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1972 by Academic in my
of reproduction
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form rrscrocd
Experimental
CATALASE
TURNOVER W. J. MELLMAN,3
Departments
Cell Research 73 (1972) 399-409
IN HUMAN R. T. SCHIMKE’
DIPLOID
CELL
CULTURES
and L. HAYFLICK
of lPharmacology and 2Medical Microbiology, Stanford University School of Medicine, Stanford, Calif. 94305, and 3Departments of Pediatrics and Medical Genetics, University qf Pennsylvania, Philadelphia, Pa 19104, USA
SUMMARY The rates of synthesis and degradation of catalase protein were measured in cultures of human diploid fibroblasts by the method of Price et al. [lo] which employs 3-aminotriazole as an irreversible inhibitor of catalase without affecting its synthesis. The half-life of catalase has been estimated at approx. 30 h, a value comparable to that obtained for rat and mouse liver catalase. The turnover of catalase is influenced by a factor present in fetal calf serum which is inactivated during incubation. Suboptimal amounts of this serum factor retard both synthesis and degradation of catalase, resulting in a reduction in the cell content of catalase. From the double isotope method of Arias et al. [18] it would appear that catalase is turning over more slowly than average cellular protein.
mammalian tissues are known to be continuously synthesized and degraded, and the rate constants of these processes vary remarkably from one protein to another [l]. There is little information about the genetic and non-genetic effecters of protein synthesis and degradation. The ability to study these dynamics with proteins which have specific genetic determinants in a homogeneous cell culture system should facilitate the identification of regulators of the cell content of these proteins. This paper will demonstrate that cultured human fibroblasts can be used to study the turnover of a specific protein, catalase, whose structural protein appears to have a single autosomal determinant [2]. Mutations affecting catalase activity occur in man and are expressed in cultured cells [3]. There are also strains of mice with mutations that affect their levels of catalase; one Proteins
26-
721808
in non-dividing
mutation has led to an altered rate constant of degradation, and the locus of this apparent degradation mutant appears to be distinct from that of the catalase structural gene [4]. Individual human diploid fibroblast cultures have a characteristic cell replication pattern consisting of a period of rapid multiplication, followed by a slow, nearly arrested, phase of confluent cell multiplication. In this latter period, the cell content of cultures is essentially static, and the non-dividing cells are viable. It is these cultural characteristics that are suited to studies of protein synthesis and degradation where a constant cell mass is assumed. The growth rates of cells in culture are known to be stimulated by labile components of serum in the culture medium [S], and these experiments that measure the dynamics of catalase metabolism will demonstrate that both the synthesis and degradation of proExptl CeN Res 73 (1972)
400
W, J. Mellman et al.
teins are also influenced bv serum factors. Moreover, such serum factors appear to affect individual proteins in different ways. d
MATERIALS AND METHODS
values were used when duplicate determinations agreed within 10 %. Optical density change was found to be proportional to the amount of cell lysate. The protein content of the lysates ranged from 1.4-4.9 mg/ml. Analysis cubation inhibitor
4f catalase synthesis and de.eradation by inoj cells with aminotriazole,an irreversible of catalase: After the cells had reached the
The method originally described by Beers & Sizer [6] was used. Peroxide disappearance was recorded at 240 nm with a Gilford model 2000 spectrophotometer in a 1 ml reaction mixture containing H,O, (Baker analyzed), 20 mM; Triton X-100 (Rohm & Haas), 0.1 %; potassium phosphate buffer, pH 7.5, 100 mM. The recorder was set at a full scale expansion of 0.2 OD units and dilutions of assay material were used so that an essentially linear decrease in OD could be recorded for at least 1 min. Activity was expressed as AOD,,,/min/mg protein in theTriton X-100 solubilized cell fraction.
stage of c&fluency and arrested multiplication (i.e. when only a rare mitotic figure was evident 24 h after a complete medium change), regular growth medium was removed and 5 ml of medium without serum and containing 3-amino 1, 2, 4-triazole (Mann Research Labs), 1 mg/ml, was added to the culture flasks. After 4 h at 37°C this medium was decanted, the monolayer washed with 20 ml of medium without serum and finally medium was added as described for individual experiments (see Results). This time of incubation and dose of aminotriazole was found to inactivate greater than 90% of the cell catalase activity (I, 2, 3, and 4 h of incubation and 0.2, 1, and 5 mg/ml of drug were tested).
Cell cultures, preparation of cell lysates and incubations with 3-aminotriazole
Preparation of human liver catalase for immunization
Human fibroblast cell cultures derived at the Children’s Hospital of Philadelphia from skin biopsies of adult females UPG 70-14 and UPG 70-39 and the fetal lung fibroblast cell strain WI-38 of Hayflick were used. Cells were passaged routinely after they had reached confluency in 250 ml plastic culture flasks (Falcon) [7]. The medium consisted of Eagle basal essential components (Grand Island Biological Co. powdered medium) supplemented with 10 % fetal calf serum (Microbiological Associates, Inc.) and aureomycin (Lederle) 50 &ml. Periodic tests of cell culturesfor mycoplasma cc&amination were negative 181.At the time of cell harvest for assay, the monolayer surface was washed once with phosphate buffered saline (PBS) (K phosphate, pH 7.5, 10 mM; NaCl, 150 mM), then 0.25 % (w/v) trypsin was layered over the monolayer surface for 1 min and decanted. When the cells detached (in 5-10 min) they were suspended in PBS, centrifuged at 500 g for 5 min and washed with 5 ml of PBS. The pelleted cells, collected after a second centrifugation, were held at 4°C until the completion of the experiment; all pellets from the same experiment were then lysed and assayed at the same time. Catalase activity was found to be stable for at least a week when the cells were keut as a nellet and stored at 4°C. Pelleted cells were lysed by freezing and thawing 5 times in 0.5 % Triton-X-100 (v/v); (for most experG ments the cell pellet from a 250 ml culture flask was lysed in 04ml). Lysates were centrifuged for 5 min in a Beckman model 152 microfuge (9 400 g) and the supernatants removed for catalase assay and protein determinations, the latter by the method of Lowry et al. 191.Deuending on the catalase activity of the preparation, 5 to l& pl of the supernatants was added to the 1 ml catalase reaction mixture. Assays were performed in duplicate, and activity
Human liver specimens (about 1 kg) obtained at autopsy were extracted initially by steps l-4 of Price et al. [IO] used for rat liver catalase. Unlike their experience with rat liver, we found that human catalase did not precipitate in 20% ethanol, 0.1 M NaCl and acetate buffer, pH 4.7. Rather, preparations dialysed in this solution, and placed at 4°C for 24 h developed a precipitate rich in ferritin while the catalase remained in solution. The major portion of the ferritin in this preparation was removed by centrifuging at 105 000 g for 4 h. This urecinitate was used lacer to adsorb firritin antibody from rabbit serum (see below). Catalase activity was completely precipitated from the 105 000 g supernatant by raising the alcohol content to 60%. After standing at 4°C overnight, the orecinitate was collected bv centrifugation at 13 od0 g for 30 min, dissolved in 25 ml Gf sodium acetate, 0.1 M, pH 4 0, and then dialysed against distilled water overnight. Catalase was reprecipitated in 26 % (w/v) ammonium sulfate at 4°C. and the precipitate ‘was again collected by centrifugation. The ammonium sulfate precipitated catalase was treated with sequential 4 ml portions of ammonium sulfate solutions of decreasing concentrations (28-20 % w/v). The precipitate was stirred with a Teflon stirring bar in each 4 ml portion of ammonium sulfate for 20 min and then centrifuged for 10 min at 13 000 g. The following total volumes of ammonium sulfate were used: 28 % 8 ml, 24 % 8 ml, 22 % 40 ml, 20 % 12 ml. Each supernatant fraction was analysed for its catalase content by measuring absorption at 280, 310 and 400 nm [lo]. Although a considerable amount of catalase was solubilized in 22 % ammonium sulfate, the purest catalase preparations by this procedure were solubilized in 20 %. This material had a 400/280 nm absorption ratio of 0.90 with peak absorption
Catalase assay
Exptl Cell Res 73 (1972)
Catalase turnover in cell culture 401 at 275 and 405 nm, and was concentrated by reprecipitating in 30% (w/v) ammonium sulfate and redissolved in PBS to a concentration of 5 mg/ml. When 20 pg of this preparation was electrophoresed in a 7.5 % polyacrylamide disc gel [ll], and then stained with 2 % amido black, a single band was observed. Table 1 presents the catalase specific activities at various steps in the purification procedure. There was 10.4 mg of protein in the final preparation, and 800 g of wet liver (approx. 40 g protein) were used in the initial extraction.
Preparation of antiserum to human liver catalase and immunoprecipitation reactions One ml of the purest catalase (5 mg) was mixed with 1 ml of Freund’s complete adjuvant (Difco) and was injected into the toe pads of a male New Zealand albino rabbit. Three weeks later blood was obtained from an ear vein and the serum was collected by centrifugation. This serum was incubated overnight at 4°C with aminotriazole, 10 mg/ml, and a concentrated preparation of ferritin obtained during the liver catalase purification described above. The amount of this ferritin preparation used was determined by sequentially adding aliquots to a test volume of serum until no further precipitation occurred. Aminotriazole was added when significant catalase activity was found in the rabbit serum presumably from contamination with red cell catalase. The serum was absorbed with ferritin when it was found to contain two precipitin lines by double immunodiffusion with a partially purified liver preparation known to contain both catalase and ferritin. On the premise that one precipitin line was formed by an antibody resulting from immunization of the rabbit with catalase contaminated with a trace amount of ferritin, the serum was absorbed with ferritin. After the ferritin treatment, one band disappeared from the immunodiffusion reaction. Following incubation of the serum with ferritin and aminotriazole, it was centrifuged at 13 000 g for 30 min at 4°C to remove the ferritin-antiferritin complexes. Partially purified gamma globulin was prepared from this serum with ammonium sulfate (40 % saturated v/v) followed by dialysis as described by Palmiter et al. [12]. This material was heated at 56°C for 30 min and stored at -20°C until used. When this antibody preparation was used as a reagent for determination of catalase equivalence units, it was first centrifuged for 5 min in a Beckman microfuge. For precipitation of isotopically-labelled cell catalase, the anti-serum was centrifuged 1 h at 105 000 g and both the precipitate and the fat layer were discarded. Catalase immunoprecipitations: Immunoprecipitations were performed with partially purified human liver preparations (using the material precipitated with 60 % ethanol), and fibroblast lysates prepared as described above for catalase assays. Quantitative reactions were performed with either constant amounts of antigen (cell lysate or semi-purified liver catalase) and increasing amounts of antibody. or a constant volume of a&body and increasing imounts of antigen. A final concentration of 0.5% (v/v) of Triton
X-100 was used in the reaction mixtures. Various incubation times were studied and two schedules were found to completely precipitate catalase from these preparations: (1) 3 h at 37°C followed by 1 h at 4°C or (2) I h at 37°C and overnight at 4°C. Isolation and analysis of radioactive catalase in cultured ceN extracts: Cell cultures were incubated with
3H and 14C amino acids as described in the Results. Cell lysates were prepared as described under assay methods extent that these nreoarations contained approx. 4 times as many cells/vol. They were centrifuged at 105 000 g for 1 h and both the nrecinitate a& the fat layer were discarded. The clear suiernatants were preincubated at 37°C for 2 h and centrifuged at 9 000 g for 5 min to remove nonspecific precipitates that develop at this temperature; then one part of 10 x PBS was added to 9 parts of the cell lysate. This cell preparation (0.5 ml) was incubated with catalase antibody (0.05 ml) at 37°C for 20 min, and then a 0.01 ml volume of semi-purified liver catalase was added for the remainder of the 3 h incubation at 37°C and 1 h at 4°C. The volume of cell lysate chosen was based on the anticipated amount of radioactivity incorporated specifically into catalase protein; the amount of liver catalase added was designed to provide a small but visible precipitate, and the amount of antibody was slightly in excess of the equivalence point of the combined antigenic sources of catalase protein. Equivalence points were established by prior titrations of both the liver and cell catalase. After incubation the tubes were centrifuged in the cold at 13 000 g for 30 min and the supernatants checked for residual catalase activity. Under the conditions of these experiments, no detectable activity remained in the supernatants. The precipitates were washed 4 times with 2 ml of PBS. The pellets were transferred to plastic ‘microfuge’ tubes (Beckman Instruments) and the precipitates solubilized in 50 ~1 of a solution containing Tris 24 mg, dithiothreitol 30 mg (CalBiothem), sodium lauryl sulfate 20 mg (Fisher), glycerol 0.2 ml, saturated bromhenol blue 0.1 ml and water 1.7 ml. Pellets were suspended in this solution by sonication. This was a&omplished by taping thk plugged microfuge tubes to the bottom of a glass Petri dish, covering the tube with water and passing the sonicator tin along the outside of the tube until the pellet was &spended. The tube was then boiled for 5 min and 40 ul of this boiled nrenaration was placed over a 16% polyacrylamidk &sc gel and electrophoresed as described by Palmiter et al. [12]. Sonication was found to be critical to this procedure; without sonication aggregated material tended to remain at the top of the gel and caused smearing of radioactivity in the region of the peak corresponding to the catalase subunit (mol. wt 60 000) [13]. Current was applied to the gels until the tracking dye reached the bottom, whereupon the gels were frozen on dry ice and cut into 2.4 mm segments and counted as described elsewhere [12]. Counting efficiency and 14C spillover into the SH channel were estimated by the channels ratio method (SH eff. 34.5 96,I’C eff. 59.5 %, spillover 15 %). A reference gel was run simultaneously with a sample of human liver catalase that had been sonicated and boiled in the same solution as the Exptl Cell Res 73 (1972)
402
W. J. Mellman et al. methyl-5-phenoxazolyl)] benzene per liter of toluene. Counting efficiency and 14C spillover into the 3H channel were estimated by the channels ratio method (JH eff. 35.5 %, 14Ceff. 64 %, spillover 12.7 %).
RESULTS
o.o5y,, , , ) , , ,I 0
20
40
Figs I, 2. Abscissae: time (hours) after end of 3-aminotriazole incubation period; ordinates: catalase activity expressed as nOD,,,/min/mg Triton X-100 soluble protein. Fig. 1. O, n, activities of cultures treated with aminotriazole; n , A, mean of 6 (WI-38) and 5 (UPG 70-14) control cultures incubated under the same conditions as the experimental cells except for aminotriazole; estimate of data points up to and including 20 h &t; ---, extension of this line to point representing activity of control cells. Measurement of catalase turnover by irreversible inactivation of enzymes with aminotriazole. Catalase activity in fetal (WI-38) and adult (UPG 70-14) human diploid fibroblast cells incubated with aminotriazole for 4 h and then incubated in complete medium containing 10 % fetal calf serum. Medium was changed 24 h before and at the end of the aminotriazole incubation. Turnover data calculated from the slope of these plots; T& based on the assumption that the controI means represent the normal steady level of the enzyme (-9: E T3 (AOD2.dmin/ (hours) k,’ mg protein) ha 21 0.057 0.033 1.72 WI-38 UPG 70-14 23 0.021 0.030 0.694 ak, and kd for this and figs 2 and 3 expressed as k,: units activity (nOD,,,/min/mg protein/h) ): 10-l; k,: h-‘.
antigen-antibody precipitate of the cell lysate. This gel was stained for protein, and the slice containing catalase was estimated by the migration position of catalase subunits on this stained gel. Determination of incorporation of radioactive amino acids into TCA-precipitable cell fraction: Lysates prepared for immunoprecipitation of catalase were assayed for total protein radioactivity by treating duplicate 10 ~1 aliquots with 50 ~1 of cold 10 % trichloroacetic acid (w/v). The precipitates were washed twice with 100 ul of 5 % trichloroacetic acid. the washed pellets were dissolved in 100 yl of NCS solubilizer and counted in scintillation fluid containing 5 g 2,5-diphenyloxazole and 0.3 g 1, 4-bis [2-(4Exptl Cell Res 73 (1972)
1. Estimation of catalase turnover from the recovery of catalase activity after inactivation of existing enzyme by aminotriazole Aminotriazole has been shown to bind in vivo to the protein portion of liver catalase [14], resulting in irreversible inactivation of the enzyme. This activity then returns to its original steady state level by new synthesis of the enzyme, and the rate constant of synthesis (k,) is constant during this period. The rate of approach to this steady state is a function of the rate constant of degradation (kd) and the return of activity following aminotriazole has been described as an exponential function [lo]. The catalase activity of cultured fibroblasts shows a similar response to aminotriazole, and in these experiments the return of activity has been plotted logarithmically. The catalase activity of human cell strains (UPG 70-14, UPG 70-39, WI-38) exposed for 4 h to aminotriazole (1 mgjml) (see Methods) was less than 10% of control cultures, treated the same way except for the absence of inhibitor in the incubation medium. Cells exposed to aminotriazole did not differ morphologically from control cells during the experiment. After incubation with aminotriazole, fresh medium containing 10 % fetal calf serum was substituted, and, for the experiment depicted in fig. 1, this medium was not changed until the cells were harvested. Fig. 1 plots the cell catalase activity (expressed as OD,,, change/min/mg Triton X-100 soluble protein) as a function of time after the aminotriazole was removed from the cell cultures. There were random variations (maximum variations of 20%) in the
Catalase turnover in cell culture total protein content of individual cultures within each experiment and total protein content was assumed to be constant between the time of aminotriazole exposure and the end of the experiment. Catalase turnover was estimated from the lines drawn through the data plotted semilogarithmically in fig. 1. The linear portions of the solid curves were drawn from a least squares analysis of the points obtained for the first 20 h after removal of aminotriazole from the cells. This line was extended (dotted line) through the point that represents the average activity of control cells. Subsequent experiments (see results presented in fig. 3) suggest that the slowed return of catalase activity found at 26 h reflects the depletion of a critical component in the medium required for catalase synthesis. The T$ was estimated from these slopes and is the time for catalase activity to rise half way to its steady state level. Steady state levels (E) are based on assays of control cultures, and k,, the zero-order rate constant of synthesis, and kd, the first-order rate constant of degradation, respectively, were estimated by formulae derived elsewhere [15] that assume random degradation of specific protein molecules and steady state kinetics (k, = .0.692/T&;k, = kdE). These calculations assume that there is essentially no change in cell mass during the period of the experiment. Values of k, and k, for these and subsequent experiments are presented in the appropriate figure captions. 2. The influence of serum on catalase turnover Experiments such as those depicted in fig. 1 revealed that the recovery of catalase activity in these cell cultures after irreversible inactivation by aminotriazole is abortive, i.e. there was a progressive return of activity for approx. 20 h, followed by a period of apparently arrested or retarded catalase synthesis. From
I
I
I
I
0
12
24
36
403
I
48
Fig. 2. The influence of serum on catalase turnover.
Cell cultures of UPG 70-14 were handled similarly to those in fig. 1 through the period of aminotriazole incubation. After 4 h exposure to aminotriazole, half the experimental cultures were fed every 12 h with complete medium (+ serum) and the other half with serum-free medium (-serum). Turnover data for the + serum experiments are: rj, 27 h; k,, 0.019; k,, 0.026; n n-v??“. I,‘.
Ilr,
these observations, we postulated that a critical component of the culture medium was being either degraded or depleted. In fig. 2 evidence was obtained that the responsible component of the medium is probably in the serum. Cultures were treated with aminotriazole as in the experiments in fig. 1 and, after the aminotriazole incubation period, half of the cultures were fed with medium containing serum (+serum) and the other half with medium without serum (-serum). -serum) were replaced comMedia (sor pletely every 12 h until the cultures were harvested. Cells fed with medium tserum demonstrated a progressive increase in catalase activity with time until they reached levels of control cultures, while the cultures fed with medium -serum showed only a modest rise in catalase activity for the first 24 h after aminotriazole incubation. We propose that the increase in catalase activity observed in the -serum cultures represents the persistent influence of this postulated serum factor still present in the cells from the period before they were exposed to aminotriazole, when the serum was present in the medium. Exptl Cell Res 73 (1972)
404
W. J. Mellman et al.
I
0
12
24
36
48
Fig. 3. Abscissa: time (hours) after end of 3-aminotriazole incubation period; ordinates: catalase activity
expressed as aOD,,,/min/mg Triton X-100 soluble protein. The effect of ‘conditioned’ medium on catalase turnover. Sixteen cultures were prepared from the same passage level of UPG 7&39, a normal adult female fibroblast cell strain. Eight cultures had a complete medium change on days 3, 6, 7 and 8 after subculture, and every 12 h after incubation with aminotriazole (fed cells). The other 8 had a complete medium change on days 3, 6, and 7 after subculture and the medium added on day 7 was saved during the aminotriazole incubation, then put back on the cells until the cultures were harvested for catalase assay. Cells from individual cultures were harvested at the times indicated after the aminotriazole incubation period of 4 h. Turnover data calculated from these slopes (based on least squares analysis) and the mean catalase activity of the four control cultures (E) are: T?$
Fed Unfed
(hours) k, 30 0.018 39 0.010
kd
0.023 0.018
&OD,,,jmin/ mg protein) 0.774 0.580
No catalase activity could be detected in undiluted samples of fetal calf serum used in these experiments, in contrast to the findings of significant amounts of catalase activity in the human sera used by Krooth et al. [3]. Control cell cultures incubated for more than 24 h without serum were noted to have decreasing amounts of total Triton X-100 soluble protein as well as decreasing catalase specific activity. 3. The effect of ‘conditioned’ medium on catalase turnover The data thus far have demonstrated that cells require frequent changes of serumcontaining medium for complete repletion Exptl Cell Res 73 (1972)
of their catalase activity after aminotriazole inactivation. In the experimental data presented in fig. 3, two groups of cultures from the same cell strain (UPG 70-39) and subcultivated simultaneously were used. A different cell strain from that in fig. 2 was used in order to test the comparability of results with different strains of adult human fibroblasts. One group had a complete medium change daily for 3 days before incubating with aminotriazole, and the medium was changed completely (+serum) every 12 h after aminotriazole removal (‘fed’). The other group did not have a medium change for 2 days before adding the aminotriazole. The complete medium removed from these latter cultures was saved before the aminotriazole incubation. The pH was adjusted with NaHCO, to that of the fresh medium, and this was added back to the cultures after the aminotriazole incubation was complete. These cultures did not have any changes of medium (‘unfed’). Both fed and unfed cultures were harvested at 6, 18, 30 and 40 h after the end of the aminotriazole incubation. The pH of all media was measured and both fed and unfed cultures did not vary among each other by more than 0.1 units. Fig. 3. shows that both synthesis and degradation of catalase are affected by cell nutrition. Fed cells appear to have both a greater k, and k, than unfed cells. There is a difference in the initial rise of enzyme activity following aminotriazole treatment, indicating that there are dissimilar rates of catalase synthesis by fed and unfed cells. The overall rates of approach to steady state levels are different for the two groups of cultures, and these slopes reflect primarily their rates of degradation. The fed cells reach a higher steady state level of catalase than the unfed cells(&, =0.774, Eunfed=0.580OD,,, change/ min/mg protein). Pan & Krooth [16] had previously found that unfed cells as defined
Catalase turnover in cell culture in this experiment have a lower specific activity than fed cells.
r I I1
catalase
4. The influence of cell culture nutrition on cell catalase content as measured immunologically In fig. 3 it was noted that frequent medium changes resulted in greater catalase activity. This enhanced activity was found to be associated with proportionate increases in immunologically reactive material. Quantitative immunoprecipitation studies of cell lysates fromfed and unfed cultures are recorded in fig. 4 and demonstrate that the equivalence point, the amount of antibody required to neutralize a unit of catalase activity, is approximately the same for lysates of fed and unfed cells when there is a two-fold difference in specific activity. 5. Turnover of catalase relative to other Triton X-100 soluble proteins The preceding experiments suggest that there is a factor in the serum component of culture medium, unstable when incubated with cells, that affects the rate of catalase turnover. Both the specific activity of catalase and the total protein content of cells decreased when cell nutrition was suboptimal. As discussed by Berlin & Schimke [17], if factors such as nutritional ones affect the synthesis and degradation of all cell proteins proportionately, proteins which are rapidly degraded (i.e. have a relatively large kd) would be expected to demonstrate greater changes in their levels than the remaining proteins when these factors are altered. The following experiment was designed to test whether the observed effect of nutrition on catalase specific activity indicates that catalase turns over more rapidly than the average protein in the fraction under study (Triton X-100 soluble), or alternatively whether the turnover of catalase is particularly
( I I I I,
405
I I I I
0.5,
04
x x
Fed
0.3 f\ Unfed \
x
0.2 \- 0
0.1. 0
‘\‘\
‘\
‘\\\ ‘l, 0 ‘x, I I I I I.3 IL I ‘-.-I I I \\
‘\
0.5
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I .5
Fig. 4. Abscksa: Catalase activity (nOD,,,/min) remaining in supernatants from 0.1 ml of cell lysate incubated with antibody for 1 h at 37°C and overnight at 4°C; ordinate: vol (~1) of antibody to human liver catalase incubated with cell lysate. Cell lysates were prepared from the same passage level of the same cell strain (UPG 70-14) after the cells had reached confluency. One was from the pooled cells of six 250 ml cultures whose medium was cLanged daily for 4 days before harvest (fed cells) and the other from 6 cultures in which the medium was left in olace for 3 days before harvest (unfed cells). The lysate from the feh cells had twice thk catalase activity of the unfed (1.06 and 0.475 AODl,,/min/mg Triton X-100 soluble protein).
sensitive to the depletion of this postulated serum factor. For this analysis the approach suggested by Arias et al. [18] that assays relative turnover by labelling cell proteins with pulses of two different isotopic forms of amino acids at two different times was used. The first pulse (3H) is given 48 h before harvest, so that there is time for the proteins labelled with this isotope to degrade. The second isotope (‘“C) is given just before harvesting cells, so that there is effectively no time for the proteins labelled with that isotope to be degraded. Therefore, the degree to which proteins are labelled with the second isotope is an index of synthetic rate. The ratio of the two isotopes for different proteins can be used to measure the relative turnover of various proteins in the same cell preparation. For the experiment recorded in table 2, four cultures of cell strain WI-38 were grown to confluence in 250 ml plastic flasks (Falcon) Exptl Cell Res 73 (1972)
406
W. J. Mellman et al.
Table 1. Purification liver
of catalase from human
ODdmin/
mg protein
Stage 1. Crude homogenate 2. Supernate after step 1 of Price et al. [lo] 3. Supernate from step 4 of Price et al. [lo] 4. Supernate from 10.5000 x g spin (major portion of ferritin in pellet) 5. Fraction extracted with 20 % ammonium sulfate after precipitation with 26 % ammonium sulfate
6.5 46.6 700 2 330 6600
and the medium was completely changed on days 3 and 5-10 following subculture. On day 8 cultures were preincubated for 30 min in medium-serum containing 1 part nutrient medium (BME) and 19 parts Hanks balanced salt solution, i.e. l/20 BME. Further incubation proceeded for 3 h in 5 ml of l/20 BME containing 3H amino acids (38.5 ,&i/ml of reconstituted protein hydrolysate, algal profile, Schwarz Bioresearch, Orangeburg, N. J.). The radioactive medium was decanted, and the cells were incubated in 20 ml of undiluted medium (1 x BME) for 30 min. This medium Table 2. Relative turnover of total protein (TCA-precipitable) and catalase (immunoprecipitable) in Triton-X soluble cell fraction dpm/ml cell extract lacb
TCA-Precipitable Catalase % Catalase in TCA-Precipitable
sH/14C
5.29 x IO6 3.47 x lo6 1.52 466 1.92 910 0.0172 %
0.0134 %
a 3 h incubation with SH-amino acids 48 h before harvest. b 3 h incubation with W-amino acids 3 h before harvest. Exptl Cell Res 73 (1972)
was replaced with fresh medium (+ serum) which was replaced completely again 18 and 30 h later. At 42 h the same cultures were again preincubated for 30 min with l/20 BME, then incubated for 3 h in l/20 BME containing 14C amino acids (5 &i/ml of reconstituted protein hydrolysate, algal profile, Schwarz Bioresearch). The l”C medium was decanted and the cultures were incubated for 90 min in 1 x BME (-serum). All cultures were incubated at 37”C, except the last which was done at room temperature. The cells from the 4 cultures were then trypsinized into a single suspension, lysed in 2 ml of 0.5 % Triton X-100, and prepared for immunoprecipitation with catalase antibody (see Methods). In table 2 the ratio (3H/14C) for catalase isolated by immunoprecipitation and electrophoresis is compared with that of the average protein in the Triton X-100 soluble fraction. In addition, table 2 records that catalase represents 0.01-0.02 Y/Oof the total protein in this cell fraction. The greater 3H/14C ratio obtained for catalase indicates that it is turning over more slowly than the average protein, since the slower a protein degrades the greater the residual 3H labelling of that molecule under the conditions of this experiment. DISCUSSION This preliminary study indicates that human diploid cell cultures can be exploited for the analysis of specific protein turnover, and emphasizes the responsiveness of such turnover, to nutritional conditions. The sensitivity of protein turnover to medium conditions suggests that these cell cultures offer a valuable tool for investigating the complex variables controlling specific protein synthesis and degradation. The catalase model has proven to be particularly useful for in vitro studies, because
Catalase turnover in cell culture there is available a highly specific inhibitor that binds irreversibly to the active site of catalase without interfering with its synthesis. The validity of analyzing catalase turnover with aminotriazole had been presented by two groups of investigators who performed their studies in vivo. [4, 191. It is interesting that the half-lives of rat and mouse liver catalase are similar to that found in these experiments for human fibroblast cells. Methods involving irreversible inhibition of enzyme activity offer two important advantages in the measurement of turnover: (1) it is possible to examine enzymes that are not inducible in the system under study; (2) the need for isolating the specific protein is obviated since only activity measurements are required. There is currently no clear evidence that the content of any enzyme can be induced in cultured human fibroblasts. Since these are the most readily cultivated normal human cells, methods of turnover analysis not requiring that the protein under study be inducible by agents, such as hormones, are therefore welcome. The procedure of measuring the synthesis and degradation of specific proteins usually necessitates the isolation of isotopitally-labelled molecules, and this technique has its perennially attendant problem of isotope reutilization [20]. Immunological analysis, as described in these experiments, has supported the assumptions of Pan & Krooth [21] using human diploid cells and DeLuca [22] using heteroploid cell lines that differences in catalase activity are associated with changes in their specific protein content. There is one potentially significant methodological difference between the cell catalase assays of other investigators and ours, which might invalidate such comparisons. We have determined that a detergent (in our experiments Triton X-100) is required to solubilize catalase from cultured fibroblasts. Liver catalase has been shown to be compartmented
407
in specific particles (peroxisomes) [23]; our experience with cultured fibroblasts suggests that the catalase activity of stationary phase cells also is sequestered in a particulate fraction, and is completely released into the supernatant by freezing and thawing in the presence of Triton X-100. From the experimental evidence described here, there appears to be a critical serum factor which decays in culture after a period of incubation with cells. This serum factor is needed for optimal catalase turnover, and its depletion has been shown to slow both the rate of synthesis and degradation of catalase. Data are offered principally as qualitative evidence for this effect, although calculated changes in turnover constants are presented. Nutritional influences on the steady state levels of specific enzymes have been described in vivo and these changes in activity may be explained by effects on either synthesis or degradation. With protein starvation the content of rat liver arginase rises because degradation is slowed without changing the rate of synthesis [24]. On the other hand, under the same conditions catalase degradation is unaffected, while synthesis is slowed [24]. Studies of the influence of dietary alterations on acetyl coenzyme A carboxylase suggest that independent factors regulate the rates of synthesis and degradation of this protein [25]. Kenney found that cycloheximide inhibited tyrosine aminotransferase, but stabilized the tissue content of this enzyme by concomitant slowing of degradation [26]. Clearly there is no way of predicting whether a factor affecting the level of an individual protein will do so by altering its rate of synthesis or degradation, or both. We are attempting to identify those serum factors which influence the turnover of catalase, and presumably other proteins, in cultured cells. Schwartz & Amos [27] have deExptl Cell Res 73 (1972)
408 W. J. Mellman et al. scribed the effect of insulin and serum on both protein and RNA synthesis by established cell lines. Foster & Pardee [28] have found a serum dependence of 3T3 cells for amino acid transport which was demonstrable for a period after the cells were removed from their serum-containing medium. These latter experiments are consistent with our finding that cells incubated in serum just prior to aminotriazole incubation continue to synthesize catalase for a period in serum-free medium. A large current literature attests to the role of serum factors in the multiplication and growth of cultured cells. Growth responses to serum stimulation are expressed after a lag of 24 h with human fibroblasts, with maximal mitotic peak at 40 h [29]. These findings are in keeping with our assumptions that total cell mass is not influenced by serum additions during the period of the described experiments. It is unresolved at this time whether the serum factors responsible for growth stimulation are separable from those that affect protein turnover. The growth factor of Todaro et al. [5] and the protein turnover factor we are studying both decay on incubation with cells. The estimated half-life of this growth factor in calf serum was found to be 4 h. The study of catalase turnover in cultured human fibroblasts by the isolation of isotopically labelled protein with specific antibody is complicated by its small fractional content (0.01-0.02 7” of solubilized protein). The catalase of mouse liver in contrast is 1 % of the total protein [4]. This technological complication requires special attention to the specificity of the immunoprecipitates as described in the Results. We suspect that isolations of these small fractions of the total protein will regularly require a further purification step such as the one we have used employing gel electrophoresis. Unlike the aminotriazole procedure, the radioimmunoExptl Cell Res 73 (1972)
logical approach offers a more general technique for studying the relative turnover of individual proteins as suggested by Arias et al. [18] m their in vivo studies of protein turnover. The isotope dilution estimate of the catalase fraction of the total cell protein (table 2) is supported by independent calculations based on the ratio of catalase specific activities of cell extracts to that of purified human liver catalase (table 1) prepared in our laboratory, and the similar estimate of Krooth et al. in which they compared cell extracts with crystalline beef liver catalase [3]. The double isotopic method of Arias et al. should be applicable to other enzymes of human cells in culture for which specific antibodies are available. A number of enzymes interesting from the point of view of genetics are known to express mutant genotypes in fibroblast cell cultures [30]. The authors thank Miss N. Pleibel and Mrs E. Pfendt for their valuable technical assistance, and Dr R. Palmiter and Dr P. Dehlinaer for their valuable SUEgestions to the senior auth& throughout the period of these studies. This work was performed when the senior author was a visiting scientist in the Department of Pharmacology, Stanford University School of Medicine, and was supported in part by USPHS grants GM 14931, HD 04004, HD 00588 and HD 15545.
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20. Koch, A L, J theor biol 3 (1962) 283. 21. Pan, Y L & Krooth, R S, J cell physiol 71 (1968) 151. 22. De Luca, C, Exptl cell res 44 (1966) 403. 23. De Duve. C & Baudhuin.,_P. Phvsiol _ rev 46 (1966) 323. 24. Schimke, R T, Ann NY acad sci 102 (1968) 587. 25. Majerus, P W & Kilburn, E, J biol them 244 (1969) 6254. 26. Kenney, F T, Science 156 (1967) 525. 27. Schwartz. A G & Amos. H. Nature 219 (1968) . 1366. 28. Foster, D 0 & Pardee, A B, J biol them 244 (1969) 2675. 29. Wiebel, F & Baserga, R, J cell physiol 74 (1969) 191. 30. Mellman, W J, Adv human genetics 2 (1971) 259.
Received December 15, 1971
Exptl Cell Res 73 (1972)