites (acetyl and malonyl groups) and the catalytic site may be very critical. That inactivation by dissociation of the fatty acid synthetase on binding of antibody does not take place is supported by the presence of an antigen-antibody complex of a molecular weight in the range of 1.2 x 106. For inhibition of other partial reactions of fatty acid synthesis, a greater conformational change and distortion of active sites would be required. Inhibition of these reactions would then require the
206
KATIYAR
binding of 18 to 30 antibody molecules per molecule of fatty acid synthetase (Fig. 4). Our results may also suggest, as an alternative explanation, that there are some antibodies which bind to antigen at critical areas around the condensing site. (This assumes that the condensing site is on the surface, or in an accessible crevice, and is more open than other active sites.) Under these conditions, the antibody would interfere with the transfer of substrates to the condensing site, and this site would, therefore be inaccessible to substrate. Such an enzyme molecule would also not be able to catalyze the overall synthesis of fatty acids. This interpretation implies that the catalytic sites for other partial reactions are either located inside the multienzyme complex and are therefore less affected by the binding of small numbers of antibody molecules, or that the greater proportion of antibodies are raised for antigenic segments located in regions remote from these catalytic sites. Further information on the antigen-antibody interaction is provided by the observed failure of dissociation of the fatty acid synthetase complex into two- halfmolecular weight subunits to affect the relative sensitivities of the partial reactions of fatty acid synthesis in the presence of antibody. This indicates either that the
ET AL.
portion of the fatty acid synthetase that is exposed after dissociation does not have antigenic determinants, or that if it does, the exposed portion is remote from the sites of component reactions. REFERENCES 1. KABAT, E. A., AND MAYER, M. M. (1961) Experimental Immunochemistry, 2nd ed. pp. 22-96, Charles C Thomas, Springfield, Ill. 2. MATSUURA, S., CHEN, H. C., AND HODGEN, B. D. (1978) Bioehemnistq 17, 575-580. 3. KUMAR, S., SRINIVASAN, K. R., AND ASATO, N. (1977) Biochim. Biophys. Acta 489, 32-47. 4. MUESING, R. A., AND PORTER, J. W. (1975) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 35, pp. 45-59, Academic Press, New York. 5. LIVINGSTON, D. M. (1974) in Methods in Enzymology (Jacoby, W. B., and Wilcheck, M., eds.), Vol. 34, Pt. B, pp. 723-731, Academic Press, New York. 6. MARCH, S. C., PARIKH, I., AND CUATRECASAS, P. (1974) Anal. Biochem. 60, 149-152. 7. WEEKE, B., (1973) in Quantitative Immunoelectrophoresis (Axelson, N. H., Kroll, J., and Weeke, B., eds.), pp. 47-59, Universitetforlaget, Oslo. 8. KUMAR, S., DORSEY, J. A., MUESING, R. A., AND PORTER, J. W. (1970) J. Biol. Chem. 245, 4732-4744. 9. NIXON, J. E., PUTZ, G. R., AND PORTER, J. W. (1968) J. Biol. Chem. 243, 5471-5478.
Affinity Purification lmmunoglobulin
of Anti-Pigeon Liver Fatty Acid Synthetase and Comparative lmmunoreactivity of the Catalytic Reaction9
SARVAGYA S. KATIYAR,2
FRANK A. LORNITZO, JOHN W. PORTER
Lipid Metabolism Laboratory, William Physiological Chemistry,
RICHARD E. DUGAN,
AND
S. Middleton Memorial Veterans Hospital, and the Department University of Wisconsin, Madison, Wisconsin 53706
Received July 6, 1979; revised
of
December 20, 1979
Rabbit anti-pigeon liver fatty acid synthetase antibody was prepared by affinity chromatography on Sepharose-fatty acid synthetase to near monospecificity (98% or more) as shown by immunodiffusion plates and rocket immunoelectrophoresis. Immunotitrations of the highly purified monospecific antibody against the overall activity and partial activities of fatty acid synthetase were then carried out. Only 6 ma1 of antibody/ma1 of enzyme was required to inactivate overall fatty acid synthetase activity and the condensation reaction, while 12 to 18 mol were required to partially inactivate the P-ketoacyl reductase and the malonyl- and acetyl-CoA transferases. Palmitoyl-CoA thioesterase (deacylase) activity was not inhibited by the antibody. The degree of inactivation of the partial reactions by antibody was not affected by dissociation of the fatty acid synthetase. Immunoprecipitation of the enzyme indicated that there are approximately 35 immunoreactive sites on the fatty acid synthetase molecule. The possible implications of these results to an understanding of the structural organization of pigeon liver fatty acid synthetase and its antigenic determinants are discussed.
It has been shown that large proteins may possess many determinants for antibody formation (1). However, little is actually known about these antigenic sites or the heterogeneity of the population of antibodies generated. At the present time, the characterization of antibody recognition sites in small proteins by methods such as those employing peptide analogs as antigen is being developed (2). If a large protein is also a multienzyme complex, then the effects of antigen-antibody interaction on the component enzymes can be compared by immunotitrations of the catalytic activities. Kumar et al. (3) raised the question of comparative immunoreactivity of the component ’ This investigation was supported in part by Grants AM 01333 and 21148 from the National Institute of Arthritis, Metabolic and Digestive Diseases of the National Institutes of Health, United States Public Health Service, and the Medical Research Service of the Veterans Administration. ’ Present address: Department of Chemistry, Indian Institute of Technology, Kanpur 208016, India.
enzymes of chicken liver fatty acid synthetase. They found that only one component activity was very sensitive to inhibition from interaction with antibody. However, their antiserum was only partially purified by ammonium sulfate precipitation. It is now well known that supposedly homogeneous preparations of antigens sometimes give rise to antisera having contaminating antibodies. Therefore, the use of monospecific IgG3 for biochemical research has become increasingly important in recent years. In the present paper we report procedures for the purification of the IgG to pigeon liver fatty acid synthetase to near monospecificity. In this procedure, the purified antibody is bound to an affinity column of purified fatty acid synthetase and then eluted with 4.5 M MgCl,. This antibody was used in this study to determine the number of antigenie sites on the pigeon liver fatty acid synthetase complex. It was also used to deters Abbreviation
199
used: IgG, immunoglobulin
G.
0003-9861/80/050199-08$02.00/O Copyright 0 1980 by Academic press, Inc. All rights of reproduction in any form reserved.
200
KATIYAR
mine the effect of antibody-antigen interaction on each of the catalytic activities present in the fatty acid synthetase complex. EXPERIMENTAL
PROCEDURE
Chemicals. Acetyl-CoA and malonyl-CoA were obtained from P-L Biochemicals, and [l-‘4C]acetyl-CoA and [2-‘4C]malonyl-CoA were purchased from New England Nuclear. NADPH, S-acetoacetyl-N-acetylcysteamine, and fatty acid-free bovine serum albumin were obtained from Sigma. Agarose (Type I), used for crossed rocket immunoelectrophoresis, was a product of Sigma. Preparation of fatty acid synthetase. Pigeon liver fatty acid synthetase was purified by the standard procedure reported by Muesing and Porter (4). Preparation of antibody. Antibody was made in response to subcutaneous injections into a rabbit of purified pigeon liver fatty acid synthetase. Doses of 5 mg of enzyme protein mixed with Freund’s adjuvant were administered at lo-day intervals. The purification of the rabbit serum IgG fraction was carried out by a procedure similar to that of Livingston (5) and included a 50% ammonium sulfate fractionation and DEAE-cellulose chromatography. This IgG fraction was further purified by immunoaffinity chromatography. Immunouf$nity chromatography. Activation of the agarose by cyanogen bromide was carried out by the method of March et a2. (6). Meanwhile, 50 mg of purified pigeon liver fatty acid synthetase (4) was dialyzed twice for 2 h each in 20 times its volume of 0.2 M potassium phosphate buffer, pH 7.0, to remove mercaptans. The pH of this solution was increased to 8.6 by the addition of 2 M sodium carbonate and the activated gel was then added to the fatty acid synthetase solution and gently stirred overnight at room temperature. The gel was washed on a sintered glass funnel with water, followed by 0.9% NaCl containing 1 mM EDTA. Unbound fatty acid synthetase was measured by biuret analysis of the filtrate and the amount of bound enzyme was calculated indirectly. The gel was packed in a column, 0.2 cm I.D., to a height of 1.5 cm, and 1 mg of bovine serum albumin in 0.9% NaCl was passed through the column prior to passage of the rabbit serum IgG. Rabbit serum IgG, purified through DEAE-cellulose chromatography as described above, was placed on the column. The flow rate did not exceed 1.2 ml/h/O.03 cm2 cross se&ion of the column. The affinity gel was washed with 0.9% NaCl containing I mM EDTA and then eluted with 4.5 M magnesium chloride which had been neutralized to an apparent pH of 6.4 with 1 M Tris-acetate. The IgG fractions were pooled, concentrated, and dialyzed in a Schleicher and Schuell collodion bag against 0.9% sodium chloride and 1 mM EDTA. A
ET AL. final dialysis was carried out against 0.02 M potassium phosphate, pH 8.0. The antibody was stored frozen at -20°C at a concentration of 3.5 mg/ml. Ouchterlon,y double diffusion studies. Immunodiffusion studies were performed by the method of Ouchterlony. Gels on glass plates were prepared from 0.5% (w/v) agarose in 0.9% NaCl. Crossed immunoelectrophoresis. Crossed immunoelectrophoresis was carried out by the method described byAxelsoneta1. (7). Gels were prepared by pouring 1% agarose in barbital buffer, pH 8.6, at an ionic strength of 0.02, onto glass plates (10 x 8 cm). After the agarose hardened, antigen (DEAE-cellulose purified fatty acid synthetase) was applied to a well punched into one corner of the gel plate, and then electrophoresis in the first dimension was carried out. A 2 x 8-cm section of the gel containing the antigen which had been electrophoresed in one dimension was allowed to remain on the glass plate and the remainder of the gel was removed from the plate with a razor blade. On the part of the glass plate from which the gel was removed, 10 ml of agarose (1Yo)containing immunoaffinity-purified anti-fatty acid synthetase IgG, was now poured and electrophoresis in the second dimension was then performed. After electrophoresis the gel was pressed and dried. Staining of the proteins in the gel was carried out with Coomassie brilliant blue for 10 min. The plates were destained in ethanol:acetic acid:water (4.5:1.0:4.5) for three time periods of 10 min each. Quantitative imrnunoprecipitation. Five milligrams of DEAE-cellulose-purified antibody protein was mixed with varying amounts of DEAE-cellulose-purified fatty acid synthetase in each of a series of test tubes containing 0.9% sodium chloride, 1.0% Triton X-100, and 10 mM potassium phosphate buffer, pH 8.0, in a volume of 2 ml. The mixtures were maintained at 22°C for 2 h, then at 4°C for 20 h. After centrifugation, the precipitates were repeatedly washed and then assayed for protein. The equivalence point of antigen and antibody was determined by the standard procedure. lmmunotitrations of enzymatic activities. Small quantities of purified fatty acid synthetase and affinitypurified anti-fatty acid synthetase antibody were incubated together in 0.2 M potassium phosphate buffer for 45 min at 30°C. A series of incubations was performed with the amount of fatty acid synthetase constant and the amount of antibody varied. In other titrations, enzyme was varied and antibody was constant. The effect of antibody on the catalytic activity of the fatty acid synthetase complex for fatty acid synthesis and its component reactions was determined by enzyme assay for each of the individual component catalytic activities. The lines and intercepts obtained from titrations with varying amounts of fatty acid synthetase were determined by linear regression. Enzyme assays. The necessary substrates were
IMMUNOREACTIVITY
OF REACTIONS
added for the determination of activity in the presence of varying amounts of antibody and in the absence of antibody. All assays except deacylase contained 200 pgiml of bovine serum albumin. In the deacyiase assay, the albumin is omitted because it prevents quantitative extraction of the product palmitic acid. Fatty acid synthesis. The concentrations of substrates in the assay mixture were malonyl coenzyme A, 100 pM; acetyl coenzyme A, 33 PM; and NADPH, 100 FM. The reaction was followed spectrophotometrically at 340 nm. Ketoreductase. The concentrations of substrates in the assay mixture were N-acetyl-S-acetoacetylcysteamine, 4.4 mgiml, and NADPH, 100 pM. NADPH oxidation was measured as for fatty acid synthesis and reaction volumes for both assays were 1 ml. Deacylase. [‘%]Palmitoyl coenzyme A was diluted with nonradioactive palmitoyl coenzyme A to a specific activity of 1 x lo4 dpm/pg. Assay mixtures for deacylase activity contained 0.01 M potassium phosphate, approximately 5 pg of fatty acid synthetase, and up to 35 pg of protein of anti-fatty acid synthetase antibody in a l-ml volume. The reaction was started by the addition of 2.16 kg of [‘%]palmitoyl coenzyme A. Incubations were carried out for 3 min at 30°C and the reaction was stopped by the addition of 30 ~1 of 60% perchloric acid. After the addition of 1 ml of ethanol, the reaction mixture was extracted three times with 2-ml portions each of petroleum ether. Radioactivity was determined in the petroleum ether extracts in a liquid scintillation spectrometer. Mixtures with a constant amount of antibody contained 12 fig of anti-fatty acid synthetase antibody and 2.5 to 15.0 kg of fatty acid synthetase. Acetyland malonyl-CoA transacylases. Assays were carried out by measuring the exchange of ‘“Clabeled acyl groups between CoA and pantetheine (8). Immunotitration mixtures with a constant amount of antigen contained 10 pg of fatty acid synthetase, 10 to 75 pg antibody, and 20 Fg albumin, in 0.20 ml of a 0.2 M potassium phosphate: 1 tIIM EDTA buffer, pH 7.0. These mixtures were incubated for 45 min at 30°C before completion of the assay. Pantetheine, 330 nmol in 50 ~1 of 0.2 M potassium phosphate buffer, was then placed in each of a series of small test tubes in an ice-water bath. A 20-~1 aliquot of immunotitration mixture (0.25 to 2.0 pg fatty acid synthetase protein) was then added to each tube. The exchange reaction was started by the addition of 40 ~1 of l“‘C]acetyl-coenzyme A (15,000 dpm, 10 nmol) or [W]malonyl-coenzyme A (20,000 dpm, 10 nmol), and the reaction was stopped after 5 min by the addition of 20 ~1 of 2 N acetic acid. The mixture was then pipetted onto a BioRad AGl-X8 (Cl-) ion exchange column 4 x 20 mm. The product was washed from the column with 4 ml of 2 N acetic acid, and an aliquot was assayed for radioactivity.
OF FATTY
ACID
SYNTHESIS
201
Condensation reaction. The condensation of acetyland malonyl-CoA thioesters was assayed by following the formation of triacetic lactone, the product formed in the absence of NADP+ and NADPH, by light adsorption at 280 nm (9). Antigen-antibody mixtures (0.225 ml) for the forward immunotitration contained a constant amount of fatty acid synthetase (3 yg) and varying amounts of antibody in the range of 1 to 100 pg of protein, whereas in the reverse titration, antibody was held constant at 1.2 Fg and fatty acid synthetase was varied between 1 and 7 pg. Each mixture was incubated for 45 min at 30°C and then centrifuged at 25009 for 5 min. The supernatant solution was placed in a microcuvette at 30°C and the substrate mixture containing acetyl-coenzyme A, 25 nmol, and malonyl-coenzyme A, 51.5 nmol, in 25 ~1 was added. Assays were carried out with a pinhole slit in a Gilford spectrophotometer. Determination of molecular weight of fatty acid synthetase:lgG complex. Fully active pigeon liver fatty acid synthetase, 100 fig of protein, was diluted with buffer to a final concentration of 0.1 M potassium chloride, 0.02 M potassium phosphate, and 1 ITIM EDTA. Two milligrams of serum albumin was added and the final volume was adjusted to 5.0 ml. An amount of monospecific antipigeon liver fatty acid synthetase IgG to just inactivate the fatty acid synthetase was then added. The antibody-antigen mixture was incubated at 30°C for 30 min and then concentrated to 1 ml with a Schleicher and Schuell dialysis membrane at 4°C. This mixture was passed without further delay into a Sepharose 6B column (1.5 x 64 cm). Eluate fractions were assayed for light absorption at 280 nm and for palmitoyl-CoA deacylase activity. The average molecular weight of the fatty acid synthetase:IgG complex was determined by extrap olation of a plot of V, against the logs of the molecular weights for thyroglobulin and rat liver fatty acid synthetase. RESULTS
Putification
of Antibody
Rabbit serum protein (10 mg), purified by ammonium sulfate fractionation and DEAEcellulose chromatography, was placed on an affinity column containing DEAE-cellulosepurified pigeon liver fatty acid synthetase as the bound ligand. Most of the protein eluted without binding. The bound protein was then eluted with 4.5 M MgC&. A typical profile of binding and elution of anti-pigeon liver fatty acid synthetase antibody is shown in Fig. 1. The yield of IgG specific for fatty acid synthetase was found to be 21%, and this compared well with the 24%
202
KATIYAR a&b,,, 1.
0
P
c
IO ELUATE (ml)
,,,b -P
ET AL.
Crossed-rocked immunoelectrophoresis of the antiserum purified on the immunoaffinity column containing bound DEAEcellulose-purified fatty acid synthetase against DEAE-cellulose-purified fatty acid synthetase, showed the presence of one major and one trace rocket (Fig. 3). This indicates that the antibody reacted with a trace (approximately 2%) of another antigen which was present in the DEAE-cellulosepurified preparation of fatty acid synthetase. However, when sucrose density gradientpurified fatty acid synthetase was bound to the affinity gel, the crossed-rocket immunoelectrophoresis of the antibody to DEAEcellulose purified enzyme showed only one rocket. 20
FIG. 1. Immunoaffinity chromatography of DEAEcellulose-purified rabbit serum immunoglobulins. Ten milligrams of protein in 0.02 M potassium phosphate, pH 8.0, solution (a) were placed on a Sepharose 4B: fatty acid synthetase affinity gel column (0.2 g of gel); the column was then washed with (b) 0.9% sodium chloride containing 1 mM EDTA, pH 7.0; (c) 4.5 M magnesium chloride neutralized to an apparent pH of 6.4, and finally with the above sodium chloride solution.
yield determined by quantitative precipitation of the antibody with fatty acid synthetase.
Quantitative
Precipitation
At the equivalence point 1.2 mg of a 5 mg aliquot of DEAE-cellulose-purified rabbit serum IgG was precipitated by purified pigeon liver fatty acid synthetase. This indicates a 24% yield of anti-pigeon liver fatty acid synthetase antibody in the IgG fraction. From the highest ratio of antibody to enzyme, it is calculated that 35 to 40 molecules of anti-fatty acid synthetase antibody are bound to one molecule of fatty acid synthetase.
Ouchterlony Double Diffusion and Crossed-Rocket ImmunoeEectroiDhoresis
The precipitin patterns obtained in Ouchterlony immunodiffusion studies are shown in Fig. 2. Affinity-purified antiserum gave a single line with DEAE-cellulosepurified fatty acid synthetase (Fig. 2, wells 1 and 2), and with the 100,OOOg supernatant solution of pigeon liver homogenate (wells 3 and 4). This indicates that the antiserum is monospecific. Cross-precipitation reactions of the antiserum, which did not bind to the immunoaffinity column, with varying concentrations of the DEAEcellulose-purified fatty acid synthetase, showed no precipitin lines indicating, therefore, that all the antiserum specific to fatty acid synthetase was bound to the column.
FIG. 2. Ouchterlony double diffusion analysis of anti-pigeon liver fatty acid synthetase antibody. The central well contained 10 pg of immunoaffinitypurified antipigeon liver fatty acid synthetase antibody. The outer wells marked 1 and 2 contained 5 and 10 pg of fatty acid synthetase, respectively, and the outer wells marked 3 and 4 contained 50 and 100 Kg of the 100,OOOg supernatant solution of the pigeon liver homogenate.
IMMLINOREACTIVITY
Comparative Immwnoreactivity Component Activities
OF
REACTIONS
OF FATTY
ACID
SYNTHESIS
203
of
The effects of increasing amounts of affinity-purified anti-fatty acid synthetase antibody on the catalytic activities of the fatty acid synthetase for fatty acid synthesis and for individual component reactions of this process are shown in Fig. 4. These results were obtained by immunotitrating a constant amount of antigen with increasing amounts of affinity-purified antibody. Inactivation patterns were similar for fatty acid synthesis and the condensation reaction, whereas transacylase and reductase activity components were progressively less sensitive to inactivation. The catalytic activity of the deacylase component was not inhibited by anti-fatty acid synthetase
MOLES ANTI-FATTY ACID SYNTHETASE MOLES FATTY ACID SYNTHETASE
FIG. 4. Immunotitration of the catalytic activities of pigeon liver fatty acid synthetase for fatty acid synthesis and its component partial reactions. Fatty acid synthetase and affinity-purified antibody in the ratios shown on the figures were mixed and incubated 45 min at 30°C before assay. (0) Fatty acid synthesis; (A) condensation reaction; (m) acetyl transacylation; (0) malonyl transacylation; (0) ketoreduction; (x) deacylation. The molecular weights used were 456,000 for the fatty acid synthetase and 150,000 for the antibody. (The amounts of antigen protein used for each titration were as follows: fatty acid synthesis, 2.5 Kg; condensation reaction, 3 pg; acetyl transacylation, 10 pg; malonyl transacylation, 10 pg; ketoreduction, 5 pg; deacylation, 5 wg.)
FIG. 3. Crossed immunoelectrophoresis of immunoaffinity-purified anti-pigeon liver fatty acid synthetase antibody. Fifty micrograms of DEAE-cellulose-purified fatty acid synthetase was applied to the gel. The first dimensional electrophoresis was carried out at 10 V/cm for 2 h at 25°C in 1% agarose in barbital buffer, pH 8.6, of an ionic strength of 0.02. The anode is to the right. The second dimensional immunoelectrophoretic run was carried out at 2 V/cm at 25°C for 18 h. The anode is at the top. The immunogel contained 1.1 fig anti-pigeon liver fatty acid synthetase antibody per cm’ gel area.
antibody. In fact, at low ratios of antibody to antigen, there appeared to be a slight increase in enzyme activity. The results were the same when DEAE-cellulosepurified antibody, rather than affinitypurified antibody was used. The effect of antibody on fatty acid synthesis and its component partial activities were also determined by titration with constant amounts of antibody and increasing amounts of enzyme. This reverse method provides a much sharper end point than the forward method of titration. The intercept on the y-axis of the linear plot of enzyme activity vs amount of enzyme protein is the amount of enzyme activity suppressed by the amount of anti-fatty acid synthetase antibody in the incubation mixture (Fig. 5).
KATIYAR
ET AL.
acylase reaction is essentially unaffected by the binding of antibody under nonprecipitation conditions. Effect of Dissociation of Fatty Acid Synthetase on Antigen-Antibody Interaction
FIG. 5. Immunotitration of the catalytic activities of pigeon liver fatty acid synthetase for fatty acid synthesis and for ketoreduction. Enzyme activity, expressed as rate of oxidation of NADPH (measured by the decrease in light absorption at 340 nm), is plotted against pg of enzyme protein. Before the spectrometric assay, affinity-purified anti-fatty acid synthetase antibody, 4.66 pg of protein, was incubated for 45 min at 30°C with the quantities of sucrose density gradient-purified fatty acid synthetase shown on the figure. (0) Ketoreduction; (0) fatty acid synthesis.
Similarly, the intercept on the x-axis is the amount of enzyme protein inactivated by the given amount of antibody. It is evident (Fig. 5) that the ketoreductase reaction is less sensitive to inactivation of catalytic activity by antibody than the fatty acid synthetase overall reaction. Table 1 lists the moles of anti-fatty acid synthetase antibody required to inactivate 1 mol of fatty acid synthetase activities for fatty acid synthesis and for the component partial reactions. The data in this table were obtained by “reverse” immunotitrations similar to the ones described above. The condensation reaction is, the most sensitive of the component reactions, requiring approximately 6 mol of antibody to inactivate 1 mol of fatty acid synthetase. The overall reaction for fatty acid synthesis is similarly inactivated. The transacylase reactions require more binding of antibody, and the ketoreductase requires near saturation binding (see quantitative precipitation) in order to reach minimum activity. The de-
Dissociation of fatty acid synthetase into half-molecular weight species affects the catalytic activities of the enzyme. Fatty acid synthesis and the condensation reaction are abolished, whereas the other component reactions such as acetyl transacylase and ketoreductase are reduced to two-thirds of their activity in the undissociated complex. However, dissociation of the enzyme to half-molecular weight subunits did not significantly change the sensitivities of the partial activities of these subunits on reaction with antibody. Molecular Weight of Antigen-Antibody Complex
The antigen-antibody complex formed by adding to pigeon liver fatty acid synthetase an amount of IgG that would just eliminate enzyme activity for fatty acid synthesis yielded one peak of protein on molecular filtration through Sepharose 6B. The molecular weight of this complex, as determined by light absorption at 280 nm and by palmitoyl-CoA deacylase activity, was TABLE
I
IMMUNOINHIBITION OF THE CATALYTIC OF FATTY ACID SYNTHETASE
Catalytic activity Fatty acid synthesis Condensation reaction Malonyl transacylation Acetyl transacylation Ketoreduction Deacylation
ACTIVITIES
Molar ratio (Antibody/antigen) 6.6 6.0 12 18 30 m
o Moles of anti-fatty acid synthetase antibody required to inhibit the catalytic activity of 1 mol of fatty acid synthetase as determined by extrapolation of the linear rate of inhibition obtained under conditions of antigen excess.
IMMUNOREACTIVITY
OF
REACTIONS
1.2 x 106. This value corresponds reasonably well with a complex containing 6 mol of IgG bound to 1 mol of undissociated fatty acid synthetase (calculated molecular weight of 1.43 x 106). There was no 280-nm absorbing material or deacylase activity in those eluate fractions which corresponded to the molecular weight of a complex between antifatty acid synthetase IgG and halfmolecular weight subunits of pigeon liver fatty acid synthetase (about half of 1.4 x 106, or 600,000 to 800,000). DISCUSSION
Studies on the rates of enzyme synthesis and degradation, especially those for antigens which vary markedly in animals in different nutritional states, are carried out by quantitative immunoprecipitation or immunotitration. The purity of the antiserum is therefore of critical importance, particularly in those studies in which immunoprecipitation is carried out. The results presented in this paper describe a general procedure for the purification of antiserum to pigeon liver fatty acid synthetase by immunoaffinity chromatography. The efficiency of the immunoaffinity column approaches 90%, as shown by the correspondence between the percentages of anti-fatty acid synthetase antibody removed by the column from the DEAEcellulose-purified antibody and by the quantitative immunoprecipitation with the antigen. The nearly monospecific antibody purified by the method indicated above has been used to study the comparative immunoreactivities of the catalytic activities of pigeon liver fatty acid synthetase. The inhibition of several of the component catalytic activities of the antigen by the antibody appears to be one of the important effects of antigenantibody interaction. The extent of inhibition due to antigen-antibody interaction was measured by the immunotitrations shown in Figs. 4 and 5. The two types of immunotitrations reported here give different types of information. The forward immunotitration (fixed amount of antigen, varying amount of antibody, Fig. 4) indicates the upper limit of inhibition reached in the
OF FATTY
ACID
SYNTHESIS
205
presence of excess of antibody. These titration curves are not linear except for a relatively short range of antibody/antigen molar ratios, as they are essentially saturation curves. On the other hand, reverse immunotitration (fixed amount of antibody, varying amount of antigen, Fig. 5) gives an essentially linear plot, which, when extrapolated to the y-axis, gives a measure of the limit of sensitivity of the catalytic sites of fatty acid synthetase to relatively small amounts of antibody. The data of Figs. 4 and 5 and Table I show the loss of activities for the component reactions as a function of antibody concentration. It is of interest to note that on the molar basis the Condensation-CO, exchange reaction is inhibited by 6 mol of purified antibody/m01 of fatty acid synthetase, whereas a larger molar excess of antibody is required for the inactivation of other partial activities; s-acetoacetyl-N-acetylcysteamine reductase required a molar excess of 30. These results, along with the quantitative precipitation data, demonstrate that while 6 molecules of antibody are sufficient to inactivate 1 molecule of fatty acid synthetase for the overall reaction, a total of 35 to 40 antibody molecules can bind to one molecule of the enzyme. The inhibition of the component reactions can be interpreted as follows: The mechanism of inhibition of the overall and partial reactions of fatty acid synthesis may involve conformational change or steric hindrance, or both, of the enzyme complex. A conformational change induced by the binding of 6 antibody molecules per fatty acid synthetase molecule might be sufficient to disrupt the condensation reaction. In this reaction, a precise geometry between the two binding sites (acetyl and malonyl groups) and the catalytic site may be very critical. That inactivation by dissociation of the fatty acid synthetase on binding of antibody does not take place is supported by the presence of an antigen-antibody complex of a molecular weight in the range of 1.2 x 106. For inhibition of other partial reactions of fatty acid synthesis, a greater conformational change and distortion of active sites would be required. Inhibition of these reactions would then require the
206
KATIYAR
binding of 18 to 30 antibody molecules per molecule of fatty acid synthetase (Fig. 4). Our results may also suggest, as an alternative explanation, that there are some antibodies which bind to antigen at critical areas around the condensing site. (This assumes that the condensing site is on the surface, or in an accessible crevice, and is more open than other active sites.) Under these conditions, the antibody would interfere with the transfer of substrates to the condensing site, and this site would, therefore be inaccessible to substrate. Such an enzyme molecule would also not be able to catalyze the overall synthesis of fatty acids. This interpretation implies that the catalytic sites for other partial reactions are either located inside the multienzyme complex and are therefore less affected by the binding of small numbers of antibody molecules, or that the greater proportion of antibodies are raised for antigenic segments located in regions remote from these catalytic sites. Further information on the antigen-antibody interaction is provided by the observed failure of dissociation of the fatty acid synthetase complex into two- halfmolecular weight subunits to affect the relative sensitivities of the partial reactions of fatty acid synthesis in the presence of antibody. This indicates either that the
ET AL.
portion of the fatty acid synthetase that is exposed after dissociation does not have antigenic determinants, or that if it does, the exposed portion is remote from the sites of component reactions. REFERENCES 1. KABAT, E. A., AND MAYER, M. M. (1961) Experimental Immunochemistry, 2nd ed. pp. 22-96, Charles C Thomas, Springfield, Ill. 2. MATSUURA, S., CHEN, H. C., AND HODGEN, B. D. (1978) Bioehemnistq 17, 575-580. 3. KUMAR, S., SRINIVASAN, K. R., AND ASATO, N. (1977) Biochim. Biophys. Acta 489, 32-47. 4. MUESING, R. A., AND PORTER, J. W. (1975) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 35, pp. 45-59, Academic Press, New York. 5. LIVINGSTON, D. M. (1974) in Methods in Enzymology (Jacoby, W. B., and Wilcheck, M., eds.), Vol. 34, Pt. B, pp. 723-731, Academic Press, New York. 6. MARCH, S. C., PARIKH, I., AND CUATRECASAS, P. (1974) Anal. Biochem. 60, 149-152. 7. WEEKE, B., (1973) in Quantitative Immunoelectrophoresis (Axelson, N. H., Kroll, J., and Weeke, B., eds.), pp. 47-59, Universitetforlaget, Oslo. 8. KUMAR, S., DORSEY, J. A., MUESING, R. A., AND PORTER, J. W. (1970) J. Biol. Chem. 245, 4732-4744. 9. NIXON, J. E., PUTZ, G. R., AND PORTER, J. W. (1968) J. Biol. Chem. 243, 5471-5478.