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[112] Transglutaminase
[112]
TRANSGLUTAMINASE
833
[ 112 ] Transglutaminase Ca++ Prot-CONH2 -~ NH~R ~- Prot-CONHR -}- NH3 (R = see text) B y HEINRICH WAELSCH AND MARY J. MYCEK
Introduction Transglutaminase, alternatively called "the amine-incorporating system, ''1 catalyzes the incorporation of primary amines, ammonia, hydroxylamine, some diamino acids, and some monoamino acid amides into certain acceptor proteins or peptides, such as casein, fl-lacteglobulin, insulin, or the A-chain of the latter. According to our present knowledge, this incorporation proceeds by a reversible replacement of the amide group of some of the protein-bound glutamine residues and results in the liberation of ammonia. 2 In the absence of added amine, the c-amino group of protein-bound lysine can replace the amide group of glutamine in what is assumed to be a reaction analogous to that occurring in the presence of amine. 2 Transglutaminase activity is Ca++-dependent.
Assay Methods On the basis of the reactions discussed above, transglutaminase activity m a y be assayed conveniently by three methods: (1) colorimetric determination of an enzymatically produced hydroxamic acid; (2) measurement of the radioactivityin the acceptor protein after incorporation of a C1~-labeled amine; and (3) determination of the ammonia produced. With acid-soluble acceptor proteins or peptides, measurement of hydroxylamine incorporation (method 1) is most convenient, since the ferric chloride reaction can be used directly.If the reaction product is acid-insoluble, the amount of hydroxylamine incorporated can be determined after hydrolysis2 It should be noted that the units of enzymatic activity are based on the hydroxylamine assay; however, the other two methods are useful in determining the specificityof the enzyme towards protein substrates. 1D. D. Clarke, M. J. Myeek, A. Neidle, and H. Waelsch, Arch. Biochem. Biophys. 79, 338 (1959). 2A. Neidle, M. J. Mycek, D. D. Clarke, and H. Waelsch, Arch. Biochem. Biophys. 77, 227 (1958). s T. Z. Csaky, Aeta Chem. ,$eand. 2, 450 (1948).
834
ENZYMES OF PROTEIN METABOLISM
[112]
Hydroxylamine Assay
Principle. The hydroxamic acid formed is assayed as a colored complex on the addition of ferric chloride reagent. Reagents N-Z Amine Type E (Sheffield Laboratories). This material is used as a source of acceptor peptides. An ethanol extract of the commercial tryptic digest of casein is prepared as follows: 100 g. of the :N-Z Amine powder is extracted with 200 ml. of 8 0 ~ ethanol (about 15% is soluble). After centrifugation, the supernatant fluid is evaporated to dryness. The residue is dissolved in water (about 25 ml.), heated with activated charcoal (1 g.), filtered, and water added (to about 45 ml.) for a final concentration of 300 mg./ml. 0.1 M CaC12. 0.2 M glutathione adjusted to pH 8 with N a 0 H . 0.1 M Tris buffer, pH 8. 1.0 M hydroxylamine-HCl. Neutralized before use with 6 N NaOH and diluted to 0.5 M with water. FeC18-trichloroacetic acid reagent. 4 Enzyme. Diluted to about 100 to 1000 units.
Procedure. To a solution containing 1.0 ml. of the ethanolic extract of N-Z Amine, 0.1 ml. of 0.1 M CaC12, 0.1 ml. of 0.2 M glutathione, 0.2 ml. of 0.5 M hydroxylamine, and 0.4 ml. of 0.1 M Tris buffer in a 15-ml. conical centrifuge tube, a suitable amount of enzyme is added. The final volume is adjusted to 2 ml. with water. After a 10-minute incubation at 37 °, 1.5 ml. of the ferric chloride-trichloroacetic reagent is added. The precipitated protein is removed by centrifugation, and the color is read immediately against a no-enzyme blank in a Coleman Junior spectrophotometer at 525 m~. A blank without Ca ++ is also incubated simultaneously, although this blank sometimes results in higher values than the no-enzyme blank, owing to the difficulty in removing traces of Ca ++ from the substrate. Definition of Unit and Specific Activity. A unit is defined as that amount of enzyme which results in an optical density of 0.0001 per minute. Specific activity is expressed as units per milligram of protein. Protein may be determined either by the biuret reaction 5 or by the Lowry procedure2 ' F . Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1954). 5j. W. Mehl, J. Biol. Chem. 157, 173 (1945). a O. It. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
[112]
TRANSGLUTAMINASE
835
Incorporation of C14-Labeled Amine Principle. The amount of radioactivity incorporated into an acceptor protein is determined at infinite thickness; the amount of amine incorporated (in micromoles) into a given amount of protein can be calculated from these data. 1 Reagents
Casein or other substrate protein. A solution containing 30 mg./ml. is dialyzed against 0.005 M Versenv, pH 8, overnight in the cold to remove calcium ions or other interfering substances such as ammonia. A dry-weight determination is carried out. 0.1 M CaC12. 0.5 M glutathione adjusted to pH 8.0 with NaOH. 0.1 M Tris buffer, pH 8.0. 0.04M C14-1abeled primary amine (0.05 microcurie/micromole). (For preparation of various C'-labeled amines, see original publication. 1) Enzyme, 2000 units/2 ml. Procedure. To 0.5 ml. of the protein substrate solution, 0.1 ml. of 0.1 M CaC12, 0.2 ml. of 0.5 M glutathione, 0.8 ml. of Tris buffer, pH 8.0, and 0.2 ml. of 0.04 M C'-labeled amine, a n amount of enzyme equivalent to 2000 units is added, and the final volume is brought to 2 ml. with water. As an appropriate blank, another tube is incubated without added Ca ++. The tubes are incubated for 1 hour at 37 °. The reaction is stopped by the addition of 0.2 ml. of 50~ trichloroacetic acid. The precipitated protein is removed by centrifugation and subsequently is washed thoroughly. (The supernatant solution can be assayed for ammonia released in the course of the reaction, provided a nonvolatile amine has been employed in the procedure.) The washing of the protein is carried out according to the method of Siekevitz. T The washed protein is plated on perforated Teflon planchets (0.8-cm. 2 cross-sectional area) and counted at infinite thickness in a thin-window gas-flow counter, to a precision of ___1%. The radioactivity of a sample of a derivative of the Cl~-labeled amine adequately diluted with the unlabeled amine derivative (e.g., cadaverine diHC1, methylamine pierate) is counted at infinite thickness. After correction for the dilution resulting from the use of the derivative in place of the amine itself, these data are used to recalculate the extent of amine incorporation as micromoles of amine incorporated per 100 mg. of protein.
' P. Siekevitz, J. Biol. Chem. 195, 549 (1952).
836
ENZYMES
OF P R O T E I N M E T A B O L I S M
[112]
Estimation of Released Ammonia
Principle. The ammonia is absorbed into boric acid after being released from the incubation mixture by saturated potassium carbonate. The procedure is carried out in a Conway microdiffusion dish. Reagents Same as above for the incorporation of a C14-1abeled amine. 2% boric acid with mixed indicator. Mixed indicator: 2 ml. of 1% methyl red in ethanol added to 10 ml. of 1 ~ bromocresol green in ethanol. This is placed into 500 ml. of 2% boric acid. Saturated KsCOs.
Procedure. The same procedure is followed as that described for the incorporation of amine. The supernatant fluid, obtained after the reaction has been stopped with trichloroacetic acid, is used in this determination. It is convenient to remove ca. 1.3 ml. of the supernatant fluid. This amount is placed in 5-ml. volumetric flasks which have been previously washed in acid. After neutralization by the dropwise addition of 6 N NaOH, water is added to volume. Aliquots of 2 ml. of each are used for the Conway method, s The diffusion vessel is prepared with 1 ml. of boric acid solution in the center well and 1 ml. of saturated K2C0s in the outer well. It is allowed to stand at room temperature for 2 hours. The center well is titrated with 1/70 N HC1. Purification Procedure The purification of transglutaminase has not progressed very far; therefore these data are presented as a means of facilitating the first steps of purification. It should be noted that transglutaminase occurs in the soluble protein fraction of the tissue after the removal of the particulates. Step I. Preparation o] Liver Supernatant. All steps are carried out in the cold. The livers from 10 guinea pigs (ca. 150 g.) are divided into 10-g. portions to each of which is added 20 ml. of 0.25 M sucrose. The tissue is minced prior to homogenization in a Potter-Elvehjem glass homogenizer. The combined homogenatos are then centrifuged at 78,000 X g in the Spinco Model L refrigerated centrifuge for 1 hour. Step 2. pH 5 Precipitation. The supernatant fluid is collected, and 1 ml. of 1 M sodium acetate is added for each 100 ml. of supernatant. Glacial acetic acid is then added dropwise with stirring until a final pH of 5.1 is obtained. The precipitate that forms is collected by centrifuga' E . J. Conway, "Microdiffusion Analysis and Volumetric Error, #' 4th ed., p. 98. Macmillan, New York, 1958.
[112]
TRANSGLUTAMINASE
837
tion in the Servall SS-1 centrifuge (18,000 X g) for 20 minutes. The supernatant fluid is discarded. Step 3. Washing and Extraction o] the pH 5 Precipitate. The precipitate is suspended in a volume of 0.05 M sodium acetate buffer, pH 4.5, corresponding to about 2 5 ~ of the volume of supernatant fluid. The suspension of the pH 5 precipitate in the acetate buffer removes some active proteins and ottier impurities. The suspension is again centrifuged for 20 minutes at top speed, and the supernatant fluid is discarded. The residue is resuspended in a volume of 0.05 M phosphate buffer, pH 6.0, corresponding to 15~ of the original supernatant fluid. Once more the suspension is subjected to centrifugation in the Servall. The precipitate is homogenized in 0.05M phosphate buffer, pH 6.5 (a volume 15% of the original supernatant fluid), and the suspension is centrifuged at 18,000 X g for 20 minutes. The supernatant fluid containing the enzyme is dialyzed overnight in the cold against 0.001 M Versene, pH 7.5. This solution contains 5 to 11 rag. of protein per milliliter. A summary of the purification procedure is given in the table. SUMMARY OF PURIFICATION PROCEDURE"
Step
Homogenate Supernatant pH 6.5 extract of pH 5 precipitate
Volume Specific of Units, Protein, activity, Yield, solution, X 101 mg. units/rag. % rid. protein 300 200 30
1053 834 376
9570 5960 330
110 140 1141
100
79 36
° Units were measured b y the hydroxylamine method with N-Z Amine as the source of accepter peptides.
Properties
Occurrence. Traces of transglutaminase activity have been found in the soluble fractions of the organs of many mammals. Guinea pig liver is the most active. Specificity. A variety of proteins can serve as amine acceptors; among the best of these are casein, fl-lactoglobulin, insulin, and its A-chain. Ovalbumin, ~-globulin, and serum albumin are among those proteins which do not serve as substrates. Most primary amines tested as well as ammonia and hydroxylamine are incorporated. However, the enzyme may exhibit some specificity toward the replacing amine; e.g., tyrosine amide is not incorporated. Furthermore, the presence of a free carboxyl function in the replacing amine interferes with its ability to participate
838
ENZYMES OF PROTEIN METABOLISM
[113]
in the reaction; e.g., glycine and ~,-aminobutyric acid are not incorporated, in contrast to glycine amide. pH Optimum. The optimal incorporation of amines into proteins occurs at pH 7.5 to 8.5; however, the pH of optimal ammonia liberation varies with the substrate. It is, therefore, possible that another reaction capable of releasing ammonia (e.g., either replacement by the c-amine group of protein-bound lysine or hydrolysis) can also take place. 9,~° Activators and Inhibitors. The enzyme has a requirement for calcium ions. Strontium, and to a lesser degree Mn ++, Ba +÷, and Mg ++, can replace the calcium requirement. Heavy metal ions, such as Cu ++, Hg ++, Zn++, and Fe ++, inhibited the reaction even in the presence of Ca +*. Reducing agents, such as glutathione, thioglycolate, cysteine, or cyanide, are also required to maintain activity. Iodoacetamide (0.002M) and p-chloromercuribenzenesulfonic acid (0.001 M) completely inhibit the enzyme. Substances capable of combining with Ca ++ (e.g., citrate, phosphate, pyrophosphate, and fluoride) inhibit the reaction; these inhibitions can be overcome by adding excess Ca++. g M. J. Mycek, D. D. Clarke, A. Neidle, and H. Waelsch, Arch. Biochem. Biophys. 84, 528 (1959). lo M. J. Mycek and H. Waelsch, J. Biol. Chem. 235, 3513 (1960).
[ 113] Serine Hydroxymethylase L-Serine~ Glycine -{-H C H 0
(I)
By K. G. SCRIMGEOURand F. M. HUENNEKENS Assay Method
Principle. Serine hydroxymethylase (also called serine aldolase ~) catalyzes the reversible hydrolytic cleavage of L-serine into glycine and formaldehyde, as shown in the above equation. In the direction of serine breakdown, the reaction may be followed by coupling the product, HCHO, with the TPN-linked NS,Nl°-methylene tetrahydrofolic dehydrogenase.2,3 The enzymatic reaction has also been measured from right to left by decomposing the product, serine, with periodate~,4 and measuring the one-carbon unit released either, as HCHO or as CO2, or by 1N. Alexander and D. M. Greenberg, J. Biol. Chem. 220, 775 (1956). 2F. M. Huennekens, Y. Hatefi, and L. D. Kay, J. Biol. Chem. 224, 435 (1957). s y . Hatefi, M. J Osborn, L. D. Kay, and F. M. Huennekens, J. Biol. Chem. 227, (}37 (1957). ' R. L. Blakley, Biochem. J. 61, 315 (1955).
TRANSGLUTAMINASE
833
[ 112 ] Transglutaminase Ca++ Prot-CONH2 -~ NH~R ~- Prot-CONHR -}- NH3 (R = see text) B y HEINRICH WAELSCH AND MARY J. MYCEK
Introduction Transglutaminase, alternatively called "the amine-incorporating system, ''1 catalyzes the incorporation of primary amines, ammonia, hydroxylamine, some diamino acids, and some monoamino acid amides into certain acceptor proteins or peptides, such as casein, fl-lacteglobulin, insulin, or the A-chain of the latter. According to our present knowledge, this incorporation proceeds by a reversible replacement of the amide group of some of the protein-bound glutamine residues and results in the liberation of ammonia. 2 In the absence of added amine, the c-amino group of protein-bound lysine can replace the amide group of glutamine in what is assumed to be a reaction analogous to that occurring in the presence of amine. 2 Transglutaminase activity is Ca++-dependent.
Assay Methods On the basis of the reactions discussed above, transglutaminase activity m a y be assayed conveniently by three methods: (1) colorimetric determination of an enzymatically produced hydroxamic acid; (2) measurement of the radioactivityin the acceptor protein after incorporation of a C1~-labeled amine; and (3) determination of the ammonia produced. With acid-soluble acceptor proteins or peptides, measurement of hydroxylamine incorporation (method 1) is most convenient, since the ferric chloride reaction can be used directly.If the reaction product is acid-insoluble, the amount of hydroxylamine incorporated can be determined after hydrolysis2 It should be noted that the units of enzymatic activity are based on the hydroxylamine assay; however, the other two methods are useful in determining the specificityof the enzyme towards protein substrates. 1D. D. Clarke, M. J. Myeek, A. Neidle, and H. Waelsch, Arch. Biochem. Biophys. 79, 338 (1959). 2A. Neidle, M. J. Mycek, D. D. Clarke, and H. Waelsch, Arch. Biochem. Biophys. 77, 227 (1958). s T. Z. Csaky, Aeta Chem. ,$eand. 2, 450 (1948).
834
ENZYMES OF PROTEIN METABOLISM
[112]
Hydroxylamine Assay
Principle. The hydroxamic acid formed is assayed as a colored complex on the addition of ferric chloride reagent. Reagents N-Z Amine Type E (Sheffield Laboratories). This material is used as a source of acceptor peptides. An ethanol extract of the commercial tryptic digest of casein is prepared as follows: 100 g. of the :N-Z Amine powder is extracted with 200 ml. of 8 0 ~ ethanol (about 15% is soluble). After centrifugation, the supernatant fluid is evaporated to dryness. The residue is dissolved in water (about 25 ml.), heated with activated charcoal (1 g.), filtered, and water added (to about 45 ml.) for a final concentration of 300 mg./ml. 0.1 M CaC12. 0.2 M glutathione adjusted to pH 8 with N a 0 H . 0.1 M Tris buffer, pH 8. 1.0 M hydroxylamine-HCl. Neutralized before use with 6 N NaOH and diluted to 0.5 M with water. FeC18-trichloroacetic acid reagent. 4 Enzyme. Diluted to about 100 to 1000 units.
Procedure. To a solution containing 1.0 ml. of the ethanolic extract of N-Z Amine, 0.1 ml. of 0.1 M CaC12, 0.1 ml. of 0.2 M glutathione, 0.2 ml. of 0.5 M hydroxylamine, and 0.4 ml. of 0.1 M Tris buffer in a 15-ml. conical centrifuge tube, a suitable amount of enzyme is added. The final volume is adjusted to 2 ml. with water. After a 10-minute incubation at 37 °, 1.5 ml. of the ferric chloride-trichloroacetic reagent is added. The precipitated protein is removed by centrifugation, and the color is read immediately against a no-enzyme blank in a Coleman Junior spectrophotometer at 525 m~. A blank without Ca ++ is also incubated simultaneously, although this blank sometimes results in higher values than the no-enzyme blank, owing to the difficulty in removing traces of Ca ++ from the substrate. Definition of Unit and Specific Activity. A unit is defined as that amount of enzyme which results in an optical density of 0.0001 per minute. Specific activity is expressed as units per milligram of protein. Protein may be determined either by the biuret reaction 5 or by the Lowry procedure2 ' F . Lipmann and L. C. Tuttle, J. Biol. Chem. 159, 21 (1954). 5j. W. Mehl, J. Biol. Chem. 157, 173 (1945). a O. It. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
[112]
TRANSGLUTAMINASE
835
Incorporation of C14-Labeled Amine Principle. The amount of radioactivity incorporated into an acceptor protein is determined at infinite thickness; the amount of amine incorporated (in micromoles) into a given amount of protein can be calculated from these data. 1 Reagents
Casein or other substrate protein. A solution containing 30 mg./ml. is dialyzed against 0.005 M Versenv, pH 8, overnight in the cold to remove calcium ions or other interfering substances such as ammonia. A dry-weight determination is carried out. 0.1 M CaC12. 0.5 M glutathione adjusted to pH 8.0 with NaOH. 0.1 M Tris buffer, pH 8.0. 0.04M C14-1abeled primary amine (0.05 microcurie/micromole). (For preparation of various C'-labeled amines, see original publication. 1) Enzyme, 2000 units/2 ml. Procedure. To 0.5 ml. of the protein substrate solution, 0.1 ml. of 0.1 M CaC12, 0.2 ml. of 0.5 M glutathione, 0.8 ml. of Tris buffer, pH 8.0, and 0.2 ml. of 0.04 M C'-labeled amine, a n amount of enzyme equivalent to 2000 units is added, and the final volume is brought to 2 ml. with water. As an appropriate blank, another tube is incubated without added Ca ++. The tubes are incubated for 1 hour at 37 °. The reaction is stopped by the addition of 0.2 ml. of 50~ trichloroacetic acid. The precipitated protein is removed by centrifugation and subsequently is washed thoroughly. (The supernatant solution can be assayed for ammonia released in the course of the reaction, provided a nonvolatile amine has been employed in the procedure.) The washing of the protein is carried out according to the method of Siekevitz. T The washed protein is plated on perforated Teflon planchets (0.8-cm. 2 cross-sectional area) and counted at infinite thickness in a thin-window gas-flow counter, to a precision of ___1%. The radioactivity of a sample of a derivative of the Cl~-labeled amine adequately diluted with the unlabeled amine derivative (e.g., cadaverine diHC1, methylamine pierate) is counted at infinite thickness. After correction for the dilution resulting from the use of the derivative in place of the amine itself, these data are used to recalculate the extent of amine incorporation as micromoles of amine incorporated per 100 mg. of protein.
' P. Siekevitz, J. Biol. Chem. 195, 549 (1952).
836
ENZYMES
OF P R O T E I N M E T A B O L I S M
[112]
Estimation of Released Ammonia
Principle. The ammonia is absorbed into boric acid after being released from the incubation mixture by saturated potassium carbonate. The procedure is carried out in a Conway microdiffusion dish. Reagents Same as above for the incorporation of a C14-1abeled amine. 2% boric acid with mixed indicator. Mixed indicator: 2 ml. of 1% methyl red in ethanol added to 10 ml. of 1 ~ bromocresol green in ethanol. This is placed into 500 ml. of 2% boric acid. Saturated KsCOs.
Procedure. The same procedure is followed as that described for the incorporation of amine. The supernatant fluid, obtained after the reaction has been stopped with trichloroacetic acid, is used in this determination. It is convenient to remove ca. 1.3 ml. of the supernatant fluid. This amount is placed in 5-ml. volumetric flasks which have been previously washed in acid. After neutralization by the dropwise addition of 6 N NaOH, water is added to volume. Aliquots of 2 ml. of each are used for the Conway method, s The diffusion vessel is prepared with 1 ml. of boric acid solution in the center well and 1 ml. of saturated K2C0s in the outer well. It is allowed to stand at room temperature for 2 hours. The center well is titrated with 1/70 N HC1. Purification Procedure The purification of transglutaminase has not progressed very far; therefore these data are presented as a means of facilitating the first steps of purification. It should be noted that transglutaminase occurs in the soluble protein fraction of the tissue after the removal of the particulates. Step I. Preparation o] Liver Supernatant. All steps are carried out in the cold. The livers from 10 guinea pigs (ca. 150 g.) are divided into 10-g. portions to each of which is added 20 ml. of 0.25 M sucrose. The tissue is minced prior to homogenization in a Potter-Elvehjem glass homogenizer. The combined homogenatos are then centrifuged at 78,000 X g in the Spinco Model L refrigerated centrifuge for 1 hour. Step 2. pH 5 Precipitation. The supernatant fluid is collected, and 1 ml. of 1 M sodium acetate is added for each 100 ml. of supernatant. Glacial acetic acid is then added dropwise with stirring until a final pH of 5.1 is obtained. The precipitate that forms is collected by centrifuga' E . J. Conway, "Microdiffusion Analysis and Volumetric Error, #' 4th ed., p. 98. Macmillan, New York, 1958.
[112]
TRANSGLUTAMINASE
837
tion in the Servall SS-1 centrifuge (18,000 X g) for 20 minutes. The supernatant fluid is discarded. Step 3. Washing and Extraction o] the pH 5 Precipitate. The precipitate is suspended in a volume of 0.05 M sodium acetate buffer, pH 4.5, corresponding to about 2 5 ~ of the volume of supernatant fluid. The suspension of the pH 5 precipitate in the acetate buffer removes some active proteins and ottier impurities. The suspension is again centrifuged for 20 minutes at top speed, and the supernatant fluid is discarded. The residue is resuspended in a volume of 0.05 M phosphate buffer, pH 6.0, corresponding to 15~ of the original supernatant fluid. Once more the suspension is subjected to centrifugation in the Servall. The precipitate is homogenized in 0.05M phosphate buffer, pH 6.5 (a volume 15% of the original supernatant fluid), and the suspension is centrifuged at 18,000 X g for 20 minutes. The supernatant fluid containing the enzyme is dialyzed overnight in the cold against 0.001 M Versene, pH 7.5. This solution contains 5 to 11 rag. of protein per milliliter. A summary of the purification procedure is given in the table. SUMMARY OF PURIFICATION PROCEDURE"
Step
Homogenate Supernatant pH 6.5 extract of pH 5 precipitate
Volume Specific of Units, Protein, activity, Yield, solution, X 101 mg. units/rag. % rid. protein 300 200 30
1053 834 376
9570 5960 330
110 140 1141
100
79 36
° Units were measured b y the hydroxylamine method with N-Z Amine as the source of accepter peptides.
Properties
Occurrence. Traces of transglutaminase activity have been found in the soluble fractions of the organs of many mammals. Guinea pig liver is the most active. Specificity. A variety of proteins can serve as amine acceptors; among the best of these are casein, fl-lactoglobulin, insulin, and its A-chain. Ovalbumin, ~-globulin, and serum albumin are among those proteins which do not serve as substrates. Most primary amines tested as well as ammonia and hydroxylamine are incorporated. However, the enzyme may exhibit some specificity toward the replacing amine; e.g., tyrosine amide is not incorporated. Furthermore, the presence of a free carboxyl function in the replacing amine interferes with its ability to participate
838
ENZYMES OF PROTEIN METABOLISM
[113]
in the reaction; e.g., glycine and ~,-aminobutyric acid are not incorporated, in contrast to glycine amide. pH Optimum. The optimal incorporation of amines into proteins occurs at pH 7.5 to 8.5; however, the pH of optimal ammonia liberation varies with the substrate. It is, therefore, possible that another reaction capable of releasing ammonia (e.g., either replacement by the c-amine group of protein-bound lysine or hydrolysis) can also take place. 9,~° Activators and Inhibitors. The enzyme has a requirement for calcium ions. Strontium, and to a lesser degree Mn ++, Ba +÷, and Mg ++, can replace the calcium requirement. Heavy metal ions, such as Cu ++, Hg ++, Zn++, and Fe ++, inhibited the reaction even in the presence of Ca +*. Reducing agents, such as glutathione, thioglycolate, cysteine, or cyanide, are also required to maintain activity. Iodoacetamide (0.002M) and p-chloromercuribenzenesulfonic acid (0.001 M) completely inhibit the enzyme. Substances capable of combining with Ca ++ (e.g., citrate, phosphate, pyrophosphate, and fluoride) inhibit the reaction; these inhibitions can be overcome by adding excess Ca++. g M. J. Mycek, D. D. Clarke, A. Neidle, and H. Waelsch, Arch. Biochem. Biophys. 84, 528 (1959). lo M. J. Mycek and H. Waelsch, J. Biol. Chem. 235, 3513 (1960).
[ 113] Serine Hydroxymethylase L-Serine~ Glycine -{-H C H 0
(I)
By K. G. SCRIMGEOURand F. M. HUENNEKENS Assay Method
Principle. Serine hydroxymethylase (also called serine aldolase ~) catalyzes the reversible hydrolytic cleavage of L-serine into glycine and formaldehyde, as shown in the above equation. In the direction of serine breakdown, the reaction may be followed by coupling the product, HCHO, with the TPN-linked NS,Nl°-methylene tetrahydrofolic dehydrogenase.2,3 The enzymatic reaction has also been measured from right to left by decomposing the product, serine, with periodate~,4 and measuring the one-carbon unit released either, as HCHO or as CO2, or by 1N. Alexander and D. M. Greenberg, J. Biol. Chem. 220, 775 (1956). 2F. M. Huennekens, Y. Hatefi, and L. D. Kay, J. Biol. Chem. 224, 435 (1957). s y . Hatefi, M. J Osborn, L. D. Kay, and F. M. Huennekens, J. Biol. Chem. 227, (}37 (1957). ' R. L. Blakley, Biochem. J. 61, 315 (1955).