THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemista, Inc.

Vol. 262, No. 4, Issue of February 5, pp. 1535-1541,1987 Printed in U.S.A.

Activation of Alkaline Phosphatase with Mg2+ and Zn2+ in Rat Hepatoma Cells ACCUMULATION OF APOENZYME*

(Received for publication, August 18, 1986)

Kenji Sorimachi From the Department of Microbiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan

In Reuber rat hepatoma cells (R-YlSlB), alkaline phosphatase activity increased without de novo en- zyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol frac- tion induced a large increase in enzyme activity (5- 10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 “C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both M 8 + above 1 mM and Zn2+ at low con- centrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly in- creased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 “C. Polyacrylamide gel electropho- resis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that “Zn2+ bound to it during enzyme activation with “Zn2+ and M 8 + . The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepa- toma cells (R-YlSlB), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity with- out new enzyme synthesis.

Recently, we reported that cycloheximide as well as acti- nomycin D increased alkaline phosphatase (EC 3.1.3.1) activ- ity in Reuber rat hepatoma cells (R-Y121B) (2). More re- cently, it was shown in a more simple cell homogenate system that alkaline phosphatase activity markedly increased during the incubation of R-Yl21B cell homogenate at 37 “C (1). These results indicate that the increase in alkaline phospha- tase activity is due to modulations of the enzyme molecule rather than to increased biosynthesis. Griffin and Cox (3) reported that prednisolone enhanced alkaline phosphatase

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

activity in HeLa cells and that this enhancement might not be due to an increased de novo synthesis of alkaline phospha- tase or a hormone-mediated decrease in the catabolism of the enzyme. However, they also reported that this induction of alkaline phosphatase was inhibited by either actinomycin D or cycloheximide (4). It was suggested that the hormone regulates the synthesis of a modifier molecule which might interact with the enzyme to produce a1 high enzyme activity (5) . Ghosh et al. (6) and Goz and Walker (7) reported similar results. On the other hand, Hanford et al. (8) and Hamilton and Sussman (9) have shown that the increased catalytic activity was a consequence of greater biosynthesis of enzyme molecules and not some subtle change in the catalytic effi- ciency of the enzyme. Thus, the regulatory mechanism of alkaline phosphatase in cultured cells remains unclear.

The present study was designed to clarify the reason why alkaline phosphatase activity increased without de novo bio- synthesis of the enzyme molecule.

EXPERIMENTAL PROCEDURES

Chemicak-Acrylamide, N,N’-methylenebis(acrylamide), N,N,- N’,N‘-tetramethylethylenediamine, ammonium persulfate, 5-bromo- 3-indolylphosphate p-toluidine salt, EDTA, disodium phenylphos- phate, 4-aminoantipyrine, potassium ferricyanide, NaCl, KC1, ZnClz, CuClz.2H20, CoCl2.6H20, FeSO4.7HzO, MgSO4.7HzO, HCl, boric acid, and sodium carbonate were purchased from Wako Pure Chem- ical Industries, Ltd. (Osaka, Japan). Tris, NiCl2.6H20 and sodium dodecyl sulfate were from Nakarai Chemical Co., Ltd. (Kyoto, Japan). Trypsin inhibitor (Type 1-S) and glutathione were from Sigma. Pepstatin and Ieupeptin were from Boehringer Mannheim GmbH (Federal Republic of Germany). Ferritin (crystalline, cadmiun free, from horse spleen) was from United States Biochemical Corp. (Cleve- land, OH). Apoferritin (from horse spleen) was from Behring Diag- nostics. Rat metallothionein was kindly supplied by Dr. Chiharu Tohyama of the National Institute for Environmental Studies, Tsu- kuba, Ibaraki, Japan. 65ZnC1z (177 mCi/mg) was from New England Nuclear.

Cell Culture-Rat hepatoma cells (R-Y121B) (lo), established from Reuber rat hepatoma cells (H4-11-E) ( l l ) , were cultured in a modified Eagle’s minimum essential medium in which glutamine was replaced with glutamic acid (12). The stock cells were cultured in glass culture flasks with a rubber stopper, and the cells used for subsequent experiments were cultured in LUX plastic culture dishes with a 6-cm diameter (Naperville, IL) in an atmosphere of 5% COS and 95% air (12). VERO cells, established from African green monkey kidney (13), were cultured in a modified Eagle’s minimum essential medium containing 0.5% calf serum. JTC-32 cells (14), established from human urinary bladder carcinoma, were cultured in Eagle’s minimum essential medium containing 10% fetal bovine serum.

Homogenization and Subcellular Fractionation-Cell pellets, kept a t -20 “C before use, were homogenized in a Teflon-glass Potter- Elvehjem type homogenizer in H20 or in 50 mM Tris-HC1, pH 7.4, with 5 mM M P at 0 “C, unless otherwise stated. The fractionation of subcellular components was done according to the previously reported method (15). The nuclear fraction was removed by centrif- ugation at 300 x g for 10 min, and then the supernatant fluids were

1535

1536 Activation of Alkaline Phosphatase centrifuged at 10,OOO X g for 20 min at 4 "C to collect the mitochon- drial fraction. The microsomal fraction and the cytosol fraction were separated by centrifugation of the postmitochondrial supernatant fluid at 100,000 X g for 1 h. These procedures were done at 4 "C.

Enzyme Extraction with 1-Butanol-The extraction of alkaline phosphatase from the cell homogenates or subcellular fractions with 1-butanol was done according to the method of Smith et al. (16) with a slight modification (1, 17). Butanol was gradually added to the cell homogenate in 50 mM Tris-HC1, pH 7.4, with 5 mM M$+ at 4 "C. Butanol addition was completed within 15 min, and its final concen- tration was 17% (v/v). The mixture was stirred for another 60 min at 4 "C, and then it was centrifuged at 38,000 X g for 30 min at 4 "C. The supernatant fluid without floating components was dialyzed against 50 mM Tris-HC1, pH 7.4, with 5 mM M$+ at 4 "C.

Gel Filtration-Sephadex G-100 (Pharmacia, Sweden) was used, and the column size was 1 X 65 cm. The elution buffer was 50 mM Tris-HC1, p H 7.4, with 5 mM M%+. The flow rate was 4 ml/h. This chromatography was done at 4 "C.

Activation of the Enzyme-In our previous study (l), the optimum pH for the enzyme activation was observed at pH 7.1, but the activated enzyme was quite unstable a t lower pH levels (below 7.5). Therefore, the activation of the enzyme localized in the subcellular fractions, or purified from them with 1-butanol, was carried out a t pH 7.4 throughout the present study. Usually, 50 pl of enzyme in 50 mM Tris-HC1, pH 7.4, was mixed with 50 p1 of other solutions containing the cytosol fraction, or various metal ions, in glass tubes (1.5 X 10.5 cm). The mixtures were used directly for the enzyme assay after treatment.

Enzyme Assay-The assay of alkaline phosphatase activity was carried out according to the method of Watanabe et al. (18) who modified the method of Kind and King (19). The procedure was described in detail in our previous papers (1, 2, 17). As a substrate of alkaline phosphatase, disodium phenylphosphate was used. The amount of phenol produced from phenylphosphate was estimated spectrophotometrically a t 500 nm by the potassium ferricyanide reaction. The protein concentration was estimated by the method of Lowry et al. (20) with bovine serum albumin as the standard.

Gel Electrophoresis-The method of Smith et al. (16) was slightly modified (1, 17). The concentration of acrylamide gel was 5% and that of N,N'-methylenebis(acry1amide) was 5% of the gel concentra- tion. The applied current was 1 mA/tube for 5 min, then increased to 3 mA/tube. It is known that alkaline phosphatase is quite stable with sodium dodecyl sulfate under certain conditions (21,22). In the present study, therefore, gel electrophoresis was also carried out in the presence of 0.1% sodium dodecyl sulfate, with the same gel concentration and buffer. However, the applied current was 2 mA/ tube. Alkaline phosphatase bands in polyacrylamide gels were stained with 5-bromo-3-indolylphosphate-p-toluidine salt (1 mg/ml) in 60 mM boric acid in which the pH was adjusted to 9.7 with KOH solution.

Metal Analysis-The measurement of metal content of proteins was done by atomic absorption spectrophotometry with a Simazu Seisakusyo model AA-610 S instrument (Japan). The protein solu- tions were used directly without incineration. All solutions were prepared with deionized and distilled water.

RESULTS

Increase in Alkaline Phosphatase Activity by 37 "C Incuba- tion' of Cell Homogemtes-In the previous study (l), the incubation of R-Y121B cell homogenates at 37 "C increased alkaline phosphatase activity. When incubated under differ- ent conditions with H20, Tris-HC1 (pH 7.4), or buffer with M$+ the activity increased 5-9-fold (Table I). In order to determine whether the increase in alkaline phosphatase activ-

ity is a general phenomenon, other cell lines were examined. We have already reported that JTC-32 cells and VERO cells had alkaline phosphatase activity (17,23). Alkaline phospha- tase activity in both cell homogenates decreased after 37 "C incubation in H20 or in buffer without M$+. In buffer con- taining M$+, however, alkaline phosphatase activity in- creased slightly (1.1-1.6-fold). The behavior of alkaline phos- phatase in R-Y121B cells is clearly different from that of alkaline phosphatase in JTC-32 cells and VERO cells.

Subcellular Fractions-In order to purify alkaline phospha- tase, subcellular components were fractionated by centrifuga- tion according to the method reported previously (15) with a slight modification. Since M$+ is an important metal ion for preserving alkaline phosphatase activity, the buffer used for the subcellular fractionation contained 5 mM M P . EDTA, which reduces alkaline phosphatase activity, was omitted from the buffer. After centrifugation at 300 X g for 10 min, 50% of the total alkaline phosphatase activity was recovered in the precipitate which contained 38% of the total cellular protein (Table 11). In our previous study on subcellular frac- tionation (15), the protein content of the nuclear fraction was 11-18% of the total cellular protein. These results suggest that the nuclear fraction prepared in the present study con- tained mitochondria and microsomes. In fact, the amounts of protein in the mitochondrial and the microsomal fractions were smaller than those in the previous work ( E ) , although the amount of protein in the cytosol fraction was similar in our two studies. In these experiments, more than 90% of the total alkaline phosphatase activity was recovered in the par- ticulate fractions.

Activation of Alkaline Phosphatase in Particulate Fractions with and without Cytosol Fraction-Nascent alkaline phos- phatase was converted to mature alkaline phosphatase by incubating the cell homogenates at 37 "C (1). An increase in alkaline phosphatase activity was observed even after incu- bation of the mitochondrial plus microsomal fractions at 37 "C (Fig. 1). Alkaline phosphatase activity reached a maximum after 9 h of incubation and then decreased slowly. The addi- tion of the dialyzed cytosol fraction caused a more rapid increase in alkaline phosphatase activity and yielded a higher level. On the other hand, the addition of the undialyzed cytosol fraction slowed the increase in alkaline phosphatase activity compared with the control, but the final level of alkaline phosphatase activity was the same. These results suggest that the cytosol fraction produces two different effects on alkaline phosphatase activation.

Activation of Purified Alkaline Phosphatase with and with- out Cytosol Fraction-When purified alkaline phosphatase was incubated at 37 "C in the presence of M$+, alkaline phosphatase activity increased %fold after 2 h of incubation (Fig. 2). However, after 5 h alkaline phosphatase activity gradually decreased and almost reached the initial level after 24 h. A similar phenomenon was observed in Escherichia coli alkaline phosphatase experiments with apoenzyme obtained with 8-hydroxyquinoline-5-sulfonic acid (24). The addition of

TABLE I Alkaline phosphatase activities before and after incubation of cell homogenates at 37 "C

Cell homogenates were incubated at 37 "C for 12-24 h. The values are the means f S.D.

Buffer before activa- tion Sample

After activation Number of experiments Hz0 Buffer Buffer + M 8 +

nmollminlmg protein -fold increase R-Y121B cells 3.1 f 1.5 5.12 & 1.63 7.69 f 2.20 8.54 f 1.75 16 VERO cells 16.1 f 0.1 0.74 f 0.16 0.86 f 0.37 1.63 & 0.16 4 JTC-32 cells 30.5 & 18.3 0.63 f 0.06 0.93 & 0.07 1.12 & 0.06 7

Activation of Alkuline Phosphatase 1537

TABLE I1 Distributions of protein and alkaline phosphatase activity in

subcellular fractions The values are the means f S.D. for triplicate analyses. The values

in parentheses are percent of whole homogenate. Sample Protein Enzyme activity

pg/50 pl nml/min/mg protein Whole homogenate 175 (100) 3.47 f 0.01 (100) Nuclear fraction 65 (38) 1.75 f 0.15 (50) Mitochondrial fraction 22 (13) 1.27 f 0.02 (37) Microsomal fraction 11 (6) 0.10 f 0.01 (3) Cytosol fraction 75 (43) 0.12 ? 0.01 (3)

lot I

+ Oialyzed cytosol

0 10 20 TIME ( h l

30

FIG. 1. Time course of the increase in alkaline phosphatase activity at 37 OC. Subcellular fractions were incubated in 50 mM Tris-HC1, pH 7.4, containing 5 mM M$+ at 37 "C for the stated periods. o " - o , the mixture of mitochondrial and microsomal frac- tions, "the particulate fraction" (35 pg, 2.3 nmol/min/50 pl); o"--c>, the particulate fraction plus the cytosol fraction (75 pg); W, the particulate fraction plus the dialyzed cytosol fraction. The cytosol fraction was dialyzed against two changes of an approx- imately 100-fold volume excess of 50 D M Tris-HC1, pH 7.4, at 4 "C for 24 h. The values are the means for duplicate analyses.

+Dialyzed cytosol I

0 10 20 TIME ( h l

30

FIG. 2. Time course of the increase in alkaline phosphatase activity at 37 "C. Solubilized alkaline phosphatase from the mito- chondrial and microsomal fractions was incubated in 50 mM Tris- HCl, pH 7.4, containing 5 mM M e at 37 'C for the stated periods. -, enzyme alone; M, enzyme plus the cytosol fraction (75 pg); W, enzyme plus the dialyzed cytosol fraction (see the legend to Fig. 1). The values are the means for duplicate analyses.

the dialyzed cytosol fraction more rapidly increased alkaline phosphatase activity, and the highest activity was about 9- fold. The appearance of the effect of the undialyzed cytosol fraction on alkaline phosphatase activation was slow, al- though the effect of two cytosol preparations on the final level of the enzyme activity was the same. These results are con- sistent with those obtained with the particulate fractions (Fig. 1). A cytosol fraction that had been treated with 1-butanol according to the method for alkaline phosphatase extraction was unable to activate purified alkaline phosphatase (data not shown). This inactivation of the cytosol fraction by 1-butanol might be due to the precipitation of cytosol proteins from which metal ions could not be released. In addition, the metal ions released from proteins by 1-butanol would be diffused

during the dialysis of the sample. Glutathione at 0.1-1 mM slightly reduced the increase in alkaline phosphatase activity in the presence of the cytosol fraction.

The alkaline phosphatase used in this experiment was purified from the mitochondrial and microsomal fractions used in the experiment whose data are shown in Fig. 1. The plateau levels of alkaline phosphatase activity were quite similar between alkaline phosphatase bound to the particulate fractions and alkaline phosphatase eluted from them after activation of the enzyme at 37 "C. These results indicate that the extraction of alkaline phosphatase with 1-butanol does not preferentially release a special alkaline phosphatase from the particulate fractions.

Effect of Different Concentrations of Cytosol on the Enzyme Actiuation-The results shown in Figs. 1 and 2 suggest that the cytosol fraction caused two different effects on alkaline phosphatase activation. One is that undialyzable cytosol com- ponents accelerate alkaline phosphatase activation (positive effect), and the other is that dialyzable cytosol components retard alkaline phosphatase activation (negative effect). Thus, it was necessary to dilute the undialyzed cytosol fraction in order to obtain the optimum concentration, as shown in Fig. 3. On the other hand, the dialyzed cytosol fraction contained no inhibitor so that its effect on alkaline phosphatase acti- vation decreased with decreasing concentration.

Gel Filtration of Cytosol Fraction-The cytosol fraction was subjected to gel filtration chromatography with Sephadex G- 100 to reveal the distribution of alkaline phosphatase activa- tion activity according to molecular weights. A single com- ponent with a high alkaline phosphatase activation activity was not observed, and the activity was widely distributed from the void volume to just before the total volume (Fig. 4).

Effects of Metal Ions on Alkaline Phosphatase Actiuation and Stability-Liver alkaline phosphatase is a metalloenzyme containing Zn2+ and Mg2+ (25, 26). Therefore, the binding of metal ions may be involved in alkaline phosphatase activation in R-Y121B cells. Purified nascent alkaline phosphatase with a low enzyme activity was incubated with various metal ions in the presence of Mg2+ (Table 111). Na+ at 10 mM strongly inhibited alkaline phosphatase activation during 6 h of incu- bation, and even at 1 mM this inhibitory effect was observed. However, after 24 h a small inhibitory effect of Na' at 10 mM on alkaline phosphatase activation was observed, and at a 1 mM concentration the effect of Na' disappeared. This time- dependent inhibitory effect was observed with the undialyzed cytosol fraction (Figs. 1 and 2). These results suggest that Na' in the cytosol fraction contributes to the retardation of

.: ._ - 1 ""__""""""""""""""""~ ""_ x 2 i J 2 L I l L

0 1 2 3 Log dilution

FIG. 3. Effect of cytosol fraction at different concentrations on alkaline phosphatase activation. Solubilized alkaline phos- phatase (ALP) was incubated in 50 mM Tris-HC1, pH 7.4, containing 5 mM M%+ at 37 "C for 4 h. M, enzyme plus the dialyzed cytosol fraction; ."-., enzyme plus the cytosol fraction (75 Kg). The dotted line represents the alkaline phosphatase activity obtained with the enzyme alone after 37 "C incubation. The values are the means for duplicate analyses.

1538 Activation of Alkaline Phosphatase I Blue dextran I

Chymotrypsinogen A

m 30 FRAC%N

"

FIG. 4. Distribution of alkalfe phosphatase-enhancing ac- tivity on Sephadex G-100 column chromatography. The cyto- sol fraction (1 ml, 4.8 mg) was applied to the column, and a portion of each fraction (50 pl) was mixed with solubilized alkaline phospha- tase (50 pl, 0.6 nmol/min) and incubated at 37 "C for 24 h. Then, alkaline phosphatase activity was measured. Another portion of each fraction (100 pl) was used for the protein assay. Each fraction con- tained 1.5 ml BSA, bovine serum albumin.

TABLE I11 Effects of metal ions on alkaline phosphatase activity in the presence

The mixture of alkaline phosphatase (50 pl) and metal solution or buffer (50 p l ) was incubated for 24 h. The values are the activity ratios to the original enzyme (immature alkaline phosphatase, 1.3 nmol/min; or mature alkaline phosphatase, 3.8 nmol/min) and are the means for triulicate analvses.

of Mg2+

Immature enzyme" Condition

Mature enzymeb

Buffer Na+

10 mM 1 mM

10 mM 1 mM

10 mM 1 mM

1 mM 0.1 mM O.OlmM

Cu2+, 1 mM Co", 1 mM Ni2+. 1 mM

K+

Ca2+

Zn2+

6 h 24h Oh

4.99 4.07 1.00

1.61 3.05 1.01 3.14 4.19 1.02

4.55 4.08 1.02 4.82 4.48 1.00

4.90 3.95 1.01 5.10 4.42 1.02

0.46 0.45 0.10 3.18 3.84 0.67 6.43 7.24 1.02 1.38 0.25 1.00 4.53 2.40 1.00 3.02 0.81 0.97

6 h

0.62

0.56 0.61

0.68 0.66

0.62 0.61

0.06 0.55 1.13 0.37 0.69 0.46

24 h

0.34

0.33 0.27

0.31 0.33

0.32 0.34

0.08 0.55 1.11 0.00 0.49 0.06

24 h (0 "C)

0.88

0.87 0.87

0.90 0.88

0.86 0.88

0.11 0.67 1.03 0.84 0.90 0.89

Fez+; 1 mM 3.10 1.27 0.98 0.27 0.09 0.81 Immature enzyme: alkaline phosphatase was prepared from the

mitochondrial and microsomal fractions of fresh R-Y121B cells in the presence of 5 mM M e .

Mature enzyme: fresh R-Y121B cell homogenates were preincu- bated at 37 "C for 24 h in 50 mM Tris-HCI, pH 7.4, containing 5 mM M F , and alkaline phosphatase was prepared in the same manner as immature alkaline phosphatase.

alkaline phosphatase activation. K+ and Ca" did not affect alkaline phosphatase activation at 10 or 1 mM. Other metal ions such as Cu2+, Co2+, Ni2+, and Fez+ inhibited more or less alkaline phosphatase activation. Zn2+ at 1 mM strongly inhib- ited alkaline phosphatase activation, and at 0.01 mM this metal ion increased alkaline phosphatase activity more than Mg2+ alone.

When nascent alkaline phosphatase is used as a starting material, two effects of the metal ions are observed on alkaline phosphatase activity; one is the effect of alkaline phosphatase activation and the other is the effect on the stability of the enzyme. The effects of metal ions on enzyme stability were examined with mature enzyme obtained from cell homoge- nates treated at 37 "C. Metal ions other than Zn2+ at 1-10

mM did not significantly affect alkaline phosphatase activity after 24 h of incubation at 0 "C, and about 90% of the initial activity remained under various conditions (Table 111). Zn2+ at 1 and 0.1 mM decreased alkaline phosphatase activity to 11 and 67% of the initial, even at 0 "C in the presence of Mg2+, but at the lower concentration of 0.01 mM, 100% activity remained. At 37 "C, 100% of alkaline phosphatase activity remained in the presence of Zn2+ at 0.01 mM with 5 mM Mg+ after 24 h of incubation. Not only Zn2+ at 0.1 or 1 mM but also other metal ions, Na+, K+, Ca2+, and Co2+, did not prevent the decrease in alkaline phosphatase activity in the presence of Mg+. Mg2+ alone did not prevent the decrease. The decrease in alkaline phosphatase activity was accelerated in the presence of Cu2+, Ni", or Fe2+ with M$+. Ohkubo et al. (25) reported that Ca2+, Na+, and Mn2+ decreased the enzyme activity of purified rat liver alkaline phosphatase. In addition, Co2+ also decreased it in rat ascites hepatoma cells, and Zn2+ at 0.01 mM greatly decreased it (22). The present results show that Zn2+ at a low concentration (0.01 mM) stabilizes alkaline phosphatase purified from R-Y121B cells.

Effects of Mg2+ on Alkaline Phosphatase Activation-In order to investigate the effect of M$+ on alkaline phosphatase activation, alkaline phosphatase with a low enzyme activity was prepared with metal-free buffer. The enzyme activity of this alkaline phosphatase was approximately one-half that of alkaline phosphatase prepared in the presence of 5 mM Mg+ (Table IV). The addition of M e increased alkaline phospha- tase activity in a dose-dependent manner at 37 "C, and little increase was observed at 0 "C. The effect of Mg2+ reached the maximal level at 1 mM. Zn2+ alone, even at low concentrations (0.01 or 0.1 mM), did not increase alkaline phosphatase activ- ity at 37 "C. In the presence of 5 mM Mg', however, Zn2+ at 0.01 mM markedly increased alkaline phosphatase activity.

The activation of alkaline phosphatase with a low enzyme activity by Mg2+ and Zn2+ was carried out in the presence of the cytosol fraction dialyzed against metal-free buffer. The cytosol fraction contains a small amount of alkaline phospha- tase activity (Table 11). In the presence of the dialyzed cytosol fraction, M$+ at 0.1-5 mM increased the enzyme activity (4.0-4.9-fold) at 37 "C. The effect of Mg2+ was multiplied with the cytosol fraction. On the other hand, Zn2+ at 0.01 mM did not increase the activity even in the presence of the cytosol fraction at 37 "C, unless Mg2+ was also present. The highest alkaline phosphatase activity was obtained from the incuba- tion of alkaline phosphatase, dialyzed cytosol fraction, 5 mM M e , and 0.01 mM Zn2+ at 37 "c.

TABLE IV Effect of Mg2+ and Zn2+ on the activation of alkaline phosphatase Alkaline phosphatase and cytosol fraction were prepared in the

absence of metal ions. The mixture of alkaline phosphatase (50 PI, 1.1 nmol/min), cytosol fraction (50 pl, 55 pg), or buffer (50 pl) and metal solution (50 pl) was incubated for 6 h. The values are the activity ratios to the enzyme (2.0 nmol/min) prepared in the presence of 5 mM Mg+ and the means for triplicate analyses.

Only enzyme Enzyme +

Condition tosol

0 ° C 3 7 ° C 0°C 37 'C

Buffer 0.54 0.27 0.66 1.82

5 mM 0.96 2.47 1.05 4.90 1 mM 0.89 2.39 1.00 4.40 0.1 mM 0.72 1.64 0.85 3.94

0.1 mM 0.46 0.13 0.58 0.10 0.01 mM 0.59 0.20 0.72 0.77

M e , 5 mM M%+ + Zn2+, 0.01 mM 1.03 4.48 1.15 5.61

M%+

Zn2+

Activation of Alkaline Phosphatase 1539

Treatment of Alkaline Phosphatase and Cytosol with EDTA-It has been shown that Mg2+ and Zn2+ in concert increase alkaline phosphatase activity in R-Y121B cells and that the addition of the cytosol fraction provides even better conditions for enzyme activation. Based on these observa- tions, the effect of EDTA on alkaline phosphatase activation by the cytosol fraction was investigated. Alkaline phosphatase was activated with Mg2' alone in the presence and absence of the dialyzed cytosol fraction, but in the presence of the EDTA- treated cytosol fraction M$+ alone did not increase the en- zyme activity. However, the presence of both ZnZ+ with Mg2+ abolished the effect of EDTA. The treatment of alkaline phosphatase with EDTA also affected the increase in the enzyme activity. Mg2+ alone did not increase the activity of the EDTA-treated alkaline phosphatase in the presence and absence of the cytosol fraction, but the presence of both Zn2+ with Mg2+ completely restored the enzyme activity of the EDTA-treated alkaline phosphatase. These results suggest that nascent alkaline phosphatase contains a small amount of Zn2+ and that the cytosol fraction supplies Zn2+ to alkaline phosphatase for the enzyme activation.

Effect of Protease Inhibitors on the Enzyme Actiuatwn-In order to investigate the contribution of proteases to the acti- vation of nascent alkaline phosphatase, the cell homogenate or the partially purified alkaline phosphatase was incubated with pepstatin, leupeptin, or trypsin inhibitor (5-500 pg/ml) at 37 "C for 6 h. However, these protease inhibitors did not show a significant effect on the enzyme activation.

Gel Electrophoresis of Alkaline Phosphatase-Although the protease inhibitors did not inhibit the increase in alkaline phosphatase activity, this does not mean that the activation of nascent alkaline phosphatase is independent of the cleavage of peptide bonds. Therefore, alkaline phosphatase was sub- jected to polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Even though nascent alkaline phosphatase was incubated with Mg2+ plus Zn2+ in the presence and absence of the cytosol fraction at 37 "C for 6 h, no new band was observed with polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (Fig. 5, A, B, and C). However, a main band with slow mobility became slightly broad after enzyme activation (Fig. 5, B and a .

In the presence of sodium dodecyl sulfate, two sharp bands were observed in every sample, with polyacrylamide gel elec-

FIG. 5. Gel electrophoretic patterns of alkaline phospha- tase. The enzyme was incubated at 37 ' C for 6 h under different conditions. The concentrations of Me and Zn2+ were 5 and 0.01 mM, respectively. The protein content of the dialyzed cytosol was 60 pg/50 pl. Gel electrophoresis was carried out in the absence of sodium dodecyl sulfate (columns A, B, and C) and in the presence of the detergent (columns a, b, and c). Columns A and a, nascent alkaline phosphatase; columns B and b, activated with metal ions; columns C and c, activated with metal ions plus cytosol fraction.

trophoresis (Fig. 5, a, b, and c). In this case, the band with fast mobility was strongly stained. There was no significant difference in the mobility of each band among the following three samples: original nascent alkaline phosphatase, acti- vated with Mg2+ plus Zn", and activated with the metal ions in the presence of the cytosol fraction. The mobility of the main band was quite similar to that of bovine serum albumin dimer without reducing agents. The molecular weights of alkaline phosphatase with fast and slow mobilities were esti- mated at 1.1 X IO5 and 1.3 X lo5, respectively, compared to the mobilities of the bovine serum albumin monomer, dimer, trimer, and tetramer. No new band was observed even after activation of the enzyme. These results suggest that the activation of nascent alkaline phosphatase in R-Y121B cells does not involve peptide bond cleavages. Kominami et al. (27) reported that there were two forms of rat liver alkaline phos- phatase with different molecular weights (1.4 X lo5 and 1.7 X 10".

Binding of to Alkaline Phosphatase-Nascent alka- line phosphatase was incubated with fi5Zn2+ in the presence of M$+ and then subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Fig. 6). A compo- nent containing radioactivity coincided with the band of alkaline phosphatase. This shows that fi5Zn2+ bound to nas- cent alkaline phosphatase during the enzyme activation. A high radioactivity was also observed at the origin of the gel, whereas the enzyme activity was not observed there.

Effects of Ferritin, Apoferritin, and Metallothionein on Al- kaline Phosphatase Actiuation-Metallothionein, ferritin, and apoferritin were used to check whether these proteins are involved in the activation of nascent alkaline phosphatase activity. Alkaline phosphatase activity was increased by the cytosol fraction alone, whereas metallothionein, apoferritin, and ferritin did not increase the enzyme activity (Table VI). On the other hand, the combination of each protein with Mg2+ (5 mM) plus zn2+ (0.01 mM) increased more the enzyme actvity than metal ions alone. These results suggest that metallothi- onein and ferritin contribute to the activation of alkaline phosphatase.

Contents of Mg2+ and Zn2+ in the Cytosol Fraction-The

r

6 -

- 4 -

- 2 -

- 0 -

- + RG. 6. Binding of ssZna* to alkaline phosphatase in gel

electrophoresis. Nascent alkaline phosphatase was incubated with 5 mM Me plus 2.1 p~ @Zn2+ (7.1 X lo' cpm) for 20 h at 37 "C. Gel electrophoresis was carried out in 0.1% sodium dodecyl sulfate. The gel length was 5.1 cm, and the gel was sliced into 28 pieces with about 1.8 mm width.

1540 Activation of Alkaline Phosphatase

TABLE V Treatment of alkalinephosphatase and cytosol with EDTA for the

enzyme activation with Mg2+ and Zn2+ Immature alkaline phosphatase and cytosol were incubated with

EDTA at 10 mM for 30 min at 37 "C, and then they were dialyzed against 50 mM Tris-HC1, pH 7.4, at 4 "C overnight. The mixture of alkaline phosphatase (50 pl), metal solution (50 pl), and cytosol or buffer (50 pl) was incubated for 6 h at 37 "C. The protein concentra- tion of the dialyzed cytosol was 50 pg/50 pl, and the final concentra- tions of M$+ and Zn2+ were 5 and 0.01 mM, respectively. The values are the activity ratios to the initial enzyme activity (0.97 nmol/min) and are the means for triplicate analyses.

Condition Alkaline EDTA-treated

phosphatase alkaline phos- phatase

Buffer Mg2f 3.60 0.26

Mg2+ 5.70 1.04

M$+ 1.17 0.73

Mg2f + Zn2+ 4.96 5.71 Dialyzed cytosol

Mg2+ + Znz+ 7.69 8.66 EDTA-treated cytosol

M$+ + ZnZ+ 7.58 8.76

TABLE VI Effects of cytosol, metallothionein, apoferritin, and ferritin on alkaline

phosphatase activation with M$+ and Zn2+ The mixture of alkaline phosphatase (50 pl), cytosol or protein

solution (50 pl) , and metal solution or buffer (50 pl) was incubated for 6 h at 37 "C. The protein concentration of the dialyzed cytosol was 60 pg/50 pl, and the amount of each protein was 25 pg/50 pl. The final concentrations of M$+ and Zn2+ were 5 and 0.01 mM, respec- tively. The values are the activity ratios to the enzyme activity at 0 "C (1.3 nmol/min) without either cytosol or protein and are the means for triplicate analyses.

0 'C 31 "C

M2+ + Zn2+ - Metal M? + Zn2+

Control 1.00 0.62 4.44 Dialyzed cytosol 1.51 2.71 9.77 Metallothionein 1.04 0.81 5.11 Apoferritin 1.12 0.76 5.58 Ferritin 1.21 0.75 6.54

TABLE VI1 Metal contents of cytosol fractions of different cell lines

The values (pglmg protein) are the means f S.D. The cytosol fractions were Drepared with metal-free buffer.

R-Y121B cells VERO cells JTC-32 cells

Zn2+ 0.25 f 0.04 0.29 f 0.02 0.36 f 0.12

Number of samdes 6 3 3 Mg2+ 3.2 f 0.5 2.3 f 0.2 2.5 f 0.8

data presented in Tables 111, IV, and V show that M$+ and Zn2+ are important metal ions in the yielding of higher alka- line phosphatase activity. Fig. 2 and Tables IV and V show that the cytosol fraction might supply Zn2+ to alkaline phos- phatase with a low enzyme activity. Therefore, the contents of Zn2+ and Mg2+ in the cytosol fractions obtained from different cell lines were measured by atomic absorption spec- troscopy. In the present study, the samples were directly applied without incineration. However, the metal content (Zn'+) in the unincinerated samples was slightly higher than that in the ash (28).

The contents of Zn2+ and M e in the cytosol fractions were almost the same among the following three samples, R- Y121B, VERO, and JTC-32 cells (Table VII), although the behavior of alkaline phosphatase in R-Y121B cell homogenate differed from that in VERO and JTC-32 cell homogenates at

37 "C incubation (Table I). This means that the accumulation of nascent alkaline phosphatase in R-Y121B cells is not due to the lack of Zn2+ and Mg2+ inside the cells. In R-Y121B cells, the binding of Zn2+ and M e appears to be prevented by an unknown factor(s). Schlesinger (29) found a mutant strain of E. coli that produced an alkaline phosphatase apoen- zyme lacking Zn'+. This apoenzyme did not form a dimer and did not show enzyme activity.

The content of M$+ in the cytosol fraction decreased to less than 10% of the initial value after dialysis with a metal- free buffer, whereas the content of Zn2+ was almost constant (data not shown). This means that M$+ in the cytosol frac- tion is readily removed by the dialysis even at 0 "C but Zn2+ is not.

DISCUSSION

Price and Joshi (30) suggested that ferritin, molecular weight 480,000, detoxifies excess Zn2+ in vivo and that this protein supplies Zn2+ to the apoenzymes of alkaline phospha- tase, yeast phosphoglucomutase, and yeast aldolase. Further- more, Udom and Brady (26) reported that zinc-thionein also contributes to the activation of the apoenzymes. In the present study, the presence of ferritin or metallothionein with M e plus Zn2+ increased nascent alkaline phosphatase activity more than metal ions alone (Table VI). These results show that metalloproteins can be involved in the regulation of metalloenzymes by a mechanism other than the detoxification of excess metal ions in cells. However, the dialyzed cytosol fraction was more effective than ferritin or metallothionein for the activation of nascent alkaline phosphatase with M e plus Zn2+. In addition, the activation activity of nascent alkaline phosphatase was widely distributed with gel filtration chromatography using Sephadex G-100 (Fig. 4). Judging from these results, not only ferritin and metallothionein but also other unidentified proteins may contribute to the activation of alkaline phosphatase.

A significant increase in alkaline phosphatase activity was observed by the addition of M F to the enzyme prepared in the absence of M e , and the further addition of dialyzed cytosol fraction further increased the enzyme activity (Table IV). However, M$+ alone did not increase the enzyme activity of the EDTA-treated nascent alkaline phosphatase (Table V). In other experiments using apoenzyme obtained with Chelex, 8-hydroxyquinoline-5-sulfonic acid, or EDTA, Mg2+ alone could not activate the enzyme of E. coli (241, kidney (31), and intestinal alkaline phosphatase (32). However, Zn2+ alone activated the former two alkaline phosphatases (24, 31) and only slightly the latter (32). These results suggest that nascent alkaline phosphatase in R-Y121B cells contains a small amount of Zn2+ (perhaps less than 2 g atoms/mol of enzyme). E. coli alkaline phosphatase with 2 g atoms of Zn2+/mo1 of enzyme showed a little enzyme activity (24), although 4 g atoms of Zn2+ and 1.3 g atoms of M$+ were needed to yield the maximal enzyme activity (28). On the other hand, even when nascent alkaline phosphatase with and without the dialyzed cytosol fraction was incubated with Zn2+ at lower concentrations, no significant increase in alkaline phospha- tase activity was observed (Table IV). This means that the presence of Zn2+ with Mg2+ is necessary for yielding the highest enzyme activity.

The reason why alkaline phosphatase with a low enzyme activity cannot interact with the metal ions in R-Y121B cells remains to be elucidated. We have already shown that cyto- toxic substances such as cycloheximide, actinomycin D, chlor- oquine, dinitrophenol, and KCN increase alkaline phospha- tase activity in R-Y121B cells cultured in a monolayer at

Activation of Alkaline Phosphatase 1541

0.5% serum (1, 2). In addition, serum or purified proteins, y- globulin, casein, and bovine serum albumin increased the enzyme activity in the same cell culture system (1). The functions of these cytotoxic substances are different from each other and from the functions of serum or proteins, although we do not well understand the functions of serum or proteins for cells. These perturbants might alter the phys- icochemical state of the plasma membrane containing the apoenzyme of alkaline phosphatase rather than directly acti- vate the enzyme molecule. Alterations in the plasma mem- brane induce the interaction of alkaline phosphatase and metal ions, resulting in the increase in alkaline phosphatase activity without de nouo biosynthesis of the enzyme molecule.

It is well known that the liver-type alkaline phosphatase is heat labile and its activity decreased rapidly at 56 "C. In the previous study (l), we have shown that the incubation of R- Y121B cell homogenates at 56 OC as well as at 37 OC increased alkaline phosphatase activity within 30 min but then de- creased it. However, this increase in alkaline phosphatase at 56 "C was not observed in the homogenates, preincubated at 37 "C for 12-24 h or obtained from the cells pretreated with cytotoxic substances. A small increase in the enzyme activity was observed in the cells pretreated with serum after 37 "C incubation of the homogenates.' Since the activation of the nascent alkaline phosphatase with Mg2+ and Zn2+ is a tem- perature-dependent phenomenon (Table IV), the incubation of the cell homogenates at 56 "C induces an increase in alkaline phosphatase activity. In the early period of the in- cubation of R-Y121B cell homogenates at 56 "C, the rate of activation of nascent alkaline phosphatase is greater than that of the inactivation of the activated enzyme. Evidently, nascent alkaline phosphatase in R-Y121B cells is heat stable.

In this study, the contents of Zn2+ and Mg2+ bound to alkaline phosphatase before and after activation could not be analyzed, since an extremely large amount of purified enzyme is necessary for the precise analysis of the metals. As we have already shown (l), serum increases the growth rate of cells accompanied by the activation of alkaline phosphatase in R- Y121B cells. However, if a perturbant increases the accumu- lation of nascent alkaline phosphatase with a low enzyme activity, the precise number of metal ions bound to the alkaline phosphatase molecule before and after activation of nascent alkaline phosphatase could be determined.

Even though the amount of metal ion bound to the alkaline phosphatase molecule has not been measured in the present study, it has been clearly shown that the increase in alkaline phosphatase activity in R-Y121B cells under various condi- tions is due to the activation of nascent alkaline phosphatase "apoenzyme" with Zn2+ and Mg2+. This increase does not require either new protein synthesis or RNA synthesis.

Acknowledgments-I am indebted to Professor Yosihiro Yasumura for the facilities that were made available in his laboratory. I also gratefully acknowledge the help of Dr. Jacob Robbins of the National

K. Sorimachi, unpublished data.

Institutes of Health in preparing this paper, Dr. Masatoshi Hayashi of this university in measuring the metal content by atomic absorp- tion spectroscopy, and Dr. Hideki Ohtake of this university in ob- taining metallothionein.

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