[110] Branched-chain amino acid aminotransferase (pig heart, soluble)

802

BRANCHED-CHAIN AMINO ACIDS

[110]

alumina step, and the overall yield is approximately 1/100 of the crude extracts. Properties

Substrate Specificity. The enzyme reacts with L-leucine, L-valine, Lisoleucine, and L-norvaline. Using Vmax for L-leucine as 100, the Vm,~x of L-valine is 80, that of L-isoleucine is 65 and that of L-norvaline is 25. DPN is absolutely specific for the enzyme. 4 Kinetic Properties. The following Michaelis constants, Kin, have been determined at pH 11.3 for the oxidative deamination reaction: 1.6 × 10 -4 M for DPN, 6.2 × 10 -3 for L-leucine, 2.0 × 10-3 for valine and 5.2 x 10-a for isoleucine. For the reverse reaction the reported K,, values are: 1.2 x 10 -4 for DPNH, 2.2 X 10-3 for ot-ketoisovaleric acid, 3.3 × 10-3 for a-ketoisocaproic acid, and 1.3 X 10 -z for NH4 +. The equilibrium constant, K'e, = (a-ketoisocaproate)(leucine)(DPNH)(DPN) +(NH4+) (H +) = (11.1 --+ 1) X 10 -14

pH Optimum and lnhibitors. The optimum pH of the oxidative deamination reaction is 11.3. The enzyme is very sensitive to sulfide and pCMB. The inhibition by the latter is completely reversed by L-cysteine. Chelating agents do not affect enzyme activity. 4M. W. Zink and B. D. Sanwal, Arch. Biochem.Biophys. 99, 72 (1962).

[110] Branched-Chain Amino Acid Aminotransferase (Pig Heart, Soluble) 1 ByW. TERRYJENKINSand ROBERTT. TAYLOR Branched-chain L-amino acids (leucine, valine, isoleucine) + a-ketoglutarate ~ branched-chain a-keto acids + L-glutamate

Assay M e t h o d s

Principle. When leucine is used as the substrate, the 2,4-dinitrophenylhydrazone of a-ketoisocaproate may be selectively extracted by cyclo1There appear to be at least two enzymes capable of transamination from leucine to a-ketoglutarate that occur together in tissues. This particular enzyme occurs in the cytoplasm (supernatant) and has a wide specificity including valine and isoleucine. Assignment of the Enzyme Commission number EC 2.6.1.6 is thus ambiguous. For descriptions of other enzymes, see this volume [111], [113]. 2R. T. Taylor and W. T.Jenkins,J.Biol. Chem. 241 4391 (1966).

[110]

AMINOTRANSFERASE(PIG HEART, SOLUBLE)

803

hexane from acidic solutions containing an excess of ot-ketoglutarate 2,4-dinitrophenylhydrazone. ~

Reagents L-Leucine, 0.02 M Tris-ketoglutarate buffer containing 0.02 M a-ketoglutaric acid and 0.2 M Tris base, pH 8.6 Dinitrophenylhydrazine reagent made as follows: 2,4-dinitrophenylhydrazine (600 mg) is thoroughly triturated in a mortar with 34 ml of concentrated HCI. T h e volume is then brought up to 200 ml by the slow addition of water, and the solution is clarified by filtration. NaOH, 1 N Na2COa, 10% w/v Procedure. The reaction mixture (3 ml) contains 20 micromoles each of leucine, ketoglutarate and enzyme. The reaction is initiated, after equilibration at 37 °, by the addition of enzyme; it is terminated 10 minutes later by the addition of 1 ml of the acidic dinitrophenylhydrazine reagent. T h e assays are conveniently carried out in 12-ml centrifuge tubes, mixing with a vortex mixer. After exactly 10 minutes at room temperature, during which a heavy precipitate of the ketoglutarate hydrazone forms, the solution is extracted with 5 ml of cyclohexane by shaking vigorously for 20 seconds. (The tubes are conveniently capped with polyethylene Caplugs)) T h e phases are separated by brief centrifugation in a clinical centrifuge; then 4 ml of the upper phase is carefully removed, with a syringe, to another 12-ml centrifuge tube. Care must be taken not to transfer any ketoglutarate hydrazone from the walls or interface. The ot-ketoisocaproate hydrazone is extracted from the cyclohexane with 1.5 ml of 10% w/v sodium carbonate. The amount is determined by mixing a 1-ml aliquot with 2 ml of 1 N N a O H and reading the absorbance at 440 m~. This assay was found to be suitable for tissue extracts. T h e amount of color developed is dependent on both the amount of ketoglutarate employed, and to some extent on the phenylhydrazine reagent. The absorbances are most readily converted into micromoles isocaproate formed by carrying a known amount through the procedure as an internal standard. One micromole gives an absorbance of about 1.4. Protein Determination. Proteins were determined by the Lowry method 3a with bovine serum albumin as a standard. aProtectiveClosures Co., 2207 ElmwoodAve., Buffalo,New York 14216. aaVol. IIl [73].

804

BRANCHED-CHAIN AMINO ACIDS

[ 110]

Preparation o f Leucine Transaminase 4

Step 1. Extraction and Heating. Forty-five pounds of pig heart, obtained from the slaughterhouse fresh on ice, are trimmed free of auricles with a pair of 6-inch scissors. The ventricles are minced through 3-mm holes in a Hobart electric meat grinder (Model 4812) and then combined with an equal Volume o f 0.05 M caproate containing 0.005 M EDTA, p H 6.1.5 The suspension of minced ventricles is then put into a 35liter stainless steel beaker and heated to 62 ° over a period of about 10 minutes, in a water bath maintained at 750; 6 it is stirred with a wooden paddle. When the temperature reaches 40 °, 100 ml of 1 M sodium aketoglutarate (or 14.6 g of ketoglutaric acid) is added to convert the pyridoxamine form of the enzyme into the pyridoxal form. When the suspension reaches 62 °, the temperature of the bath is lowered to 63 ° by the addition of ice, and the heat treatment is continued at 62 ° for a further 20 minutes. Immediately after this period, the denatured material is removed by filtration through four layers of cheesecloth fashioned into a 150 × 14 cm diameter sock. This filtration and subsequent steps were carried out in a 0.5 ° cold room. Step 2. Ammonium Sulfate Fractionation. The filtrate (about 18 liters ~) is clarified by centrifugation for 10 minutes at top speed in a Sorvall GSA rotor (10,000 g) and then treated with ammonium sulfate (290 g/liter). The precipitate after 1.5 hours is collected by centrifugation, as before, and dissolved in a minimum of 0.02 M Tris caproate, containing 2 mM EDTA, pH 6. After dialysis overnight against this same buffer the pH is adjusted to pH 8.5 by the addition of 1 M Tris base (about 20 ml); any precipitate remaining after 20 minutes is removed by centrifugation. Step 3. DEAE-Sephadex Chromatograph),. The enzyme solution is allowed to pass slowly (1 ml/minute) through a large column (10 × 30 cm) of DEAE-Sephadex (A50) previously equilibrated with 0.05 M Tris-caproate buffer (0.05 M with respect to Tris) containing 0.005 M EDTA, pH 8.5. 8 The enzyme is eluted with a linear concentration gradient at a rate of about 1 ml per minute. The gradient is made by having a 2-liter mixer containing the equilibration buffer joined by a siphon to an iden4This is a modification of the earlier procedure. R. T. Taylor and W. T. Jenkins, J. Biol. Chem. 241 4396 (1966). SThe solution is essentially sodium caproate. T o make 1.5 liters of 0.5 M caproate, 0.05 M EDTA, mix 28.5 g of disodium EDTA, 88 g of caproic acid (about 95 ml), and 54 g (about 35 ml) of sodium hydroxide solution; 50% w/w. T h e r e should be only a trace of undissolved caproic acid on the surface. eGroen Model D30 steam-jacketed kettle. ~For assay add 25- and 50-/~1 aliquots to a 3-ml volume and incubate for 10 minutes at 37 °. ' I t is easy to overload this column; check to see that all the enzyme has been adsorbed.

[110]

AMINOTRANSFERASE (PIG HEART, SOLUBLE)

805

tical reservoir containing 2 liters of this buffer together with 0.5 M sodiu m chloride. Tubes containing the leucine transaminase are pooled. 9 Step 4. Chromatography on Hydroxylapatite. For this step, it is essential to have a large (4.5 x 40 cm) column o f hydroxylapatite ~° which possesses good flow characteristics. Both alanine and leucine transaminases are adsorbed onto the hydroxylapatite from the DEAE-column eluate. T h e E D T A in the eluate protects the enzymes on the column, but it also slowly dissolves some o f the hydroxylapatite. T h e transaminases are eluted by a linear gradient m a d e f r o m a l-liter mixer containing 0.025 M Tris-caproate, 0.0025 M EDTA, pH 6.8, and an identical reservoir containing this buffer together with 0.5 M potassium phosphate, pH 6.8. T h e first yellow peak to emerge is alanine transaminase? ~ T h e leucine transaminase, which is well separated, is associated with the latter part of a subsequent red peak. Step 5. CM-Sephadex Chromatography. Tubes containing the leucine transaminase are pooled, precipitated with a m m o n i u m sulfate (275 g/liter), and then dialyzed against 0.05 M caproate, pH 6.0 overnight. This dialyzed solution was then put onto a 2.5 × 30 cm column o f CMSephadex previously equilibrated with 0.02 M caproate buffer, pH 6.0. T h e leucine transaminase is eluted with 0.05 M caproate, pH 6.0. Tubes containing the yellow transaminase are pooled, a n d the enzyme is precipitated by a d d i n g a m m o n i u m sulfate (280 g/liter). T h e precipitate was dissolved in a m i n i m u m of 0.005 M caproate pH 6.0 a n d dialyzed against this solution to remove the a m m o n i u m sulfate. T h e caproate may be removed by dialysis against distilled water, but the enzyme is then unstable to prolonged storage. This product contains less PLP/protein than that from the earlier procedure 4 but does not show the stimulation by mercaptoethanol which was previously observed. It is p r e s u m e d therefore to be a superior, largely u n d e n a t u r e d , product.

9Large quantities of L-alanine aminotransferase (this volume [7]) are eluted just prior to the ieucine aminotransferase. If these fractions are pooled with the leucine aminotransferase, and then carried through step 4, both enzymes may be isolated from one preparation. See footnote 11. 1°0. Levin, Vol. V [2]. See also footnote 20 in Vol. V [94a]. 11Recovery of alanine transaminase: Tubes containing the alanine transaminase are pooled, and the enzyme is precipitated with ammonium sulfate (350 g/liter). The precipitate is dissolved in 0.04 M caproate containing 0.004 M EDTA, adjusted to pH 6 with Tris, and dialyzed against this same buffer. When passed through a 2.5 x 30 cm column of CM-Sephadex, equilibrated and eluted with this buffer, colored impurities are removed. The bright yellowproduct is approximately 25% pure alanine transaminase.

806

BRANCHED-CHAIN AMINO ACIDS

[1 10]

Reaction Properites Specificity. The relative rates with 45 mM concentrations of amino acids and ketoglutarate, at pH 8.3, were isoleucine (100), leucine (75), and valine (90). A modification of the routine assay must be used with isoleucine and valine as substrates, z The enzyme was also active with alloisoleucine (75), norvaline (44), norleucine (22), a-aminobutyrate (18), a-aminopimelate (12), L-methionine (11), S-methylcysteine (8), a-aminoadipate (7), and DL-allylglycine (6). Little if any activity was observed with D-amino acids or other natural amino acids. pH Activity Curve. With 6.7 mM substrate concentrations, the assay shows a sharp optimum at about pH 8.3. No marked buffer effects were noted. Further analysis of this pH activity curve showed that the decrease on the acid side of the optimum was due to both a decrease in maximum velocity and an increase in the Km for leucine. The decrease on the basic side of the optimum, which was due to an increase in the Km for ketoglutarate, does not occur when transamination from leucine to a-ketoisovalerate is studied. Inhibitors. The enzyme is sensitive to sulfhydryl reagents. The loss of activity upon storage at high pH values may be recovered by treatment with thiols, 4"1. Like other transaminases, the enzyme is inhibited by carboxylic acid substrate analogs and by a variety of carbonyl reagents, especially phenylhydrazine and hydroxylamine. Equilibrium. At pH 8.3 the equilibrium constant for the reaction was found to be 1.75 for the reaction as written above. Substrate Michaelis Constants. At pH 8.3 in 0.067 M sodium pyrophosphate buffer the Michaelis constants for leucine and ketog!utarate were approximately 5 mM. Physical Properties. The calculated sedimentation constant for zero protein concentration was 5,1 S. A molecular weight determination on a highly purified sample, assuming a partial specific, volume of 0.74, was 75000 - 4000 based on the sedimentation equilibrium distribution of the chromophore. Pyricloxal Phosphate Content. One molecule o f pyridoxal phosphate was found per molecule of protein. This gave the protein two absorption bands at 414 and 326 m/~. The best preparations gave--z/280/~414 14.4; however the A414/A3zeratio varied with the preparation. This enzyme is distinct from an enzyme found in rat liver, which is specific for leucine, a~'13 In m a n y respects it resembles an enzyme in =

12A. Ichihara and E. Koyama,J. Biochem. (Tokyo) 59, 160 (1966). 13A. Ichihara, H. Takahashi and K. Aki and A. Shirai, Biochem. Biophys. Res. Commun. 26, 674 ( 1967); see also this volume [ 113].

[ 111]

AMINOTRANSFERASE (PIG HEART, MITOCHONDRIA)

807

T h e r e is a separate enzyme with comparable specificity in mitochondria, t5

Escherichia coli) 4

14D. Rudman and A. Meister,J. Biol. Chem. 200, 591 (1953). tSK. Aki, K. Ogawa, A. Shirai and A. Ichihara, J. Biochem. (Tokyo) 62,610 (1967); see also this volume [ 111 ].

[111] B r a n c h e d - C h a i n

Amino Acid Aminotransferase

(Pig Heart Mitochondria) 1

By K. AKI and

A. ICHIHARA

Branched-chain L-amino acids (valine, leucine, and isoleucine) + tr-ketoglutarate branched-chain ,t-keto acids (a-ketoisovalerate, a-ketoisocaproate, and a-keto-/~-methylvalerate) + L-glutamate

Assay Method

Principle. T h e branched-chain a-keto acid formed was converted to the 2,4-dinitrophenylhydrazone, which was selectively extracted with toluene. T h e hydrazone was then transferred to sodium carbonate solution; the color was developed by addition of NaOH and measured at 440 mtt. This method TM is based on Friedemann and Haugen's method 2 with slight modifications, a Although cyclohexane is a more specific solvent for extraction of the hydrazone of a-ketoisocaproate, 4 toluene is routinely used because all hydrazones of these branched chain a-keto acids can be extracted equally well by toluene and contamination with the hydrazone of a-ketoglutarate is essentially negligible. Reagents Branched-chain L-amino acid, 0.1 M a-Ketoglutarate, 0.1 M. Adjust pH to 7.4 with sodium hydroxide solution and store in refrigerator. Pyridoxal phosphate, 0.002 M 1For the preparation of the branched-chain amino acid transferase from the soluble fraction of pig heart, see article [110]. The preparation from hog brain supernatant is described in article [112]. The leucine-specific aminotransferase from rat liver is described in article [113]. taA. Ichihara and E. Koyama, J. Biochem. 59, 160 (1966). 2T. E. Friedemann, Vol. III [66]. SH. Wada and E. E. Snell, J. Biol. Chem. 237, 127 (1962). 4R. T. Taylor and W. T. Jenkins, J. Biol. Chem. 241, 4391 (1966).