to the monomeric form. The isoelectric point was determined to be ph 5.2 by ph

Transcription

1 J. Biochem., 75, (1974) L-Histidine Ammonia-lyase in Rat Liver I. Purification and General Characteristics Haruki OKAMURA,* Teruo NISHIDA, and Hachiro NAKAGAWA Division of Protein Metabolism, Institute for Protein Research, O saka University, Suita, Osaka 565 Received for publication, July 2, 1973 Histidine ammonia-lyase [EC ] was purified approximately 390 fold from female rat liver. The enzyme preparation was shown to be homogeneous and its S20, w value was 11.6 by ultracentrifugal analysis. The molecular weight was determined to be 190,000 by the sedimentation equilibrium method. On disc-electrophoresis, purified enzyme separated into one major and two minor components having enzyme activity, but after pretreatment with reduced glutathione the preparation showed only one band. This suggests that the enzyme preparation was pure and that a thiol group is involved in the conversion of polymeric forms to the monomeric form. The isoelectric point was determined to be ph 5.2 by ph gradient electrophoresis. On immunoelectrophoresis, the purified enzyme gave a single band against anti histidine ammonia-lyase serum. The enzyme activity was highest between ph 8.8 and 9.0. The K, value for histidine was calculated to be 1.2 ~10-3 M. Thiol compounds, such as glutathione and mercaptoethanol, considerably acti vated the enzyme. Parachloromercuribenzoate completely inhibited histidine ammonia lyase activity at a concentration of 10-5M and this inhibition was partially reversed by addition of the substrate, histidine. The results indicate that a thiol is involved, not only in the conversion of the associated forms to the dissociated form, but also in the increase in catalytic activity of the enzyme. EDTA markedly inhibited enzyme activity and this inhibition was reversed effec tively by Mn2+, Mg2+ Zn2+, and Cd2+ and less effectively by Ca2+ and Nit+. The activating effect of glutathione was lost in the presence of EDTA and reversed by addition of Mn2+. These results suggest that a divalent metal ion is essential for the catalytic activity of histidine ammonia-lyase and that thiol is intimately related with its function. Sodium borohydride and nitromethane, which are known to inhibit bacterial histidine ammonia-lyase activity by reacting with dehydroalanine in the active center, considerably inhibited the enzyme from rat liver., * Present address : Division of Food Chemistry Osaka 565., Institute for Industrial Science, Osaka University, Suita, Vol. 75, No. 1,

2 140 H. OKAMURA, T. NISHIDA, and H. NAKAGAWA Sex-steroids such as estradiol, progesterone, and testosterone showed no effect on enzyme activity at concentrations of 10-1 M to 10-4M. Immunochemical analyses of histidine ammonia-lyase indicated that the histidine ammonia-lyase content of liver extracts of female rats was more than that of similar extracts from male rats. This suggests that sex-dependent histidine ammonia-lyase activity is expressed by a dif ference in the quantity of enzyme protein. It is well known that there is striking sexdifference in the activity of liver histidine ammonia-lyase [EC ] in adult rats. Feigelson (1) ascribed this sex-difference to induction of the enzyme forming system by estrogen during pubertal development. Later, Feigelson (2, 3) provided evidence that factors of hypophyseal origin counteracted the action of estrogen on histidine ammonia-lyase. We also presented data suggesting that other hormones, such as thyroxine and growth hormone, or dietary factors might be involved in expression of the sex-difference in this en zyme activity (4). It is not yet clear, however, whether this sex-difference is based on a difference in the structure, or in the turnover of the en zyme molecule. To approach these problems, we attempted to purify liver histidine am monia-lyase from female rat liver and suc ceeded in obtaining a pure preparation. This paper reports the purification and general characteristics of the enzyme. Studies on the immunochemical properties of histidine am monia-lyase in crude extracts of both sexes are also reported. EXPERIMENTAL Animals-Female Sprague-Dawley rats, weighing approximately 200g, were obtained from the Junkei Dobutsu Jigyojo (Pure Animal Strain Supplier) of Osaka University and main tained on laboratory chow pellets (MF, from Oriental Yeast Co., Ltd., Osaka). Male New Zealand strain rabbits, weighing approximately 1.5 kg, were purchased from Japan Rabbit Co., Osaka. Assay of Histidine Ammonia-lyase-Histidine ammonia-lyase was assayed spectro photometrically by the method of Tabor and Mehler (5). The assay mixture contained 100 ƒêmoles of glycylglycine buffer, ph 8.7, 60 ƒê moles of histidine-hc1 (previously adjusted to ph 8.7 with KOH), 100 ƒêmoles of reduced glutathione and enzyme solution in a final volume of 2.0 ml. After equilibration at 37 Ž for 5 min, the reaction was initiated by addition of the substrate, histidine. Incubation was done at 37 Ž for 10min and was termi nated by addition of 2.0 ml of ice cold 10% HC1O4. A control without histidine was in cubated in the same way. The precipitate was removed by centrifugation for 10 min at 3,000 x g and urocanic acid in the supernatant was determined from the optical density at 265 mp. No correction was made for degrada tion of the reaction product by urocanase even when enzyme preparations at early steps of purification were used, since urocanase activity was negligible under the standard assay conditions. The concentration of uro canic acid was calculated taking its molecular extinction coefficient at 265 mp as One unit of enzyme was defined as the amount producing 1 ƒêmole of urocanic acid per min under the standard assay conditions. Specific activity was defined as units per mg of protein. Protein concentration was determined by the method of Lowry et al. (6) with bovine serum albumin as a standard. Ultracentrifugal Analyses-Ultracentrifugal analyses were performed in a Spinco Model E Ultracentrifuge by courtesy of Dr. K. Kakiuchi, Division of Physical Chemistry in our institute. The molecular weight was determined by the sedimentation equilibrium method (7, 8). Electrophoretic Analyses-Analytical poly acrylamide gel electrophoresis was carried out by the procedure of Davis (9) at 5 ma per column in Tris-glycine buffer, ph 8.3. Amido Black was used to stain protein bands, J. Biochem.

3 RAT LIVER HISTIDINE AMMONIA-LYASE 141 and was removed by electrophoresis in another column in 7% acetic acid. For determination of enzyme activity, the gel was sliced into widths of 1 mm from the origin with the aid of a gel cutter (Asai Seisakusho, Osaka) im mediately after electrophoresis. Each slice was transferred to a test tube, crushed with a glass rod and soaked overnight in 0.5 ml of 2 X 10-2 M potassium phosphate buffer, ph 7.4, in the cold. Then 0.2 ml of the extract was used for enzyme assay. ph-gradient electrophoresis was performed in an Ampholine Electrofocusing Apparatus, LKB 81000, devised by Svensson (10). Immunological Analyses-One milligram of purified histidine ammonia-lyase in 10-1M potassium phosphate buffer, ph 7.4, was thoroughly emulsified with I ml of Freund's complete adjuvant with the aid of a syringe. This emulsion was injected into the foot pads of rabbits. After 3 weeks, an additional in jection was given. Two weeks after the second injection, a blood sample was taken from the heart. The blood was stood for 2 hr at room temperature and then overnight at 4 Ž and then centrifuged for 10 min at 3,000x g. The antibody in the supernatant was used for experiments. Immunoelectrophoretic analyses were car ried out in agarose gel. The gel contained 17o agarose, 10-2M potassium phosphate buf fer, ph 7.5, 1.5 x 10-1 M NaC1 and 0.02% NaNs. Electrophoresis was run for 3 hr at 200 V and 5 ma per cm. Ouchterlony gel double diffusion analyses of the enzyme were carried out by a modifica tion of the method of Ouchterlony (11). Chemicals L-Histidine-HC1 and glycine were kindly supplied by Tanabe Amino Acid Research Foundation, Osaka. Glycylglycine and reduced glutathione were products of the Peptide Research Foundation, Mino, Osaka. Whatman DE-52 (microgranular DEAE-cellu lose) was a product of W. and R. Balston Ltd., Kent., England. TEAE-cellulose was from Serva Entwicklungslabor, Heidelberg, Germany, Hypatite C (hydroxylapatite) from Clarkson Chemical Co., Inc., Pa., U.S.A., Sephadex G-25 and Sepharose 4B and 6B from Pharmacia Fine Chemicals, Uppsala, Sweden and Carrier ampholite for ph gradient electro phoresis from LKB-Produkter AB, Stockholm, Sweden. Bact-adjuvant (complete Freund) was purchased from Difco Laboratories,. Detroit, U.S.A. Agarose A-37 was a product of I.B. Francaise, France. Other chemicals used were standard commercial products. RESULTS Purification of Histidine Ammonia-lyase All procedures were carried out at 0 to 4 Ž. Step I. Preparation of crude extract: Eighty to one hundred rats were used for one experiment. Animals were killed by a blow on the head and their livers were quickly removed and chilled on ice. Pooled livers were blotted with filter papers, weighed and homogenized with 3 volumes of 5X10-'M potassium phosphate buffer, ph 7.4, in a Virtis type homogenizer for 2 min at 12,000 rpm, The homogenate was centrifuged for 20min at 15,000 X g. Step If. Heat treatment : Portions of 200 ml of the supernatant (Fraction I) were trans ferred to 500 ml flasks and placed in a water bath at 60 Ž. After equilibration for 2 min, the flasks were immersed in an ice bath. The contents of several flasks were collected and centrifuged for 20 min at 15,000 x g. The precipitate was resuspended in an equal volume of 5 X 10-2 M potassium phosphate buffer, ph 7.4, and recentrifuged. The washing fluid was combined with the supernatant (Fraction II). Step III. First ammonium sulfate frac tionation : Solid ammonium sulfate was added with mechanical stirring to the heat-treated extract to a final saturation of 33%. The mixture was adjusted to ph 7.4 by dropwise addition of a 10% solution of ammonium hydroxide. After 30 min, the precipitate formed was removed by centrifugation for 20, min at 15,000>< g and the supernatant wass brought to 55% saturation with ammonium sulfate. The mixture was again centrifuged and the precipitate was dissolved in 2 x 10-1 Mm potassium phosphate buffer, ph 7.4 (Fraction III). Step IV. DEAE-cellulose column chroma- Vol. 75, No. 1, 1974

4 142 H. OKAMURA, T. NISHIDA, and H. NAKAGAWA tograph y : Fraction III was placed on a column (7 ~40 cm) of Sephade ~ G-25 equili brated with 2 ~10-2M potassium phosphate buffer, ph 7.4, and eluted with the same buffer. Fractions containing reddish brown chromoprotein were pooled and then applied to a column (4 ~50 cm) of DEAE-cellulose (Whatman DE-52) equilibrated with 2 ~10-2M potassium phosphate buffer, ph 7.4. The column was washed with 600 ml of 2 ~10-2 M potassium phosphate buffer, and then eluted with a linear concentration gradient of 2 ~10-2 M to 2 ~10-1M potassium phosphate buffer, ph 7.4, achieved with 1,000 ml of each buffer, at an elution velocity of appro ~imately 60 ml per hr. Fractions with histidine ammonia lyase activity were pooled (Fraction IV). Step V. Second ammonium sulfate frac tionation : Solid ammonium sulfate was added to Fraction IV and material precipitating between 43% and 60% saturation was dissolved in a small volume of 5 ~10-2M potassium phosphate buffer, ph 7.4 (Fraction V). Step VI. Hydro ~ylapatite column chroma tography: Fraction V was placed on a column (2.5 ~40 cm) of Sephade ~ G-25 equilibrated with 2 ~1O-2M potassium phosphate buffer, ph 7.4, and eluted with the same buffer. Fractions with absorbance at 280 raft were pooled and applied to a column (3 ~30 cm) of hydro ~ylapatite equilibrated with 4 ~10-2 M potassium phosphate buffer, ph 7.4. Material was eluted with a linear concentration gradi ent of 4 ~10-2M to 1.6 ~10-1M potassium phosphate buffer, ph 7.4, achieved with 400 ml of each buffer. Fractions with histidine, ammonia-lyase activity were pooled (Fraction VI). Step VII Sepharose 6B column chroma tography: Fraction VI was precipitated with 60% saturation of solid ammonium sulfate, dissolved in a small volume of 2 ~10-2M potassium phosphate buffer, ph 7.4, containing 10-3M reduced glutathione, and applied to a column (3 ~104 cm) of Sepharose 6B equilibrated with 2 ~10-2M potassium phos phate buffer, ph 7.4, containing 10-3M gluta thione. Elution was carried out with the same buffer at a flow rate of 20 ml per hr. Fractions with enzyme activity were pooled (Fraction VII). Step VIII. TEAE-cellulose column chro matography: Fraction VII was concentrated with solid ammonium sulfate and desalted on a Sephade ~ G-25 column by the method de scribed in Step VII. Then the preparation was applied to a column (2 ~30 cm) of TEAE cellulose equilibrated with 8 ~ 10-2 M potassium phosphate buffer, ph 7.4. The column was eluted with a linear concentration gradient of 8 ~10-2 M to 2 ~10-1M potassium phosphate buffer, ph 7.4, achieved with 400 ml of each Fig. 1. Elution pattern on TEAE-cellulose column chromatography. ü, enzyme activity (optical density at 265 m u) ; œ protein concentration (ƒêg per ml); specific activity. Histidine ammonia-lyase was eluted with about 1.3 ~10-1M potassium phosphate buffer.,. J. Biochem,.

5 RAT LIVER HISTIDINE AMMONIA-LYASE 143 TABLE I. Summary of purification. buffer. Figure 1 shows the elution pattern at the final step of purification. The specific activities of fractions No. 50 to No. 60 were almost the same, suggesting that the purified histidine ammonia-lyase was homogeneous. The enzyme was purified approximately 390 fold with 6% yield. An attempt to crystallize histidine ammonia-lyase at this step, using finely powdered ammonium sulfate, was unsuccessful. A summary of the purification is given in Table I. Purity of the Purified Preparation of Histidine Ammonia-lyase-Ultracentri fugal an alyses : The Schlieren pattern of the enzyme preparation after pretreatment with 10-2M reduced glutathione exhibited a single, symmetrical peak with the sedimentation coeffi cient (S20, w value) of 11.6, as shown in Fig. 2. The molecular weight was determined by the sedimentation equilibrium method. Figure 3 shows the effect of the protein concentra tion on the molecular weight. From these data, the molecular weight of histidine am monia-lyase was calculated as 190,000 at zero protein concentration, taking the partial spe cific volume of the enzyme as 0.72 cm3 per g. Electrophoretic analyses : Purified histidine ammonia-lyase was subjected to disc-electro phoresis. The enzyme activity on the gel was determined after cutting the gel into 1 mm slices, as described in the experimental. As shown in Fig. 4A, when the preparation Fig. 2. Ultracentrifugal pattern of purified histidine ammonia-lyase. The protein concentration was 6.0 mg per ml of 10-2M potassium phosphate buffer, ph 7.4. Photographs were taken 7 min (right) and 27 min (left) after the rotor reached 50,000 rpm at 20 Ž Fig. 3. Effect of protein concentration on the molecular weight of histidine ammonia-lyase. Histi dine ammonia-lyase was dialyzed twice against 1,000 ml of 5 X 10-2 M potassium phosphate buffer containing 10-3 M reduced glutathione. Centrifugation was carried out at 20 Ž at 5,227 rpm for 12 hr. The protein concentrations examined were 1.00, 1.75, and 2.5 mg per ml. The linear plot was obtained by the method of least squares. Vol. 75, No. 1, 1974

6 144 H. OKAMURA, T. NISHIDA, and H. NAKAGAWA had not been treated with reduced glutathione, it separated into 3 bands, one major band running nearest to the anode and 2 minor ones. All 3 bands had enzyme activity (Fig. 5A). However, as shown in Fig. 4B, after treating the enzyme preparation with 10-2M reduced glutathione for 10 min at 37 Ž before electrophoresis, only one band corresponding in position to the major band was observed on the disc-electrophoretogram, and activity correspond to minor bands disappeared (Fig. 5B). The ratio of the minor bands to the major band increased on aging the enzyme form a single precipitin line with anti-serum added to a parallel slit after the electrophoretic run. These results indicate that the purified histidine ammonia-lyase was immunochemi cally homogeneous. Enz)molog,cal Properties of Purified Liver Histidine Ammonia-lyase-Stability : Histidine ammonia-lyase at every step of purification lost little activity on storage at -20 Ž (Table II). At 0 Ž, however, the purified preparation tended to firm a precipitate and lost its ac tivity within 1 week, whereas preparations at preparation. These results suggest that purified histidine ammonia-lyase tends to as sociate by forming disulfide bridges and that the associated forms can dissociate in the presence of a thiol compound. Immunochemical analyses : Purified his tidine ammonia-lyase gave a single precipitin line against anti-rabbit histidine ammonia-lyase serum in the Ouchterlony double diffusion test. As shown in Fig. 6, on immunoelectrophoresis, the enzyme preparation moved to the anode under the conditions employed and reacted to Fig. 5. Distribution of activity after electrophoresis of histidine ammonia-lyase with or without glutathione-treatment. Gels were run simultaneously with the samples described in the legend to Fig. 4 and then sliced into 1 mm sections. Slices were soaked in 0.5 ml of 2x10-2M potassium phosphate buffer, ph 7.4, for 15 hr. Aliquot of 0.2 ml of extracts were used for assay. Fig. 4. Pattern of purified histidine ammonia-lyase on analytical polyacrylamide gel electrophoresis Approximately 30 beg of purified histidine ammonia lyase were applied to 7.5% polyacrylamide gel. Electrophoresis was run at 5 ma per column in Tris-glycine buffer, ph 8.3. (A) Preparation not treated with reduced glutathione. (B) Preparation treated with 10-2M reduced glutathione for 10 min at 37 Ž. Details are described in the text.. Fig. 6. Immunoelectrophoretic pattern of purified histidine ammonia-lyase. Immunoelectrophoresis was carried out as described in the text. The gel was stained with Amido Black, dried and then photographed. J. Biochem.

7 RAT LIVER HISTIDINE AMMONIA-LYASE 145 TABLE II. Stability of enzyme activity at each purification step. The eluate from a TEAE-cellulose column was stored for 1 week at 0 Ž and centrifuged for 10 min at 3,000 ~g. The resulting precipitate was suspended to an equal volume of 2 ~10-2 m potassium phosphate buffer, ph 7.4. Enzyme activity is expressed as the absorbance at 265 m,a under the standard assay conditions. For comparison, the same volume of enzyme solution (or suspension) was taken out for assay at each purification step. purification steps V to VII were stable. This suggests that during purification histidine am monia-lyase loses some protective factor(s) or undergoes some change in catalytic properties, but the reason for this change in stability was not investigated. During storage for 24 hr at 0 Ž between ph 7.0 and 10.0, the purified enzyme retained full activity. However, at more acidic or more alkaline ph values, the enzyme activity decreased considerably and was completely lost at ph 3.0 or At 37 Ž, the enzyme lost its activity faster, but was still stable between ph 7.0 and 9.0 (Table III). Optimum ph: The enzyme activity was determined at various ph values obtained with glycylglycine-koh and glycine-koh buffer. As shown in Fig. 7, the enzyme activity was maximal between ph 8.8 and 9.0 and decreased sharply on both the acidic and alkaline side. This optimum ph is consistent with the sta bility of the enzyme at ph 9.0 at 37 Ž. TABLE III. Stability of enzyme at various ph's. The ph was carefully adjusted to various ph values with KOH or HCl. After storage, an aliquot con taining 0.3 beg of purified enzyme protein was used for assay. Activity is expressed as indicated in the legend to Table II. Fig. 7. Effect of ph on histidine ammonia-lyase activity. Enzyme activity was assayed in glycyl glycine-koh buffer ( ü) or glycine-koh buffer ( œ) and is expressed as indicated in the legend to Table II. Vol. 75, No. 1, 1974

8 146 H. OKAMURA, T. NISHIDA, and H. NAKAGAWA Fig. 8. ph-gradient electrophoresis of histidine ammonia-lyase. Fraction VI (protein, 10 mg) was applied on a column and run for 48 hr at 500 V. Fractions of 2 ml were collected and 0.2 ml aliquots were used for assay. Enzyme activity is expressed as indicated in the legend to Table II. pi: The enzyme preparation at step VII (eluate from a hydroxylapatite column) was subjected to ph gradient electrophoresis with carrier Ampholyte from ph 3.0 to As shown in Fig. 8, 3 protein peaks were sepa rated by this procedure, but most protein was found, coagulated at ph 5.2. The coagulated material hardly dissolved in phosphate buffer showed some activity though most was lost during electrophoresis. This loss of activity was not reversed by addition of divalent ions, such as Mn2+, Mg2+, Zn2+, Co2+, and Ni2+, or reduced glutathione. Michaelis constant : Figure 9 shows a double reciprocal plot of histidine ammonia lyase activity against histidine concentration. From this relationship, the K, value for his tidine was calculated to be 1.2 ~10-3M. This value is of the same order as that reported by Cornell and Villie (12) for rat liver en zyme of approximately 70% purity (2 ~10-3M), Effect of thiol compounds: Reduced glu tathione activated the purified histidine am monia-lyase 3 fold or more, but had almost no effect on enzyme preparations at early purification steps (Table IV). It caused max- Fig. 9. Effects of concentrations of histidine on enzyme activity. Enzyme activity (V) is expressed as indicated in the legend to Table II. imum stimulation at a concentration of 10-2M (Fig. 10). For full activation, the enzyme did not require the continuous presence of glutathione during incubation, but only prein cubation with it. Moreover, after activation, the activity did not decrease even on dialysis against phosphate buffer containing no gluta thione (Table IV). 2-Mercaptoethanol also J. Biochem.

9 RAT LIVER HISTIDINE AMMONIA-LYASE 147 TABLE IV. Effect of reduced glutathione (GSH) on crude and purified histidase activities. 1) 10-2M GSH was included in the assay system. 2) Enzyme was preincubated with 10-2M GSH and dialyzed against 1,000 volumes of 10-2M potassium phosphate buffer, ph 7.4, containing no GSH. In this case, GSH was omitted from the assay system. TABLE V. Effect of histidine in reversing inhibition of histidine ammonia-lyase activity by p-chloromer curibenzoate (PCMB). Histidine ammonia-lyase was preincubated with 10-I m PCMB in the presence or absence of histidine for 5 min at 37 Ž. An aliquot of 0.25 beg of enzyme protein was used for assay. Enzyme activity is expressed as indicated in the legend to Table II. Fig. 10. Stimulation of purified histidine ammonia lyase activity by reduced glutathione. Enzyme ac tivity is expressed as indicated in the legend to Table II. activated histidine ammonia-lyase and activa tion was proportional to its concentration up to 10-2M, during incubation. At concentra tions above 10-2M, mercaptoethanol inhibited enzyme activity. However, this inhibition was prevented by adding a metal ion such as Mn2} to the assay mixture at a concentration of 5 ~10-5 M (Fig. 11A). The effect of cysteine on the enzyme was similar to that of mer captoethanol, but cystine did not affect his tidine ammonia-lyase activity at a concentra tion of 10-2 M. In contrast, dithiothreitol was inhibitory rather than activatory even at a concentration of 10-3M. This inhibition was also entirely reversed by addition of metal ion (Fig. 11B) or by dialysis against potassium phosphate buffer containing no dithiothreitol. Parachloromercuribenzoate completely in hibited histidine ammonia-lyase activity at a concentration of 10-5M (Table V). However, the loss of the enzyme activity was partially prevented by addition of the substrate, his tidine. These findings indicate that the thiol group is present in the active center and is essential for catalytic activity. Effects of divalent metal ions : EDTA lowered histidine ammonia-lyase activity to 25% of the control level when preincubated with the enzyme preparation at a concentration of 2 ~10-4 M for 10 min at 37 Ž. This Vol. 75, No. 1, 1974

10 148 H. OKAMURA, T. NISHIDA, and H. NAKAGAWA Fig. 11. Effect of divalent metal ion on histidine ammonia-lyase inhibited by thiol compounds. Various concentrations of mercaptoethanol and dithiothreitol were included in the assay system in experiment (A) and (B), respectively. Mn2+ was added at a concentration of 5 ~10-5M. TABLE VI. Reactivation of histidine ammonia-lyase by metal ions after inhibition by EDTA. Enzyme was preincubated for 10 min with various concentrations of divalent metal ions in the presence of 10-4M EDTA at 37 Ž. Enzyme activity is expressed as indicated in the legend to Table II. suggests that metal ions contribute to the catalytic activity. Accordingly, various metal ions were tested to see if they could reverse the inhibition by EDTA. As shown in Table VI, none of the metal ions tested had any effect at a concentration of 5 x 10_6 M, but Mn2+, Mg2+, Zn2+, and Cd2+ were active at a concentration of 10-4 M. At a concentration of 10-3M, Ca2 { and Ni2+ also caused activation. Kinetic studies were made to elucidate the mechanism of inhibition by EDTA. The Vmax value was found to be the same in the presence and absence of EDTA, but typical curves of competitive inhibition were observed (Data not shown), Accordingly divalent metal ions seem to associate with the site which combines with substrate. As mentioned previously, glutathione con siderably activated histidine ammonia-lyase activity, but its activating effect was lost in the presence of 5 x 10-4 M EDTA and reversed by addition of a divalent cation, such as Mn2+ (10-3M), as shown in Table VII. Together with the above findings, these results suggest that the site involving a thiol group is inti mately related with that combining with J. Biochem,

11 RAT LIVER HISTIDINE AMMONIA-LYASE 149 TABLE VII. Effect of GSH in the presence of EDTA. Enzyme activity is expressed as indicated in the legend to Table II. TABLE VIII. Effects of sex-steroids on purified histidase. Enzyme was preincubated for 5 min with sex-steroids in the presence or absence of GSH at 37 Ž. Enzyme activity is expressed as indicated in the legend to Table II. Fig. 12. Effects of sodium borohydride and nitro methane on enzyme activity. Histidine ammonia lyase was preincubated with sodium borohydride and nitromethane for 20 min at 37 Ž. An aliquot con taining 0.3ƒÊg of enzyme protein was added to the standard assay mixture. sex-steroid might inhibit it. However, estra diol, progesterone and testosterone at concen divalent ion. Effects of sodium borohydride and nitro methane : Histidine ammonia-lyase of bacterial origin is inhibited by sodium borohydride or nitromethane, since dehydroalanine, which is involved in the active center of the enzyme, reacts with these compound (13, 14). Thus the effects of these compounds on rat liver histidine ammonia-lyase were examined. As shown in Fig. 12, sodium borohydride mark edly inhibited enzyme activity of the rat liver enzyme when was preincubated with it for 20 min at 37 Ž. Nitromethane also inhibited histidine ammonia-lyase activity, but less than sodium borohydride. Sex-difference-Effects of sex-steroids : It seemed possible that the sex-difference in the enzyme activity in crude liver extract might be because female sex-steroid stimulates his tidine ammonia-lyase activity, whereas male trations of 10-9M to 10-4 m did not affect histidine ammonia-lyase activity, irrespective of the presence or absence of reduced gluta thione, as shown in Table VIII. Quantitative sex-difference in histidine am monia-lyase in liver extracts : The anti-his tidine ammonia-lyase mentioned above was antibody causing neutralization and precipita tion. Using this, the amounts of histidine ammonia-lyase protein in crude liver extracts of female and male rats were titrated. For this purpose, various amounts of crude extract and a fixed amount of anti-serum were incu bated for 30 min at 37 Ž and then stood overnight in a cold room. The reaction mixtures were then centrifuged for 10 min at 3,000 x g. The supernatants were used for enzyme assay and the precipitates were dissolved in 0.5N NaOH solution and their protein concentra tions were determined. Vol. 75, No. 1, 1974

12 150 H. OKAMURA, T. NISHIDA, and H. NAKAGAWA Fig. 13. Difference in the amount of enzyme pro tein, which was precipitated with anti-histidine am monia-lyase, in crude extracts of both sexes. Fifty microliter of anti-serum was added to various vol umes of liver extract and the total volume was adjusted to 1.0 ml with Krebs-Ringer phosphate buffer. The mixtures were incubated for 30 min at 37 Ž, followed by standing for 24 hr in a cold room. Then they were centrifuged for 10 min at 3,000 ~g. The precipitate was dissolved in 0.5 N NaOH and used for determination of protein concentration. The supernatant was used for enzyme assay. As shown in Fig. 13, the amount of protein precipitated increased with increase in the amount of added liver extract either of female or male rats, but it was constantly more with extract from female rats than with that from male rats. Moreover, the amount of liver extract required to give histidine am monia-lyase activity in the supernatant was far less with extract from female rats than with that from male rats. These findings suggest that the sex-difference in histidine ammonia-lyase activity is due to a difference in the amount of enzyme protein in the liver extract. DISCUSSION A number of investigators have attempted to purify histidine ammonia-lyase from mammalian liver to study its unique function of catalyzing direct elimination of ammonia from the substrate (12, 15, 16). Kato et al. (15) in our laboratory reported about 40 fold purifi cation of histidine ammonia-lyase from guineapig liver. Cornell and Villie (12) reported approximately 200 fold purification and 70% purity of enzyme from rat liver. However, no pure preparation has yet been reported. Our enzyme preparation purified from adult female rat liver was shown to be homogeneous in three ways ; ultracentrifuga tion, electrophoresis, and immunochemical an alyses. Cornell and Villie (12) estimated the sedimentation coefficient and the molecular weight of rat liver histidine ammonia-lyase to be 9.70 and 226,000, respectively, by zonal gradient centrifugation. With more accurate methods, we calculated the sedimentation coefficient as 11.6 and the molecular weight as 190,000, with extrapolation to zero protein concentration. However, the orders of the values of our preparation were similar to those of Cornell and Villie. With histidine ammonia-lyase from Pseu domonas, Klee (17) demonstrated that the thiol group of the enzyme is important in activation as well as in conversion of the polymeric forms to the monomeric form of the enzyme. Spolter and Baldridge (18) stated that thiol compounds did not increase histidine ammonia-lyase activity in a crude extract of rat liver. In contrast, Kato et al. (15) demonstrated that glutathione and cys teine stimulated the enzyme activity of a partially purified preparation from guinea-pig liver. Cornell and Villie (12) observed a similar phenomenon with partially purified enzyme from rat liver. Consistent with these findings, the thiol compound, glutathione, considerably increased the enzyme activity of our purified preparation, but had no effect on cruder preparations. These results suggest that a thiol group is removed from the en zyme protein during purification, but that thiol compounds reversibly interact with the active center of the enzyme to reduce a disulfide bridge and so cause full activation. Our purified preparation separated into 3 J. Biochem.

13 RAT LIVER HISTIDINE AMMONIA-LYASE 151 components when subjected to disc-electro phoresis without previous treatment with glutathione. Each component showed histidine ammonia-lyase activity, and also cross-reacted with the other components without any spurformation in the Ouchterlony double diffusion test (Data not shown). Thiol-treatment re sulted in the conversion of the two minor components to the major component, located nearer to the anode than the minor compo nents. If the bands represent oligomeric and polymeric forms of the enzyme and if all three components are composed of the same unit, a polymeric form would probably migrate more slowly than an oligomeric form under the conditions employed for electrophoresis. Thus it seems quite likely that the major component and minor components are the monomeric form and polymeric forms, re spectively, of the enzyme. If this is so, the effects of thiol on mammalian histidine am monia-lyase are analogous its effect on bac terial enzyme. Mehler and Tabor (19) reported that partially purified histidine ammonia-lyase from guinea-pig liver was inhibited by EDTA and this inhibition was overcome by divalent metal ions such as Mn2+ Mg2+ and Cat+, Kato et al. (15) also reported that Cd2+ was a cofactor of histidine ammonia-lyase of guinea-pig liver. Cornell and Villie (12) reported that divalent ions, such as Mn2+, Mg2+, Zn2+, and Fee+, were required for full activation of dialyzed enzyme from rat liver. We confirmed this. Moreover, we found that the inhibition by EDTA is due to competition with histidine, suggesting that divalent metal ion is involved in the active center. However, it is still unknown what metal is the true cofactor. Klee (20) demonstrated that the effective concentration of Mn2+ or Cd2+ for activation of bacterial histidine ammonia-lyase was far less for the reduced form than for the oxidized form and these divalent metal ions protected the enzyme from inhibition by 5, 5 L-dithiobis- (benzoic acid). From these results, he con cluded that the increase in activity after re duction by a thiol compound was based on increase in affinity of the enzyme for metal ions. Metal ions are required for activation of mammalian histidine ammonia-lyase by glutathione and for overcoming the inhibition by mercaptoethanol or dithiothreitol. These results are consistent with those on the bac terial enzyme. Givot et al. (13) reported that histidine ammonia-lyase from Pseudomonas was inac tivated with nitromethane and after nitro methane-treatment they obtained 2, 4-diamino butyric acid, 4-amino-2-hydroxybutyric acid and ƒà-alanine by catalytic reduction and acid hydrolysis of the enzyme. Wickner (14) also isolated tritiated alanine from an acid hydrol ysate of bacterial histidine ammonia-lyase which was incubated with tritium-labeled sodium borohydride. These results suggest that dehydroalanine is involved in the active site of the bacterial enzyme. These findings tempted us to examine whether dehydroalanine contributed to the catalytic activity of mam malian histidine ammonia-lyase. Givot and Abeles (21) reported that the same products could be obtained as those from the bacterial enzyme after incubating rat histidine am monia-lyase with 14C-nitromethane. Their preparation was only 20 to 30% pure, but our data with highly purified enzyme also showed that histidine ammonia-lyase from rat liver was considerably inactivated by sodium boro hydride or nitromethane. It seems highly probable from these findings that mammalian histidine ammonia-lyase contains dehydro alanine in its active site. Cornell and Villie (12) suggested that the sex-difference in histidine ammonia-lyase ac tivity was due to the presence of an inhibitor in extract of male liver, since the total en zyme activity tended to increase during puri fication of histidine ammonia-lyase from the extract. In this work, we excluded the pos sibility that this inhibitor was male sex steroid. We demonstrated that the sex-difference in histidine ammonia-lyase activity was due to a difference in the quantity of enzyme protein. Our results also suggested that some factor(s) was present in crude enzyme prep arations which protected the enzyme from precipitation and inactivation at 0 Ž. How ever, further studies are required on whether Vol. 75, No. 1, 1974

14 152 H. OKAMURA, T. NISHIDA, and H. NAKAGAWA there really is an inhibitor only in liver extract of male rats and protecting factor(s) only in that of female or whether there is a qualitative difference between the enzyme pro teins in the two sexes. We express our deep gratitude to Prof. M. Suda for his valuble advice and continuous encouragement during this work. We also thank Dr. K. Kakiuchi, Division of Physical Chemistry of our institute for performing ultracentrifugal analyses and Dr. S. Utsumi, Department of Bacteriology, Osaka Univer sity School of Medicine, for help in preparing anti histidine ammonia-lyase serum. This work was supported in part by Tanabe Amino Acid Research Foundation, Osaka. REFERENCES 1. M. Feigelson, J. Biol. Chem., 243, 5088 (1968). 2. M. Feigelson, Biochim. Biophys. Acta, 230, 296 (1971). 3. M. Feigelson, Biochim. Biophys. Acta, 230, 309 (1971). 4. K. Noda and H. Nakagawa, Endocrinology, 90, 207 (1972). 5. H. Tabor and A.H. Mehler, "Methods in Enzymology," ed S.P. Colowick and N.O. Kaplan, Academic Press, New York, Vol. II, p. 228 (1955). 6. O.H. Lowry, N.J. Rosebrough, A.L. Farr, and R.J. Randall, J. Biol. Chem., 193, 265 (1953). 7. "Instruction Mannual," Beckman Instruments Inc., Spinco Division, California, Part úh, p. 3. (1961). 8. D.A. Yphantis, Biochemistry, 3, 297 (1964). 9. B.J. Davis, Ann. N.Y. Acad. Sci., 121, 404 (1964). 10. H. Svensson, Acta Chem. Scand., 16, 456 (1962) O. Ouchterlony, "Handbook of Experimental. Immunology," ed. by D.M. Weir, Blackwell Sci entific Publications, Oxford and Edinburgh, p. 655 (1967). 12. N.W. Cornell and C.A. Villie, Biochim. Biophys Acta, 167, 172 (1968). 13. I.L. Givot, T.A. Smith, and R.H. Abeles, J. Biol. Chem., 244, 6341 (1969). 14. R.B. Wickner, J. Biol. Chem., 244, 6550 (1969). 15. A. Kato, Y. Yoshioka, M. Watanabe, and M_ Suda, J. Biochem., 42, 305 (1955). 16. K. Hayashida and M. Uchida, Seikagaku (J. Japan_ Biochem. Soc.), 43, 736 (1971) (in Japanese). 17. C.B. Klee, J. Biol. Chem., 245, 3143 (1970). 18. P.D. Spolter and R.C. Baldrige, J. Biol. Chem., 238, 2071 (1963). 19. A.H. Mehler and H. Tabor, J. Biol. Chem., 201, 775 (1953). 20. C.B. Klee, J. Biol. Chem., 247, 1398 (1972). 21. I.L. Givot and R.H. Abeles, J. Biol. Chem., 245, 327 (1970). J. Biochem.