THEJOURNAL OP B~LO~~CALCHEHI~TRY Vol. 237, No. 11, November1062

Printed in U.S. A.

The Kinetics of Catalase Svnthesis and Destruction in Viva* J VISCEST E. PHICE,~ WILLIAM R. STERLING,~ VINCENT A. TARAXTOLA,~ ROBERT W. HARTLEY, JR.,

AKD MILOSLAV RECHCIGL, JR.

From the Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda l/t, Maryland

(Received for publication, March 23, 1962)

Until recently, the principal mrthods for measuring the rate of protein turnover in uiuo have required the use of radioisotopes. Many of these studies have involved measurcmcnt of the turn- over of the total protein of an organ, which is the mean value for the turnover of the multitude of different proteins present within the organ (3-X). I\ number of stud&, notably thosr of Velick et al. (9-11) on proteins of muscle, of -ikeson et al. (12) on myoglobin, and of Schapira et ul. (13) on muscle aldolase, and numerous studies on collagen (14-19) and on the plasma proteins (4, 2&24) have been made to detrrminr the rates of turnover of individual proteins. The use of radioisotopes re- quires laborious fractionation and purification of the individual proteins to constant spwific radioactivity.

Recently Peigelson, Dashman, and hlargolis (25) have dctrr- mined the half-life of the enzyme, tryptophan pyrrolase, by studying the rate of disappearance of the induced enzyme. Such techniques are valuable but cannot readily be upplicd to condi- tions under which a given enzyme is present at a strady state level.

We have been interested in studying the rates of synthrsis and destruction of catalasc, which is markedly depressed in the livers of tumor-bearing animals. Although previous studies (26) suggested that a highly active form of ratalase was present in the liver of normal rats but absent in rats bearing the Sovikoff hepatoma, this was later shown to be an artifact of thr purifica- tion procedure (27), and when catalase of livers from tumor- bearing animals was isolated directly from the particulate frac- tions in which it is located, a highly active enzyme was obtained for which the specific activity was nearly the samr as that ob- tained from normal animals.’ This meant that the lowering of liver cat&se in the tumor-bearing host must result from a change in the rate of synthesis or dcsbrurtion of the enzyme, rathw than from an alteration in structure resulting in a lowered specific activity of the enzyme. Initial attempts to measure thr rate of catalasc synthesis in normal and tumor-bearing animals by use of radio-iron met with considrrable difficulty because of marked alterations of the iron pools in the tumor-bearing animals rrsult-

*A portion of this work was presented at the 51st Annual Meeting of the American Society of Biological Chemists, Chicago, Illinois, April 12,1966 (1). A preliminary note has been published (2).

t Present address, Division of General Medical Sciences, Na- tional Institutes of Health, Bethesda 14. Maryland.

$ Present address, Department of Pharmacology, George Wash- in&on Universitv. School of Medicine. Washinaton 6. D. C.

j Deceased, Sebtember 1, 1961. ’ 1 Unpublished data.

- ’

ing from a markrd anemia produced by hemorrhage into the tumor (28).

In 1955, Heim, Appleman, and l’yfrom (29) showed that 3-amino-1,2,4-triazole produced a rapid fall in the liver catalase activity when injected intrapcritoneally in a dosage of 1 g per kg of body weight. This precipitous fall was followed by a pro- grcssive return of the catalase activity to normal levels in 4 or 5 days. If the return of liver catalaw activity was the result of new synthesis of the enzyme, rather than of a reversal of the inhibitory effect produced by 3-amino-l ,2,4-triazole, it eccmed possible that the rate of catalase return might be used to deter- mine the rates of catalase synthesis and destruction in tivo.

First, of course, it had to be shown that 3-amino-1,2,4-tria- zole itself did not significantly alter the rates involved. The present report concerns the dcvelopmmt of the method for determining the kinetics of catalase synthesis and destruction in Go, proofs as to its validity, and a check of the results ob- tained by use of an alternative technique involving allylisopro- pglacetamide (30, 31), a derivative of allylisopropylacetyl- carhamidc (Sedormid), which was shown (32, 33) to product an acute porphyrinuria, a marked rlevation in liver porphyrins, and a fall in liver catalase resulting from a block in catalase synthesis.

METHODS

.4 ssay Procedure

Catalasr was assayed spectrophotomrtrically on a continuously recording spectrophotometer with a log absorbance attachment? by modification (27) of the method of Beers and Sizer (34). To carry out the assays during the catalase purification proce- dure, 2.9 ml of 0.02 M phosphate buffer at pH 7.0 and 22” were pipettcd into two clean cuvettes. The enzyme was diluted with phosphate buffer at 0” to give a concentration in the range of 0.5 to 5 units per ml. ;iftcr addition of 75 ~1 of the diluted enzyme to both cuvettes, the pen of the spectrophotomrtcr was set at the base line, and 30 ~1 of 1.0 Y hydrogen peroxide were rapidly added to the experimental cuvette with the adder-miser described by Royer and Segal (35). Recording was started immediately, and the decrease in optical density at 230 rnp was traced directly onto semilogarithmic paper.a From the slope

* Cary model 11 MS recording spcctrophotometer with log absorbance attachment; Applied Physics Corporation, Pasadena, California.

3 Chart No. 1092, Applied Physics Corporation, Pasadena, California.

3468

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November 1962 Price, Sterling, Tarantola, Hartley, and Rechcigl

of the line obtained, k,,, the first order rate constant for a given determination, was calculated. One unit of catalase is sufficient enzyme to cleave 63.2% of a given concentration of H&z in I second and is equivalent to 5.85 pg of purified rat liver catalase.’ One unit of rat liver cat&se contains 0.99 pg of nitrogen and 0.60511 pg of Fe and is equivalent to 22.9 ppmoles of the enzyme based on a molecular weight of 256,000 (27).

For the assay of liver homogenates, the livers were ground for 2 minutes in a Waring Mendor with 49 volumes of cold distilled HzO, and a 50-~1 aliquot was used in the assay procedure as described before. Rat liver contains I60 to 220 units of catalase per g, varying with the species, ses, and season of the year.

Purif~atlon of Catalase from Rat Liver

Step l--Liver, 45 g, was homogenized in a Waring Mendor with 383 ml (8.5 ml per g of liver) of a solution containing 235 ml of absolute ethanol a.nd 47 ml of 1.0 M acetate buffer at pH 4.0 per liter, and precooled to 0”. .ifter 1 minute, 22.5 ml of chlo- roform (0.5 ml per g of liver) were added to denature the hemo- globin, and homogenization was continued for a second minute, at the end of which time the temperature had risen to approxi- mately 10” and the pH was 4.7. The homogenate was immedi- ately centrifuged at 29,000 x g in the Spinco model L ultracen- trifuges for 30 minutes, and the pellet, P1, was resuspended, assayed, and discarded.

Step %--To the supernatant, Si, with a volume of 400 ml, were added 8 ml of 0.5 11 YaSO4, which produced a marked turbidity. After 30 minutes at O”, the precipitate, P*, was collected in a Servall model SS-1 centrifuge6 and the inactive supernatant, Sp, was assayed and discarded. Frequently, it is convenient to store the active pellets in a freezer at this point and to perform the subsequent steps simultaneously on batches collected over a number of days. If kept in a Dry Ice box at -57”, the pellets can be stored for a year without significant loss of activity.

Step S-The active pellet, Pp, was suspended in 18 ml of 0.1 nr phosphate buffer at pH 7.8, allowed to extract for 4 hours at O”, and then centrifuged.6 The pellet, Pa, was suspended, assayed, and discarded.

Step C-The supernatant, Ss, was assayed and dialyzed at 0” overnight against 109 volumes of a solution containing 20% ethanol, 0.1 Y NaCl, and 0.1 XI acetate buffer at pH 4.7. The sac contents were then removed and centrifuged in the Servall centrifuge.6 The active pellet, P4, was saved and fractionated further as in Step 6.

Step S-The supernatant, S,, containing ferritin and some globulins, was assayed for catalase content and centrifuged for 4 hours at 30,000 X g in the Spinco model L ultracentrifuge. The resulting pellet, containing ferritin, was suspended in 0.1 ,\I phosphate buffer at pH 7.8 and centrifuged in the Servall appa- ratus, and this supernatant was used as the fcrritin fraction for radioisotope studies.

4 As determined from an arbitrary catalase standard which was selected from a large number of enzyme preparations as having the highest specific activity and the best, optical and sedimenta- tion characteristics.

5 Although the Spinco ultracentrifuge is convenient in that a tightly packed pellet is rapidly formed, any refrigerated cent.rifuge can be used for this purpose.

6 Ivan Sorvall, Inc., Norwalk, Connecticut. Centrifuged at a rheostat. setting of 90 volts for 10 minutes, during which the cen- trifuge accelerated approximately to 7000 X g.

Step &-The pellet, P4, containing catalase and some ferritin, was suspended in 1.35 ml of 0.02 M acetate buffer at pH 4.7 and dialyzed against 100 volumes of this solution for 4 hours at 0”. The sac contents were removed and centrifuged in the Servall centrifuge.6 The pellet, Ps, was assayed and discarded.

Step r--The active supernatant, Sb, was dialyzed against 100 volumes of solution containing 20% ethanol, 0.1 M NaCI, and 0.1 M acetate buffer at pH 5.7 at 0” overnight, removed from the sac, and centrifuged in the Servall centrifuge.6 The supernatant, Se, containing ferritin and traces of catalase, was assayed and discarded.

Siep g--The active pellet, P6, containing catalase and small amounts of ferritin, was extracted with 1.8 ml of a solution containing 10% ethanol, 0.1 11 SaCl, and 0.1 M acetate buffer, pH 5.7, at 0” for 30 minutes with frequent stirring and then centrifuged in the Servall centrifuge.6 The pellet, Pi, was then extracted with 1.8 ml of a solution containing the acetate buffer and sodium chloride but no ethanol and centrifuged.6 The supernatants, Sr and Se, containing ferritin and some cntalase were assayed and discarded.

Step g-The active pellet, Ps, was suspended in 1.35 ml of 0.02 Y acetate buffer, pH 4.7, dialyzed against 100 volumes of this solution overnight at O”, removed from the sac, and cen- trifuged in the Servall centrifuge.6 The pellet, Pg, was discarded. The supernatant, Ss, containing nearly pure and highly active cataluse,? was assayed. The overall yield was approximately 50% of the catalase in the original homogenate.

Spectropholumetry

The ultraviolet and visible spectra of SP were determined in the Cary spectrophotometer. The spectrum of rat liver catalase, shown in Fig. 1, has maxima at 276 and 407 rnl.r and minima at 252 and 312 rnp. The maxima, having molar extinction co- efficients of 397,000 and 430,000 at 276 and 407 mp, respectively, are useful for calculating the specific enzyme activities of the purified catalase preparations. This is particularly true of the Soret band absorption at 407 ml.r, since this peak has the least likelihood of being contaminated by other proteins with physical properties similar to catalase.

The ratios of the masima and minima are of great value for determining the purity of ratalase. In our best catalase prepa- rations, the ratio of absorption at 407 rnp to that at 276 rnb (&7:&6) was 1.085, DsIz:D~~ WBS 0.19, D252:D~i6 was 0.65, and D312:D4~7 was 0.175 (27). Whereas catalase has a minimal value at 312 rnp, ferritin absorbs strongly at this wave length. A contamination of I.07c ferritin will lower the &:D2r6 ratio to 1.02 and raise the Ds12: 401 ratio to 0.23. Protein contami- nants other than ferritin produce a marked lowering of the DN7:Dne ratio with almost no effect on the D812:D4~7 ratio. By suitable calculations, using the data on catalase presented above and those on ferritin to be presented later, one could readily calculate from the spectrum of a given catalase preparation how

7 Purity of 97 to 98c/0, calculated from optical data as described in the following section. The optical data indicate that prac- tically all of the impurity is ferritin. The catalase preparations from normal liver had a L 4*7 of 4.52 X 10’ based on their specific activity per unit of absorbancy at 407 rnM (27). Since the normal catalase preparations had an average impurity of 1.73% ferritin, the actual specific activity, k,, would be 4.44 X lo’, which is equivalent to a Kat. f. of 73,300.

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Kinetics of G’ataluae SynM and De&u&m in Vivo Vol. 237, No. 11

t

\ 0.r ‘\

‘\ ‘\ \

0.6 \ \

,I 1 I I I I I I I IJ 240 280 320 360 400 440

WAVELENGTH IN mp

Fro. 1. Spectra of catalase and ferritin prepared from rat liver. Catalaae (-) concentration, 360 rg per ml; ferritin (- - -) con- taining 10.8 pg of Fe per ml; in 0.02 M phosphate buffer at pH 7.0.

much absorption at a given wave length is due to catalase, to ferritin, and to other proteins.

Ferritin merita further consideration because of its hetero- geneity, which makes it difficult to separate completely from the purified catalase preparations. In most fractionation proce- dures, and in calcium phosphate gel columns as well (26), apoferritin and ferritin molecules with relatively low amounts of iron are found on one side of the cat&se fraction and ferritin molecules with higher amounts of iron are found on the other side. Therefore, in almost any procedure, some ferritin, with intermediate concentrations of iron, is found contaminating the catalase fraction.

Ferritin has a strong ultraviolet absorption, which increases progressively as the wave length decreases, as shown in Fig. 1. This absorption is largely due to the ferric ion, and the small absorption due to the protein moiety is shown by the slight elevation at 280 mp. The concentration of iron in a given ferritin preparation can readily be determined, since at 312 mp an optical absorbancy (or optical density) of 1.0 in a l-cm light path represents 22.5 pg of iron.8 In ferritin, DS1: Dsll is approxi- mately 1.51, Dna:Dalz is 1.36, and D4~:D~I~ is 0.31. Ferritin absorbs approximately 5 times ‘as much light per mg at 276 rnp and 20 time as much per mg at 312 mp as does catalase.9 It is for this reason that optical methods are so sensitive to the pres- ence of ferritin in catalase preparations.

EXPERIMENT8 ANTI RESULTS

Rate of C&use Inactiuation by Aninoltiole-Heim, Apple- man, and Pyfrom (29) showed that the liver catalase activity

* When the purity of ferritin fractions is examined, 330 ma is a preferable wave length since it is further from the absorption band of proteins at 276 to 280 w. At 330 Q, an optical absorb- ancy of 1.0 represents 26.7 pg of iron.

9 With an impurity of 2% ferritin in a catalase preparation, approximately 10% of the absorbancy at 276 ~J.I, 4oo/o at 312 mp, and only 2.3% at 407 w will be due to the ferritin component. These data are only approximate because of the vnrying iron content of ferritin.

LIVER CATALASE ACTIVITY FOLLOWING

INJECTION OF 3-AMINO-1,2,4-TRIAZOLE wq , I I I I I I , I I]

l9m/K9BW-XC

1

,.t,l lo 20 30 40 SC 60 70609090

MINUTES

FIG. 2. Kinetics of catalase inactivation in rat liver after in- jection of 3-amino-1,2,4-triazole, 1 g per kg of body weight, intraperitoneally. 0, actual data, showing descent to a plateau of approximately 22 units per g, which represents erythrocyte cataltise that is unaffected by aminotriazole (36). A, catalase activity per g of liver after correction for the erythrocyte cata- lase, showing approximate first order kinetics.

reached a minimal point in the liver 30 minutes after AT*0 wag injected in a dosage of 1 g per kg of body weight intraperitoneally into rata. These experiments were repeated in order to obtain more accurate data on the over all kinetics of the process. When the data were plotted in a semilogarithmic plot as shown in Fig. 2, the curve obtained approximated first order kinetics during the early phase but then veered off to a minimal value of 2% of the total cat&se present. This represents catalaae of erythro- cytcs remaining in the liver. Feinstein and Dainko (36) have shown that the catalase of red cells is unaffected, since AT does not penetrate the membrane of the cells. When the above data were corrected for this artifact by subtracting the minimal value from all of the points, a first order curve was obtained as repre- sented by the triangles and solid line in Fig. 2. From the slope of thiscurve, it was calculated that the time required for catalase activity to fall to one-half of a given value, the half-time, is approximately 8 minutes.

Return of Cafahe Activity after Aminotriazole-After reaching the minimal point approximately 1 hour after the injection of AT, catabe activity remains very low for 2 hours, after which it slowly starts to return, as shown in Fig. 3, reaching a maximal rate at 24 to 36 hours and then mounting to a maximal value approximately 5 days after the initial injection.

Did this return of catalase activity represent new synthesis of the enzyme, or was it simply a progressive reversal of the inhibitory process that produced the initial disappearance of catalase activity? In order to approach this problem, six groups of five male Buffalo rats weighing an average of 311 g received injections of an aqueous solution of AT, 50 mg per ml, in a dosage of 2 ml per 100 g of body weight. Six more groups of rats of the same average body weight were given injections of a similar

I0 The abbreviations used are: AT, 3-amino-1,2,4-triazole; AIA, allylisopropylacetamide.

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November 1962 Price, Sterling, Tarantda, Hartley, and Rechcigl 3471

FALL AND RETURN OF LIVER CATALASE ACTIVITY FOLLOWING AMINOTRIAZOLE

--- -__ --- ____ --- --- -- -- E 200 > i 180 i

8 4 160 c

9 ” 140

5 c. 120

.

. .

/

. . .

HOURS AFTER AMINOTRIAZOLE

FIG. 3. Disappearance and return of liver catalase activity after injection of 3-amino-1,2,4-triazole. 0, experimental data; -, theoretical curve to fit Equation 2 in text on the assumption of a rate of eynthesis of 4.8 units of catalaae per hour and a rate of destruction of 2.259& of the catalaee molecules present per hour; -, period of inactivation and lag period before experi- mental data approaches theoretical curve; - - -, plateau value of animals not given injection8 of aminotriaeole.

volume of 0.6 M NaCl, which is approximately equimolar to the AT solution.

Two hours after the injection of aminotriazole, at a time when catalase activity was at a minimal value, all of the animals were given intraperitoneal injections of 5 /.IC per 100 g of body weight of Fe”9 11 in the form of ferric ammonium citrate.

At daily intervals thereafter, for 6 days, one group each of the rats that had received injections of AT and NaCl solution were subjected to the purification procedure for cat&se as outlined before under “Methods.” Steps 1 and 2 were performed daily, and the active pellets, Pr, were then stored in a deep freeze until the fractions of all 6 days had been obtained. The following week, all I2 of the fractions were simultaneously subjected to Steps 3 through 9 of the procedure, so that as far as possible all fractions were subjected to identical conditions during the purification procedure.

The final fraction, So, was nearly pure by spectrophotometric criteria (27), but traces of ferritin were still present which were difficult to remove because of the marked heterogeneity of the ferritin molecules. These traces of ferritin could cause significant errors in the radioactivity measurements because they contain relatively high concentrations of iron (up to 30%) as compared with catalase (0.09%).

In order to remove the final traces of ferritin iron, l-ml aliquotz of the Sp catalase fractions were placed in small dialysis sacs and inserted in a 2-liter glass-stoppered graduate that was filled nearly to the top with 0.1 r&acetate buffer at pH 4.7, 5”. Sufh- cient powdered sodium hydrosulfite, Na&Oc, was added to bring the solution to 0.1 M, and the graduate was rapidly stoppered to exclude excess oxygen. The graduate was then placed in a rotating dialyzer in a 5“ cold room overnight. The following morning, the dialyzed sacs were placed in 4ml glass vials, and

11 Oak Ridge National Laboratory, Oak Ridge, Tennessee. Prepared from FeWI, with a specific activity of 2254 mc per g.

LIVER CATALASE ACTIVITY AND RACdOACTlVITy IN NORMAL RATS FOLLOWING IP INJECTION OF SALlNE AND

6WO( 3-AMINO-1.2.4-TRIAZOLE

, 125

0’ ’ ’ 0 ‘0 0 2 3 4 5 6

DAYS

Fro. 4. Liver catalase activity and radioactivity in rats after injection of NaCl solution and AT, followed 2 hours later by 6 pc of Fe60 (in the form of ferric ammonium citrate).

the radioactivity was measured in a well-type scintillation counter.12 The sacs were then removed from the vials, dialyzed again in a fresh solution of sodium hydrosulfite, and recounted. This procedure was repeated a third time. No significant differ- ence was observed in the radioactivity of the saos between the second and third counts. The sacs were then dialyzed three times against 1206 ml of 0.02 M acetate buffer at pH 4.7 to remove all traces of hydrosulfite. They were then opened into 4ml vials and carefully rinsed with 1 ml of the acetate buffer to remove the catalase as quantitatively as possible. The vials were then counted a final time under standard conditions of geometry. These values provided a reliable measure of the radioactivity of the catalase contained in 1 ml of Sp. These values were then related to the catalase activity and the Soret band absorption at 407 rnp of the SO fraction, which thus provided a measure of the specific radioactivity of the catalase.

By multiplying the ratio of radioactivity to catalase activity in So by the total catalase activity in the original homogenate, a value could be obtained which represents the total radioiron incorporated into the catalase.18 These values were then divided by the specific activity of the iron in the ferritin pool to minimize possible differences in the iron pools of the various animals. These data are presented in Pig. 4 along with the total units of catalase activity that were present on the respective days. It will be seen that radioiron was incorporated into the catalase at a rate that directly paralleled the reappearance of catalase activ- ity in the aminotriazole groups. These findings indicate that the reappearance of catalase activity is accompanied by a corre- sponding uptake of Fe69 into the cat&se, presumably during the synthesis of new catalase. The almost identical uptake of Fe60 into the catalase of animals given injections of AT and in the

I* Model DS-3, Nuclear Instrument and Chemical Corporation, Chicago, Illinois.

18 This assumes that the catalase of the purified E$ fraction was representative of all of the catalase in the homogenate. Subae- quent studies have shown a small fraction of approximately 10% of the total liver catalase in which synthesis appears not to be inhibit.ed by allylisopropylacetamide. These findings are being explored further and may introduce some error into the above assumption.

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3472 Kinetics of Catulase Synthesis and Destruction in Vivo

RELATIONSHIP BETWEEN SORET BAND ADSORPTION AND CATALASE ACTIVITY FOLLOWING INJECTION OF

3-AMINO-1.2.4-TRIAZOLE

I I 2 3 4 5 z

DAYS AFTER AMINOTRIAZOLE

FIG. 5. Relationship between Soret band absorption and cata- lase activity after injection of 3-amino-1,2,4-triazole based on the relationship that a solution containing 100 units, or 585 pg, of catalase per ml haa an absorbancy at 407 rnp of 0.965 cm-r.

NaCl solution controlsi provides evidence that AT itself has little, if any, effect on the radioiron incorporation into catalase.

Further evidence that the uptake of Fe60 represents new synthesis of catalase may be inferred from Fig. 5. In this figure, the actual amount of cat&se isolated on a given day, as repre- sented by its Soret band absorbancy at 467 mp, is compared with the absorbancy that one would predict from the catalase activity present on that day. It will be seen that the Soret band absorp- tion of the catalase falls during the first day to approsimately one-half the initial value, after which it slowly returns back up to the normal plateau. The differenre between the actual ab- sorbancy and the absorbancy expected from the catalase activity represents inactive catalase which is progressively disappearing from the livers of the animals treated with injections of AT. These data are consistent with the findings of hlargoliash, Novo- grodsky, and Schejter (37) that aminotriazole binds to the protein moiety of cat&se to form an irreversible complex. If this binding of AT to protein is truly irreversible, it would naturally follow that any reappearance of enzyme activity would have to result from the synthesis of new catalase.

Kinetics of Return of Cata2ase Activity-The rate at which the rising catalase activity returns to the normal plateau was first examined by making certain assumptions and determining empir- ically whether they would fit the data obtained. The validity of these assumptions was then checked by an alternate method, in which cat&se synthesis was blocked by use of allylisopropyl- acetamide.

The assumptions that were made are these: (a) that the cata- lase is being synthesized at a constant rate, ks, and (b) that a constant fraction, kn, of the active ratalase molecules present in the liver is being destroyed per unit of time.

From these assumptions, the following equations can be de- rived.

r4 It will be observed that NaCl solution itself produces some lowering of the catalase activity. This is usually a reduction of approximately 15%; the mechanism involved is not known at this time.

Vol. 237, No. 11

If C = 0 at t - 0, then

Cc = 5 (1 - 6-b’) (2)

At the plateau value, CN, the amount of catalase being syn- thesized per unit of time equals the amount being destroyed. Hence, if kD is known, ks can readily be calculated from the equation, k8 - kDCN.

In Fig. 3 are shown the catalase activities of 60 individual rats at various times after the injection of aminotriazole. The solid line represents the theoretical curve that was obtained from the above assumptions if 4.3 units or 28 /Ig of catalase per hour were being synthesized and 2.25 y0 of the catalase molecules pres- ent were being destroyed, with an initial lag period of 13 hours for escretion of the aminotriazole, since catalase formed during this initial period will be inactivated by the drug.

If the reappearance of catalase activity is plotted semilogarith- mically, as the difference between CN, the normal catalase level, ad CAT, the catalase level in the aminotriazole animals, as shown in Fig. 6, a reasonably straight line is obtained with a slope is equal to kD, the rate of catalase destruction,

Kinetics of Cat&se Disappearance Based on Allylisopropyl- acetamide-.Uthough the data obtained fit the equations baaed on the assumptions presented, it is possible that another group of assumptions and equations could be developed that would also fit the data. A separate line of evidence was needed, there- fore, to check the validity of the previous assumptions. This was obtained by using allylisopropylacetamidc to block catalase synthesis and by then following the disappearance of catalase from the liver over the next few days.

5

2 0

a t.

Log Period 13 Hours w

-, K. = .0226

i

HOURS AFTER AMINOTRIAZOLE

FIG. 6. Kinetics of liver catalase destruction in tico by 3-amino- 1,2,4-triazole. Semilogarithmic plot of data in Fig. 3 presented a8 CN - CAT yersua time, in which CN is the normal level of liver catalaae and CAT is the catalase level at various times after ad- ministration of aminotriazole. Each point represents the average of two animals. kD is the first order constant for catalase de- struction, aa determined by the slope of the solid line (----) drawn to fit the experimental points. ka, the rate of catalase synthesis, equals k&N. The solid line extrapolates back to the dashed line (- - -), which represents CN, at 13 hours. This is equivalent to the lag period or time required for the excretion of aminotriazole, catalase formed during this period being inacti- vated.

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November 1962 Price, Sterling, Tarantola, Hartley, and Rechcigl 3473

The experiment was performed in such a way as to determine simultaneously the rate constant for catalase disappearance ob- tained with MA and the rate constant obtained with AT. For t.he experiment, four groups of male Sprague-Dawley rats, 6 weeks old, weighing approximately 140 g were used. Throughout these studies, animals were fed a pelleted diet, the composition of which was described before (38). One group n-as given a single injection of AT, 1 g per kg intraperitoneally, the second group was given MA, 200 mg per kg of body weight twice daily intraperitoneally, the third group was given both drugs in the dosage described, and the fourth group was given no drugs and used as controls. The data obtained (Fig. 7) show that whereas the catalase in the AT group was rising, that in the AIA group was falling. The group given both drugs was of particular interest, since it would show whether or not catalase synthesis had been completely blocked by the AIA. Instead of staying at the minimal value expected from red cell catalase (2x.), catalase activity showed an early increase to a low plateau of about 10% of the normal value. This difference of approxi- mately 8% represents catalase that can be destroyed by amino- triazole, but in which synthesis is not blocked by AIA. If the rate at which the AT curve in Fig. 7 approaches the control values is compared with the rate at which the MA curve approaches the low plateau of the group given both drugs, in a semilogarithmic plot of the data as shown in Fig. 8, it will be seen that the slopes are nearly equal. This gives reasonably secure evidence that the initial assumptions are correct and that catalasc molrcules are being destroyed in a random fashion without regard to their age, so that in a given period of time, newly formed catalase molrcules have the same risk of destruction as older ones.

Comparison of Rates of Catalase Synthesis and Destruction in Liver and Kidney-The level of catalase activity in the liver is approsimately 3.5 times that found in the kidney. In order to compare the relative rates of catalase synthesis and destruction in the two organs, a number of male Sprague-Hawley rats nrigh-

LIVER CATALASE SYNTHESIS AND DESTRUCTION IN V/V0

t 2;;j

3-AMINO-1.2.4-TRIAZULE

1 I1 II 4 41 1 I I 2 3 4

DAYS AFTER AMINDTRIAZOLE

Fro. 7. J,iver catnlase activity in mt.s nfter injection of 3-amino-1,2,&triazole and allylisopropylacetamide.

KINETICS OF LIVER CATALASE DESTRUCTION IN V/V0 I I 1 I

IO’ I I I I I I 2 3 4

DAYS

FIG. 8. Kinetics of liver catalase destruction in viuo by 3- amino-l ,2,4-triazole (AT) and allylisopropylacetamide (AZA). Semilogarithmic plot of CN - CAT nnd of CAIA - CAT+AIA versus time, where CN is the normal level of liver cntalase, CAT is the cat.alase level at various t.imes after the administration of nmino- triazole (1 g per kg of body weight), CAI., is the catalase level during administration of allylisopropylacetamidc (200 mg per kg of body weight twice daily), and C r\T+AIA is the liver catalnse level of animals given both drugs.

SEMILOG PLOT C,-CAT VS.TlME I I I I

-I

Ktdney Kdz.0227 KS= I.18

24 48 72 96

HOURS AFTER AMINOTRIAZOLE

FIG. 9. Kinetics of wt.&se destruction in liver and kidney by 3-amino-1,2,4-triuzole. Semilogarithmic plot of C,,. - c.4~ versus t.ime. l nnd n , data from two separate experiments.

ing 200 g were given injections of .\T, 1 g per kg of body weight, and an equal number wrre used as controls. At intervals for 4 days, five animals of each group were killed, and assays were performed on the liver and kidneys. The AT values were then subtracted from the normal value for that day, and the data were plotted semilogarithmically as shown in Fig. 9. It will be seen that the slope of the two curl-es is nrarly equal and that the rate constants for catalase destruction were nearly identical in the two

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Kinetics of Catalme Synthesis and Destruction in Vioo Vol. 237, No. 11

organs. The higher level of catalase in the liver, therefore, results from a greater rate of catalase synthesis, which is approxi- mately 3.5 times that of the kidney.

DISCUSSION

The aforementioned method provides a means for determining the rate of catalase synthesis and destruction in t&o. The evi- dence for synthesis comes ffom two sources.

1. The incorporation of P’P into catalase at a rate parallel to the rate at which catalase activity reappears after the adminis- tration of 3-amino-1,2,4-triazole. Although it is conceivable that this incorporation of Febg may represent a turnover of Fe’9 in the aminotriazole-inactivated catalase, this is unlikely in view of the rather rapid disappearance of Soret band absorption after the administration of aminotriazole and the observations by Margoliash, Novogrodsky, and Schejter (37) of the irreversible binding of aminotriazole to the protein moiety of catalase.

2. The disappearance of cat&se activity in animals treated with allylisopropylacetamide, which is known to modify por- phyrin metabolism in the liver (30-32, 39) and has been shown to interfere with catalase synthesis (32, 33). In the present studies, it was shown that when AIA is administered after the injection of AT, catalase rises from a minimal value of 2% of normal to a level of 107U, thus showing that cat&se synthesis in the liver is almost, but not completely, blocked.

The evidence for catalase destruction is (a) that catalase dis- appears from the liver after administration of allylisopropyl- acetamide (32) and (b) that the rate of disappearance observed was that which would be required in a steady state to compensate for the rate of catalase synthesis as measured by its reappearance after aminotriazole.

Although the catalase activity disappears after allylisopropyl- acetamide, we do not know that it represents catalase destruction per se; it may instead represent the rate of passage of catalase from the liver (cells) into the circulation. If one considers that the aforementioned data represent a steady state condition, how- ever, then the rate of disappearance from the liver would be identical with the rate of its destruction; otherwise, a considerable level of catalase activity should be observed in the blood plasma, where negligible levels are found (40, 41). The use of the term, destruct.ion, therefore implies that this is the rate at which catalase disappears from the liver and that the actual site of destruction may be either in the liver or at some other site within the body. It is interesting to note, in this regard, that the rate of catalase destruction in the kidney is the same as that in the liver This suggests that the process of inactivation may be the same in both organs.

It was considered significant that, although aminotriazole inactivated all of the catnlase in the liver except for that present in red cells, allylisopropylacetamide did not completely inhibit the formation of all catalase within the liver, since after the administration of both AT and AIA, the catalase activity in the liver rose from 2 70 up to a level of 10 70 of the normal value. The rapid rate of this ascent, within 24 hours, suggests that it may have a more rapid rate of turnover than the bulk of the liver cat&se and could t.herefore constitute an important contribution to the total amount of catalase being synthesized and destroyed in a given unit of time. This .4IA-resistant catalase may be kJcated in 3. different fracti@n of the cell or it may be located in cells other than the hepatic crlls of the liver, which may be im- permeable to .41,4. In this connrction, it is interesting to men-

tion that the catalase activity of the Morris 5123 hepatoma (42) responds to aminotriazole but not to allylisopropylacetamide.*~ Further studies on the AIA-resistant catalase in both normal liver and a number of different hepatomas is underway.

The methods described for the measurement of the rate of cat&se synthesis and destruction in tico promise to be of con- siderable value for studying the rates and synthesis of this enzyme in tumors,rb in the organs of the tumor-bearing host, in starvation (43), in protein deficiency (44), and in hormonal regulation, aging, etc. It seems quite likely that similar ap- proaches can be made with other enzymes that can be irrevers- ibly inactivated or in which synthesis can be blocked by drugs or known inhibitors. The judicious use of these approaches, with suitable controls, andappropriatecheckswith radioisotopes, alter nate methods, or both, may greatly enhance our understand- ing of the rates of enzyme formation and destruction in c+o.

SUMhIARY

1. The kinetics of catalase synthesis and destruction in Go has been studied by use of 3-amino-I,2,4-triazole, which irre- versibly inactivates catalase, and allylisopropylacetamide, which blocks catalase synthesis.

2. .4fter the administration of 3-amino-I,2,4-triazole, the return of catalase activity is paralleled by a corresponding uptake of Fe*9 into the catalase, indicating that the return of catalase results from the formation of new enzyme.

3. From kinetic studies on the rate of return of catalase activ- ity after the administration of aminotriazole, it was calculated that the observed data could be accounted for by the synthesis of 28 pg of catalase per hour per g of rat liver if 2.25% of the catalnse molecules present were being destroyed in that time.

4. By use of allylisopropylncetamide to block the formation of new catalase, it was shown that the rate of catalase disap- pearance was nearly the same as that calculated from the amino- triazole data.

5. A comparison of the kinetics of catalasc return after amino- triazole administration showed that similar rates of destruction were observed in the liver and kidney. The higher levels of catalase activity in the liver (3.5-fold), therefore, result from a greater rate of catalase synthesis in that organ.

6. After the administration of both aminotriazole and allyliso- propylacetamide, the catalase activity rapidly rose to a low plateau of approximately 8Y0 of the normal levels, suggesting the existence of a second catalasc which can be inactivated by aminotriazole but in which synthesis is not blocked by allyliso- propylacetamide.

Acknozllledgments-ffe wish t.o express our sincere thanks to Dr. Robert E. Greenfield for his interest and valuable criticisms during this work and to Mrs. Bessie Watkins for skilled technical assistance. Thanks are also extended to Dr. W. E. Scott of Hoffman-La F&he, Inc., Nutley, New Jersey, for a generous gift of allylisopropylacetamidc.

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and Miloslav Rechcigl, Jr.Vincent E. Price, William R. Sterling, Vincent A. Tarantola, Robert W. Hartley, Jr.

in VivoThe Kinetics of Catalase Synthesis and Destruction

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