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Acid phosphatase of HeLa cells: Properties and regulation of lysosomal activity by serum
ARCHIVES
OF BIOCHEMISTRY
AND
172,
BIOPHYSICS
191-201 (1976)
Acid Phosphatase of HeLa Cells: Properties and Regulation of Lysosomal Activity by Serum1 CHIH-CHENG Department
of Molecular
WANG2
Biology,
AND
Vanderbilt
OSCAR TOUSTER
University,
Nashville,
Tennessee 372S5
Received June 27, 1975 Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal(70-80%) and supernatant forms (2030%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane. The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, pglucuronidase and N-acetyl-P-n-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomytin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately,, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de nouo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.
Although acid phosphatase (EC 3.1.2) has been studied extensively as a lysosoma1 enzyme, its physiological substrates and functions are basically unknown. Previous reports have suggested that multiple forms of this enzyme are present in a number of tissues (l-16). In HeLa cells the activity of acid phosphatase was influenced by serum (17-20). Rose (171, using human serum in the media of HeLa cell cultures, noted the appearance of phaseblack granules in the cytoplasm. Gropp (18) found that sera of varying origin (human, bovine, and calf) did not differ in the ability to produce granules in the cells. Later, Ahearn (19) reported that HeLa 1 This study was supported in part by Grant GB33176X from the National Science Foundation. 2 Present address: Department of Biochemistry, Southern Research Institute, Birmingham, Alabama 35205. Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
cells respond to an optimum concentration of human serum in their medium not only by greater pinocytotic activity, but also by the formation of an increased number of lysosomes, as indicated by cytochemical detection of acid phosphatase-containing granules. The process was found to be sensitive to actinomycin D (20). The aims of the present study were: (a) to determine the subcellular distribution and properties of acid phosphatase in HeLa cells, (b) to determine the effect of serum on the synthesis of acid phosphatase in the cells, and (c) to estimate the minimum stability of the mRNA for acid phosphatase. MATERIALS
AND
METHODS
Cell cultures and homogenization. Suspension cultures of HeLa cells were routinely grown in Eagle’s minimal medium without calcium ions, supplemented with glutamine, 5% calf serum, and 0.1% 191
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“Pluronic” (Wyandotte Chemical Co., Wyandotte, Mich.) (21). These cultures were maintained and furnished by Drs. D. Friedman and K. V. Kumar, tc whom we are very grateful. After centrifugation, the cells were washed three times with 5 mM TrisHCl-0.15 M NaCl, pH 7.5, and resuspended in 10 volumes of 0.25 M sucrose that had been adjusted to pH 7.0 with 1 N NaOH. Most biochemical studies were performed on cells grown in 5% serum. When other concentrations of serum were required, the cells were grown for 3 days in the medium supplemented with the desired amount of serum. Cell homogenization. The cell suspensions were homogenized in 10 volumes of 0.25 M sucrose in a Dounce homogenizer with a tight fitting pestle until 95% or more of the cells were broken; 100 strokes were required. Differential centrifugation. All centrifugations were performed at 4°C. Differential centrifugation was performed in polycarbonate tubes. The homogenates were centrifuged at 80% for 10 min in a Sorvall SS34 centrifuge to precipitate nuclei. The supernatant fraction was further centrifuged at 59OOg for 10 min to yield mitochondria. To prepare the lysosoma1 fraction, the postmitochondrial supernatant fraction was centrifuged at 26,OOOgfor 10 min in a Spinco L2 centrifuge. The lysosomal pellet was a brownish layer with a small loose white layer on top. The white layer was scraped off and removed with a pipette. The microsomal fraction was prepared from the postlysosomal supernatant fraction by centrifugation at 105,OOOgfor 60 min. Each pellet isolated, comprising nuclear, mitochondrial, lysosomal, and microsomal fractions, was resuspended in 0.25 M sucrose in volume ratios of 1:4, 1:2, 1:2, and 1:4, respectively. The supernatant solution from isolation of the microsomal fraction constituted the supernatant fraction. Density gradient centrifugation. Density gradient centrifugation was performed in sucrose-water gradients (25 ml) extending between densities of 1.06 114% (w/v)] and 1.18 140% (w/v)] and resting on a cushion (3 ml) of sucrose solution of density of 1.20 143% (w/v)]. The homogenates derived from 1 ml of packed cells were centrifuged at 105,OOOg for 60 min. The pellet containing the cell particulates (nuclear, mitochondrial, lysosomal, and microsomal fractions) was resuspended in 4 volumes of 0.25 M sucrose. Two milliliters of the suspension was layered onto the gradient. Centrifugation was performed in the SW 25.1 rotor of a Spinco Model L2 ultracentrifuge at 20,000 rpm for 3 h. Each tube was divided into approximately 15 2-ml fractions (starting from the top of the gradient) by aspiration with a glass syringe. Determination of densities of gradient fractions. The refractive index of each fraction from the gradient was determined with a Bausch and Lomb refractometer. The sucrose concentration and density
TOUSTER were calculated
from
the data compiled
by Sober
(22). Enzyme assays. All enzymatic assays were performed at 37°C. Unless otherwise stated, acid phosphatase was measured in 0.05 M acetate buffer, pH 5.0, with 5 mMp-nitrophenyl phosphate (Sigma 104) as substrate in a final volume of 0.5 ml. The pnitrophenol was determined spectrophotometrically at 400 nm after the reaction was stopped with 1.5 ml was used of 0.05 N NaOH. When P-glycerophosphate as the substrate, the inorganic phosphate released was determined according to the method of Dryer et al. (23). /3-glucuronidase (EC 3.2.1.31) was determined according to Stahl and Touster (24). N-acetylp-n-hexosaminidase (EC 3.3.1.30) was assayed in 0.5 ml of 0.05 M citrate buffer, pH 5.0, which was 5 mM N-acetyl-p-n-glucosaminide p-nitrophenyl i&hwartziMann). The reaction was stopped with 1.0 ml of 0.05 N NaOH, followed by 1.0 ml of stopping reagent which consisted of 0.133 glycine, 0.067 M NaCl, and 0.983 M Na,CO,, pH 10.7 (25). The pnitrophenol was determined as described above. In this paper, the term “free activity” of a lysosoma1 enzyme denotes the percentage activity of the enzyme in a lysosome-containing fraction relative to the activity of this fraction observed in the presence of 0.1% Triton X-100. Glucose-6-phosphatase activity (EC 3.1.3.9) was determined according to the method of Swanson (261, with phosphate determined by the method of Dryer et al. (23). E&erase activity (EC 3.1.1) was measured by the Gomori method (27) as modified by Schiff et al. (28). NADPH-cytochrome c reductase (EC 1.6.2.1) was assayed as described previously (29). Succinate-cytochromec reductase (EC 1.3.99.1) was determined according to Green et al. (30). Protein was measured by the method of Lowry et al. (311, as modified by Miller (32). Radioactive isotope incorpomtion studies. At time intervals, 5 ml of cell suspensions were incubated in duplicate with 0.5 @i of [3H]uridine (Schwarz! Mann) for 30 min at 37°C in a prewarmed tube gassed with 5% CO,. The pulse was stopped by the addition of 25 ml of cold Spinner salt solution (33), pH 7.4. Cells collected by centrifugation at 60% for 10 min were washed once with cold 5 mM Tris-HCl0.15 M NaCI, pH 7.4, and resuspended in 2.0 ml of cold 5% trichloroacetic acid for 15 min at 5°C. The resulting precipitate was collected on a Millipore filter. Radioactivity was determined by liquid scintillation counting in 10 ml of a solution of 0.1 g ofpbis[2-(5-phenyl-oxazolyl)lbenzene and 4 g of 2,5-diphenyloxazole per liter of toluene. The background radioactivity was about 6 cpm. RESULTS
Measurement of acid phosphatase. In the cell homogenates the amount ofp-nitro-
ACID
PHOSPHATASE
phenol released under the assay conditions varied linearly with the amount of protein at concentrations of 20 pg or greater, but not below this amount. A similar nonlinear relationship was noted with the supernatant fraction as the source of enzyme, but not with the lysosomal fraction. This may be due to the presence of a dissociable activator or coenzyme (34) in the supernatant. Alkaline phosphatase of renal cells also exhibits this type of abnormal kinetics (35). In the present work, the acid phosphatase activity was determined routinely with 25-100 pg of protein per assay. To assess the specificity of the method employingp-nitrophenyl phosphate, determinations of acid phosphatase were also made by an assay using p-glycerophosphate as substrate. While the latter was hydrolyzed, at a concentration (50 mM) generally used (251, inhibition was observed. The relative activities in the homogenate at 50, 10, and 5 mM concentrations were 1, 12.0, and 12.5, respectively, taking the activity at 50 mM substrate concentration as unity. Since the results obtained using 5 mM P-glycerophosphate were generally similar to, but less reproducible than, those obtained with nitrophenyl phosphate, the latter substrate was employed, unless otherwise stated. Differential centrifugation. Fig. 1 shows the results of differential centrifugation of a homogenate of HeLa cells. The distribution pattern of acid phosphatase expressed as relative specific activity is similar to that of P-glucuronidase and N-acetyl-p-nhexosaminidase and distinct from that of succinate-cytochrome c reductase. The profile of the acid phosphatase shows the highest peak. in the lysosomal fraction and the second highest peak in the mitochondrial fraction, a result typical of true lysosomal enzymes (36). However, there is no clear separation between the lysosomal and microsomal marker enzymes, although the microsomal marker enzymes glucose-6-phosphatase and NADPH-cytochrome c reductase show their second highest peak in the microsomal fraction. It would appear that endoplasmic reticulum fragments of HeLa cells sediment at a similar rate to the lysosomal population and appear in the lysosomal fraction. This sug-
OF HeLa
CELLS
x Protaln FIG. 1. Subcellular distribution patterns of six marker enzymes in subcellular fractions from HeLa cells. Fractions plotted in order of collection, from left to right, are nuclear (N), mitochondrial (M), lysosomal (L), microsomal (P), and supernatant (S). Ordinate: relative specific activity for each fraction. Abscissa: percentage total protein per fractions.
gests either that only a small difference in sedimentation coefficients exists between the microsomes and lysosomes of HeLa cells or that the marker enzymes used are less suitable for these cells than for other mammalian cells. As shown in Fig. 1, the relative specific activities of the acid phosphatase in the lysosomal and supernatant fractions are, respectively, lower and higher than those of the other two lysosomal enzymes, pglucuronidase and N-acetyl-fi-n-hexosaminidase. These results can be accounted for by the presence of an extralysosomal acid phosphatase in the supernatant fraction, which hydrolyzesp-nitrophenyl phosphate. Density
gradient
centrifugation.
The
above experiments suggested that HeLa cells contain at least two forms of acid phosphatase with specific intracellular locations. The percentage of acid phospha-
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tase present in the supernatant fraction essentially the same distribution pattern was studied by centrifuging the homogeas p-glucuronidase and N-acetyl-p-n-hexnate at 105,OOOgfor 60 min to separate the osaminidase and it is different from microsupernatant from the pellet fractions. In somal and mitochondrial enzymes. six experiments, the recoveries of activity The apparent position of the lysosomal in the supernatant fraction were found to enzymes at equilibrium densities lower range from 20 to 33% of total recovered than that of the mitochondria, i.e., at activity. That the activity associated with 1.11-1.15 g cmP3, represents a situation the pellet fraction is present in lysosomes similar to that found for Ehrlich ascites was shown by density gradient centrifugatumor cells (371, beef heart (381, and rat tion as follows. lymphoid tissues (39), but opposite to those Since differential centrifugation was rel- of liver and kidney. Differentiation of theproperties of lysosoatively ineffective in separating lysosomes from endoplasmic reticulum, density gra- ma1 and soluble cytoplasmic acidphospharesults were obtained dient centrifugation was employed. In a tases. Similar preliminary experiment in which the su- whether the cells were grown in 5 or 30% serum. crose gradient (25 ml) extended between pH optima. Soluble acid phosphatase densities 1.10 and 1.25 and rested on a cushion (3 ml) of a solution of density 1.27, and lysosomal acid phosphatase showed the equilibrium density of mitochondria distinct pH activity profiles (Fig. 3). The (as represented by succinate-cytochrome c lysosomal enzyme has a broad activity optireductase) was 1.16 g cm-3, while acid phos- mum near pH 4, with activity rapidly dephatase was found between 1.10 and 1.13. clining above pH 5. The supernatant enFigure 2 shows the profiles for acid phos- zyme displays a relatively sharp pH optiphatase, p-glucuronidase, N-acetyl-p-nmum at pH 5.7 with the activity falling off hexosaminidase, glucose-6-phosphatase, on either side of this pH. These differences e&erase, and succinate-cytochrome c re- in pH requirements are similar to those ductase in a density gradient extending reported by Cristofalo et al. (10) for acid from 1.06 to 1.18. Acid phosphatase has phosphatases of human WI-38 cells, by Nelson (6) for rat liver, by Yu et al. (8) for leucocytes, and by Nicholson and Davies (15) for rat mammary carcinoma. Sensitivity to inhibitors. The effects of ntartrate and fluoride on acid phosphatase in the lysosomal and supernatant fractions were determined. Fluoride inhibited the lysosomal activity 81 and 76% at 2 and 20 mM concentrations, respectively, whereas the inhibition of the supernatant activity was only 10 and 9% at these fluoride concentrations. Similarly, 20 mM tartrate inhibited the lysosomal fraction 43%, but inhibited the supernatant activity only 7.5%. Thus, these results confirm previous findings (5-7) from other tissues indicating differences between the acid phosphatase FIG. 2. Distribution of enzymes after centrifugaof lysosomal and soluble fractions. tion of a HeLa cell homogenate in a sucrose gradient Thermal stability. The differences beat 25,000 rpm for 3 h in a Spinco SW 25.1 rotor. tween the lysosomal and supernatant acid Details are described under Materials and Methods. phosphatases were further revealed by The recoveries for the enzyme activities are: acid heating at 54°C the lysosomal and supernaphosphatase, 77.8%; p-glucuronidase, 99.5%; N-acetant fractions (see Materials and Methtyl-p-n:hexosaminidase, 112%; glucose-6-phosphaods). The lysosomal enzyme showed a tase, 118%; e&erase, 70.1%; succinate-cytochrome c reductase, 72.3%; protein, 110%. rapid loss of about 35% activity after 5 min
ACID
PHOSPHATASE
\ PH
FIG. 3. Effect of pH on the hydrolysis of p-nitrophenyl phosphate by lysosomal, soluble cytoplasmic, and serum acid phosphatases. The buffers were 0.05 M sodium acetate.
of incubation, which was followed by a gradual decline in activity to 35% of its original activity after 2 h. In contrast, the acid phosphatase from the supernatant fraction showed a very rapid inactivation to 15% of the original activity within a few minutes; further loss of activity occurred with prolonged incubation. The initial sharp drop in the activities of both enzymes suggests that more than one acid phosphatase may be present in both the lysosomal and supernatant fractions. Kinetics. Increasing the concentration of p-nitrophenyl phosphate from 0.05 to 5 mM had no inhibitory or activating effects on acid phosphatase of the supernatant fraction. On the other hand, lysosomal acid phosphatase is “activated” by high substrate concentrations. Lineweaver-Burk plots (Fig. 4) show the normal and anomalous kinetics displayed by the supernatant and lysosomal acid phosphatase, respectively. The apparent K, values calculated from the linear portion of the plot were 0.11 mM for the supernatant enzyme (or enzymes) and 0.09 mM for the lysosomal enzyme (or enzymes). Distinction between lysosomal acidphosphatase and glucose&phosphatase activities. The experimental results reported above constituted strong evidence for the presence of distinctive lysosomal and soluble cytoplasmic acid phosphatase activities. It is also of interest to record an experiment bea.ring on the question ‘as to whether there is any overlap in reactivity between a-nitronhenvl nhosnhatase and
OF HeLa
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CELLS
Alactivities. glucose-6-phosphatase though fresh cells showed, after homogenization and density gradient centrifugation, only one glucose-6-phosphatase peak (Fig. 2), it was accidentally discovered that cells, when stored for several days at -70°C before homogenization and centrifugation, showed two peaks of glucoseSphosphatase activity (Fig. 51, a slight difference in the apparent equilibrium positions for both acid phosphatase and glucase-6-phosphatase being observed between cells grown in 5% and 30% serum. The appearance of the peak of acid phosphatase at the trough of glucose-6-phosphatase (Fig. 5, lower graph) makes the possibility rather unlikely that acid phosphatase hydrolyzed glucose-gphosphate or that glucose-6-phosphatase hydrolyzes pnitrophenyl phosphate to an appreciable extent. Structure-lined latency of the lysosomal enzymes. Since the subcellular distribution pattern indicated that the three lysosoma1 marker enzymes are contained in particles with similar sedimentation coefficient and density, it remained to be established that these enzymes also exhibit the structural latency expected for lysosomal LYSOSOMaL
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FIG. 4. Lineweaver-Burk plots showing the effect of p-nitrophenyl phosphate concentrations on the rate of its hydrolysis by lysosomal and soluble cytoplasmic enzymes. The proteins used in each assay were 25 and SO fig for the lysosomal and soluble fractions. resoectivelv.
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ma1 acid phosphatase. The biochemical properties of the bound and soluble forms were similar, no differences being observed in pH optima, kinetics, or response to inhibition by sodium fluoride and sodium tartrate.
Thermal activation of enzymes in lysosomes. Aliquots of the lysosomal fraction 30%
Serum
r
in 0.25 M sucrose were adjusted to contain 0.1% Triton X-100 and then incubated for varying lengths of time at 37°C and pH 5.0. Assays for free activity of acid phosphatase, P-glucuronidase, and N-acetyl-p-Dhexosaminidase gave similar results, namely, about 40% free activity at zero time, about 80% at 60 min, and about 90% at 90 min.
The influence of serum concentrations on acid phosphatase content. The previous FIG. 5. Distribution of acid phosphatase and glucase-8phosphatase after homogenization of HeLa cells which have been frozen for 5 days at -70°C. The serum concentrations used for growing the cells were 5 and 30%.
enzymes. Triton X-100 (0.1%) was found to unmask the latent activity of the three enzymes in the lysosomal fraction. However, the percentage of the free activity of each enzyme was found to vary somewhat in different experiments, probably as a result of variations in the handling and assay of the isolated fractions. Free activity for acid phosphatase ranged from 41 to 52%, for p-glucuronidase from 32 to 48% and for N-acetyl-p-n-hexosaminidase from 22 to 37%. The three lysosomal enzymes are released in an approximately parallel fashion by repeated freezing and thawing (Fig. 6); after five cycles no additional activity could be released. Further release of about 35% p-glucuronidase could be achieved by washing the sediment with 0.4 M NaCl. Similar treatment caused the release of 4 and 17% of acid phosphatase and N-acetylp-n-hexosaminidase, respectively. These results suggest that the major lysosomal acid phosphatase activity is associated with the membrane. In contrast to freezing and thawing, Triton X-100 (0.1%) was able to release more than 95% of the lysoso-
hi&chemical findings (17) suggested that serum played an important role in the formation of phase-black granules. To examine this phenomenon in more detail, quantitative biochemical studies were carried out on HeLa cell suspension cultures in media containing various grown amounts of calf serum. In a preliminary experiment, cells were grown for 3 days in Eagle’s minimal medium supplemented with 1, 10, and 30% serum, and the acid
.“” 80 !
! FIG. 6. Release of acid phosphatase, p-glucuronidase and N-acetyl-P-n-hexosaminidase from HeLa cell iysosomes (L fraction) upon freezing and thawing. After each thawing a portion of the fraction was removed, centrifuged (105,OOQg for 30 min) and assayed for the enzyme activities. The recoveries for the enzyme activities (including residual membrane); after five cycles of freezing and thawing, were: N-acetyl-p-n-hexosaminidase, 36%; p-glucuronidase, 81%; acid phosphatase, 36%.
ACID
PHOSPHATASE
phosphatase was assayed using either pnitrophenyl phosphate or P-glycerophosphate as the substrate. The patterns (Fig. 7) obtained with homogenate, mitochondrial plus lysosomal fraction, and the pellet fractions were almost identical with the two substrates, but a small difference was observed with the supernatant fraction, which could be due to the occurrence, in this fraction, of a minor form of acid phosphatase which has different specificity for p-nitrophenyl phosphate and /3-glycerophosphate. Because of the similarity in these results and because of the simplicity of assays employing p-nitrophenyl phosphate, this substrate was used in subsequent studies involving the effect of serum. In an extended study, including estimations of p-glucuronidase and N-acetyl-p-nhexosaminidase, as well as acid phosphatase, the content of all these enzymes, as well as cellular protein, was found to increase with increasing concentrations of serum in the medium (Fig. 8a). However, the increase in p-glucuronidase and N-acetyl-p-n-hexosaminidase was much less than that of protein, whereas the increase in acid phosphatase was much greater. Thus, the concentration of serum had opposite effects on the relative specific activity of acid phosphatase and the two lysosomal of enzymes, taking the specific activity each enzyme of the cells grown in 1% serum as unit,y (Fig. 8b). This fact seems to suggest that acid phosphatase and the other two lysosomal enzymes are present in different lysosomal particles or are controlled by different mechanisms. Although the hexosaminidase seems to be affected only to a minor extent (Fig. 8a), its activity in low serum (1%) was found in many experiments to be consistently less than that in high serum (30%). The increase in acid phosphatase activity in HeLa cells with high concentration of serum cannot be due to specific uptake of serum acid phosphatase since (a) the amount of acid phosphatase initially present in the serum is too low to account for the activity increase in the cells (see legend to Fig. 81, and (b) the serum acid phosphatase shows a pH activity profile differ-
OF HeLa
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FIG. 7. Effect of serum concentrations (o/o)on acid phosphatase activity in HeLa cells. The cells were grown in serum for 3 days. Ordinate: relative specific activity for each fraction taking the activity of the cells grown in 1% serum as unity. Abscissa: fractions assayed; H, homogenate; M + L, mitochondria and lysosomes; Pt, 105,OOOgpellet (i.e., H-S); S, supernatant. The substrates used were p-nitrophenyl phosphate and P-glycerophosphate.
ent from that of the phosphatase in HeLa cells (Fig. 3). The high activity of acid phosphatase at high serum concentration cannot be explained by the possible existence of activators or inhibitors of enzyme activity, since enzyme activities in mixtures of homogenates of 1 and 30% serum induced cells were additive. Effect of actinomycin D, cordycepin, and cycloheximide on serum induction of acidphosphatase. Fig. 9a shows the effects of actinomycin D and cordycepin at 5.0 and 25 pug/ml, respectively, on the serum induction of acid phosphatase. Actinomycin D added 24 h after serum induction was found to reduce L3H]uridine incorporation into nucleic acids by 95% (see Materials and Methods for isotope incorporation studies). However, the rate of acid phosphatase synthesis was unaffected during the following 8 h (Fig. 9a). Beyond 8 h, the data are unreliable because of irreversible cell damage. Similarly, cordycepin had no effect on acid phosphatase for 8 h, although it may be noted that it reduced radioactive uridine incorporation into the nucleic acids of HeLa cells only by an initial 65% at 0.5 h. Figure 9b shows that cycloheximide added 58 h after serum induction at 5 pg/ml markedly reduced the acid phospha-
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FIG. 8. Effect of serum concentration on the activity of acid phosphatase, N-acetyl-P-nhexosaminidase, and P-glucuronidase and on protein in HeLa cell homogenates. The cells were grown in serum for 4 days. On the ordinates are plotted relative specific activity for each enzyme. (a) The levels of activity in the cells obtained from 1, 5, 10, 20, and 30% serum were: acid phosphatase, 0.52, 0.74,1.16,2.42, and 2.36; N-acetyl-P-n-hexosaminidase, 1.07,1.21, 1.41, 1.35, and 1.42; p-glucuronidase, 0.016,0.015, 0.021,0.023, and 0.028 unit&O6 cells, respectively. One unit corresponds to the activity hydrolyzing 1 pmol of substrate/h. The levels of enzyme activity in calf serum are 0.24, 5.22, and 0.0059 units/ml for acid phosphatase, N-acetyl-P-nrespectively. (b) The actual specific activities hexosaminidase and P-glucuronidase, (pmol/h/mg protein) of the homogenates from 1, 5, 10, 20, and 30% serum are: acid phosphatase, 1.98, 2.15, 2.54, 2.87, and 3.31; N-acetyl-p-n-hexosaminidase, 3.30, 2.79, 2.49, 2.20, and 1.60; /3-glucuronidase, 0.062, 0.043, 0.047, 0.048, and 0.039, respectively. The specific activities of the enzymes in the calf serum used are: acid phosphatase, 0.0024, N-acetyl-P-n-hexosaminidase, 0.052; P-glucuronidase, 0.00006, respectively. In these experiments each 8 x lo6 cells was initially exposed to 100 ml of medium containing the desired amount of serum in a given flask. On Day 3, 50 ml of fresh medium was added to each flask. The cell concentrations at the time of harvest (Day 4) were 247,000,411,000,337,000, 346,000, and 250,000 cells/ml for the cells grown in 1, 5, 10, 20, and 30% serum, respectively.
tion to the active site. This theory was tase activity, whereas cordycepin, again, had little effect on acid phosphatase for 12 used to explain the abnormal kinetics of h. some enzymes (42-44) until the concept of negative cooperativity was developed (40, DISCUSSION 45). In the case of the lysosomal acid phosThe present report presents evidence phatase in HeLa cells, no distinction that HeLa cells contain both lysosomal among the three possibilities can be made and soluble cytoplasmic acid phosphatase without the availability of pure enzyme. activities that exhibit different pH-activThe third explanation seems most likely in ity profiles, heat inactivation rates, inhibiview of the reported multiplicity of acid tion by fluoride and tartrate, and enzyme phosphatase in mammalian lysosomes kinetics. There are three possible interpre(e.g., 1). Indeed, we have preliminary inditations for the biphasic curve in the Line- cations that HeLa cells also contain more weaver-Burk plot (Fig. 4) observed for ly- than one phosphatase. Upon DEAE-cellusosomal acid phosphatase: (a) enzyme acti- lose3 chromatography of 0.1% Triton Xvation at high substrate concentration 100~solubilized lysosomes at pH 7.8, three (401, 6) negative cooperativity (401, and (c) enzyme peaks were eluted from the cola mixture of two or more enzymes each umn, with unfortunately only lo-20% rewith different affinity for substrate (41). The first possibility implies that there may 3 Abbreviation used: DEAE-cellulose; 0-(diethylbe a second activator substrate site in addi- aminoethyl) cellulose.
ACID
PHOSPHATASE
OF HeLa
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FIG. 9. (al Effect of cordycepin (25 pg/ml) and actinomycin D (5 pg/ml) on the induction of acid phosphatase by 30% serum. Cells were previously grown in 1% serum for 4 days. Arrow indicates the addition of drugs. One unit corresponds to one OD,J30 min. (b) Effect of cycloheximide (5 wg/ml) and cordycepin _ _ (25 &ml) on the induction of acid phosphatase by 30% serum. Other details as for Fig. 9a.
covery in total activity. Each of these peaks displayed normal Michaelis-Menten kinetics. More information on this aspect must await further purification of the enzymes. It is interesting that only a small percentage of lysosomal acid phosphatase can be released from the lysosomes by freezing and thawing followed by washing with salt. An inability to release all acid phosphatase from rat liver lysosomes by physical disruption was reported by Shibko and Tappel (7) and by Allen and Gockerman (46). However, in the latter cases, it was not clear whether the acid phosphatase associated with the sediment represented adsorption of the enzyme onto the membrane fragments, since no washing with salt was performed. Some lysosomal enzymes, particularly, N-acetylhexosaminidase (47) an.d deoxyribonuclease II of rat liver (48) appear to adsorb readily to the membranes and are released by washing with salt. Serum is generally required by mammalian cells for growth in culture. In addition to its influence on protein and nucleic acid synthesis, serum also regulates alkaline phosphatase in KB cells (49), modulates pyruvate kinase in RLC cells (501, enhances the hormonal induction of tyrosine aminotransferase in HTC cells (51), and influences catalase turnover in human diploid cell culture (52). Calf serum, but not human serum, causes the formation of Nglycolylneuraminic acid in HeLa cells (53).
Our observations that serum stimulates acid phosphatase in an established cell line of human origin represent another example of such action. Because serum is a complex material, it is possible that stimulation is the result of several independent or interdependent actions of various serum components. The kinetics of serum induction of acid phosphatase in HeLa cells is similar to the kinetics of acid hydrolase production in mononuclear phagocytes after adding serum (54); the curves for HeLa cells vary from linear to slightly concave. The results presented in this study suggest that de nouo protein synthesis is involved in the induction of acid phosphatase by serum. Cycloheximide (5 pg/ml), an inhibitor of protein synthesis, immediately blocks further serum induction of acid phosphatase. On the other hand, cordycepin (25 pglml) or actinomycin D (5 pglml), added during the period of enzyme increase, do not interfere with the enzyme for at least 8 h after their addition to the culture. In HeLa cells, cordycepin, at the concentration used, appears to cause a marked suppression of the synthesis of cytoplasmic mRNA (55). Actinomycin D is also known to markedly inhibit messenger RNA synthesis (56, 57). Preexisting messenger is insensitive to the effects of cordycepin and actinomycin D and is capable of directing further protein synthesis until it is inactivated. The lifespan of this mRNA (or mRNA’s) can be realistically estimated
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WANG
AND
to be at least 8 h, since both cordycepin and actinomycin D, which inhibit mRNA synthesis by different mechanisms, do not interfere with enzyme induction for this period of time. It appears that messenger RNA synthesis is not rate-limiting in lysosomal acid phosphatase synthesis, at least under the specific conditions of these experiments involving induction by serum. ACKNOWLEDGMENTS We are indebted to Mrs. Vera Coleman for extensive laboratory assistance. REFERENCES 1. ARSENIS, C., AND TOUSTER, 0. (1968) J. Biol. Chem. 243, 5702-5708. 2. GOODLAD, G. A. J., AND MILLS, G. T. (1957) B&hem. J. 66, 346-354. 3. BARKA, T. (1961) J. Histochem. Cytochem. 9, 564-571. 4. GEORGATSOS,J. G. (1965) Arch. B&hem. Biophys. 110,354-356. 5. MOORE, B. W., AND ANGELEITI, P. U. (1961) Ann. N. Y. Acad. Sci. 94, 659-667. 6. NELSON, B. D. (1966) hoc. Sot. Exp. Biol. Med. 121,998-1001. 7. SHIBKO, S., AND TAPPEL, A. L. (1963) Biochim. Biophys. Acta 73, 76-78. 8. Yu, B. P., KUMMEROW, F. A., AND NISHIDA, T. (1966) Proc. Sot. Exp. Biol. Med. 122, 10451048. 9. NEIL, M. W., AND HORNER, M. W. (1964) Biothem. J. 92, 217-224. 10. CRISTOFALO, V. J., KABAKJIAN, J. R., AND KRITCHEVSKY, D. (1967) Proc. Sot. Exp. Biol. Med. 126, 649-653. 11. DIPIETRO, D. L., AND ZENGERLE, F. S. (1967) J. BioZ. Chem. 242, 3391-3396. 12. SMITH, J. K. K., AND WHITBY, L. G. (1968) Biochim. Biophys. Acta 151,607-618. 13. HEINRIKSON, K. L. (1969) J. BioZ. Chem. 244, 299-307. 14. VANHA-PERTTULA, T. (1970) Biochim. Biophys. Acta 227,390-401. 15. NICHOLSON, R. I., AND DAVIES, M. (1974) Eur. J. B&hem. 44, 25-35. 16. GONZALES, L. W., AND MEIZEL, S. (1973) Biochim. Biophys. Acta 320, 166-179. 17. RQSE, G. G. (1957) Biophys. Biochem. Cytol. 3, 697-704. 18. GROPP, A. (1963) in Cinemicrography in Cell Biology (Rose, G. G., ed.), Academic Press, New York/London. 19. AHEARN, M. J. (1962) M. A. thesis, University of Texas, Austin. 20. AHEARN, M. J., HAMILTON, T. H., AND BIESELE,
TOUSTER
21. 22. 23. 24. 25. 26. 27. 28.
29. 30. 31.
32. 33. 34. 35. 36.
37. 38.
39. 40. 41. 42. 43. 44. 45. 46.
J. J. (1966) Proc. Nat. Acad. Sci. USA 55, 852-857. SWIM, H. H., AND PARKER, R. F. (1960) Proc. Sot. Exp. BioZ. Med. 103, 252-254. Handbook of Biochemistry (1968), The Chemical Rubber Co., Cleveland, Ohio, p. j-248. DRYER, R. L., TAMMES, A. R., AND ROUTH, J. I. (1957) J. BioZ. Chem. 225, 177-183. STAHL, P. D., AND TOUSTER, 0. (1971) J. BioZ. Chem. 246,5398-5406. GIANETTO, R., AND DEDUVE, C. (1955) Biochem. J. 59,433-438. SWANSON, M. A. (1955) Methods Enzymol. 2, 541-543. GROMORI, G. (1953) J. Lab. CZin. Med. 42,445453. SCHIFF, R., BRUNSTETTER, M. A., HUNTER, R. L., AND CROSS, C. E. (1970) J. His&hem. Cytochem. 18, 167-177. HORVAT, A., BAXANDALL, J., AND TOUSTER, 0. (1969) J. Cell BioZ. 42, 469-479. GREEN, D. E., MII, S., AND KOHOUT, P. M. (1955) J. BioZ. Chem. 217, 551-567. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioZ. Chem. 193, 265-275. MILLER, G. L. (1959)AnaZ. Chem. 31, 964. EAFLLE, W. R. (1943) J. Nat. Cancer Inst. 4,165212. DIXON, M., AND WEBB, E. C. (1964) Enzymes, 2nd ed., p. 61, Academic Press, New York. Cox, R. P., AND MACLEOD, C. M. (1962) J. Gen. Physiol. 45, 439-485. DEDUVE, C., PRESSMAN, B. C., GIANETTO, R., WATTIAUX, R., AND APPELMANS, F. (1955) Biothem. J. 60,604-617. HORVAT, A., AND TOUSTER, 0. (1967) Biochem. Biophys. Acta 148, 725-740. ROMEO, D., STAGNI, N., SOTTOCASA, G. L., PUGLIARELLO, M. C., DE BERNARD, B., AND VITTUR, F. (1966) B&him. Biophys. Acta 130, 64-80. BOWERS, W. E., AND DEDUVE, C. (1967) J. Cell BioZ. 32, 339-348. CONWAY, A., AND KOSHLAND, D. E., JR. (1968) Biochemistry. 7, 4011-4023. THOMPSON, W. J., AND APPLEMAN, M. M. (1971) Biochemistry 10, 311-316. OLSON, J. A., AND ANFINSEN, C. B. (1953) J. BioZ. Chem. 202, 841-856. OKAZAKI, R., AND KORNBERG, A. (1961) J. BioZ. Chem. 239, 275-284. NISSELBAUM, T. A., AND BODANSKY, 0. (1961) J. BioZ. Chem. 236, 323-327. LY~TER, R. L. J., AND WATTS-TOBIN, R. J. (1968) J. Mol. BioZ. 31, 617-619. ALLEN, J. M., AND GQ~KERMAN, J. (1964) Ann. N. Y. Acad. Sci. 121, 616-633.
ACID 47. BACCINO, F. M.,
B&him. 48. Dummy,
PHOSPHATASE
AND ZURETTI, M. F. (1971)
Biophys. A&
235,353-357.
J. T., AND TOUSTER, 0. (1972) J. Biol.
Chem. 246, 1424-1432. 49. HERZ, F., KAPLAN, E., AND FINEMAN, E. L. (1973) Biochim. Biophys. Actu 304,660-668. 50. GERSCHENSON, L. E., AND ANDERSSON, M. (1971) Biochem. Biophys. Res. Commun. 43, 12111218. 51. GELEHRTEI~, T. D., EMANUEL, J. R., AND SPENCER, C. J. (1972) J. Biol. Chem. 247, 6197-
6203. 52. MELLMAN, W. J., SCHIMKE, R. T., AND HAY-
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CELLS
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FLICK, L. (1972) Exp. Cell Res. 73.399-409. 53. HOF, L., AND FAILLARD, H. (1973) Biochim. Biophys. Actu 297, 561-563. 54. COHN, Z. A., AND BENSON, B. (1965) J. Exp. Med. 121,835~848. 55. PENMAN, S., ROSBASH, M., AND PENMAN, M. (1970) Proc. Nat. Acad. Sci. USA 67, 18781885. 56. REICH, E., FRANKLIN, R. M., SHATKIN, A. J., AND TATUM, E. L. (1962)Proc. Nat. Acad. Sci. USA 48, 1238-1245. 57. FRANKLIN, R. M. (1963) Biochim. Biophys. Actu
72, 555-565.
OF BIOCHEMISTRY
AND
172,
BIOPHYSICS
191-201 (1976)
Acid Phosphatase of HeLa Cells: Properties and Regulation of Lysosomal Activity by Serum1 CHIH-CHENG Department
of Molecular
WANG2
Biology,
AND
Vanderbilt
OSCAR TOUSTER
University,
Nashville,
Tennessee 372S5
Received June 27, 1975 Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal(70-80%) and supernatant forms (2030%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane. The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, pglucuronidase and N-acetyl-P-n-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomytin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately,, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de nouo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.
Although acid phosphatase (EC 3.1.2) has been studied extensively as a lysosoma1 enzyme, its physiological substrates and functions are basically unknown. Previous reports have suggested that multiple forms of this enzyme are present in a number of tissues (l-16). In HeLa cells the activity of acid phosphatase was influenced by serum (17-20). Rose (171, using human serum in the media of HeLa cell cultures, noted the appearance of phaseblack granules in the cytoplasm. Gropp (18) found that sera of varying origin (human, bovine, and calf) did not differ in the ability to produce granules in the cells. Later, Ahearn (19) reported that HeLa 1 This study was supported in part by Grant GB33176X from the National Science Foundation. 2 Present address: Department of Biochemistry, Southern Research Institute, Birmingham, Alabama 35205. Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
cells respond to an optimum concentration of human serum in their medium not only by greater pinocytotic activity, but also by the formation of an increased number of lysosomes, as indicated by cytochemical detection of acid phosphatase-containing granules. The process was found to be sensitive to actinomycin D (20). The aims of the present study were: (a) to determine the subcellular distribution and properties of acid phosphatase in HeLa cells, (b) to determine the effect of serum on the synthesis of acid phosphatase in the cells, and (c) to estimate the minimum stability of the mRNA for acid phosphatase. MATERIALS
AND
METHODS
Cell cultures and homogenization. Suspension cultures of HeLa cells were routinely grown in Eagle’s minimal medium without calcium ions, supplemented with glutamine, 5% calf serum, and 0.1% 191
192
WANG
AND
“Pluronic” (Wyandotte Chemical Co., Wyandotte, Mich.) (21). These cultures were maintained and furnished by Drs. D. Friedman and K. V. Kumar, tc whom we are very grateful. After centrifugation, the cells were washed three times with 5 mM TrisHCl-0.15 M NaCl, pH 7.5, and resuspended in 10 volumes of 0.25 M sucrose that had been adjusted to pH 7.0 with 1 N NaOH. Most biochemical studies were performed on cells grown in 5% serum. When other concentrations of serum were required, the cells were grown for 3 days in the medium supplemented with the desired amount of serum. Cell homogenization. The cell suspensions were homogenized in 10 volumes of 0.25 M sucrose in a Dounce homogenizer with a tight fitting pestle until 95% or more of the cells were broken; 100 strokes were required. Differential centrifugation. All centrifugations were performed at 4°C. Differential centrifugation was performed in polycarbonate tubes. The homogenates were centrifuged at 80% for 10 min in a Sorvall SS34 centrifuge to precipitate nuclei. The supernatant fraction was further centrifuged at 59OOg for 10 min to yield mitochondria. To prepare the lysosoma1 fraction, the postmitochondrial supernatant fraction was centrifuged at 26,OOOgfor 10 min in a Spinco L2 centrifuge. The lysosomal pellet was a brownish layer with a small loose white layer on top. The white layer was scraped off and removed with a pipette. The microsomal fraction was prepared from the postlysosomal supernatant fraction by centrifugation at 105,OOOgfor 60 min. Each pellet isolated, comprising nuclear, mitochondrial, lysosomal, and microsomal fractions, was resuspended in 0.25 M sucrose in volume ratios of 1:4, 1:2, 1:2, and 1:4, respectively. The supernatant solution from isolation of the microsomal fraction constituted the supernatant fraction. Density gradient centrifugation. Density gradient centrifugation was performed in sucrose-water gradients (25 ml) extending between densities of 1.06 114% (w/v)] and 1.18 140% (w/v)] and resting on a cushion (3 ml) of sucrose solution of density of 1.20 143% (w/v)]. The homogenates derived from 1 ml of packed cells were centrifuged at 105,OOOg for 60 min. The pellet containing the cell particulates (nuclear, mitochondrial, lysosomal, and microsomal fractions) was resuspended in 4 volumes of 0.25 M sucrose. Two milliliters of the suspension was layered onto the gradient. Centrifugation was performed in the SW 25.1 rotor of a Spinco Model L2 ultracentrifuge at 20,000 rpm for 3 h. Each tube was divided into approximately 15 2-ml fractions (starting from the top of the gradient) by aspiration with a glass syringe. Determination of densities of gradient fractions. The refractive index of each fraction from the gradient was determined with a Bausch and Lomb refractometer. The sucrose concentration and density
TOUSTER were calculated
from
the data compiled
by Sober
(22). Enzyme assays. All enzymatic assays were performed at 37°C. Unless otherwise stated, acid phosphatase was measured in 0.05 M acetate buffer, pH 5.0, with 5 mMp-nitrophenyl phosphate (Sigma 104) as substrate in a final volume of 0.5 ml. The pnitrophenol was determined spectrophotometrically at 400 nm after the reaction was stopped with 1.5 ml was used of 0.05 N NaOH. When P-glycerophosphate as the substrate, the inorganic phosphate released was determined according to the method of Dryer et al. (23). /3-glucuronidase (EC 3.2.1.31) was determined according to Stahl and Touster (24). N-acetylp-n-hexosaminidase (EC 3.3.1.30) was assayed in 0.5 ml of 0.05 M citrate buffer, pH 5.0, which was 5 mM N-acetyl-p-n-glucosaminide p-nitrophenyl i&hwartziMann). The reaction was stopped with 1.0 ml of 0.05 N NaOH, followed by 1.0 ml of stopping reagent which consisted of 0.133 glycine, 0.067 M NaCl, and 0.983 M Na,CO,, pH 10.7 (25). The pnitrophenol was determined as described above. In this paper, the term “free activity” of a lysosoma1 enzyme denotes the percentage activity of the enzyme in a lysosome-containing fraction relative to the activity of this fraction observed in the presence of 0.1% Triton X-100. Glucose-6-phosphatase activity (EC 3.1.3.9) was determined according to the method of Swanson (261, with phosphate determined by the method of Dryer et al. (23). E&erase activity (EC 3.1.1) was measured by the Gomori method (27) as modified by Schiff et al. (28). NADPH-cytochrome c reductase (EC 1.6.2.1) was assayed as described previously (29). Succinate-cytochromec reductase (EC 1.3.99.1) was determined according to Green et al. (30). Protein was measured by the method of Lowry et al. (311, as modified by Miller (32). Radioactive isotope incorpomtion studies. At time intervals, 5 ml of cell suspensions were incubated in duplicate with 0.5 @i of [3H]uridine (Schwarz! Mann) for 30 min at 37°C in a prewarmed tube gassed with 5% CO,. The pulse was stopped by the addition of 25 ml of cold Spinner salt solution (33), pH 7.4. Cells collected by centrifugation at 60% for 10 min were washed once with cold 5 mM Tris-HCl0.15 M NaCI, pH 7.4, and resuspended in 2.0 ml of cold 5% trichloroacetic acid for 15 min at 5°C. The resulting precipitate was collected on a Millipore filter. Radioactivity was determined by liquid scintillation counting in 10 ml of a solution of 0.1 g ofpbis[2-(5-phenyl-oxazolyl)lbenzene and 4 g of 2,5-diphenyloxazole per liter of toluene. The background radioactivity was about 6 cpm. RESULTS
Measurement of acid phosphatase. In the cell homogenates the amount ofp-nitro-
ACID
PHOSPHATASE
phenol released under the assay conditions varied linearly with the amount of protein at concentrations of 20 pg or greater, but not below this amount. A similar nonlinear relationship was noted with the supernatant fraction as the source of enzyme, but not with the lysosomal fraction. This may be due to the presence of a dissociable activator or coenzyme (34) in the supernatant. Alkaline phosphatase of renal cells also exhibits this type of abnormal kinetics (35). In the present work, the acid phosphatase activity was determined routinely with 25-100 pg of protein per assay. To assess the specificity of the method employingp-nitrophenyl phosphate, determinations of acid phosphatase were also made by an assay using p-glycerophosphate as substrate. While the latter was hydrolyzed, at a concentration (50 mM) generally used (251, inhibition was observed. The relative activities in the homogenate at 50, 10, and 5 mM concentrations were 1, 12.0, and 12.5, respectively, taking the activity at 50 mM substrate concentration as unity. Since the results obtained using 5 mM P-glycerophosphate were generally similar to, but less reproducible than, those obtained with nitrophenyl phosphate, the latter substrate was employed, unless otherwise stated. Differential centrifugation. Fig. 1 shows the results of differential centrifugation of a homogenate of HeLa cells. The distribution pattern of acid phosphatase expressed as relative specific activity is similar to that of P-glucuronidase and N-acetyl-p-nhexosaminidase and distinct from that of succinate-cytochrome c reductase. The profile of the acid phosphatase shows the highest peak. in the lysosomal fraction and the second highest peak in the mitochondrial fraction, a result typical of true lysosomal enzymes (36). However, there is no clear separation between the lysosomal and microsomal marker enzymes, although the microsomal marker enzymes glucose-6-phosphatase and NADPH-cytochrome c reductase show their second highest peak in the microsomal fraction. It would appear that endoplasmic reticulum fragments of HeLa cells sediment at a similar rate to the lysosomal population and appear in the lysosomal fraction. This sug-
OF HeLa
CELLS
x Protaln FIG. 1. Subcellular distribution patterns of six marker enzymes in subcellular fractions from HeLa cells. Fractions plotted in order of collection, from left to right, are nuclear (N), mitochondrial (M), lysosomal (L), microsomal (P), and supernatant (S). Ordinate: relative specific activity for each fraction. Abscissa: percentage total protein per fractions.
gests either that only a small difference in sedimentation coefficients exists between the microsomes and lysosomes of HeLa cells or that the marker enzymes used are less suitable for these cells than for other mammalian cells. As shown in Fig. 1, the relative specific activities of the acid phosphatase in the lysosomal and supernatant fractions are, respectively, lower and higher than those of the other two lysosomal enzymes, pglucuronidase and N-acetyl-fi-n-hexosaminidase. These results can be accounted for by the presence of an extralysosomal acid phosphatase in the supernatant fraction, which hydrolyzesp-nitrophenyl phosphate. Density
gradient
centrifugation.
The
above experiments suggested that HeLa cells contain at least two forms of acid phosphatase with specific intracellular locations. The percentage of acid phospha-
194
WANG
AND
TOUSTER
tase present in the supernatant fraction essentially the same distribution pattern was studied by centrifuging the homogeas p-glucuronidase and N-acetyl-p-n-hexnate at 105,OOOgfor 60 min to separate the osaminidase and it is different from microsupernatant from the pellet fractions. In somal and mitochondrial enzymes. six experiments, the recoveries of activity The apparent position of the lysosomal in the supernatant fraction were found to enzymes at equilibrium densities lower range from 20 to 33% of total recovered than that of the mitochondria, i.e., at activity. That the activity associated with 1.11-1.15 g cmP3, represents a situation the pellet fraction is present in lysosomes similar to that found for Ehrlich ascites was shown by density gradient centrifugatumor cells (371, beef heart (381, and rat tion as follows. lymphoid tissues (39), but opposite to those Since differential centrifugation was rel- of liver and kidney. Differentiation of theproperties of lysosoatively ineffective in separating lysosomes from endoplasmic reticulum, density gra- ma1 and soluble cytoplasmic acidphospharesults were obtained dient centrifugation was employed. In a tases. Similar preliminary experiment in which the su- whether the cells were grown in 5 or 30% serum. crose gradient (25 ml) extended between pH optima. Soluble acid phosphatase densities 1.10 and 1.25 and rested on a cushion (3 ml) of a solution of density 1.27, and lysosomal acid phosphatase showed the equilibrium density of mitochondria distinct pH activity profiles (Fig. 3). The (as represented by succinate-cytochrome c lysosomal enzyme has a broad activity optireductase) was 1.16 g cm-3, while acid phos- mum near pH 4, with activity rapidly dephatase was found between 1.10 and 1.13. clining above pH 5. The supernatant enFigure 2 shows the profiles for acid phos- zyme displays a relatively sharp pH optiphatase, p-glucuronidase, N-acetyl-p-nmum at pH 5.7 with the activity falling off hexosaminidase, glucose-6-phosphatase, on either side of this pH. These differences e&erase, and succinate-cytochrome c re- in pH requirements are similar to those ductase in a density gradient extending reported by Cristofalo et al. (10) for acid from 1.06 to 1.18. Acid phosphatase has phosphatases of human WI-38 cells, by Nelson (6) for rat liver, by Yu et al. (8) for leucocytes, and by Nicholson and Davies (15) for rat mammary carcinoma. Sensitivity to inhibitors. The effects of ntartrate and fluoride on acid phosphatase in the lysosomal and supernatant fractions were determined. Fluoride inhibited the lysosomal activity 81 and 76% at 2 and 20 mM concentrations, respectively, whereas the inhibition of the supernatant activity was only 10 and 9% at these fluoride concentrations. Similarly, 20 mM tartrate inhibited the lysosomal fraction 43%, but inhibited the supernatant activity only 7.5%. Thus, these results confirm previous findings (5-7) from other tissues indicating differences between the acid phosphatase FIG. 2. Distribution of enzymes after centrifugaof lysosomal and soluble fractions. tion of a HeLa cell homogenate in a sucrose gradient Thermal stability. The differences beat 25,000 rpm for 3 h in a Spinco SW 25.1 rotor. tween the lysosomal and supernatant acid Details are described under Materials and Methods. phosphatases were further revealed by The recoveries for the enzyme activities are: acid heating at 54°C the lysosomal and supernaphosphatase, 77.8%; p-glucuronidase, 99.5%; N-acetant fractions (see Materials and Methtyl-p-n:hexosaminidase, 112%; glucose-6-phosphaods). The lysosomal enzyme showed a tase, 118%; e&erase, 70.1%; succinate-cytochrome c reductase, 72.3%; protein, 110%. rapid loss of about 35% activity after 5 min
ACID
PHOSPHATASE
\ PH
FIG. 3. Effect of pH on the hydrolysis of p-nitrophenyl phosphate by lysosomal, soluble cytoplasmic, and serum acid phosphatases. The buffers were 0.05 M sodium acetate.
of incubation, which was followed by a gradual decline in activity to 35% of its original activity after 2 h. In contrast, the acid phosphatase from the supernatant fraction showed a very rapid inactivation to 15% of the original activity within a few minutes; further loss of activity occurred with prolonged incubation. The initial sharp drop in the activities of both enzymes suggests that more than one acid phosphatase may be present in both the lysosomal and supernatant fractions. Kinetics. Increasing the concentration of p-nitrophenyl phosphate from 0.05 to 5 mM had no inhibitory or activating effects on acid phosphatase of the supernatant fraction. On the other hand, lysosomal acid phosphatase is “activated” by high substrate concentrations. Lineweaver-Burk plots (Fig. 4) show the normal and anomalous kinetics displayed by the supernatant and lysosomal acid phosphatase, respectively. The apparent K, values calculated from the linear portion of the plot were 0.11 mM for the supernatant enzyme (or enzymes) and 0.09 mM for the lysosomal enzyme (or enzymes). Distinction between lysosomal acidphosphatase and glucose&phosphatase activities. The experimental results reported above constituted strong evidence for the presence of distinctive lysosomal and soluble cytoplasmic acid phosphatase activities. It is also of interest to record an experiment bea.ring on the question ‘as to whether there is any overlap in reactivity between a-nitronhenvl nhosnhatase and
OF HeLa
195
CELLS
Alactivities. glucose-6-phosphatase though fresh cells showed, after homogenization and density gradient centrifugation, only one glucose-6-phosphatase peak (Fig. 2), it was accidentally discovered that cells, when stored for several days at -70°C before homogenization and centrifugation, showed two peaks of glucoseSphosphatase activity (Fig. 51, a slight difference in the apparent equilibrium positions for both acid phosphatase and glucase-6-phosphatase being observed between cells grown in 5% and 30% serum. The appearance of the peak of acid phosphatase at the trough of glucose-6-phosphatase (Fig. 5, lower graph) makes the possibility rather unlikely that acid phosphatase hydrolyzed glucose-gphosphate or that glucose-6-phosphatase hydrolyzes pnitrophenyl phosphate to an appreciable extent. Structure-lined latency of the lysosomal enzymes. Since the subcellular distribution pattern indicated that the three lysosoma1 marker enzymes are contained in particles with similar sedimentation coefficient and density, it remained to be established that these enzymes also exhibit the structural latency expected for lysosomal LYSOSOMaL
7 6 5 -,;
4 3 2
P
I , I 01234567
1
I
$mM-'1
FIG. 4. Lineweaver-Burk plots showing the effect of p-nitrophenyl phosphate concentrations on the rate of its hydrolysis by lysosomal and soluble cytoplasmic enzymes. The proteins used in each assay were 25 and SO fig for the lysosomal and soluble fractions. resoectivelv.
196
WANG
AND
TOUSTER
ma1 acid phosphatase. The biochemical properties of the bound and soluble forms were similar, no differences being observed in pH optima, kinetics, or response to inhibition by sodium fluoride and sodium tartrate.
Thermal activation of enzymes in lysosomes. Aliquots of the lysosomal fraction 30%
Serum
r
in 0.25 M sucrose were adjusted to contain 0.1% Triton X-100 and then incubated for varying lengths of time at 37°C and pH 5.0. Assays for free activity of acid phosphatase, P-glucuronidase, and N-acetyl-p-Dhexosaminidase gave similar results, namely, about 40% free activity at zero time, about 80% at 60 min, and about 90% at 90 min.
The influence of serum concentrations on acid phosphatase content. The previous FIG. 5. Distribution of acid phosphatase and glucase-8phosphatase after homogenization of HeLa cells which have been frozen for 5 days at -70°C. The serum concentrations used for growing the cells were 5 and 30%.
enzymes. Triton X-100 (0.1%) was found to unmask the latent activity of the three enzymes in the lysosomal fraction. However, the percentage of the free activity of each enzyme was found to vary somewhat in different experiments, probably as a result of variations in the handling and assay of the isolated fractions. Free activity for acid phosphatase ranged from 41 to 52%, for p-glucuronidase from 32 to 48% and for N-acetyl-p-n-hexosaminidase from 22 to 37%. The three lysosomal enzymes are released in an approximately parallel fashion by repeated freezing and thawing (Fig. 6); after five cycles no additional activity could be released. Further release of about 35% p-glucuronidase could be achieved by washing the sediment with 0.4 M NaCl. Similar treatment caused the release of 4 and 17% of acid phosphatase and N-acetylp-n-hexosaminidase, respectively. These results suggest that the major lysosomal acid phosphatase activity is associated with the membrane. In contrast to freezing and thawing, Triton X-100 (0.1%) was able to release more than 95% of the lysoso-
hi&chemical findings (17) suggested that serum played an important role in the formation of phase-black granules. To examine this phenomenon in more detail, quantitative biochemical studies were carried out on HeLa cell suspension cultures in media containing various grown amounts of calf serum. In a preliminary experiment, cells were grown for 3 days in Eagle’s minimal medium supplemented with 1, 10, and 30% serum, and the acid
.“” 80 !
! FIG. 6. Release of acid phosphatase, p-glucuronidase and N-acetyl-P-n-hexosaminidase from HeLa cell iysosomes (L fraction) upon freezing and thawing. After each thawing a portion of the fraction was removed, centrifuged (105,OOQg for 30 min) and assayed for the enzyme activities. The recoveries for the enzyme activities (including residual membrane); after five cycles of freezing and thawing, were: N-acetyl-p-n-hexosaminidase, 36%; p-glucuronidase, 81%; acid phosphatase, 36%.
ACID
PHOSPHATASE
phosphatase was assayed using either pnitrophenyl phosphate or P-glycerophosphate as the substrate. The patterns (Fig. 7) obtained with homogenate, mitochondrial plus lysosomal fraction, and the pellet fractions were almost identical with the two substrates, but a small difference was observed with the supernatant fraction, which could be due to the occurrence, in this fraction, of a minor form of acid phosphatase which has different specificity for p-nitrophenyl phosphate and /3-glycerophosphate. Because of the similarity in these results and because of the simplicity of assays employing p-nitrophenyl phosphate, this substrate was used in subsequent studies involving the effect of serum. In an extended study, including estimations of p-glucuronidase and N-acetyl-p-nhexosaminidase, as well as acid phosphatase, the content of all these enzymes, as well as cellular protein, was found to increase with increasing concentrations of serum in the medium (Fig. 8a). However, the increase in p-glucuronidase and N-acetyl-p-n-hexosaminidase was much less than that of protein, whereas the increase in acid phosphatase was much greater. Thus, the concentration of serum had opposite effects on the relative specific activity of acid phosphatase and the two lysosomal of enzymes, taking the specific activity each enzyme of the cells grown in 1% serum as unit,y (Fig. 8b). This fact seems to suggest that acid phosphatase and the other two lysosomal enzymes are present in different lysosomal particles or are controlled by different mechanisms. Although the hexosaminidase seems to be affected only to a minor extent (Fig. 8a), its activity in low serum (1%) was found in many experiments to be consistently less than that in high serum (30%). The increase in acid phosphatase activity in HeLa cells with high concentration of serum cannot be due to specific uptake of serum acid phosphatase since (a) the amount of acid phosphatase initially present in the serum is too low to account for the activity increase in the cells (see legend to Fig. 81, and (b) the serum acid phosphatase shows a pH activity profile differ-
OF HeLa
CELLS
197
FIG. 7. Effect of serum concentrations (o/o)on acid phosphatase activity in HeLa cells. The cells were grown in serum for 3 days. Ordinate: relative specific activity for each fraction taking the activity of the cells grown in 1% serum as unity. Abscissa: fractions assayed; H, homogenate; M + L, mitochondria and lysosomes; Pt, 105,OOOgpellet (i.e., H-S); S, supernatant. The substrates used were p-nitrophenyl phosphate and P-glycerophosphate.
ent from that of the phosphatase in HeLa cells (Fig. 3). The high activity of acid phosphatase at high serum concentration cannot be explained by the possible existence of activators or inhibitors of enzyme activity, since enzyme activities in mixtures of homogenates of 1 and 30% serum induced cells were additive. Effect of actinomycin D, cordycepin, and cycloheximide on serum induction of acidphosphatase. Fig. 9a shows the effects of actinomycin D and cordycepin at 5.0 and 25 pug/ml, respectively, on the serum induction of acid phosphatase. Actinomycin D added 24 h after serum induction was found to reduce L3H]uridine incorporation into nucleic acids by 95% (see Materials and Methods for isotope incorporation studies). However, the rate of acid phosphatase synthesis was unaffected during the following 8 h (Fig. 9a). Beyond 8 h, the data are unreliable because of irreversible cell damage. Similarly, cordycepin had no effect on acid phosphatase for 8 h, although it may be noted that it reduced radioactive uridine incorporation into the nucleic acids of HeLa cells only by an initial 65% at 0.5 h. Figure 9b shows that cycloheximide added 58 h after serum induction at 5 pg/ml markedly reduced the acid phospha-
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FIG. 8. Effect of serum concentration on the activity of acid phosphatase, N-acetyl-P-nhexosaminidase, and P-glucuronidase and on protein in HeLa cell homogenates. The cells were grown in serum for 4 days. On the ordinates are plotted relative specific activity for each enzyme. (a) The levels of activity in the cells obtained from 1, 5, 10, 20, and 30% serum were: acid phosphatase, 0.52, 0.74,1.16,2.42, and 2.36; N-acetyl-P-n-hexosaminidase, 1.07,1.21, 1.41, 1.35, and 1.42; p-glucuronidase, 0.016,0.015, 0.021,0.023, and 0.028 unit&O6 cells, respectively. One unit corresponds to the activity hydrolyzing 1 pmol of substrate/h. The levels of enzyme activity in calf serum are 0.24, 5.22, and 0.0059 units/ml for acid phosphatase, N-acetyl-P-nrespectively. (b) The actual specific activities hexosaminidase and P-glucuronidase, (pmol/h/mg protein) of the homogenates from 1, 5, 10, 20, and 30% serum are: acid phosphatase, 1.98, 2.15, 2.54, 2.87, and 3.31; N-acetyl-p-n-hexosaminidase, 3.30, 2.79, 2.49, 2.20, and 1.60; /3-glucuronidase, 0.062, 0.043, 0.047, 0.048, and 0.039, respectively. The specific activities of the enzymes in the calf serum used are: acid phosphatase, 0.0024, N-acetyl-P-n-hexosaminidase, 0.052; P-glucuronidase, 0.00006, respectively. In these experiments each 8 x lo6 cells was initially exposed to 100 ml of medium containing the desired amount of serum in a given flask. On Day 3, 50 ml of fresh medium was added to each flask. The cell concentrations at the time of harvest (Day 4) were 247,000,411,000,337,000, 346,000, and 250,000 cells/ml for the cells grown in 1, 5, 10, 20, and 30% serum, respectively.
tion to the active site. This theory was tase activity, whereas cordycepin, again, had little effect on acid phosphatase for 12 used to explain the abnormal kinetics of h. some enzymes (42-44) until the concept of negative cooperativity was developed (40, DISCUSSION 45). In the case of the lysosomal acid phosThe present report presents evidence phatase in HeLa cells, no distinction that HeLa cells contain both lysosomal among the three possibilities can be made and soluble cytoplasmic acid phosphatase without the availability of pure enzyme. activities that exhibit different pH-activThe third explanation seems most likely in ity profiles, heat inactivation rates, inhibiview of the reported multiplicity of acid tion by fluoride and tartrate, and enzyme phosphatase in mammalian lysosomes kinetics. There are three possible interpre(e.g., 1). Indeed, we have preliminary inditations for the biphasic curve in the Line- cations that HeLa cells also contain more weaver-Burk plot (Fig. 4) observed for ly- than one phosphatase. Upon DEAE-cellusosomal acid phosphatase: (a) enzyme acti- lose3 chromatography of 0.1% Triton Xvation at high substrate concentration 100~solubilized lysosomes at pH 7.8, three (401, 6) negative cooperativity (401, and (c) enzyme peaks were eluted from the cola mixture of two or more enzymes each umn, with unfortunately only lo-20% rewith different affinity for substrate (41). The first possibility implies that there may 3 Abbreviation used: DEAE-cellulose; 0-(diethylbe a second activator substrate site in addi- aminoethyl) cellulose.
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FIG. 9. (al Effect of cordycepin (25 pg/ml) and actinomycin D (5 pg/ml) on the induction of acid phosphatase by 30% serum. Cells were previously grown in 1% serum for 4 days. Arrow indicates the addition of drugs. One unit corresponds to one OD,J30 min. (b) Effect of cycloheximide (5 wg/ml) and cordycepin _ _ (25 &ml) on the induction of acid phosphatase by 30% serum. Other details as for Fig. 9a.
covery in total activity. Each of these peaks displayed normal Michaelis-Menten kinetics. More information on this aspect must await further purification of the enzymes. It is interesting that only a small percentage of lysosomal acid phosphatase can be released from the lysosomes by freezing and thawing followed by washing with salt. An inability to release all acid phosphatase from rat liver lysosomes by physical disruption was reported by Shibko and Tappel (7) and by Allen and Gockerman (46). However, in the latter cases, it was not clear whether the acid phosphatase associated with the sediment represented adsorption of the enzyme onto the membrane fragments, since no washing with salt was performed. Some lysosomal enzymes, particularly, N-acetylhexosaminidase (47) an.d deoxyribonuclease II of rat liver (48) appear to adsorb readily to the membranes and are released by washing with salt. Serum is generally required by mammalian cells for growth in culture. In addition to its influence on protein and nucleic acid synthesis, serum also regulates alkaline phosphatase in KB cells (49), modulates pyruvate kinase in RLC cells (501, enhances the hormonal induction of tyrosine aminotransferase in HTC cells (51), and influences catalase turnover in human diploid cell culture (52). Calf serum, but not human serum, causes the formation of Nglycolylneuraminic acid in HeLa cells (53).
Our observations that serum stimulates acid phosphatase in an established cell line of human origin represent another example of such action. Because serum is a complex material, it is possible that stimulation is the result of several independent or interdependent actions of various serum components. The kinetics of serum induction of acid phosphatase in HeLa cells is similar to the kinetics of acid hydrolase production in mononuclear phagocytes after adding serum (54); the curves for HeLa cells vary from linear to slightly concave. The results presented in this study suggest that de nouo protein synthesis is involved in the induction of acid phosphatase by serum. Cycloheximide (5 pg/ml), an inhibitor of protein synthesis, immediately blocks further serum induction of acid phosphatase. On the other hand, cordycepin (25 pglml) or actinomycin D (5 pglml), added during the period of enzyme increase, do not interfere with the enzyme for at least 8 h after their addition to the culture. In HeLa cells, cordycepin, at the concentration used, appears to cause a marked suppression of the synthesis of cytoplasmic mRNA (55). Actinomycin D is also known to markedly inhibit messenger RNA synthesis (56, 57). Preexisting messenger is insensitive to the effects of cordycepin and actinomycin D and is capable of directing further protein synthesis until it is inactivated. The lifespan of this mRNA (or mRNA’s) can be realistically estimated
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to be at least 8 h, since both cordycepin and actinomycin D, which inhibit mRNA synthesis by different mechanisms, do not interfere with enzyme induction for this period of time. It appears that messenger RNA synthesis is not rate-limiting in lysosomal acid phosphatase synthesis, at least under the specific conditions of these experiments involving induction by serum. ACKNOWLEDGMENTS We are indebted to Mrs. Vera Coleman for extensive laboratory assistance. REFERENCES 1. ARSENIS, C., AND TOUSTER, 0. (1968) J. Biol. Chem. 243, 5702-5708. 2. GOODLAD, G. A. J., AND MILLS, G. T. (1957) B&hem. J. 66, 346-354. 3. BARKA, T. (1961) J. Histochem. Cytochem. 9, 564-571. 4. GEORGATSOS,J. G. (1965) Arch. B&hem. Biophys. 110,354-356. 5. MOORE, B. W., AND ANGELEITI, P. U. (1961) Ann. N. Y. Acad. Sci. 94, 659-667. 6. NELSON, B. D. (1966) hoc. Sot. Exp. Biol. Med. 121,998-1001. 7. SHIBKO, S., AND TAPPEL, A. L. (1963) Biochim. Biophys. Acta 73, 76-78. 8. Yu, B. P., KUMMEROW, F. A., AND NISHIDA, T. (1966) Proc. Sot. Exp. Biol. Med. 122, 10451048. 9. NEIL, M. W., AND HORNER, M. W. (1964) Biothem. J. 92, 217-224. 10. CRISTOFALO, V. J., KABAKJIAN, J. R., AND KRITCHEVSKY, D. (1967) Proc. Sot. Exp. Biol. Med. 126, 649-653. 11. DIPIETRO, D. L., AND ZENGERLE, F. S. (1967) J. BioZ. Chem. 242, 3391-3396. 12. SMITH, J. K. K., AND WHITBY, L. G. (1968) Biochim. Biophys. Acta 151,607-618. 13. HEINRIKSON, K. L. (1969) J. BioZ. Chem. 244, 299-307. 14. VANHA-PERTTULA, T. (1970) Biochim. Biophys. Acta 227,390-401. 15. NICHOLSON, R. I., AND DAVIES, M. (1974) Eur. J. B&hem. 44, 25-35. 16. GONZALES, L. W., AND MEIZEL, S. (1973) Biochim. Biophys. Acta 320, 166-179. 17. RQSE, G. G. (1957) Biophys. Biochem. Cytol. 3, 697-704. 18. GROPP, A. (1963) in Cinemicrography in Cell Biology (Rose, G. G., ed.), Academic Press, New York/London. 19. AHEARN, M. J. (1962) M. A. thesis, University of Texas, Austin. 20. AHEARN, M. J., HAMILTON, T. H., AND BIESELE,
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