Intracellular catalase function: Analysis of the catalatic activity by product formation in isolated liver cells

ARCHIVESOFBIOCHEMISTRYAND BIOPHYSICS Vol. 214, No. 2, April 1, pp. 806-814, 1982

Intracellular

Catalase Function: Analysis of the Catalatic Product Formation in Isolated Liver Cells’

Activity by

DEAN P. JONES Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgin SOS22 Received August 21, 1981, and in revised form November 16, 1981

Intracellular catalase activity was measured in isolated rat hepatocytes by adding HzOz under anaerobic conditions and measuring O2 evolution. Hydrogen peroxide was introduced either by continuous infusion or by pulse injection. Continuous infusion at a rate similar to the endogenous HzOz production rate provided results that 60-70s of the HzOz was metabolized by the catalatic reaction. Comparison of rates of 0, evolution to estimated rates of H202 metabolism obtained by the methanol-titration method (H. Sies and B. Chance, 1970, FEBS L&t. 11, 1’72-176) indicated that the contribution of the peroxidatic reaction of catalase was small. The intracellular activity of glutathione peroxidase was estimated as the catalase-independent metabolism and used to determine the rate of intracellular Hz02 metabolism by the peroxidase. The results provide a quantitative basis for analysis of the physiological and toxicological aspects of H202 metabolism by liver. In recent studies of intracellular generation and catabolism of H202 in liver, the functions of catalase and glutathione peroxidase have been related to their intracellular localizations (1, 2). Catalase catalyzes two types of reactions, which are called the catalatic reaction (2 HzOz + O2 + 2 H,O) and the peroxidatic reaction (DH2 + Hz02 - D + 2 HzO). The intracellular studies rely principally on the peroxidatic reaction mode by measuring the steady-state catalase Compound I concentration as a function of exogenous hydrogen donors (3-5). A direct analysis of the intracellular catalatic reaction mode is not available. Such an analysis is needed because of the recognition that normal intracellular function of catalase principally involves the catalatic reaction (4, 5). 1This research was supported by NIH Grant GM 28176and American Heart Association Grant-in-Aid 80-902 with funds contributed in part by the Georgia Affiliate. 0003-9861/82/040806-09$02.00/O Copyright All rights

0 1982 by Academic Press. Inc. of reproduction in any form resewed.

In the following study of the catalatic reaction, oxygen formation from H202 is measured in suspensions of isolated liver cells under initially anaerobic conditions. The approach is specific for the catalatic activity because the peroxidatic reaction of catalase and the glutathione peroxidase reaction do not yield OZ. Analysis under anaerobic conditions allows measurement of O2 production in a very sensitive manner either by a Clark-type O2 electrode or by oxidation of mitochondrial cytochromes. Introduction of H202 by two different methods, continuous infusion and pulse injection, provides a quantitative comparison of the steady-state O2production from a known infusion rate of H202 and the total O2 produced from a known amount of H202. By establishing the quantitative relationship between H202 metabolized and O2produced these results also provide a means to estimate the turnover of glutathione peroxidase with H202 as substrate in intact cells.

806

INTRACELLULAR fate of H,O, Infusion a (nmOl/lObcells per mtn) g I

cc nj

i

CATALASE

y

FIG. 1. Oxidation of cytochrome aaa during steadystate infusion of HzOz. Isolated liver cells (lo6 cells/ ml) were allowed to become anaerobic under prepurified argon while the absorbance change at 605630 nm was recorded (A). After maximal absorbance change was attained, infusion of HzOz (7 mM) was begun at the flows indicated. Simultaneous polarographic recording was also made (B).

MATERIALS

AND METHODS

Preparations. Isolated hepatocytes were prepared from male white rats (Charles Rivers, 180-250 g, fed ad l&turn) as described by Mold&s et aL (6). This preparation previously has been characterized extensively with regard to a variety of parameters (6, 7). Cells were quantitated by counting on a hemocytometer in the presence of 0.2% trypan blue. Routinely, 95-99% of the cells exclude trypan blue. No change in cell viability occurred under the conditions used. Incubations were performed in Krebs-Henseleit medium containing 12.5 mM Hepes (N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid), pH 7.4, at room temperature. Spectrophotomet~. Spectral studies were performed on a Aminco DWZA spectrophotometer equipped with a 3.9-cm light-path incubation vessel and a magnetic stirrer (1). An oxygen electrode (Clark-type, Yellow Spring Instruments) was inserted through the cover of the vessel. The aqueous phase was normally 20 ml. The gaseous phase was continuously flushed with prepurified argon that was hydrated by passing through a bubbling tower. Anaerobic conditions eliminated endogenous HzOz production and resulted in conversion of endogenous Compound I to free catalase. This was substantiated by addition of 26 mM methanol (which stimulates conversion of Compound I to free catalase) to suspensions of cells under anaerobic conditions, which resulted in no detectable change in absorbance at 660-630 nm.

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FUNCTION

Addition of 1.2 mM KCN to inhibit catalase resulted in complete inhibition of Oz production from HzOz as measured polarographically. More specific inhibition of catalase was obtained by preincubation of cells with 5 mM 3-amino-1,2,4-triazole and 10 mM glycolate for 45 min. Under these conditions, catalase activity was inhibited by greater than 80% and cyanide-detectable catalase was less than 10% of control. Mitochondrial cytochromes and cytochrome bs are unaffected by this treatment; however, there is some loss of cytochrome P-450 (2). Production of Oz from HzOz in these cells during perfusion is inhibited as evidenced by decreased oxidation of mitochondrial cytochromes (data not shown), but not to the extent expected by an 80% inhibition of activity. However, because of the possibility that a higher steady-state HzOz concentration could compensate for diminished total activity, a better indication of inhibition was obtained by pulse injection studies (see Results). These results indicated that most (and probably all) of the H202 metabolism to Oz is catalyzed by catalase. Catalase heme concentration was determined after addition of 26 mM methanol by recording the absorbance change at 660-630 nm following addition of 1.2 mM KCN, and using the extinction value 5.4 mM-’ cm-’

(8).

Assays. A Clark-type electrode was calibrated with air-saturated distilled water relative to the dithionite zero and used for Oz assay. Additional calibration at low oxygen concentrations was obtained by injections of known volumes of Oz-saturated water to an anaerobic solution. HzOz was standardized relative to AB0 using the extinction value, 43 M-’ * cm-‘. GSH was measured as total acid-soluble thiol by the method of Saville (9). GSH loss was equivalent to half the GSSG released from the cells (1, 2). Materials. Collagenase (Type IV), aminotriazole, and glycolic acid were obtained from Sigma Chemical Company, St. Louis, Missouri. All other chemicals were of reagent grade and purchased locally. RESULTS

Steady-state analysis. Oxygen production was measured either polarographitally or optically (in terms of oxidation of mitochondrial cytochrome) while infusing standardized H202 solutions into cell suspensions (Fig. 1). A constant steady-state oxidation was obtained after approximately 100 s, provided that the rate of infusion was less than twice the maximal O2 consumption rate (E-20 nmol O&O6 cells/ min). The steady-state level of cytochrome oxidation and Oz concentration increased with the increased rate of infusion. At

808

DEAN

P. JONES

1

5

10 02(uM)

FIG. 2. Calibration of response of cytochrome ooa to additions of known concentration of Oa to suspensions of isolated hepatocytes. (A) Cytochrome m oxidation as a function of known O2 concentration. Cells were allowed to become anaerobic as in Fig. 1, and pulse injections of buffer equilibrated with 100% Oa were made. Absorbance change was measured at 605-630 nm. (B) O2 consumption vs O2 concentration. Pulse injections of 02-saturated buffer to give final concentrations of 21 and 35 pM Oa were made successively to anaerobic suspensions and the time courses for oxidation/reduction were recorded. The difference in length of time for reduction was the time required to reduce the increment of O2 (14 pM) and thus allowed calculation of O2 consumption rate. Similarly, the increment of time to go between any two states of oxidation (as in A) during the reduction curve allowed calculation of O2 consumption over that range.

sitive to O2concentration than cytochrome c at the low oxygen concentration. Estimation of O2 concentrations from the oxidation of cytochrome aa3 was obtained by calibration relative to additions of known concentrations of O2 (Fig. 2A). Respiration rate as a function of O2 concentration was obtained from the time course of reduction (Fig. 2B) following pulse injection of O2 with calibration relative to the extent of oxidation obtained

rates greater than twice the maximal respiration rate, the oxidation of cytochrome ua3 or cytochrome c rapidly reached maximal values and the polarographic trace demonstrated a continued increase in O2 concentration. Following termination of flow, the period required for reduction of cytochromes back to anaerobic levels was dependent upon the O2concentration. Data are presented only for cytochromes aa3 because this measurement was more senTABLE O2 PRODUCTION

HzOz infusion rate (H,Oa/ml/min)

RATE DURING

Steady-state cytochrome aa oxidation (%)

12.8

28

18.8

53

I

STEADY-STATE

HzOz INFUSIONS

O2 concentrationb (IL@ (I;) 2.6

Steady-state O2 production rateC (nmol/106 cells/min) 4.3 8.0

(2.1) 37.6

84

(iF.1)

16.8

a Cells (10s cells/ml) were incubated under prepurified argon until anaerobic, and infusion of HzOz (7 mM) was begun. Cytochrome w oxidation was measured at 605-630 nm. *O, concentration was estimated from the oxidation of cytochrome ou3 as calibrated in Fig. 2. Direct polarographic measurements are given in parentheses. ‘Steady-state O2 production rate was estimated as 02 consumption rate from calibrated curve in Fig. 2.

INTRACELLULAR TABLE

CATALASE

II

HzOz METABOLISM

RATE BY CATALASE DURING STEADY-STATE HsOs INFUSIONS

l&O2 infusion rate (nmol/ ml/min) 12.8 18.8 37.6

Steady-state Compound I (B of maximal) 31 52 70

H,OI metabolism rate* (nmol/lO’ cells/min) 8.34 17.8 37.0

*Isolated liver cells (10’ cells/ml; 20 ml total volume) were allowed to become anaerobic under prepurified argon before beginning infusion of HzOI (7 mu). Total eat&we beme was 0.21 nmol/lO’ cells. bHIOp metabolism rate for infusion rate of 37.6 was determined by methanol titration. Other values obtained from calibration curve (Fig. 3) as described in text.

in Fig. 2A. Under steady-state conditions, the rate of OZ production from H,O, is equal to the respiration rate (Table I). Data are presented for only one cell preparation since respiration rate varied up to 30% between preparations and introduced variations in steady-state levels of oxidation. Complete analysis of data for four cell preparations provided results similar to those presented here. The results of this analysis indicate that at an HzOz-infusion rate similar to the estimated maximal rate of endogenous HzOz production, i.e., 12.8 nmol/106 cells/ min (2), about 60-70% of the total HzOz is metabolized by the catalatic reaction. At higher rates of infusion, the fraction of HeOz metabolized by the catalatic reaction increases to over 90% (Table I). These results can be directly compared to rates of HzOz metabolized by catalase as estimated by the methanol-titration method. Steady-state levels of Compound I are achieved in about 50-60 s after initiation of infusion of H202. The level achieved is dependent upon the infusion rate (Table II). Methanol titration was performed for systems where the steadystate Compound I was greater than halfmaximal Compound I. At an infusion rate of 37.6 nmol/106 cells/min, the concentration of methanol required to decrease Compound I to the half-maximal value (a& was 4.2-6.0 mM methanol. For the experiment described in Table II, the alI2 was 5.6 mM. This corresponds to metabolism of HzOz by catalase at the rate of 37

809

FUNCTION

nmol/106 cells/min (0.21 nmol catalase heme/106 cells), and agrees with the above calculation for the rate of HzOz metabolized to O2 (Table I). An unusual feature of the methanol titration was that with continuous infusion of Hz02, the Compound I was not completely depleted by 30 mM methanol. This was indicated by a further decrease of 5-10% of the total AAsso-s30 following termination of flow in the presence of 30 mM methanol. A continued steady-state oxidation of mitochondrial cytochromes of about 20-30% and a corresponding concentration of Oz (measured polarographically) was present. Calculations indicated that Compound I depletion should be essentially complete under these conditions. Experiments with introduction of H202 by glucose plus glucose oxidase confirm this (data not shown). Since the dissolved oxygen in the H202 solution was only 4% of the total that could be formed by metabolism of H202, it is possible that the fraction of Oz generation that could not be eliminated by addition of methanol was due to a localized high concentration of HzOz at the site of infusion and could result in an overestimate of catalatic reaction. At lower rates of infusion, the steadystate Compound I levels are not sufficient to allow direct estimation of H,O, metabolism by the methanol-titration method. An alternate approach involves use of the relationship (5)

-0.5u

2.5

FIG. 3. Calibration of HzOz metabolism by catalase relative to the steady-state level of Compound I. A complete description of this figure is given in the text. Abbreviations: p,,,, Compound I concentration; pm maximal Compound I concentration; dx,/dt, rate of HcOa infusion (open circles) or theoretical rate of Hz02 metabolism by catalase (closed circles); e, catalase heme concentration.

810

DEAN

log -E!!Z-

PM - Pin

where p, is the steady-state concentra-

tion of Compound I, pu is the maximal Compound I concentration, dx,,/dt is the rate of H202 metabolism by catalase, (dx,/ dt)llz is the rate of HzOz metabolism by catalase, where p, = 0.5%, and e is the catalase heme concentration. With purified catalase, the plot of log (pJ(w - p,,,)) against log (dx,/dt)- (l/e) is linear with a slope of 1 (5). With isolated liver cells where dx,/dt is taken to be the rate of HzOz infusion under anaerobic conditions, this plot is nonlinear (open circles, Fig. 3). However, since the dx,/dt can be measured at high rates of infusion, a theoretical plot can be constructed relative to this value for other p, values (solid line, Fig. 3). With this approach, the measured p,, put and e values can be used to estimate the rate of H202 metabolism from the theoretical curve without addition of an exogenous donor (filled circles, Fig. 3). The values obtained (Table II) correspond well with the estimates of O2 production under the same conditions (Table I). The agreement of these values supports the conclusion that the peroxidatic reac-

P. JONES

tion of catalase in liver is small without an exogenous hydrogen donor (5). Thus, the balance of HzOz metabolism is due to other H202 metabolizing enzymes. In liver, glutathione peroxidase appears to account for most of the noncatalasedependent HzOz metabolism (for review, see (10)). Assay of intracellular glutathione peroxidase activities has been obtained by measuring GSH oxidation to GSSG and the loss of GSSG from the cell (2). Assuming that the non-catalase-dependent H202 metabolism is totally catalyzed by glutathione peroxidase, one can obtain a ratio, GSSG released/HzOz metabolized (noncatalase), to allow an estimation of HzOz metabolism by glutathione peroxidase during endogenous H202 production. GSSG release, which is equivalent to the loss of two molecules of GSH (2), increases with increased HzOz infusion rate (Fig. 4A). At high rates of infusion, the rate of GSH loss decreases after the first 2 min, which indicates that the contribution of glutathione peroxidase also diminishes rapidly at these high rates. Thus, the above experiments which were performed after 5-10 min of HzOz infusion, show a smaller contribution of noncatalase H202 metabolism than those performed during the initial 2 min. The best comparison was obtained at an infusion rate of 12.8nmol Hz02/106 cells/min, where 7 E

6-

E! .

t a =; ” p a E 5

-

4 .

“, ,”

.

2/

=, 0 i 5 Time

(min)

10

I 20 40 H,02 Infuilon

Rate

60

(nmolilObcells

per

mln)

FIG. 4. GSH oxidation during infusion of H,O, into suspensions of isolated hepatocytes. (A) Time course of GSH loss. (B) GSH loss as a function of HzOp infusion rate. Conditions as in Fig. 1. Control (0), 12.8 nmol/106 cells/min (O), 18.8 nmol/106 cells/min (A), 37.6 nmol/106 cells/min (A), 63 nmol/106 cells/min (0).

INTRACELLULAR

CATALASE

about 4 nmol H202/106 cells/min (range, 3.4-5.1) was metabolized by reactions other than catalase. The rate of GSH loss was 1.4 nmol/106 cells/min (range, 1.1-1.8) which is equivalent to 0.7 nmol GSSG released/lo6 cells/min. The resulting ratio, 0.7 nmol GSSG released/4 nmol H202 metabolized, can be used to provide an estimate of endogenous HzOz metabolism under conditions where GSSG efflux occurs. A plot of the initial rates of GSH loss as a function of Hz02 infusion indicates that this process is well below saturation under these conditions (Fig. 4B). Pulse injection studies. Injection of known concentrations of H202 resulted in rapid formation of Compound I; however, saturation did not occur until concentrations greater than 20 PM were added (Fig. 5). This concentration is very high relative to that observed with purified catalase under similar conditions. Chance et al. (11) found that saturation occurred below 1 PM HzOz for 1 PM purified catalase (expressed in terms of heme). Although this difference could be explained in terms of limited accessibility of exogenous Hz02 to catalase (12), calculations based upon cellular catalase content and several experimental results (see below) indicate that the measured Compound I following pulse injection of Hz02 is due to a steady-state

I 30

60

“200

HzOz ( uM)

FIG. 5. Formation of catalase Compound I in isolated hepatocytes following addition of exogenous HaOa. Cells (lOa/ml) were placed in cuvette as indicated in Fig. 1 and observed at 660-630 nm. About 1 min after polarographic measurement indicated anaerobic conditions, AGso-ssoreached a stable minimum. Addition of HaOa was made by injection of 1 to 10 ~1 of 7.0 mM H202 or 2 to 40 ~1 of 66 mM HaOa. Oa introduction under these conditions was less than 10 nM final concentration per microliter of addition and contributed negligibly to the signals recorded.

FUNCTION

811

FIG. 6. Comparison of cytochrome c oxidation by pulse injections of Oa-containing and HaOa-containing solutions. Absorbance changes were recorded at 550-540 nm under conditions as described in Fig. 5. Each data point represents the mean of 11 cell preparations for O2 additions (0). Cumulative data for 4 cell preparations are given for HZ02 additions (0). Greater separation of curves at lower values indicates a larger proportion of glutathione peroxidase at the lower HZ02 concentrations.

production of HzOB by intracellular oxidases which use the O2produced by an initial burst of catalase activity. In this regard, a pulse of H202 is essentially equivalent to a pulse of oxygen into the cell suspension. A direct comparison of the extent of oxidation of cytochrome c as a function of O2 concentration and l/2 H,Oz concentration (Fig. 6) demonstrates this relationship.2 The small displacement of the HzOz curve from the O2 curve appears to be due principally to the contribution of the glutathione peroxidase reaction to the total H202 metabolism, and thus the amount of O2 produced from H202 is always less than half the H202 added. Total O2 produced was obtained from the measurement of A[O,]/At + v,- At, summed over the entire curve. Estimates of total 0, production approach the expected stoichiometry of the catalatic mode (2 H,O,/O,) measured by either the change in oxidation of cytochrome aa or the polarographic tracing. For instance, at 36 PM H202, the estimated O2 production was 15.2 PM for the cytochrome ua3 tracing and 15.6 PM for the polarographic tracing. The contribution of the peroxidatic re‘In Figs. 6-8, data are presented for cytochrome c because it was more sensitive than cytochrome aa, at the high Oa concentrations involved in these pulse experiments.

812

DEAN

action mode following pulse injection of HzOz was assessed by measuring the oxidation of cytochrome c and steady-state levels of Compound I after addition of H202 to cells incubated with or without 25 mM methanol. The methanol had no detectable effect on the extent of cytochrome c oxidation at either l&36, or 54 PM Hz02, while the steady-state level of Compound I was lowered at least 90% (Fig. 7). These results show that the peroxidatic reaction makes a relatively minor contribution to the initial activity following pulse injection. However, the subsequent HzOz production from Oz is insufficient to maintain maximal Compound I concentration. The calculated activity of glutathione peroxidase under these conditions was 25 nmol HzOz metabolized/ml/s based upon a total activity of 2 pmol GSH oxidizedimg protein/min (13, 14) and 1.5 mg cell protein/ lo6 cells. Irreversible inhibition of catalase by preincubation with 5 mM aminotriazole plus 10 mM glycolate for 45 min resulted in a dramatic inhibition of cytochrome oxidation following pulse injection with HzOz (Fig. 8). Under these conditions, the fractional contribution of noncatalase metabolism of HzOz was increased and both the maximal oxidation of mitochondrial cytochromes as well as the integrated value for total Oz production were de550.540nm

I

660-630nm

w

+

-

CH,OH

2005

T 102

f 34

T 17

IL

?‘,

T

17 nlM

HZOZ

FIG. ‘7. Effect of methanol on cytochrome c oxidation and Compound I level following pulse injection of HzOz. Absorbance changes were measured at 550540 and 660-630 nm, respectively, under conditions as described in Fig. 5.

P. JONES

FIG. 8. Effect of preincubation with aminotriazole on O2 evolution from HzOz in liver cells as measured by oxidation of cytochrome e. Catalase was inhibited by a 45-min incubation of cells in the presence of 5 mM aminotriazole and 10 mM glycolate (curve B). Control cells (A) were incubated without aminotriazole. Cells were initially aerobic, and allowed to become anaerobic. Pulse injection of 36 PM Hz02 final concentration resulted in oxidation of cytochrome c.

creased. These results demonstrate that although the catalase turnover number is very high and the hepatic concentration is very high, inhibition by aminotriazole results in marked inhibition of intracellular function. DISCUSSION

In the current analysis of intracellular catalase function, measurement of Oz formation from exogenous HzOz provides a quantitative noninvasive probe of catalase activity. This approach measures only the catalatic reaction mode, but comparison to the methanol-titration method indicates that under these conditions the peroxidatic reaction is minimal. The agreement of values obtained by the two methods suggests that the quantitation of intracellular H202 production under aerobic conditions by the methanol-titration method is reasonably accurate. Assuming uniform accessibility of exogenous H202 to catalase, estimation of steady-state peroxisomal HzOz concentration can be obtained from the rate equation (11)

INTRACELLULAR

CATALASE

dx $ = 2k&[H202lbml, since dx,/dt has been measured as l/2 02 production, &dis known (2.6 X lo7 M-’ S-‘), and Compound I (p,,,) concentration has been measured (Table II). At an infusion rate of 12.8 nmol Hz02/106 cell/min, the peroxisomal H202 concentration is about 100 nM. Previous estimates of peroxisomal Hz02 concentrations during endogenous production range up to 100 nM for perfused liver in the presence of glycolate (5) and 20-50 nM during drug-stimulated Hz02 production (2). Since the contribution of the peroxidatic reaction is small, the difference between total H202 infusion and that metabolized to O2under steady-state conditions can be attributed to glutathione peroxidase. The data are consistent with major roles for both catalase and glutathione peroxidase when the rate of HzOz infusion is similar to expected physiological rates of H202 generation. Assuming simple MichaelisMenten kinetics for H202 dependence of glutathione peroxidase and the experimental values of 1 pM for K, (15), 20 pmOl/ g tissue/min for V at room temperature, and 4.3 nmol/106 cells/min for v (Table I), one can calculate that the H202 concentration in the cytosolic fraction is in the range of 20-30 nM. Although this estimate does not precisely agree with the H202 concentration estimate made from the catalase reaction, the two estimates together set a concentration range of 20-100 nM for HzOz in the cytosolic and peroxisomal compartments during metabolism of HzOz at 12.8 nmol/106 cells/min. In previous studies, Hz02 production in the endoplasmic reticulum of liver cells was measured in terms of GSSG release (2). Because of the contribution of glutathione reductase, GSSG release is an underestimate of the turnover of the glutathione peroxidase. In the presence of ethylmorphine, GSSG release from phenobarbital-pretreated cells was 2 nmol/106 cells/min and from control cells 0.5 nmol/ lo6 cells/min at 3’7°C. Although the rates may be lower at room temperature, com-

FUNCTION

813

parison of these values to the data in Fig. 4 and Table II suggests that in cells incubated with ethylmorphine, the rate of H202 metabolism in the endoplasmic reticulum is in the range of 2 nmol/106 cells/ min for control cells and 6 nmol/106 cells/ min for phenobarbital-treated cells. These values approximate the estimates of Hz02 metabolism obtained by the methanol titration method (l-4 nmol/106 cells/min) in cells depleted of GSH (1). The ratio of GSSG released to HzOz metabolized (0.18) is approximately an order of magnitude higher than the ratio of GSSG released to hydroperoxide metabolized (0.01-0.03) (10). The reason for this large discrepancy is not clear but may be related to the rapid metabolism of hydroperoxides by mitochondria (16) or to differences in effects on membrane structure on transport of GSH or GSSG. Studies of mitochondrial HzOz metabolism are complicated by contamination of isolated mitochondria with catalase from peroxisomes, but this analysis is needed to determine the contribution of mitochondrial glutathione peroxidase to the total cellular metabolism of Hz02. Similarly, additional details on the transport of GSH and GSSG are needed to determine whether transport is sensitive to peroxides. In a detailed theoretical treatment of diffusion effects on the metabolism of HzOz by peroxisomes, Poole (12) presented two conditions in which the latency of catalase activity in intact peroxisomes could be explained. These conditions involved either (i) a membranal permeability barrier to H,O, with the intraperoxisomal diffusion coefficient for Hz02 similar to that for HzOz in water and (ii) no significant membrane permeability barrier but a very low diffusion coefficient for Hz02 in peroxisomes relative to that for H202 in water. The present results demonstrate that HzOz, infused to cells at rates similar to estimated intracellular rates of generation, is effectively metabolized by catalase. The involvement of essentially all of the cellular catalase in metabolism of exogenous H202 is indicated by the magni-

814

DEAN

P. JONES

tude of the AA660-630 relative to the total cellular catalase concentration (1). Thus, exogenously supplied HzOz is readily accessible to intraperoxisomal catalase in hepatocytes. This observation, along with previous results which indicate that a substantial portion of H202 generated in peroxisomes diffuses out (2,1’7), suggests that the peroxisomal membrane does not offer a significant diffusion barrier to HzOz. (See Ref. (10) for further discussion.) The alternative model (12) in which the diffusion coefficient for H202 in peroxisomes is significantly lower than that for H202 in water, may therefore be more correct. This interpretation would be consistent with recent studies of O2 metabolism in hepatocytes which indicate that the intracellular diffusion coefficient of O2 is only l-2% of the extracellular value (Jones and Kennedy, manuscript submitted). If this relatively small diffusion coefficient is due to high intracellular viscosity, then a similarly small value would be expected for Hz02. The evidence for quantitatively significant roles of both catalase and glutathione peroxidase in H202 metabolism in liver corresponds to similar evidence for red blood cells (18) with the difference that subcellular compartmentation is important in determining relative functions in hepatocytes while H202 concentration is important in red cells. The direct demonstration of inhibition of O2 evolution in cells pretreated with aminotriazole provides a simple approach to assess intracellular catalase function that may be useful in measuring catalase function in other cell types as well as in toxicological conditions where inhibition of catalase may occur (19, 20). Finally, application of this approach to cells with varying catalase content, especially those with low concentrations, may provide a further understanding of the normal role of catalase in cellular H202 metabolism.

ACKNOWLEDGMENTS This research was supported by NIH Grant GM 28176 and by American Heart Association Grant-inAid 80 902, with funds contributed in part by the Georgia Affiliate. I would like to thank Drs. D. B. McCormick and D. E. Edmondson for their review of this manuscript during its preparation. REFERENCES 1. JONES, D. P., THOR, H., ANDERSSON, B., AND ORRENIUS, S. (1978) J. Biol. Chem 253,6931-6037. 2. JONES, D. P., EKL~W, L., THOR, H., AND ORRENIUS, S. (1981) Arch Biochem. Biophys. 210,505-516. 3. SIES, H., AND CHANCE, B. (1970) FEBS ktt. 11, 172-176. 4. SIES, H., BUCHER, T., OSHINO, N., AND CHANCE, B. (1973) Arch Biochem Biaphys. 154,106-1X 5. OSHINO, N., CHANCE, B., SIES, H., AND B~CHER, T. (1973) Arch. Biochem Biophys. 154,117-131. 6. MOLD&S, P., H~GBERG, J., AND ORRENIUS, S. (1978) in Methods in Enzymology (Fleisher, S., and Packer, L., eds.), Vol. 52, pp. 60-71, Academic Press, New York. 7. JONES, D. P., AND MASON, H. S. (1978) J. Biol. Chew. 253,4874-4880. 8. OSHINO, N., OSHINO, R., AND CHANCE, B. (1973) B&hem. J. 131,555-567. 9. SAVILLE, B. (1958) Analyst 83, 670-672. 10. CHANCE, B., SIES, H., AND BOVERIS, A. (1979) Physiol Rev. 59,527-605. 11. CHANCE, B., GREENSTEIN, D. S., AND ROUGHTON, F. J. W. (1952) Arch. Biochem Biophys. 37, 301-331. 12. POOLE, B. (1975) J. Theor. Biol. 51, 149-167. 13. PINTO, R. E., AND BARTLEY, W. (1969) Biochem. J. 112,109-115. 14. LANE, H. W., SHIRLEY, R. L., AND CERDA, J. J. (1979) J. Nutr. 109,444-452. 15. FLOH~, L., AND BRAND, I. (1969) B&him Bie phys. Acta 191,541-549. 16. JOCELYN, P. C., AND DICKSON, J. (1980) Biochim. Biophys. Acta 590, l-12. 17. BOVERIS, A., OSHINO, N., AND CHANCE, B. (1972) B&hem. .J. 128, 617-630. 18. COHEN, G., AND HOCHSTEIN, P. (1963) Biochemistry 2,1420-1428. 19. JONES, D. P., MEYER, D. P., ANDERSSON, B., AND ORRENIUS, S. (1981) Mol. P~UTVZUCOL20, 159164. 20. JONES, D. P. (1981) Res. Commun Chem Pathol Pharmacol. 33,215-222.