Effects of Amino Acids and Glutathione on Rat Liver Histidase Activity in vitro

Transcription

1 [Agr. Biol. Chem., Vol. 34, No. 5, p , 1970] Effects of Amino Acids and Glutathione on Rat Liver Histidase Activity in vitro By Katuhiko NODA Department of Nutrition, School of Medicine, Tokushima University Received September 10, 1969 The activity of histidase (L-histidine ammonia-lyase EC ) of rat liver was in hibited by cystine competitively with the substrate histidine. Histidase was inactivated by preincubation with cystine, but the inactivated enzyme was reactivated by the addition of glutathione to the reaction mixture. Cysteine, at a lower level, partly reactivated the enzyme inactivated by cystine, but, at a higher level, did not reactivate it. Cysteine itself acted inhibitory on the enzyme. Glutathione did not reactivate histidase activity inhibited by cysteine. No other amino acid affected histidase activity at the level of 2 mm. Apparent Km of rat liver histidase was 1.8 ~10-3M. Neither inhibitory nor activating effect on histidase activity was observed by the addition of liver homogenate obtained from rats fed a histidine imbalanced diet to that from those fed a basal diet and vice versa. Long before, histidase was studied by Takeuchi," and intensively investigated by Tabor," and Peterkofskys3) with Pseudomonas. However, there have been few descriptions"" on liver histidase of mammals. On the way of the investigation of histidine imbalance, the author found that liver histidase activity of rats fed a histidine imbalanced diet was higher than that of the animals fed a basal diets' The author also noticed, during the determination of apparent Km of crude liver histidase obtained from both groups of rats, that the relative velocity-substrate curve showed no first-order expression. The experi ments described in this paper were therefore conducted to investigate some effectors on the activity of histidase of rat liver, and whether or not there is any difference in the properties of histidase of rats fed either an imbalanced or a basal diet. MATERIALS AND METHODS Animals and diets. Young male rats of Wistar strain were used throughout the experiments. The animals were fed either a histidine imbalanced, a basal,7) or u stock diet ad libitum. Reagents. L-Histidine HCI EH2O, L-cystine E2HCl, L-cysteine and reduced glutathione were purchased from Ajinomoto K. K., Ishizu Pharmachemical Co., Nakarai Chem. Ltd., and Boehringer & Soehne, re 1) M. Takeuchi, J. Biochem. Japan, 34, 1 (1941). 2) H. Tabor and O. Hayaishi, J. Biol. Chem., 194, 171 (1952). 3) A. Peterkofsky, J. Biol. Chem., 237, 787 (1962). 4) "Methods in Enzymology," Vol. II, ed. by S. P. Colowick, Academic Press Inc., 1966, p ) N. W. Cornell and C. A. Villee, Biochem. Biophys. Acta, 167, 172 (1968). 6) K. Noda, Y. Kusaka and A. Yoshida, Agr. Biol. Chem., 31, 217 (1967). spectively. Other L-amino acids used were purchased from Kyowa Hakko Kogyo Co. Assay methods. Procedure for preparation of liver homogenate and assay method for histidase were de scribed in the previous papers) In the case of ad dition of amino acids or glutathione to the reaction 7) K. Noda and A. Yoshida, Agr. Biol. Chem,, 32, 1414 (1968).

2 Effects of Amino Acids and Glutathione on Rat Liver Histidase Activity in vitro 711 mixture, the compounds to be tested were dissolved or suspended in 0.1 M pyrophosphate buffer (ph 9.2) immediately before the assay and added to the reac tion mixture at the expense of the buffer solution. Final concentration of amino acids was 2 mm. For the test of effectors in the liver homogenate, 5 ml of the liver homogenate obtained from rats either a histidine imbalanced or a basal diet was heated in a boiling water bath for 5 min, and centrifuged at 3000 rpm for 5 min; then 0.2 ml of the supernatant solution was added to the incubation mixture. Unheated homogenate (0.1 ml) was added to the incuba tion mixture to test the presence of heat unstable effectors. cally affected the activity of histidase. Only cystine inhibited about 95%of the activity at the concentration of 2 mm. Purification of liver histidase. The liver homogenate from rats fed the stock diet was heated in a hot water bath at 65 C for 5 min and centrifuged at 3000 rpm for 10 min. The supernatant obtained was fraction ated with ammonium sulfate at 50% saturation. The precipitate was dissolved in pyrophosphate buffer and the solution was used as an enzyme source. Ultra-centrifugation analysis. The enzyme solution obtained by ammonium sulfate fractionation was cen trifuged with histidine and/or cystine by sucrose density gradient (5-15%) method in a Hitachi Model RPS- 40 ultracentrifuge, at 38, 500 rpm (1.65 x 104 g) for 5 hr. Lactic dehydrogenase (LDH) was used as a marker enzyme, the activity of which was measured by the method of Dennis.8) FIG. 1. Effect of Dialysis on Histidase Activity of Rat Liver Crude Homogenate. Five ml of crude homogenate was dialysed overnight against 1 liter of 1% KCl solution at 4 C; non dialysed œ- œ, dialysed -. RESULTS Effect of dialysis and free cystine The velocity-substrate curve obtaine with the crude homogenate did not show a firstorder reaction pattern, but after dialysis against 1% KCl solution, a first-order expression was obtained (Fig. 1). The above observation indicated the presence of some factor(s) of low molecular weight affecting the activity of histidase. Each amino acid was added to the incubation medium at the level of 2 ~ 10-3M. The results were shown in Fig. 2. No single amino acid tested except cystine practi 8) D. Dennis and N. 0. Kaplan, J. Biol. Chem., 235, 810 (1960). FIG. 2. Effect of Amino Acids on Liver Histidase (crude homogenate) Activity in vitro; Level of Each Amino Acid Added in the Incubation Medium was 2mM.

3 712 K. NODA Partial purification To know the more detailed character of this enzyme, the crude homogenate was partially purified. The method, specific activity and recovery were shown in Table I. As the en zyme in rat liver was less stable to heat treat ment than that of Pseudomonas, lower tempera ture and shorter heating time were employed ultracentrifugal analysis. The result is shown in Fig. 4, and no obvious change in molecular weight of the enzyme was observed by the addition of histidine and/or cystine. The mole cular weight of the enzyme was estimated to be more than 100,000 as compared with the marker enzyme, LDH (molecular weight 100,000). TABLE I. PURIFICATION PROCESS OF RAT LIVER HISTIDASE a) Rat liver homogenate was used as the starting material.b) The value in parentheses was that obtained without glutathione in the incubation medium. in the process. By 50% saturation of ammo nium sulfate, the activity was collected in the precipitate, and the specific activity increased about 8 times as much as that of the original crude homogenate. By the purification process the requirement for glutathione appeared a little. Crude homogenate showed no require ment for glutathione. Recovery at this step was about 700%. Effect of cystine, cysteine and glutathione The relationship between histidine, as sub strate, and cystine, as an effector, was observed on dialyzed liver homogenate from rat fed the stock diet. The inhibitory effect of cystine on the activity of histidase was illustrated in Fig. 3. Cystine seemed to act as a competi tive inhibitor (Fig. 3-A). As shown in Fig. 3-B, an allosteric like behavior was observed at the lower substrate levels with the presence of cystine. So that the effect of cystine on molecular weight of histidase was tested by FIG. 3. Inhibitory Effect of Cystine on Partially Purified Histidase Activity of Rat Liver in vitro; without Cystine œ- œ, with Cystine (10-3 M) -. Cystine in an incubation medium inhibited the enzyme activity with competitive behavior (Fig. 3), but if the enzyme solution was prein cubated with cystine in the absence of histi dine, the type of inhibition was no longer competitive (Fig. 5). Negative linear relation-

4 Effects of Amino Acids and Glutathione on Rat Liver Histidase Activity in vitro 713 In the case of cysteine, a lower level of cys teine (1.0mM) partly reactivated the activity of histidase inactivated by the preincubation with cystine, but the addition of more than 1 mm of cysteine diminished the activity of histidase rather than activated. These relationships were shown in Fig. 7. Partially purified histidase was slightly activated by the addition of glutathione, i.e. from 0.7 of absorbance without glutathione to 1.0 with glutathione. Even in the presence of glutathione, histidase ac- FIG. 4. Ultra-centrifugation Pattern of Partially Purified Liver Histidase of Rat with or without Histidine and/or Cystine; Activity of Histidase œ- œ LDH (marker enzyme) -. FIG. 6. Effect of Glutathione on Histidase (crude liver homogenate) Inactivated by Preincubation with Cystine; 2mM œ- œ and 5mM -. The point on a vertical line was the value ob tained by the incubation medium containing nei ther cystine nor glutathione. FIG. 5. Histidase (crude homogenate) Activity after Preincubation with Cystine in vitro. ship was obtained between cystine content in the preincubated solution and the enzyme ac tivity remained. Though the preincubated enzyme solution inactivated by cystine was not reactivated by histidine, the enzyme could be reactivated by the addition of glutathione to the incubation medium along with histidine as shown in Fig. 6. Almost all the activity, though not completely, reappeared by the ad dition of glutathione (final concentration 2mm). FIG. 7. Effect of Cysteine on Partially Purified Liver Histidase Activity in vitro; No Preincubation -, Preincubated with Cystine (2mm) œ- œ Supple mentation of GSH (4mM) _??_.

5 714 K. NODA tivity was completely inhibited by a higher level (10 mm) of cysteine. incubation medium. Addition of the homo genate did not affect at all each other. Ap pearent Km of liver histidase was 1.8 ~10-3M in both homogenates (Fig. 8), and this figure suggested that no difference in enzyme affinity to histidine might exist between the two homo genates. FIG. 8. Lineweaver-Burk Plot of Dialyzed Liver Histidase of Rats Fed a Histidine Imbalanced œ- œ, a Basal -, or a Stock Diet x-x. DISCUSSION Lysine and glycine4) were reported as in hibitors of rat liver histidase, but in the ex Effect of liver homogenate from rats fed a histidine imbalanced or a basal diet perimental conditions adopted in this study, either lysine nor glycine seemed specifically Table II showes the results of addition of to inhibit the activity of histidase; e.g. iso heated or unheated liver homogenate from leucine, tyrosine, serine and glutamic acid rats fed an imbalanced or a basal diet to the showed same tendency. The amount of lysine TABLE II. EFFECTS OF LIVER HOMOGENATE used in a reference book" was 48 mm and at FROM RATS FED A HISTIDINE IMBALANCED such a higher level, the degree of inhibition OR A BASAL DIET ON LIVER HISTIDASE reported was 50%. The concentration of lysine seemed too high to compare with levels ACTIVITY in vitro in rat liver. It was reported` that tyrosine and p-hydroxyphenylpyruvate inhibited histi dase from Pseudomonas. There may be the possibility that tyrosine inhibits the activity of histidase from rat liver. It is well known that histidase was activated by -SH groups."" Judging from the inactiva tion by cystine and the reactivation by gluta tione, histidase might have -SH group(s) as one of the functional site of the enzyme. Glutathione can reactivate the enzyme inac tivated by the preincubation with cystine, but histidine cannot, oxidized glutathione also ac tivates histidase,5) cysteine at higher levels in hibits the enzyme activity, and glutathione cannot overcome the inhibitory effect of cys teine. Above facts indicate some complex mechanisms in activation-inactivation of histi dase activity. No change in apparent molecular weight of histidase was detected with or without cys tine regardless of the presence of histidine. The facts that liver histidase of rats fed a basal diet and of those fed a histidine im balanced diet had same Km value and that the liver homogenates from rats of these two groups showed neither inhibitory nor activat ing effect on the enzyme activity of each other suggest that cystine might not be responsible for the difference in liver histidase activity between the two groups of rats. 9) D. H. Hug and D. Roth, Biochem. Biophys. Res. Comm., 30, 248 (1968).