studies of the stability in vito and in vitro of rat liver ... · the fact that its substrate...

THE JOURNAL OF BIOLOGICAL CHE~STRY Vol. 240, No. 12, December 1965

Printed in U.S. A.

Studies

of

of the Stability in Vito and in Vitro

Rat Liver Tryptophan Pyrrolase

ROBERT T. SCHI~IKE, E. W. SWEENEY, AND C. M. BERLIN

From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland WOOi.

(Received for publication, March 31, 1965)

Recent evidence has indicated that virtually all proteins of rat liver, including various enzymes, are continually synthesized and degraded (I, 2) at rates that differ for each specific protein (3-6). Knowledge of the nature of mechanisms involved in the physiological degradation of tissue proteins, however, is extremely rudimentary. One of the enzymes with a most rapid rate degradation is tryptophan pyrrolase, which has a half-life that has been estimated by various techniques to be 2 to 2; hours (4,7,8). This may be compared with a mean half-life of 2 to 3 days for total liver protein (I, 2, 5). Tryptophan pyrrolase would therefore appear to be a suitable liver protein for a study of the process of protein in degradation because of its rapid turnover in viva and the fact that its substrate stabilizes the enzyme both in viva (4, 9) and in vitro (10, 11).

In the present study we have therefore investigated certain properties of the stability in viva and in vitro of this enzyme in an attempt to answer the following questions. (a) What is the nature of the system that degrades tryptophan pyrrolase in VWO, and can this degradation be reproduced in vitro? (b) What is the function of tryptophan in stabilizing tryptophan pyrrolase in systems in vitro and preventing its degradation in viva? As a part of this study we have compared the effects of tryptophan analogues on the stabilization of tryptophan pyrrolase in viva and in various systems in vitro. This was performed because of reported differences between the ability of tryptophan analogues to stabilize tryptophan pyrrolase in homogenates at 2” and their ability to induce the enzyme in viva (12, 13).

The results indicate that the rapid loss in viva of tryptophan pyrrolase, as determined by immunological criteria as well as enzyme activity, cannot be duplicated in liver homogenates or livers devoid of a normal blood supply. The simplest system capable of this degrading action involves the use of liver slices. The properties of the degradation process in liver slices are similar to those described by Simpson (14) and Steinberg and Vaughan (15) for total liver protein and indicate that the conditions for the physiological process of protein degradation have not yet been reproduced outside of structurally and metabolically intact tissues.

L-Tryptophan exerts a marked stabilizing effect on the purified enzyme, whether in the presence of heat, organic solvents, urea, or proteolytic enzymes. Although a series of tryptophan analogues exerts some stabilizing effect during heating, only 01- methyltryptophan is effective against other forms of inactivation. Studies with the use of the analytical ultracentrifuge indicate that tryptophan does not act’ by altering the aggregational state of the enzyme. Based on studies of kinetics with a-methyltryptophan, it is proposed that two substrate-binding sites exist:

one the catalytic site, the other a site that mediates changes in the conformation of the enzyme which affect its stability.

A number of differences were encountered in the stability properties of tryptophan pyrrolase in viva and in various systems in vitro. These differences indicate the problems associated with applying directly the results from systems in vitro to the stability in viva of an enzyme.

EXPERIMENTAL PROCEUURE

Treatment of Animals-Male, Osborne-Mendel rats weighing 120 to I40 g each were adrenalectomized 4 to 5 days prior to use. Such animals were used in all experiments. They were main- tained on Purina laboratory chow and 0.85% NaCl for drinking water. L-Tryptophan and all tryptophan analogues were administered intraperitoneally in doses of 1 mg per g of body weight in 10 ml of 0.85% NaCl as outlined in the legends to tables.

Enzyme Assays-Tryptophan pyrrolase was assayed in liver homogenates by the method of Knox and Mehler (16) as modified by Feigelson and Greengard by the addition of hematin (17). In extracts and purified enzyme preparations, ascorbate was added as described by Tokuyama and Knox (18) to diminish the lag in onset of formylkynurenine formation. With purified enzyme preparations, continuous recordings of the increment in optical density at 321 rnp resulting from the formation of formylkynurenine were made with a Gilford recording attachment for the Beckman model DU spectrophotometer. All assays were performed at 37”. Rates were determined from the initial, linear portion of the optical density increment. Tyrosine-glutamic transaminase was measured by the method of Rosen et al. (19). Arginase was assayed as described previously (20). Trypsin was assayed with the use of N-benzoyl-n-arginine ethyl ester as substrate (21). Chymot.rypsin was assayed with benzoyl-r-tyrosine ethyl ester as substrate (22). Enzyme activity is described in terms of a unit which is defined as that amount of enzyme that results in the formation of 1 kmole of product per hour at 37”. ilfateriala-cr-Chymotrypsin, crystallized three times, and

salt-free trypsin, crystallized two times, were products of Wort,h- ington. Crystalline Streptomyces proteinase (Nagarase) (23) was obtained from the Teikoku Chemical Industry Company, Ltd., Osaka, Japan. Arginase was purified from rat liver as described previously (5). oc-Methyl-m-tryptophan was a gift of Dr. K. Pfister of Merck Sharp and Dohme. 911 other compounds were the highest grade obtainable from commercial sources. Uni- formly labeled 14C-r,-leucine was purchased from New England Nuclear.

Immunologic Analyses-The rabbit antidody specific for rat liver tryptophan pyrrolase has been described previously (4).

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4610 Stability of Tr yptophan Pyrrolase Vol. 240, No. 12

Equivalence point determinations and quar?titative precipit,in \\-as at most a 15 to 20% loss of total activity by this procedure. reactions were performed as outlined by Kabat and Mayer (24) The best preparations of enzyme had a specific activity of 180 to and as described in the legends to figures. 220 units per mg of protein.

PuriJcation of Tryptophan Pyrrolnse-Tryl)to~)han pyrrolase was purified from rat liver by procedures described previously (4). For most, of the experiment,s enzyme with specific activities of 40 to 160 units per mg of protein was used. For the studies involving the use of t,he analytical ultracentrifuge, the enzyme preparations were subjected to an additional purification step involving rhromabography on Sephadex G-200 (25). Trypbo- phan pyrrolase is retarded on passage through Sephadex G-200 but not by Scphadex G-100. A column (4 X 70 cm) of Sephadex G-200 was equilibrated with 0.005 RI sodium phosphate, pH 7.0, cont,aining 0.005 M L-t,ryptophan. To the column were applied 5 to 10 ml of enzyme solution from the ammonium sulfate precipitation step (4). The column was developed at 25” with the equilibrating solution at a flow rate of 50 ml per hour. The first fractions of enzyme appeared in 6 to 8 hours. Tryptophan pyrrolase was eluted just after the void volume and generally constituted the only protein peak &ted from the column. The peak fractions of tryptophan pyrrolase were combined and chilled to 4”, and the enzyme was precipitated by the addition of satu- rated ammonium sulfate adjusted to pH 7.0 with concentrated i\-H,OH to a final concentration of 70% of saturation. There

Tryptophan pyrrolase purified by t,he above procedure had no appreciable hematin requirement for activity (17). I’repara- tions of the apoenzyme were obtained by performing the ent.ire purification procedure in the presence of 0.8 mM sodium ascorbate. Such preparations had a hematin requirement ranging from 70 to 100%. However, yields of enzyme purified in the presence of ascorbate were low, ranging from 5 to 15%) of initial activity, as compared with yields of 35 to 50% when the purifica- tions were performed in the absence of ascorbate. In some experiments (Table VI) enzyme preparations containing both tryptophan pyrrolase and tyrosine-plutamic transaminasc were used. Such preparations were made by omitting the calcium phosphate gel and the Sephadex G-200 chromatography steps.

Purified preparations of tryptophan pyrrolasc were used on the day of preparation, or they were stored in the presence of 0.005 M L-tryptophan or 0.005 M c+methyl-nL-tryptophan at 4” or at -20” without altering the stability or kinetic properties of the

enzyme for up to 7 days. However, freezing the enzyme in the absence of a stabilizing agent affect’ed both the kinetic and stability properties of the enzyme as discussed under “Results.”

TABLE I

Comparison of deca!/ in vivo and in vitro of tryptophan pyrrolase and tyrosine-glzciamic transaminase activities

,411 enzyme preparations were initially freed of tryptophan before use by passage through a column of Sephadex G-25 (bead form) equilibrated with 0.05 M sodium phosphate, pH 7.0, the bed volume of which was at least 20 times that of the applied enzyme solution. This was performed at 4”.

Adrenalectomieed rats received 5 mg of hydrocortisone 21. phosphate intraperitoneally. Some of the animals were’killed 5; hours after the injection, designated as zero time in the experiment. The livers of these rat,s were homogenized in 3 volumes of 0.15 M KC1 containing 0.005 N NaOH. Aliquots of t.his homogenate were stored at -20” immediately and used for the zero time assay, or placed in flasks (20 ml in 125-ml Erlenmeyer flasks) and incubated aerobically with shaking or placed in stoppered flasks (20 ml in 50.ml Erlenmeyer flasks) and incubated anaerobically without shaking at 37”. The livers of other rats were removed and incubated without. homogenization at 37” in moist Petri dishes. At the end of 8 hours, livers of intact animals which had been treated previously with hydrocortisone as well as the livers incubated in Petri dishes were homogenized as described above. Colltrol animals received no injections. All homogenates were thereafter stored for 16 hours at -20” before assay. All activities are averages of duplicate determinations (homogenates) or of three separate livers (intact animals and excised livers) and are expressed as units per g of liver, wet weight.

Protein-l’rotein was estimated by the biuret method (26) when tryptophan was present in t,he solutions or by t,he method of Lowry et al. (27).

RESULTS

Comparison of Loss of Truptophan Pyrrolase and Tjjrosine- Glutamic Transaminase Activities in Viva and in Liver Ilomoge- nates-In Table I are shown results of representat,ive experiments on comparisons of the loss of activity in vivo and in vitro of tryptophan pyrrolase and tyrosine-glutamic transaminase in crude liver homogenates. Enzyme activity had been increased to high levels by single administrations of hydrocortisone to intact rats. In intact rats there was a rapid decay of bot,h tryptophan pyrrolase and tyrosine-glutamic transaminase activities (4, 7, 28). In homogenates from similarly treated animals, on the other hand, tryptophan pyrrolase activity was lost only when the homogenate was shaken in an aerated flask and was entirely stable for up to 8 hours when the homogenate was incubated under partially anaerobic conditions. Likewise trypt,ophan pyrrolase was entirely stable when the liver was simply removed from the animal and incubated for 8 hours at 37”. Tyrosine- glutamic transaminase, which is equally as unstable as tryptophan pyrrolase in vivo (3, 28), was entirely stable in homogenates irrespect,ive of the incubation condit’ions.

Treatment

Zero time, pretreated with hydrocortisone.

8 hours In vivo _., In vitro.

Excised liver.. Homogenate

Aerobic.. Anaerobic.

Control, no treatment..

Tryptophan Tyrosine-glutamic

transaminase pyrrolase

(Experiment 1) Exp$nent Experiment

III

15.5 420 480

5.2 115

17.7 I I

2.9 477 18.2 473

4.0

The stimulation of tryptophan pyrrolase activity by anaerobic incubation of homogenates at 37” (zero time versus anaerobic or excised liver in Table I) has been observed consistently. Such activation was maximal with a 3- to 4-hour incubation. The artivation was most readily observed with homogenates from untreated animals, in which it, resulted in an increase of 50% in observed enzyme activity. The basis for this activation is not clear. It may represent a more complete activation with hematin than can be accomplished in the standard assay as dtsc,ribed


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R. T. Xchimlce, E. W. Sweeney, and C. M. Berlin 4611

I I I I

0

Serum Control -

0 4 8 12 16

ADDED ENZYME UNITS

Fro. 1. Immunological analysis of decay in viva and in vitro of trypt,ophan pyrrolase activity. Adrenalectomized rats were given two series of intraperitoneal administrations at 4-hour intervals of 5 mg of hydrocortisone 21.phosphate and 1 mg of L-tryptophan per g of body weight. The livers of six animals were homogenized 4 hours after the last administration in 3 volumes by weight of 0.15 M KC1 containing 0.005 M NaOH. One-half of this homogenate was stored at -20” immediately. The other portion was incubated for 3+ hours with shaking in a bath at 37”. An additional three animals were killed 73 hours after the last series of iniections (decay in viva). All homogenates were stored for 1G hours at, -20” before enzyme assay and immunological analysis. For immunological analysis, the thawed homogenates were centrifuged for 30 min at 48,000 X 9 and then for GO min at 105,000 X g. The resulting supernatant fluids were used directly. The activities of the extracts resulting from the procedures outlined above were as follows: ApA, zero time, 8G units per g of liver, wet weight; n--n, decay in viva, 42 units per g; O--O, decay in v&o, l-1 units per g. The percentage values indicate the percentage loss of activity from zero time. A, activity in supernatant. fluid with extracts of zero time or decay in vivo samples with the

by Feigelson and Grecngard (17), or it may involve a time-de-

pendent association of inactive subunits. Whatever the cause,

it is of interest that such activation was not observed with ho- mogena.tes or cstracts from animals that had been t,reat,ed previously with trypt,ophan.

The loss of tryptophan pyrrolase activity in viva is associated with a comparable loss of immunologically reactive protein (4). Experiments were therefore undertaken to determine whether the loss of this activity in homogenates incubated in an aerobic atmosphere was also associated with a loss of immunological reactivity. The upper part of Fig. 1 depicts results of equivalence point, dctcrminations with extracts in which there has been a loss up to 49y0 of original enzyme act’ivity in viva and a loss up to IS!& of enzyme activity in vitro in crude homogenates incubated aerobically. Experimental details are given in the legend.

The loss in viva is associated with a comparable loss of immunologically reactive protein. Thus it required the same number of added enzyme units to neutralize a constant amount of antibody as indicated by the point at which enzyme activity first appeared in the supernatant fluid. This result is in contrast with that obtained with the extract from homogenate incubated in an aerobic atmosphere, in which enzyme activity appeared at a far lower amount of added enzyme units. Thus it can be concluded

that an immunological reaction occurred with inactive enzyme.

0

x4

- t z c I / I

4 I I I

0 0.25 0.5 0.75 1.0

ML 4DDED EXTRACT

specified amounts of added enzyme activity. Upper, equivalence point determinations. Rabbit antitryptophan pyrrolase antiserum (0.3 ml) was added to increasing amounts of extracts. The tubes were incubated at 37” for 30 min and thereafter at 4” for 1G hours. The tubes were centrifuged and the supernatant fluids were assayed for tryptophan pyrrolase activity. Comparable amounts of enzyme activity were recovered with all extracts incubated with nonimmune serum. The serum control indicated was obtained with extracts from the decay in vitro (O---O) incubated with the nonimmune serum. Lower, quantitative precipitin analyses. Zero time extracts and extracts from homogenates incubated aerobically were added to 0.3 ml of either trvptophan pyrrolase antiserum or nonimmune serum in the amount indicated in the figure. After incubation as described for the equivalence point determinations, the precipitates were collected by centrifugation and washed twice with chilled 0.85% NaCl, and the resulting protein content was determined (27). Protein precipitation in the extracts incubated with nonimmune serum amounted at most to 10% of that formed with the tryptophan pyrrolase antiserum.

That the inactivated enzyme was fully active immunologically is shown by the quantitative precipitin analysis of t’he lower part of Fig. 1. Thus when the immunological reaction was determined by the quantity of antigen-antibody precipitate and was based on addition of equal amounts of extract to the constant amount of antibody, the immunological reactions were identical.

In a large series of other experiments no loss of tryptophan pyrrolase activity could be demonstrated n-hen liver homogenates were incubated anaerobically and were supplemented with cysteine or glutathione in concentrations known to activate catheptic activit.y (29). Furthermore, the addition of lysed lysosomal preparations from rat livers as described by Sawant, Desai, and Tappel (30) to crude homogenates or purified preparations of tryptophan pyrrolase at pH 7.0 was without effect on enzyme activity.

Tryptophan Pyrrolase Inactivation in Liver Slices-In view of the inability to demonstrate degradation of tryptophan pyrrolase in homogenates as determined by the double criteria of enzyme activity and immunological react’ivity, experiments were undertaken with liver slices. In Table II are shown the results of the inactivation of trypt,ophan pyrrolase and tyrosine-glutamic transaminase in liver slices of rats that had received single injections of hydrocortisone to increase the levels of these enzymes. Tryptophan pyrrolase was inact,ivated in an aerobic at,mosphere.


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4612 Stability of Tryptophan Pyryolase Vol. 240, Xo. 12

In contrast with the results in crude homogenates (Table I), tyrosine-glutamic transaminase was also inactivated in liver

slices incubated aerobically. Anaerobic incubation of liver slices inhibited the inact,ivation of both enzymes.

Extracts from slices incubated in an aerobic atmosphere were

analyzed for immunological reactivity by equivalence point

determinations similar to those of Fig. 1. These results are summarized and compared with those of Fig. 1 in Table III. Xs in

the case in v&o, the loss of t,ryptophan pyrrolase activity in liver slices was associated with a loss of immunological reactivity.

Thus the decay of enzyme activity in liver slices is comparable

to that which occurs in viva as opposed to the loss of enzyme activity which occurs in the homogenized tissues, where immuno-

logical reactivity is not lost’. Comparison of Tryptophan Analogues as Stabilizers in Vivo

and as Inducers of Tryptophan Pgrrolase-Feigelson et al. (12, 13)

found that a large series of tryptophan analogues stabilized trypt,ophan pyrrolase activity in crude homogenates incubated

at 2” for 36 hours. Since many of the analogues that stabilized

the enzyme in t,heir system did not induce trypt’ophan pyrrolase

TABLE II

Inactivation of tryptophan pyrrolase and tyrosine-glutamic transaminase in liver slices

At 4-hour intervals four rats were each given two injections of 5 mg of hydrocortisone 21.phosphate. The animals were killed 6 hours after the last injection and the livers were cut into 0.5-mm slices nit.h a Stadie-Riggs microtome and equally distributed among eight flasks. A total of 0.0 to 0.8 g of liver slices was placed in 25-ml Erlenmeyer flasks in 5.0 ml of Krebs-Ringer- phosphate, pH 7.4 (31). The pH at the end of the 4-hour period was 7.4. The flasks incubated in nitrogen were flushed four times ait,h nitrogen, evacuated to remove oxygen, and then stoppered. The flasks were incubated with shaking at 37” for 4 hours. At the termination of the experiment 1.0 ml of 0.03 M L-tryptophan was added to each flask, after which the total contents were homogenized and assayed for enzyme act,ivity. Each value repre-

sents the mean of duplicate flasks and is expressed as units per g of liver, wet weight.

Conditions

Zero time. 4 hours at 37”

Tryptophan Tyrosine-glutamic pyrrolase transaminase

(units/g Z&r)

17.0 472

Aerobic. 3.7 206 Anaerobic. 11.7

I 434

TABLE III

Summary of loss o.f tryptophan pyrrolase in vivo, in liver slices and in liver homogenates

Values are expressed as the percentage of original or zero time values. See “Experimental Procedure” and legend to Fig. 1 for details.

Enzyme Activity Immunological reactivity

% In vivo

Intact animal. In vitro

4G

Liver slice (air). 33 Liver homogenate (air). 16

%

45

35 100

TABLE IV

Comparison of tryptophan analogues as stabilizers in vivo and as inducers of rat liver tryptophan pyrrolase

The description of the rats and dosages of tryptophan annlogues are described under “Experimental Procedure.” For the stabilization experiments the rats received 5 mg of hydrocortisone 21.phosphate and 1 mg of L-tryptophan per g of bodg weight intraperitoneally on two occasions at 4-hour intervals. The animals were given the listed t.rypt.ophan analogues 5 hours after the last injections, denoted zero time in the table. Livers were assayed for tryptophan pyrrolase activity 43 hours later. For the induction studies, the livers were assayed 4 hours after the listed compounds were administered to previously untreated animals. All values represent the mean of three to six animals and are expressed as units per g of liver, wet, weight. Compara- tive values for inducting and stabilizing abilities are expressed as the percentage of those produced by L-t,ryptophan as follows.

Test X 100

Tryptophan-treated - control

where control activities are those from sodium chloride-treated (stabilization) or zero time (induction) animals.

Compound

Zero time. Sodium chloride. L-Tryptophan o-Tryptophan a-Methyl-DL-trypto-

phan N-Acetyl-uL-trvpto-

phan 5-Methyl-uL-trppto-

phan G-Methyl-uL-trypto-

phan

Stabilization

uds/g

72.5 16.8 81.0 47.0

rryptophs.1 stabiliza-

tion

YO

0 100 47

71.3 j 85

23.6 11

15.8 0

14.9 i O

Induction -.

1

Activity

units/g

4.2 10.6

9.1

9.8

5.8

3.6

3.2

‘ryptophan induction

1 Y”

100 76

87

25

0

0

in the intact animal, they concluded that tryptophan stabiliza-

tion was not involved in the induction in vivo of the enzyme. We have therefore re-examined the effects of various tryptophan

analogues as inducers of tryptophan pyrrolase and as stabilizers in vivo as opposed to in vitro. In Table IV the abilities of trypto-

phan analogues to stabilize tryptophan pyrrolase in vivo and to induce t,he enzyme in intact animals are compared. Stabiliza-

tion in vivo was determined by the ability to prevent the rapid

decay of enzyme activity in animals in which tryptophan pyrrolase levels had been previously increased 15 to 20.fold by

repeated administrations of both hydrocortisone and L-tryptophan. Details are given in the text to Table IV. c+Methyl-

nL-tryptophan, the tryptophan analogue most effective in pre-

venting the rapid fall of tryptophan pyrrolase activity, was 85% as effective as L-tryptophan. o-Tryptophan and N-acetyl-nL- tryptophan are partially effective, whereas 5-methyl-m-trypto-

phan and 6-methyl-nL-tryptophan are totally ineffective. It can also be seen that there is a good correlation between the abilities

of these various analogues to stabilize tryptophan pyrrolase in vivo and their abilities to induce the enzyme. The comparative effects of tryptophan analogues as stabilizers in vivo are similar

to those observed by Civen and Knox (II), who studied the in-


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December 1965 R. T. Schimke, E. W. Sweeney, and C. M. Berlin

activation of enzyme activity in crude homogenates during incubation at 37”.

In other experiments with the liver slice system described in Table II, among the compounds used in Table IV, only n-tryptophan and cu-methyl-nn-tryptophan prevented inactivation of the enzyme observed under aerobic conditions (Table II). In these same experiments, none of the compounds was effective in preventing the loss of tyrosine-glutamic transaminase activity. Thus the effect of tryptophan in preventing the loss of tryptophan pyrrolase in the liver slice system would appear to be relat.ively specific for tryptophan pyrrolase.

proteolysis at pH 7.4 in homogenates, slices of liver incubated aerobically underwent proteolysis amounting to 4% in 4 hours. Furthermore this measure of total protein degradation was not affected by the presence of 0.005 M n-tryptophan. Thus it is concluded that L-tryptophan does not affect tryptophan pyrrolase degradation by preventing protein degradation in general. dnaerobic conditions during incubation of liver slices diminished markedly the formation of trichloroacetic acid-soluble radioactivity. The results of degradation of protein in liver slices and the effect of anaerobiosis are in agreement with those of Simpson (14) and Steinberg and Vaughan (15).

EJect of L-Tryptophan on Endogenous Proteolysis of Liver Slices and Homogenates-To ascertain the specificity of L-tryptophan in preventing the degradation of trypt.ophan pyrrolase, experiments were undertaken on t.he effect of L-tryptophan on total protein degradation in homogenates and liver slices labeled in viva with i4C-n-leucine. These results are summarized in Table V. Degradation of protein was measured as the increase in radioactivity soluble in 10% trichloroacetic acid. There was no demonstrable degradation of protein in crude homogenat.es incubated at pH 7.4. In homogenates adjusted to pH 5.5, on the other hand, prot.eolysis did occur and was not affected by the presence of 0.005 M L-tryptophan. In contrast with the lack of

TABLE \

Studies on Stability of Purified Tryptophan Pyrrolase--In order to determine the nature of the stabilizing effect of n-tryptophan on tryptophan pyrrolase, experiments were performed on t.he effects of various agents on its inactivation. The tryptophan pyrrolase preparations for these experiments had been purified from 40- to 400.fold and were prepared from livers of animals in which the level of enzyme had been increased 30-fold over basal levels by repeated administrations of L-tryptophan and hydrocortisone (4).

1. Heat inactivation: Purified preparations of tryptophan pyrrolase were rapidly inactivated by heat. Fig. 2 shows the temperature dependence of such inactivation. The inclusion of

Effects of L-tryptophan on proteolysis of liver homogenates and liver slices

Rats weighing 130 to 140 g each were given intraperitoneal separate flasks of liver slices. The material was homogeuized injections of uniformly labeled “C-r,-leucine (specific activity, and centrifuged, and the resulting precipitate was extracted with 80 mC per mmole) in 1.0 ml of 0.85’% N&l. The dosage u-as 20 5% trichloroacetic acid containing 0.01 M L-leucine by heating and 30 MC of l”C-id-leucine in Experiments 1 and 2, respectively. at 80” for 30 min. The extracts and washes were combined, and The livers were removed after 3 hours in Experiment 1 and after suitable aliquots were counted by the liquid scintillation tech- 60 hours in Experiment 2. Some livers were homogenized in 2 nique. The protein precipitates were dissolved in GO% formic volumes by weight of 0.15 M KC1 and adjusted to the desired pH acid, and suitable aliquots were counted. The results are given v,ith 0.2 M Tris or 0.2 M acetic acid. Other livers were sliced as t,ot,al counts per min soluble in trichloroacetic acid, and as the and incubated aerobically and anaerobically as described in percentage of total counts present at zero time (counts per min Table II. The homogenates were incubated in stoppered flasks. in soluble and insoluble fractions). The results are all normalized L-Tryptophan or 0.15 M KC1 diluent was added as indicated to to 1 g of liver, wet weight. l’alues for homogenate studies are slices and homogenates to a final concentration of 0.005 M. The the average of duplicate determinations. Values for liver slices incubation medium for the slices also contained 0.01 M 'VI!-L- are the result of individual flasks. Recorded pH values of ho- leucine to prevent reutilization of released iQleucine. Incuba- mogenates are those found at the end of the 4-hour incubation tions were carried out at 37” with shaking in a water bath. At period. The pH of the liver slice incubation medium remained zero time and at the end of 4 hours trichloroacetic acid (final 7.4 in all experiments. concentration, loci,) was added to samples of homogenates or

Conditions Total

Experiment 1 Homogeuate

Zero time 4 hours

pII 5.5 pH 5.5 + trypto-

pha11

pH 7.4 pH 7.4 + trypto-

phan Slices

Zero time 4 Hours

Aerobic

cfim

212,000

259,000 18,300 237,000 17,920

0% Trichlo- roacetic

acid-soluble

cpm

16,500

57,200 59,100

16,690 17,200

29,000 29,400

Ratio of soluble cpm to

total cplr

%

7.8

27.0 27.9

7.9 8.1

7.8

11.9

19.2 20.1

0.1 0.3

4.1

T

!9 Conditions Total

Experiment 1 Slices-continued

Aerobic + tryptophan

Anaerobic Experiment 2

Slices Zero time

208,000 212,000

4 hours Aerobic

Aerobic + tryptophan

Anaerobic

-

I 0% Trichlo

roacetic acid-soluble

cpm

28,700 29,500 22,300

8,700 8,520

18,200 16,500 15,500 17,000 11,300 12,800

! t ::%l:f cpm to otal cpm

YO

r lifference, test -

zero

YO

11.8

9.1

4.0

1.3

4.1

7.9 3.8

7.8 3.7 5.7 1.6


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4614 Stability of Tryptophan Pyrrolase Vol. 240, No. 12

L-tryptophan at 0.005 M essentially prevented this inactivation.

L-Tryptophan was equally as effective with preparations of

apoenzyme as with the holoenzyme. The inclusion of mercapto- ethanol or cysteine at concentrations varying from low5 to 1OW

I I I

-A 2"

I

0 30 60 90

MINUTES OF INCUBATION

FIG. 2. Effect of heat on inactivation of tryptophan pyrrolase. Tryptophan pyrrolase (specific activity, 40 units per mg of protein), 1.0 mg per ml, was incubated in 0.05 M sodium phosphate, pH 7.0, at the temperatures indicated for periods up to 60 min. --- . absence of L-tryptophan; -, presence of 0.005 M L-tryptophan.

; 50 0

%

25

I I

UREA CM)

0

I

2

4

3

0 30 60

MINUTES AT 25’

FIG. 3. Effect of L-tryptophan and differing urea concentrations on the inactivation of tryptophan pyrrolase. Tryptophan-free tryptophan pyrrolase was incubated at 25” in the presence or absence of 0.005 M L-tryptophan at the specified concentrations of urea. The final protein concentration was 2.0 mg per ml in 0.05 M sodium phosphate, pH 7.0. The enzyme was diluted 50.fold for subsequent enzyme assays. --, presence of 0.005 M L-tryptophan; - - -, no added L-tryptophan; 0 and 0, no urea; 0 and n , 1 M urea; n and A, 2 M urea; V and V, 3 M urea; 0 and +, 4 M urea.

r

0 20 40 60

MINUTES OF INCUBATION

FIG. 4. Effect of L-tryptophan and hematin on trypsin proteolysis of tryptophan pyrrolase. Tryptophan pyrrolase (specific activity, 30 units per mg of protein), 2 mg per ml in 0.05 M sodium phosphate, pH 7.0, was incubated at 25” with 10 pg of trypsin per ml in the presence or absence of 0.0025 M L-tryptophan, 0.5 PM hematin, or both, and subsequently assayed for enzyme activity after a 60.fold dilution under standard assay conditions (see “Experimental Procedure”). The enzyme preparation was 507, apoenzyme.

M, or addition of 0.8 mM sodium ascorbate, was without effect on

heat inactivation. 2. Urea inactivation: As shown in Fig. 3, tryptophan pyrrolase

was rapidly inactivated by urea. Such inactivation occurred at urea concentrations as low as 1 M. The rate of inactivation in-

creased with increasing urea concentrations up to 3 M. L-Trypto- phan prevented inactivation at urea concentrations up to 3 M

but was only partially protective at 4 M. In 5 M urea, L-tryptophan exerted no protective effect. As with heat inactivation, L-tryptophan prevented inactivation by urea as well with apoenzyme as with holoenzyme. The n-tryptophan protection was equally effective at all temperatures studied from 16 to 44”.

3. Inactivation by proteolytic enzymes: Tryptophan pyrrolase was extremely sensitive to proteolysis by a variety of proteolytic enzymes, including trypsin, chymotrypsin, and a proteinase (Nagarase) (23). In contrast with stabilization against heat and urea inactivation, stabilization of tryptophan pyrrolase

against trypsin was most complete when it was in the holoenzyme form. Thus (see Fig. 4) stabilization was most effective when both tryptophan and hematin were included in the incubation with trypsin and a preparation of tryptophan pyrrolase that was 50 y0 apoenzyme. L-Tryptophan protected partially, whereas hematin alone imparted no protective effect. In Table VI are shown the effects of L-tryptophan on proteolytic inactivation of tryptophan pyrrolase, tyrosine-glutamic transaminase, and arginase as produced by trypsin, chymotrypsin, and bacterial proteinase (23). L-Tryptophan exerted a protective effect on tryptophan pyrrolase irrespective of the proteolytic enzyme. These same enzymes had no effect on the activity of tyrosine- glutamic transaminase and arginase. The lack of effect on tyrosine-glutamic transaminase is of interest in view of the loss of enzyme activity in viva at a rate comparable to that of tryptophan pyrrolase (3, 28). In other experiments trypsin and chy-


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TABLE VI TABLE VII

Effect of trypsin, chymotrypsin, and bacterial proteinase on Effects of tryptophan analogues on inactivation of purified tryptophan pyrrolase, tyrosine-glutamic transaminase, tryptophan pyrrolase by heat, ethanol, urea, and

and arginase trypsin proteolysis

Purified arginase (5) and partially purified liver extracts containing both tryptophan pyrrolase and tyrosine-glutamic transaminase prepared as described in “Experimental Procedure” were passed through Sephadex G-25 equilibrated with 0.01 M

sodium phosphate, pH 7.0. L-Tryptophan was added to a final concentration of 0.005 M. The data for tyrosine-glutamic transaminase and arginase were similar whether or not L-tryptophan was present. Trypsin, chymotrypsin, or proteinase was added (10 fig per ml of enzyme solut’ion) and incubated at 25” for. 30 min, at which time samples were assayed for enzyme activity after a 60.fold dilution. All results are expressed as the percentage of initial enzyme activity.

The tryptophan pyrrolase preparations had specific activities varying from 30 to 60 unit’s per mg and concentrations of 1 my per ml in 0.05 M sodium phosphate, pH 7.0. All additions were made to give 0.005 M concentrations. Conditions for inactivation were as follows: (a) heat, incubation at 44” for 30 min.; (b) ethanol, incubation at 25” with 20yo ethanol for 30 min; (c) urea, incubation in 3 M urea at 25” for 30 min; and (d) trypsin, incubation with 15 rg of trypsin per ml at 25” for 30 min. All preparations were diluted at least 60-fold during subsequent assay. All values are expressed as the percentage of protection imparted by L-trypt,o- phan.

Compound Enzyme

Tryptophan pyrrolase + L-Trypt,ophan.. - L-Tryptophan

Tyrosine-glutamic transaminase.....................

Arginase................... / -

Proteolytic enzyme

Trypsin Chymotrypsin Proteinase

y. initial activity

81 49 81 25 13 15

100 100 100 100 100 100

L-Tryptophan u-Tryptophan e-Methyl-nr,-tryptophan iv-Acetyl-nL-tryptophan Tryptophol. 5-Methyl-DL-tryptophan 6-Methyl-DL-tryptophan Kynurenine Indole. L-Phenylalanine L-Histidine

44” heat 21

%

100 28 85 10 52 56

38 25 16

8

- ilyo ethano

%

100 GO

100 0 0 7 7 0 0

3 M urea

%

100 0

94 0

10

-

i --

-

‘roteolysis ,y trypsin

%

100 0

84 0 0 0 0 0 0 0 0

FIG. 5 (upper). Sedimentation properties of tryptophan pyrrolase in the presence and absence of or-methyl-DL-tryptophan. Tryptophan pyrrolase (holoenzyme) (specific activity, 180 units per mg of protein), 2.5 mg per ml in 0.05 M sodium phosphate, pH 7.0, was sedimented in a Spinco model E ultracentrifuge at 30” in the presence of 0.005 M cu-methyl-nL-tryptophan (top) and in its absence (bottom). Sedimentation is from left to right. Pictures were taken at 8-min intervals after attaining a speed of 59,780 rpm. Enzyme in the absence of a-methyltryptophan had lost 62% of its activity by the end of the sedimentation run. The apparent peaks other than the major one are artifacts resulting from convection disturbances within the sedimentation cell.

FIG. 6 (lower). Sedimentation properties of tryptophan pyrro-

lase in 3 M urea in the presence and absence of a-methyl-DL-tryptophan. The enzyme preparation and conditions of centrifugation are similar to those of Fig. 5. The preparation of tryptophan pyrrolase, 3.0 mg per ml, was treated with 3 M urea in the absence (top) and presence (bottom) of 0.005 M a-methyl-oI;-tryptophan for 30 min at 37” before the sedimentation run was performed. In the absence of or-methyl-DL-tryptophan, the enzyme solution in 3 M urea was visibly cloudy. At the end of the run, enzyme activities were 10% and 90% of initial enzyme activity in the absence and presence of a-methyltryptophan, respectively. The majority of the protein in the absence of a-methyl-DL-tryptophan (top) had aggregated and sedimented to the bottom of the cell before the first picture was taken.


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4616 Stability of Tryptophan Pyrrolase Vol. 240, Ko. 12

motrypsin activities with amino acid esters as substrates were not inhibited by the concentrations of L-tryptophan and hematin used for the stabilization experiments. Thus it is concluded that L-t.ryptophan did not affect the proteolytic enzyme, but. rather the t.ryptophan pyrrolase.

In order to compare and contrast further the various forms of inactivation of tryptophan pyrrolase, experiments were undertaken on the abilities of tryptophan analogues to prevent inactivation by heat, urea, trypsin, and ethanol. These are summarized in Table VII. There are striking differences in the abilities of tryptophan analogues to protect tryptophan pyrrolase depending on the inactivat.ing agent. employed. Thus an entire series of compounds partially stabilize tryptophan pyrrolase against heat inactivation, including indole, and kynurenine, as well as several amino acids. In contrast with heat inactivation, virtually the only analogue that prot.ects against inactivation by ethanol, urea, and proteolysis at a concent,ration of 0.005 M was oc-methyltryptophan. ol-Methyltryptophan would therefore appear t’o be unique among tryptophan analogues in its ability to protect purified tryptophan pyrrolase against various inactivating agents and as the best stabilizer in v&o.

Immunological analyses comparable to those of Fig. 1 were made with heat- and trypsin-inactivated enzyme preparations. As nit.h crude homogenates, heat inactivation of purified enzyme did not alter the immunological reactivity. On the other hand, treat’ment with trypsin abolished t,he immunological reactivity of the enzyme.

In other experiments the inclusion of 0.005 M L-tryptophan did not alter the reactivity of the tryptophan pyrrolase-antibody reaction.

In a large series of experiments with heat and urea-inactivated tryptophan pyrrolase preparations, we have been unable to restore activity reproducibly following dilution of urea, or subse-

I I

L-Tryptophon

5 X 10-3M

I x 10-3

5x 10-4

I x 10-4 5x10-5

1

0 30 60

MINUTES AT 37’C

FIG. 7. Effect of L-tryptophan concentration on the heat inactivation of tryptophan pyrrolase. Hematin-free tryptophan pyrrolase (specific activity, 120 units per mg of protein) was incubated at 37” in 0.01 M sodium phosphate, pH 7.0, at a final concentration of 1.0 mg per ml with the indicated concentrations of L-tryptophan. Aliquots of enzyme were assayed for enzyme activity at 30 and 60 min.

3. ‘0, \ \

‘. \ \ \ \ \

l \. \ I I ,\I

lO-2 lo-3 iO-4 lO-5

MOLARITY OF aMETHYL-DL-TRYPTOPHAN

FIG. 8. Effect of a-methyl-nL-tryptophan concentration on the stability of tryptophan pyrrolase in 3 M urea. To tryptophan-free tryptophan pyrrolase (specific activity, 90 units per mg of protein) was added a-methyl-DL-tryptophan to the indicated concentrations. The final concentration of enzyme was 2 mg per ml in 0.05 M sodium phosphate, pH 7.0. Urea (10 M) was added to a final concentration of 3 M. The solutions were incubated at 37” for 15 min and then assayed after dilution of the urea to 0.02 M. In the absence of any tryptophan or analogue, 75yc of the initial enzyme activity was lost. The results are presented as the percentage of protection imparted by L-tryptophan at 0.005 M, which completely protected the enzyme activity.

quent addition of L-tryptophan, or both. With an occasional preparation of urea-inactivated enzyme, a 5 to 1O70 restoration has been observed. The difficulty in restoring enzyme activity after urea denaturation may in part be due to the tendency of such an inactivated enzyme to form large, visible aggregates.

Studies on Mechanism of Protective Effect of z-Tryptophan--l. Sedimentat,ion studies: on the basis of studies with Sephadex gel filtration and with sucrose gradient studies,’ it has been estimated that tryptophan pyrrolase has a molecular weight of from 160,000 to 180,000. One possible effect of L-tryptophan or oc-methyltryptophan in imparting stability could involve a change in the aggregational state of the enzyme or other gross configurational properties that could be demonstrated by alterations in sedimentation characteristics. Fig. 5 shows sedimentation velocity st,udies with preparations of tryptophan pyrrolase in the presence and absence of 0.005 M a-methyltryptophan. ar-Methyltrypto- phan was used for these experiments because it is not metabolized by the enzyme. ar-Methyltryptophan did not alter the sedimentation properties of tryptophan pyrrolase. Both preparations of enzyme sedimentated at the same rate, with an .s~,~ value of 7.2. At the end of the run the enzyme in the absence of a-methyltryptophan had lost 65 ‘% of its initial enzyme activity, whereas in its presence no activity was lost. In other experiments with sucrose gradients (32)) no change in sedimentation characteristics would be shown to result from the presence of r,-tryptophan. We therefore conclude that stabilization of trypt,ophan pyrrolase does not involve a physical change in the enzyme sufficient to

1 C. ill. Berlin and R. T. Schimke, unpublished observations.


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affect its sedimentation characteristics. That a stabilizer does affect. the enzyme, however, is shown in Fig. 6, in which t.he enzyme was treated with 3 M urea at 30” in the presence or absence of 0.005 M cr-methyl-m-tryptophan before and during the centrifugation. In t.he presence of oc-methyltryptophan the sedimentation properties of the enzyme were similar to those of the untreated enzyme (Fig. 5), whereas in its absence the enzyme formed large aggregates that centrifuged rapidly to the bottom of the cell. Because of the formation of such aggregates, it has been impossible to determine whether or not urea first cleaved the enzyme into subunits.

2. Concentration dependence of stabilizing effect: L-Trypto- phan imparts its stabilizing effect against heat denaturation at extremely low concentrations. In Fig. 7 it can be seen that at concent,rations as low as 5 x lo+ M it produced half-maximal stabilization of a preparation of the apoenzyme. This is of particular note because the K, of L-tryptophan for the catalytic reaction under the specific conditions of temperature and ionic strength used for the heat inactivation is 3 to 5 X lop4 M. Fig. 8 depicts the concentration dependence of the stabilization by cr-methyl-DL-tryptophan of tryptophan pyrrolase against inactivation by 3 M urea. Half-maximal stabilization of the enzyme occurs at concentrations of 4 X 10e4 M oc-methyl-DL- tryptophan.

.05

04

03

.02

.Ol

0

I I I

IOmM a- Methyl-DL-Tryptophan

.

I 2 3

&

FIG. 9. Kinetics of inhibition of tryptophan pyrrolase by 01- methyl-DL-tryptophan. Tryptophan pyrrolase (specific activity, 120 units per mg of protein) was made tryptophan-free by passage through Sephadex G-25. Assays were performed at 37” in a total volume of 3.0 ml in 0.05 M sodium phosphate, pH 7.0, with 25 pg of enzyme, 0.5 PM hematin, 0.8 PM sodium ascorbate, and the stated concentrations of L-tryptophan (O---O) and 10 PM a-methyl- DL-tryptophan (0- - -0). The additions were made in the order given. The results are plotted as described by Lineweaver and Burk (34) in which the concentration of L-tryptophan (6’) is expressed as millimoles per liter, and the velocity (v) is expressed in absorbance units as measured with a Gilford recording attachment to a Beckman spectrophotometer. A unit corresponds to an optical density increment of 0.004/3 min at 321 ml*.

0.3

0.2

t

0.1

I I I I I

Added ~-Methyl- Tryptophan

I I I I I

0 2 4 6 8 IO 12

FIG. 10. Stimulation of tryptophan pyrrolase activity by 01- methyl-DL-tryptophan at low concentrations of L-tryptophan. The preparation of tryptophan pyrrolase in this experiment had been frozen and thawed in the absence of L-tryptophan and had a specific activity of 104 units per mg of protein. The enzyme was assayed at 37” in a total volume of 3.0 ml, with 25 pg of enzyme, 0.5 PM hematin, 0.8 PM sodium ascorbate, and the stated concentrations of L-tryptophan (O--O). The additions were made in the order given. a-Methyl-DL-tryptophan was added immediately before the addition of L-tryptophan at a concentration 2 times that of the L-tryptophan A- - -A. No lag periods were observed with this enzyme at any concentrations of L-tryptophan. The results are plotted as described by Lineweaver and Burk (34) in which the concentration of L-tryptophan (S) is expressed as millimoles per liter and the velocity (v) is expressed in absorbance units as defined in the legend to Fig. 9. At the lowest concentration of L-tryptophan (9 X low5 M), at most 22% of the L-tryptophan had been consumed during the period of assay. No increment in optical density at 321 rnH was observed with enzyme plus e-methyl-m- tryptophan without I,-tryptophan, or with L-tryptophan plus a-methyl-DL-tryptophan without enzyme under any conditions of assay.

3. Effects of cr-methyltryptophan on kinetics of tryptophan pyrrolase: The unique property of cr-methyltryptophan as an excellent stabilizer of tryptophan pyrrolase both in viva and in vitro is somewhat surprising in view of its inability to act as a substrate (11, 33) and its relatively poor ability to act as a competitive inhibitor (33). The competitive nature of this inhibition is shown in Fig. 9. The K, of ol-methyl-DL-tryptophan is 1 X

10-2M. In addition to the competitive inhibition of tryptophan pyrro-

lase, oc-methyltryptophan has a stimulatory effect on this enzyme under special conditions. During the course of experiments on its kinetic behavior, typical Michaelis-Menten kinetics for enzyme activity versus substrate concentration was not observed with certain preparations below a substrate concentration of 2 x 1O-4 M L-tryptophan, as shown in Fig. 10. This observation was found in enzyme preparations that had been frozen and


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Xtability of Tryptophan Pyrrolase Vol. 240, No. 12

thawed several times in the absence of L-tryptophan. Further- more, such enzyme preparations were far more unstable to heat and urea than enzyme preparations frozen in the presence of n-tryptophan or stored at 4“ with or without L-tryptophan. In these preparations the addit,ion of a-methyl-m-tryptophan at concentrations 2 times those of the added n-tryptophan stimu- lated enzyme activity in such a fashion as to normalize the kinetics, as seen in Fig. 10. The effect of a-methyltryptophan was evident only when the concentration of L-tryptophan was below 2 X 1O-4 M and Ras not observed with added n-tryptophan or 5-methyl-nn-tryptophan. The activating effect was most marked when the enzyme was incubated at 25” with 4 to 8 x 10v4 M oc-methyl-m-tryptophan before dilution into the assay medium. This activation was complete within 2 min. The added a-methyltryptophan was not simply preventing heat inactivation of the enzyme during the assay; thus the subsequent addition of n-tryptophan to saturating levels resulted in equal activities in each of two parallel assays in which a stimulatory effect of a-methyltryptophan was observed at low concentrations of L-tryptophan. Likewise the act’ivating effect of cu-methyltryptophan was not a result of altering the Km for hematin. Feigelson and Greengard showed that t,he K, of L-tryptophan is affected by the concentration of hematin in the assay medium, and vice versa (35). In t,he experiments described in Fig. 10 an enzyme preparation containing 50% apoenzyme was used. In all cases maximal amounts of hematin were added to the assay medium. Furthermore the extent of activation was equal in both the absence and presence of added hematin in the assay medium.

DISCUSSION

A number of observations that have been made in this paper indicate the apparent complexity of the degradation in viva of proteins. Thus in spite of the instability of tryptophan pyrrolase in viva, the enzyme protein is entirely stable in crude homogenates for up to 8 hours at 37” at pH 7 to 7.4 as determined by the double criteria of immunological reactivity and enzymatic activity. The simplest system capable of inactivating tryptophan pyrrolase, as well as tyrosine-glutamic transaminase, involves the use of liver slices. Studies on the degradat,ion of liver protein as measured by release of acid-soluble fragments showed good agreement with studies on the loss of activity of specific enzymes. In both cases degradation did not occur in homogenized tissues at neutral pH. On the other hand degradation did occur in liver slices and was inhibited by anaerobic conditions. These findings, then, are consistent with and confirmatory of the results of Simpson (14) and Steinberg and Vaughan (15) in showing the requirement for metabolically active liver tissue for protein degradation.

The similarity of the effect of anaerobiosis in inhibiting the degradation of total liver protein and the inactivation of an enzyme activity do not necessarily indicate that inactivation is associated with the complete breakdown of the enzyme protein to amino acids. At the present time it can be stated that the process whereby tryptophan pyrrolase is changed to a form that is neither enzymatically active nor reactive with a specific antibody requires a metabolically intact tissue.

Several explanations for the apparent energy requirement for protein degradation might include (a) a requirement for a necessary cofactor, (5) a requirement for removal of degradation products that inhibit further degradation, and (c) a requirement for

maintaining the integrity of a specific particle or organelle involved in protein degradation. A requirement for CoA and ATP was reported by Penn for the degradation of albumin by a rat liver particulate system (36). An inhibition of degradation by small molecular weight products was proposed by Mandelstam for the effects of chloramphenicol and dinitrophenol on degradation in Escherichia coli (37). It is of note that the tissue ca- thepsins heretofore identified reside in lysosomal particles (38, 39). Although it has been considered that lysosomal enzymes are active primarily when released from the lysosomal particle (38), some evidence suggests that active degradation may occur within such particles (39). I f the latter concepts were correct, an energy requirement for protein degradation could be explained on the basis of an energy requirement for the transport of proteins destined for degradation int,o the lysosomal particle; or possibly energy may be necessary in some other manner for the maintenance of the metabolic and structural integrity of such particles.

The activity of n-tryptophan to stabilize purified preparations of tryptophan pyrrolase in various systems in vitro agreed well with its stabilizing and inducing ability in viva. Studies with tryptophan analogues indicated the unique ability of ol-methyltryptophan to stabilize tryptophan pyrrolase in all systems studied in vitro and in viva. The small effect observed in viva with n-tryptophan and N-acetylt’ryptophan may result from con- version to n-tryptophan since these analogues can substitute for the growth requirements for n-tryptophan in rats (40, 41). On the other hand, a direct effect of u-tryptophan in viva cannot be ruled out in view of the apparent lack of racemization of n-tryptophan reported by Guroff and Udenfriend (42).

The studies with purified tryptophan pyrrolase showed a number of differences in properties of the inactivation depending on the agent employed. Of particular note were the differences in specificity of tryptophan analogues in protection against heat as opposed to urea, ethanol, or proteolysis. Proteolytic inactivation of tryptophan pyrrolase appeared to bear the closest correlation to the loss of enzyme in viva because of the loss of immunological reactivity and t,he tryptophan analogues that stabilized. On the other hand, the use of proteolysis does not constitute an appropriate model for the study of tyrosine- glutamic transaminase degradation in viva since this enzyme was not inactivated in vitro under conditions effective with tryptophan pyrrolase. These various results indicate the dangers in applying results with a single form of inactivation of an enzyme in vitro to the phenomenon of stability in viva.

The physical changes in tryptophan pyrrolase imparted by L-

tryptophan that increase enzyme stability are not known. Inas- much as the sedimentation characteristics of the enzyme were not affected by the presence of stabilizers, it is concluded that the stabilizing effect is not associated with a gross change in enzyme structure. Although stabilization of L-tryptophan against. inactivation by heat, urea, and ethanol may be associated only with an increase in the strength of the intramolecular bonding forces of the enzyme, it is difficult to conceive of protection against proteolytic cleavage being accomplished without altering the conformational state of the enzyme in such a manner that a proteolytic enzyme no longer has access to critical peptide bonds. It is therefore significant that the requirements for maximal stabilization of tryptophan pyrrolase against proteolysis included the presence of the enzyme cofactor, hematin. Thus, complex interactions involved in the combination of substrate and co-


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factor would appear to be important in imparting stability against proteolysis, just as they have been shown by Feigelson and Greengard to affect the kinetic properties of the enzyme (35). That conformational changes in prot,ein structure affect the susceptibility to proteolysis has recently been demonstrated by studies on the resistance of native hemoglobin to carboxypepti- dase as a function of conformational changes imparted by oxy- genation (43). Among other examples is the protective effect of glucose on trypsin inactivat#ion of hexokinase (44).

A consideration of the mechanism of the stabilizing effect of L- tryptophan and a-methyltryptophan would appear to involve a discrepancy between the K, of L-tryptophan for the catalytic site (3 to 5 X 1O-4 M) and the concentration resulting in half- maximal stabilization against heat inactivation (5 X lop5 M).

A similar discrepancy is observed between the Ki for Lu-methyltryptophan (1 X lop2 M) and that concentration resulting in half-maximal stabilization against urea inactivation (4 x 1O-4 M). It is suggested that these observations may be explained by the presence of two sites for t.he binding of tryptophan to tryptophan pyrrolase: one being the catalytic site to which CC- methyltryptophan binds poorly, and a second site with an affinity for L-tryptophan greater than the catalyt,ic site and to which o(- methyltryptophan can attach. This proposal is based on the kinet.ic studies of Fig. 10. Thus at concentrations of L-tryptophan in the range that impart stability to tryptophan pyrrolase (Fig. 7), there was an aberration from normal Michaelis-Menten kinetics. This aberration was corrected by the concomitant presence of small amounts of cY-methyltryptophan. The apparent stimulatory effect occurred only at concentrations of L-tryptophan where the abnormal kinet)ics is observed. Further- more, this effect of a-methyltryptophan was observed only in preparations of tryptophan pyrrolase rendered particularly unstable by freezing and t,hawing in the absence of L-tryptophan. Thus it, is suggested that such treatment has altered the conformational structure of the enzyme such as to increase its instability and at the same time decrease affinity for L-tryptophan at the catalytic site. The addition of a-methyltryptophan, then, could be doing one of two t’hings: (a) binding at the catalytic site such that the enzyme conformation is changed, increasing the affinity for L-tryptophan which can now more readily displace the a-methyhryptophan, or (b) attachment at. a second site to increase affinity of the catalytic site for L-tryptophan. Although a definitive conclusion as to the presence of one or two sites on the enzyme must await more extensive studies, the lower concentration of L-tryptophan required for stability as opposed to that required for catalysis would lead us to conclude that the two-site mechanism is the more likely explanation.

An alternative explanation for the effect of L-tryptophan in stabilizing tryptophan pyrrolase in &JO, involving the inhibition of a specific degradative or inactivating enzyme, has not been rigorously excluded as the nature of this inactivating system is as yet unknown. It is clear, however, that L-tryptophan does not affect the degradation of total liver protein, and the effect of L- tryptophan in preventing trypsin proteolysis of tryptophan pyrrolase cannot be ascribed to direct affect on trypsin activity. Furthermore, if one proposed that L-tryptophan affects a specific proteolytic enzyme involved in tryptophan pyrrolase degradation, a further problem would arise. Because evidence indicates that all proteins are continuously degraded (2, 5), it would therefore require an infinitely large group of proteolytic enzymes, i.e. one proteolytic enzyme to degrade another to degrade an-

other, etc. It would seem more reasonable to suggest that a limited number of proteolyt’ic enzymes exist, and the inherent or imparted stability of an enzyme is determined by its structural properties.

The significance of protein-degrading systems in control of physiological and developmental processes has been largely over- looked. Studies by Wright wit’h differentiation in slime molds (45), as well as those of Krimsky and Racker (46) and of Pogell and Gilvery (47), attest to the potent.ial, widespread importance of degradative systems in physiological control mechanisms.

SUMMARY

Degradation of tryptophan pyrrolase as measured by both enzyme activity and immunological react,ivity occurs only in metabolically and structurally intact liver tissue. Degradation does not occur in crude liver homogenates, but it can be demonstrated in liver slices. The degradation of tryptophan pyrrolase and of total liver protein have certain properties in common: (a) they do not occur in crude homogenates at pH 7.4; (b) they do occur in liver slices at pH 7.4; and (c) both are inhibited by incubation under anaerobic conditions. The degradation of tyrosine-glutamic transaminase, another enzyme of rat liver with a rapid turnover, has characteristics similar to those of tryptophan pyrrolase.

Tryptophan pyrrolase is stabilized in viva by n-tryptophan. Among a series of tryptophan analogues only Lu-methyltryptophan imparts stability in viva similar to L-tryptophan. There is a good correlation between the abilities of tryptophan analogues to stabilize tryptophan pyrrolase in viva and to induce the enzyme in adrenalectomized rats.

Purified preparations of tryptophan pyrrolase are rapidly inactivated by heat, urea, ethanol, and proteolysis by trypsin, chymotrypsin, and a bacterial proteinase. Although a number of tryptophan analogues stabilize against heat inactivation, only L-tryptophan and cu-methyltryptophan are effective against urea, ethanol, and prot,eolysis. Maximal stabilization against proteolysis, as opposed to urea and heat, occurrs only with the holoenzyme.

Stabilization of the purified enzyme occurs without altering its gross structure as determined by sedimentation and immunological criteria. Based on studies on t’he concentration dependence of the stabilizing effect of n-tryptophan and the observation of a stimulatory effect of a-met.hyltryptophan on certain preparations of tryptophan pyrrolase at very low concentrations of L-tryptophan, it is suggested that two sites exist on tryptophan pyrrolase for L-tryptophan: the catalytic site, and a second site which imparts stability to the enzyme.

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Robert T. Schimke, E. W. Sweeney and C. M. Berlin of Rat Liver Tryptophan Pyrrolasein Vitro and in VivoStudies of the Stability

1965, 240:4609-4620.J. Biol. Chem.

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studies of the stability in vito and in vitro of rat liver ... · the fact that its substrate...

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