Adaptive response of tryptophan pyrrolase and tyrosine transaminase in rat liver after partial hepatectomy and laparotomy

218

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 25 996 ADAPTIVE RESPONSE OF T R Y P T O P H A N PYRROLASE AND TYROSINE TRANSAMINASE IN RAT LIVER A F T E R PARTIAL HEPATECTOMY AND LAPAROTOMY K I N J I TSUKADA, H I K O K I C H I OURA, SHOICHI NAKASHIMA AND NOBUKO HAYASAKI

Department of Biochemistry, Drug Research Institute, Faculty of Pharmaceutical Science, Toyama University, Gofuhu, Toyama (Japan) (Received April i6th, 1968)

SUMMARY

Tryptophan pyrrolase (L-tryptophan:H20~ oxidoreductase, EC 1.11.1.4) and tyrosine-a-ketoglutarate transaminase (L-tyrosine: 2-oxoglutarate amino transferase, EC 2.6.1.5) activities of rat liver homogenate were increased about 2-2.5 fold in 4-6 h after laparotomy, and 4-6 fold in 8-1o h after partial hepatectomy, without loading of glucocorticoid or substrate. The rise of tryptophan pyrrolase was inhibited by actinomycin D administered immediately after operation. In adrenalectomized rats, the induced tryptophan pyrrolase activity was inhibited. The increased activity in tryptophan pyrrolase after partial hepatectomy has been attributed to change in supernatant fraction, and was not affected by administration of cortisone.

INTRODUCTION

Tryptophan pyrrolase (L-tryptophan:H20 2 oxidoreductase, EC I.II.I.4) and tyrosine-a-ketoglutarate transaminase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) in the liver of intact rats are increased several-fold by the injection of glucocorticoids or various amino acids 1-4. However, different mechanisms appear to be involved in the action of the two substances, glucocorticoids and L-tryptophan 5-s. SHIMAZU9,1° reported that the activity of tryptophan pyrrolase of rabbit liver was elevated after electric stimulation of the hypothalamus. In the present investigation, it was observed that tryptophan pyrrolase and tyrosine-~-ketoglutarate transaminase activities in liver were increased immediately after laparotomy or partial hepatectomy, irrespective of administration of glucocorticoid or L-tryptophan. The possible mechanism of these effects is discussed. MATERIALS AND METHODS

Wistar rats weighing about 7o-9 ° g, obtained locally, received food and water ad libitum at all times. Male rats were used. L-Tryptophan, L-tyrosine and hydro-

cortisone acetate were products of Sigma Chemical Company. Actinomycin D was kindly provided by Dr. _A_LMERALPERT, Merck Sharp and Dohme Research LaboraBiochim. Biophys. Acta, 165 (1968) 218-224

TRYPTOPHAN PYRROLASE AND TYROSINE TRANSAMINASE

219

tories. All other compounds were of the highest purity available from commercial sources. Surgery was performed under ether anesthesia. Laparotomy was performed by cutting through abdominal skin, including peritoneum, about 6-8 cm along the median, and partial hepatectomy refers to the removal of 67 % of liver (median and left lateral lobes) n. The animals were fasted following surgery. At various intervals, the animals were killed by decapitation after being stunned b y a blow on the head. Livers, excised from rats immediately after death, were homogenized in a glass homogenizer fitted with a motor-driven plastic pestle in 5 vol. of o.14 M KC1. Subcellular fractions were prepared from the whole homogenate b y differential centrifugation at 4 °. Centrifugation for IO rain at 6000 × g yielded a sediment fraction containing mostly mitochondria. The remaining supernatant fluid was centrifuged for 60 min at lO5 ooo × g to give the microsomal fraction and supernatant fraction. The tryptophan pyrrolase assay procedure of KNOX AND MEHLERTM was modified as described below. Whole homogenate, I.O ml was added to a solution consisting of 0.5 ml of 0.2 M sodium phosphate buffer (pH 7.o), 0.2 ml of 0.05 M L-tryptophan to make a total volume 2 ml. Supplements of microsomes, mitochondria, or other materials were suspended in o.14 M KC1, maintaining a constant total volume. The mixture was shaken for 6o rain in a 37 ° water bath in air. The reaction was stopped with 15 % m-phosphoric acid or by boiling for 3 rain, and the mixture centrifuged at 300 × g for 5 min. The supernatant solution was adjusted to p H 7.0 with NaOH, and the kynurenine produced was assayed spectrophotometrically at 365 m~. Tyrosine transaminase activity was assayed colorimetrically according to ROSEN et al. 13. The reaction mixture (I.O ml) contained 0.25 ml of 0.2 M sodium phosphate buffer (pH 7.5), 0.3 ml of o.oi M L-tyrosine, o.15 ml of o.I M ~-ketoglutarate, o.04 ml of 0.00025 M pyridoxal 5'-phosphate and 0.2 ml of whole homogenate. The mixture was incubated aerobically at 37 ° for 20 rain, the reaction was stopped with 5 % trichloroacetic acid, and the mixture was centrifuged as before. The supernatant solution was analyzed for p-hydroxyphenylpyruvic acid colorimetrically at 71o m~. A unit of enzyme was defined as the amount that caused the production of I ~mole kynurenine or p-hydroxyphenylpyruvic acid formation per h per g wet weight of liver. RESULTS

Course of tryptophan pyrrolase and tyrosine transaminase induction after operation The activities of both enzymes were determined at several time periods following partial hepatectomy or laparotomy. As shown in numerous previous studies 5-8, the intraperitoneal injection of hydrocortisone to adrenalectomized animals resulted in a rapid increase in the level of tryptophan pyrrolase and tyrosine transaminase in the liver. In the present experiments (Figs. I, 2), the enzyme activity increased about 2-2.5-fold in 4-6 h after laparotomy, and 4-6-fold in 8-1o h after partial hepatectomy. In partial hepatectomized rats, courses of the activities of two enzymes were somewhat different. E t h e r anesthesia, even for extended periods of time (IO rain), had no effect on increase in tryptophan pyrrolase and tyrosine transaminase as measured after 6 h.

Effect of actinomycin D and adrenalectomy on tryptophan pyrrolase activity after operation Actinomycin D, which is known to inhibit RNA synthesis, was used to determine the respective contributions made b y either new enzyme synthesis or enzyme Biochim. Biophys. Acta, 165 (1968) 218-224

1(, TSUKADA et al.

220

~2.C

5oo ~

_~5.0

4oo b

~ ~o

~

~ ,.oi

,.~ 3o

~

~ 2C

~

~_

T 2

I 4

T 6

I 8

c

~

~00 ~

~oo ~

5

°

•

5 ~o

I

HOURS AFTER OPERATION

~

600 _~

200 ~'

I0

~00 ~

4

'

~ ' ,~ ,6 ~o HOURS AFTER 09ER~TION

2~o

o~ ~

Fig. I. Induction of tryptophan pyrrolase and tyrosine transaminase in rat livers as a function of time after laparotomy. Each point represents the average of 2 or 3 rats. 0 - - 0 , tryptophan pyrrolase ; © - - O, tyrosine transaminase. Fig. 2. Induction of tryptophan pyrrolase and tyrosine transaminase in rat livers as a function of time after partial hepatectomy. Each point represents the average of 2 or 3 rats. 0 - - 0 , tryptophan pyrrolase; O - - O , tyrosine transaminase. TABLE I EFFECT OF ACTINOMYCIN D OPERATION

A N D A D R E N A L E C T O M Y ON T R Y P T O P H A N

PYRROLASE ACTIVITY AFTER

Actinomycin D, 200/zg per ioo g of body weight, was given intraperitoneally immediately after operation. At 6 h, animals were killed and their livers ~vere analyzed. Each determination represents the average of the results obtained with 2 rats.

Treatment

Operative treatment Laparotomy

Partial hepatectomy

Tryptophan pyrrolase activity (units) At o h At 6 h Plus actinomycin D Plus adrenMectomy

1.48 3.13 1.45 1.59

1.48 5.90 1.58 1.68

a c t i v a t i o n to the t o t a l a c t i v i t y of the induced t r y p t o p h a n pyrrolase and tyrosine transaminase. Based on the value obtained for the i n t a c t control animals, the induced a c t i v i t y of t r y p t o p h a n pyrrolase at 6 h after operation was almost c o m p l e t e l y inhibited, when 2oo Fg of a c t i n o m y c i n D was administered i m m e d i a t e l y after the operation (Table I). In bilaterally adrenalectomized rats, the induced t r y p t o p h a n pyrrolase a c t i v i t y was inhibited (Table I). Similar results were obtained with tyrosine transaminase.

Subcellular distribution and reconstruction of liver tryptophan pyrrolase activity I t has been shown recently t h a t t r y p t o p h a n pyrrolase a c t i v i t y of r at liver h o m o g e n a t e exists exclusively in the s u p e r n a t a n t fraction and t h a t liver mierosomes contain an iron-porphyrin cofactor which is released into the s u p e r n a t a n t fraction during the incubation period 14. In this e x p e ri m en t , the a c t i v i t y of t r y p t o p h a n pyrrolase in the s u p e r n a t a n t fraction was studied with or w i t h o u t r at liver mierosomes

Biochim. Biophys. Acta, 165 (1968) 218-224

TRYPTOPHAN PYRROLASE AND TYROSINE TRANSAMINASE

221

or mitochondrial fraction. As shown in Table II, the activity of tryptophan pyrrolase in the supernatant fraction prepared from the liver at 6 h after partial hepatectomy, though less than that in the total liver homogenate, was elevated to about 5-6 times that of normal rat. In the presence of I equivalent of rat liver microsomes or mitochondria, the activity of tryptophan pyrrolase of the supernatant fraction was increased to that of the total liver homogenate. The cofactor activity of microsomes or mitochondria prepared from control or partially hepatectomized liver was equivalent. From these results it was concluded that the enhancement in tryptophan pyrrolase activity at 6 h after partial hepatectomy was due to a change in supernatant fraction. TABLE II SUBCELLULAR DISTRIBUTION AND RECONSTRUCTION OF LIVER TRYPTOPttAN I~YRROLASE ACTIVITY T r y p t o p h a n p y r r o l a s e a c t i v i t y w a s m e a s u r e d in t h e l i v e r s from n o r m a l a n d p a r t i a l l y h e p a t e c t o m i z e d r a t s a t 6 h a f t e r o p e r a t i o n . One e q u i v a l e n t of m i c r o s o m e s or m i t o c h o n d r i a w a s us e d a n d refers to t h e n o r m a l r a t i o of m i c r o s o m e s or m i t o c h o n d r i a t o s u p e r n a t a n t f r a c t i o n in liver. 5 a n i m a l s w e r e u sed r e s F e c t i v e l y in t h i s e x p e r i m e n t .

Operative treatment Normal

Partial hepatectomy

Tryptophan pyrrolase activity (units) Whole homogenate Supernatant fraction Mitochondria Microsome Supernatant fraction Supernatant fraction Supernatant fraction Supernatant fraction Whole homogenate Supernatant fraction Mitochondria Microsome Supernatant fraction Supernatant fraction Supernatant fraction Supernatant fraction

and and and and

1.38 o.41 < o.o I < o.oi mitochondria o.71 microsomes 0.78 mitochondria (hepatectomy) 0.75 microsomes (hepatectomy) 0.82

and and and and

5.21 2.21 < o.oI o.oi 3.62 4.02 3.5° 3.68

mitochondria microsomes mitochondria (normal) microsomes (normal)

Pyridoxal phosphate requirement of rat liver tyrosine transaminase The maximal activity of the tyrosine transaminase of liver homogenate is obtained in the presence of 10-5 M pyridoxal phosphate. Neither increased amounts of pyridoxal phosphate nor preincubation of the enzyme and substrate without cofactors elicited greater activity. Table I I I shows the effect of pyridoxal phosphate on tyrosine transaminase activity. The added pyridoxal phosphate had converted apotyrosine transaminase into active holoenzyme. A decreased pyridoxal phosphate requirement is detectable at the maximal activity of this enzyme,4 h after laparotomy, and at 8 h after partial hepatectomy, although the latter is more striking. At later time, at I0, 24 h as shown in Table hi , the cofactor requirement was gradually increased.

Biochim. Biophys. Acta. 165 (1968) 218-224

222

K. TSUKADAet al.

TABLE III PYRIDOXAL PHOSPHATE AFTER OPERATION

REQUIREMENT

OF RAT LIVER TYROSINE

TRANSAMINASE

INDUCTION

PROCESS

Time of assay d e n o t e s t h e t i m e w h e n l i v e r s a m p l e s were r e m o v e d for t h e e s t i m a t i o n of t y r o s i n e t r a n s a m i n a s e a c t i v i t y a n d t h e e n z y m e a c t i v i t y was e s t i m a t e d w i t h o u t (--) a n d w i t h ( + ) i o s M a d d e d p y r i d o x a l p h o s p h a t e . E a c h v a l u e is t h e a v e r a g e of t h e r e s u l t s o b t a i n e d w i t h 3 ra t s . Operative treatment

Time of assay (h)

Tyrosine transaminase

Stimulation by pyridoxal-P (%)

Pyridoxal-P (units)

+ Pyridoxal-P (units)

Laparotomy

o 4 IO

63 276 lO2

226 435 258

259 58 153

Partial hepatectomy

o 8 24

82 76o 17o

265 87o 282

223 15 7°

T A B L E IV EFFECT OF CORTISONE ON TRYPTOPHAN PYRROLASE AND TYROSINE TRANSAMINASE INDUCED AFTER PARTIAL HEPATECTOMY Cortisone was i n j e c t e d i n t r a p e r i t o n e a l l y i m m e d i a t e l y on u n o p e r a t e d n o r m a l rats, or a f t e r operatio n. T h e r a t s were killed a t 6 h a f t e r a d m i n i s t r a t i o n of cortisone.

Operative treatment

Cortisone treatment

Normal

None Cortisone

Partial hepatectomy

Tryptophan pyrrolase (units)

Tyrosine transaminase (units)

5 mg

1.22 6.46

198 805

None Cortisone 5 m g Cortisone IO m g

5.68 5.90 5.81

829 835 796

Effect of cortisone on partially hepatectomized rats In rat livers the activity of tryptophan pyrrolase and tyrosine transaminase rose significantly within a few hours after administration of glucocorticoids. In this experiment cortisone was administered into partially hepatectomized rats. As shown in Table IV, induced response in both enzymes at 6 h after partial hepatectomy was not affected by exogeneous cortisone. DISCUSSION

The specific activities of a number of enzymes in animal tissues can be increased by the administration of hormones 15-17, by specific substrates15, TM, or by alterations in the nutritional state of the animal 19,2~. In some of these cases, the increased specific activities of these enzymes have been shown to represent protein formation de novo, as opposed to an activation phenomenon 2a-.6. The cortisone-induced elevation of liver Biochim. Biophys. Acta, I65 (I968) 218-224

TRYPTOPHAN PYRROLASE AND TYROSINE TRANSAMINASE

223

tryptophan pyrrolase and tyrosine transaminase is inhibited by actinomycin D, whereas the substrate induction of tryptophan pyrrolase is noW. The data presented in this report indicate that tryptophan pyrrolase and tyrosine transaminase activities were increased after laparotomy or partial hepatectomy without administration of hormones or substrates, and that this increase was due to newly-synthesized enzyme protein. In adrenalectomized rats, the increased tryptophan pyrrolase activity was inhibited in comparison with non-adrenalectomized controls. It would appear that the effective regulation of the induction of tryptophan pyrrolash and tyrosine transaminase in hepatectomized rats must involve a secreted, endogenous, cortisone-like substance from adrenal glands caused by the operative stress. In partially hepatectomized rats, however, the increased total enzyme activity was not affected by administration of cortisone. A decreased pyridoxal phosphate requirement is detectable at the time of maximum enzyme activity. These results as shown by GREENGARD AND GORDONus may suggest that the cofactor levels, in addition to regulating the activity of existing enzymes, may influence, in vivo, the amount of the protein moiety of appropriate enzyme systems. It has been shown that the events that lead to the alteration in liver microsomes or polyribosomes after partial hepatectomy are similar to the ones that result in the rise in RNA-polymerase activity 29-33, and in laparotomy or acute stress the increased ability of protein synthesis is occurring immediately after operation 34 with the similar time course of tryptophan pyrrolase activity. In view of these results, an increased rate of enzyme synthesis might become operative only under more drastic or stressful conditions (maybe hormonal process) in which a rapid increase in enzyme content would be required. It becomes important to extend this correlation and to define the role of these enzymes in the metabolic or synthetic response in partial hepatectomy. REFERENCES i E. C. C. LIN AND W. E. KNOX, Biochim. Biophys. Acta, 26 (1957) 85. 2 F. SERENI, F. T. KENNEY AND N. KRETCHMER, J. Biol. Chem., 234 (1959) 6o9. 3 W. E. KNox, Brit. J. Exptl. Pathol., 32 (1951) 462. 4 W. E. KNOX AND V. H. AUERBACH,J. Biol. Chem., 214 (1955) 3o7. 5 M. CIVEN AND W. E. KNOX, J. Biol. Chem., 234 (1959) 1787. 6 0 . GREENGARD AND P. FEIGELSON, Nature, 19o (1961) 446. 7 P. FEIGELSON AND O. FEIGELSON, in G. PINCOS, Recent Progress in Hormone Research, Vol. 18, Academic Press, New York, 1962, p. 491. 8 R. T. SCHIMKE, E. W. GWEENEY AND C. M. BERLIN, J. Biol. Chem., 24o (1965) 322. 9 T. SBIMAZU, Biochim. Biophys. Acta, 65 (1962) 373. IO T. SHIMAZU, J. Biochem., 55 (1964) 163. i i G. M. HIGGINS AND R. M. ANDERSON, Arch. Pathol., 12 (1931) 186. 12 W. E. K N o x AND A. H. ME~LER, J. Biol. Chem., 187 (195o) 419. 13 F. ROSEN, H. R. HARDING, R. J. MILHOLLAND AND C. A. NICHOL, J. Biol. Chem., 238 (i963) , 3725 • 14 P. FEIGELSON AND O. GREENGARD,J. Biol. Chem., 236 (1961) 151. 15 W. E. KNOX AND A. n . MEHLER, Science, xi 3 (1951) 237. 16 E. C. C. LIN AND W. E. KNOX, J. Biol. Chem., 233 (1958) I186. 17 F. RosEN, N. F. ROBER*S AND C. A. ~ICOL,J. Biol. Chem., 234 (1959) 476. 18 A. H. CONNEY AND A. G. GILMAN, J. Biol. Chem., 238 (1963) 3682. 19 R. T. SCI~IMKE, J. Biol. Chem., 237 (1962) 459. 20 W. E. KNOX, V. H. AUERBACH AND E. C. C. LIN, Physiol. Revs., 36 (1956) 164. 21 R. A. EREEDLAND AND A. E. HARPER, J. Biol. Chem., 234 (1959) 135o. 22 G. T. WEBER AND H. MACDONALD, Exptl. Cell Res., 22 (I961) 292.

Biochim. Biophys. Acta, i65 (I968) 218-224

K. TSUKADA et al.

224 23 24 25 26 27 28 29 3° 31 32 33 34

F. T. KEN~!ERY, J. Biol. Chem., 237 (1962) 161o. H. L. SEGEL, 1~. G. ROSSO, S. HOPPER AND M. M. WEBER, J. Biol. Chem., 237 (1962) PC 3303 . 1~. FEIGELSON AND O. GREENGARD, J. Biol. Chem., 237 (1962) 3714 . t~. T. SCHIMKE, J. Biol. Chem., 239 (1964) 3808. O. GREENGARD, M. A. SMITH AND G. ACS, J. Biol. Chem., 238 (1963) 1548. O. GREENGARD AND M. GORDON, J. Biol. Chem., 238 (1963) 3708. K. TSUKADA AND I. LIEBERMAN, J. Biol. Chem., 239 (1964) 2952. t(. TSUKADA AND I. LIEBERMAN, J. Biol. Chem., 240 (1965) 1731. ~5~.TSUKADA AND I. LIEBERMAN, Biochem. Biophys. Res. Commun., 19 (1965) 702. K. TSUKADA, T. ~VIORIYAMA,O. DOI AND I. LIEBERMAN, J. Biol. Chem., 243 (1968) 1152. K. TSUKADA, T. ]~ORIYAMA, T. ~.~MEDA AND I. LIEBERMAN, J. Biol. Chem., 243 (1968) 116o. C. MAJUMDAR, K. TSUKADA AND I. LIEBERMAN, J. Biol. Chem., 242 (1967) 700.

Biochim. Biophys. Acta, 165 (1968) 218-224