The Autorelease of Alkaline Phosphatase from the Plasma Membrane during the Incubation of Cultured Liver Cell Homogenates

Transcription

1 J. Biochem. 103, (1988) The Autorelease of Alkaline Phosphatase from the Plasma Membrane during the Incubation of Cultured Liver Cell Homogenates Kenji Sorimachi and Yosihiro Yasumura Department of Microbiology, Dokkyo University School of Medicine, Mibu, Tochigi Received for publication, September 7, 1987 When a rat hepatoma cell (R-Y121B) homogenate was incubated at 37 C, 30-70% of the total alkaline phosphatase was released into the supernatant fluid from the precipitate fractions. The release reached a plateau level after 10 h of incubation at 37 C. The optimum ph value for the release was 7.4. Alkaline phosphatase activity increased during the incubation of the cell homogenates, but this increase was independent of the enzyme release. Serum increased not only alkaline phosphatase activity in the cultured cells but also enzyme release in their homogenates. In addition, we examined a rat liver homogenate and the following 11 cell lines: 3 hepatoma cell lines, including the R-Y121B cell line, 4 liver cell lines, 2 human urinary bladder carcinoma cell lines, a kidney cell line, and a mouse adrenal tumor cell line. Only in the cultured liver cell line and hepatoma cell lines, 30-60% of the total enzyme was released into the soluble fraction from the precipitate fractions; the release was not observed in the other cell lines, nor in the rat liver homogenate. The release of alkaline phosphatase took place in both heat-stable and heat-labile alkaline phosphatases. Alkaline phosphatase, extracted from cell homogenates, showed two bands during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The mobilities of the two bands changed inversely with or without sodium dodecyl sulfate. In general, the alkaline phosphatase which showed slow mobility with sodium dodecyl sulfate was more readily released from the plasma membrane. It is suggested that the physicochemical state of the form of the enzyme which binds to the plasma membrane of cultured liver cells might be different from that of the other tissue cell lines and rat liver homogenate. Recently we showed that incubation of a rat hepatoma cell (R-Y121B) homogenate at 37 C increased alkaline phosphatase [EC ] activity (1). More recently, it was shown that this increase in alkaline phosphatase activity was due to activation of the nascent enzyme molecule; the apoenzyme of alkaline phosphatase was accumulated in R-Y121B cells and Zn 2+ and Mg 2+ bound to it. This was accompanied by an increase in enzyme activity during the incubation of the cell homogenate at 37 C (2). The activation of the enzyme was observed with both the cell homogenate and the partially purified enzyme (2). During these studies, we found that the incubatin of R-Y121B cell homogenate at 37 C for alkaline phosphatase activation induced the release of the enzyme molecule from the plasma membrane. These data cannot determine whether enzyme activation takes place only in the soluble enzyme released from the plasma membrane, or release itself causes activation. We also showed that the treatment of R-Y121B cells with 10% serum increased alkaline phosphatase activity {1, 3). In our previous study, alkaline phosphatase activity in the serum-treated cells was much higher than that in the control cells continuously cultured at 0.5% serum before 37 C incubation of the cell homogenates. During 37 C incubation, alkaline phosphatase activity in the control cell homogenate markedly increased, but it was slightly lower than that in the serum-treated cell homogenate. In addition, both cycloheximide and actinomycin D partially inhibited the increase in alkaline phosphatase activity by serum (3). These results indicate that a large population of the alkaline phosphatase molecules in the control cells are due to nascent enzyme, and that serum not only activates the nascent enzyme but also enhances the synthesis of the alkaline phosphatase molecule. Comparison of alkaline phosphatase activation between the control and serumtreated cells could determine whether the nascent alkaline phosphatase molecule is activated even in the form bound to the plasma membrane. Thus the present study was designed to clarify this point, and to determine whether or not the release of the alkaline phosphatase molecule is specific to the R-Y121B cell line or universal for other cell lines. As far as we know, this report is the first to conclude that the alkaline phosphatase molecule is released during the incubation of cell homogenates at 37 C. MATERIALS AND METHODS Chemicals Prednisolone, sodium butyrate, disodium phenylphosphate, and 4-aminoantipyrine were purchased from Wako Pure Chemical Industries Ltd. (Osaka). Bt 2 camp was purchased from Yamasa Shoyu Co., Ltd. (Choshi, Chiba). Other chemicals were commercial products of reagent grade. Cell Culture The rat hepatoma cell line (R-Y121B) (4), established from Reuber rat hepatoma cells (H4-II-E) (5), was continuously cultured in a modified Eagle's minimum essential medium containing 0.5% fetal bovine serum (Lot No. 29K5542) obtained from GIBCO Laboratories (Cha- Vol. 103, No. 1,

2 196 K. Sorimachi and Y. Yasumura grin Falls, Ohio, U.S.A.). The rat ascites hepatoma cell line (AH-601) and rat liver cell lines, RLC-10 (6), RLC-18 (7), and M (8), were kindly supplied by Dr. Toshiko Takaoka of this university. The human urinary bladder carcinoma cell lines, HUB-4 and HUB-15 (9), were kindly supplied by Dr. Takahide Kakuya of Utsunomiya University. The monkey hepatocarcinoma cell line (NCLP-6E) (20), chimpanzee liver cell line (CLI) (22), monkey kidney cell line (V ER0 ) (12), and the mouse adrenal tumor cell lines (Y-l) (13) were maintained in our laboratory. These cell lines were cultured in Eagle's minimum essential medium or a modified Eagle's minimum essential medium (12) with and without serum. The stock cells, cultured in glass culture flasks, were inoculated in plastic culture dishes with a 6-cm diameter (Corning, N.Y., U.S. A.) and cultured in an atmosphere of 5% C0 2 and 95% air at 37 C. Cell Homogenization and Centrifugation Ce\\ pellets were homogenized in a Teflon-glass Potter-Elvejhem type homogenizer in 50 mm Tris-HCl, ph7.4, at 0 C. This buffer contained 5 mm Mg* +, but did not contain sucrose or EDTA. As previously reported (2), in the absence of chelating agents, the subcellular components were not well fractionated by differential centrifugations. Therefore, 50 and 37% of the total alkaline phosphatase activity were detected in the nuclear and mitochondrial fractions, respectively (2). However, it is well known that alkaline phosphatase is localized mainly in the plasma membrane. Thus, in the present study, the cell homogenates were centrifuged at 100,000 Xg for 1 h at 4 C to divide the subcellular components into the following two groups: the precipitate fractions containing more than 95% of the total enzyme activity and the supernatant fluid. Alkaline Phosphatase Release Cell homogenates in 50 mm Tris-HCl, ph 7.4, with 5 mm Mg* + were incubated at 37 C for h, unless otherwise stated (2). Mg 2+, which is important for the maintenance of alkaline phosphatase activity, was added to the buffer solution. In order to measure the distribution of alkaline phosphatase activity between the precipitate and supernatant fluid fractions, enzyme activity in the supernatant fluid fraction was divided by that in the cell homogenate; the values were based on the same volume. Enzyme Extraction Alkaline phosphatase was extracted with 1 -butanol according to the method of Smith et al. (14), with a slight modification (1,2). The extraction was carried out at 4 C. The 1-butanol used for alkaline phosphatase extraction was removed from the sample by dialysis at 4 C (2, 2). Enzyme Assay Alkaline phosphatase activity in cell homogenates and in the soluble fraction was measured by the method of Watanabe et al. (15), with a slight modification (1, 2). The substrate was phenylphosphate. The protein concentration was estimated by the method of Lowry et al. (16) with bovine serum albumin as the standard. Polyacrylamide Gel Electrophoresis The procedures have been described in detail in our previous papers (1, 2). The gel concentration was 5% and the iv,at-methylenebis(acrylamide) concentration was 5% of the gel concentration. When bromophenol blue reached the bottom of the gels, electrophoresis was stopped. The band of alkaline phosphatase in the gels was enzymatically stained with 5-bromo-3-indorylphosphate-p-toluidine salt (1, 2). RESULTS The increase in serum concentration to 10% from 0.5% in the culture medium increased alkaline phosphatase activity in the whole cell homogenates (Table I). A consistent result was obtained in the precipitate fractions. A small enzyme activity was detected in the supernatant fluid fraction (cytosol fraction) in both control and serum-treated cell homogenates. The release of the enzyme did not take place at 0'C. After the control cell homogenate had been incubated at 37 C for h, a 10-fold increase in alkaline phosphatase activity was observed. The enzyme activity increased not only in the precipitate fractions but also in the supernatant fluid fraction (Table I). In our previous study (2), the TABLE I. Increase in alkaline phosphatase activity and autorelease of the enzyme by 37'C incubation of R-Y121B cell homogenates. Control cells were continuously cultured at 0.5% serum. Serum-treated cells were cultured at 10% serum for 1-2 days at 37'C. The cell homogenates were left at 0 C and 37'C for h and then centrifuged at 100,000 x </ for 1 h to separate the precipitate and supernatant fractions. The values are the means±s.d. for 3 or 4 samples. O'C incubation Control cells Serum-treated cells 37'C incubation Control cells Serum-treated cells Enzyme activity (nmol/min/mg protein) / / ± 13 8± 3 263± ± 85 21± ± ± ± ± ±144 ( h ) - -o 25 Enzyme release (% of total) 2±1 3±1 41±5 67±3 Fig. 1. A time course of the distribution change in alkaline phosphatase activity between the precipitate and supernatant fractions during the incubation of the R-Y121B cell homogenate at 37'C. Control R-Y121B cells were continuously cultured at 0.5% serum (o); serum-treated R-Y121B cells were treated with 10% serum for 2 days ( ). They were homogenized in 50 mm Tris-HCl, ph 7.4, containing 5 mm Mg 2 * and then the homogenates were incubated at 37'C for the stated periods. Thefinalprotein concentrations of the cell and serum-treated cell homogenates were and mg/ml, respectively. The values are the means for two analyses. J. Biochem.

3 Autorelease of Alkaline Phosphatase in Cell Homogenates i8r I 30 ^ (ph) Fig. 2. The effects of ph on the increase in the release of the enzyme into the cytosol fraction during the incubation of R-Y121B cell homogenates at 37'C. Upper panel, change in enzyme activity; lower panel, release of the enzyme. Cell homogenates were incubated at different ph values in the presence of 5 mm Mg*\ Cell homogenate in H 2 O (100^1, 0.14mg) was mixed with buffer containing 5 mm Mg 2 * at different phs (2 ml) and then the mixture was incubated at 37'C for 24 h. Maleate buffer was used for ph and Tris-HCl for ph (o), R-Y121B cells; ( ), R-Y121B cells pretreated with 10% serum for 2 days. The values are the means for three samples. incubation of the cytosol fraction alone at 37 C did not increase alkaline phosphatase activity. Thus this large increase in the enzyme activity in the supernatant fluid fraction indicates that enzyme molecules might be released from the precipitate fractions. Using the serum-treated cell homogenates, a 1.5-fold increase in enzyme activity was observed in the whole homogenate after 37 C incubation. In addition, a greater increase in enzyme release into the supernatant fluid fraction was observed, when compared with the control cell homogenate. Even when the precipitate fractions, resuspended in fresh buffer, were incubated at 37 C, alkaline phosphatase was similarly released. When the control R-Y121B cell homogenates were incubated at 37 C for the stated periods, the release of alkaline phosphatase into the supernatant fluid from the precipitate fractions was observed, as shown in Fig. 1. The plateau level was obtained after 10 h of incubation, and at the maximal level 30% of the total enzyme was detected in the soluble fraction. In the serum-treated cell homogenate, the release of alkaline phosphatase was also observed, although its maximal level was much higher than that in the control cell homogenate. The time course of th» enzyme release was quite similar to that of the enzyme activation (2). Judging from these results, it is possible that these two reactions were related to each other. Protease inhibitors, leupeptin, pepstatin, and trypsin inhibitor, did not inhibit the alkaline phosphatase release at ^g/ml (data not shown). We have shown that the optimum ph value is 7.1 for alkaline phosphatase activation in R-Y121B cell homogenates (i). In the present study, similar results were obtained, and the optimum ph value was in the range of 6 to 7, as shown in Fig. 2, upper panel. However, the optimum ph value for the release of alkaline phosphatase was around 7.4, as shown in Fig. 2, lower panel. Therefore, 197 TABLE II. Alkaline phosphatase activity and the release of the enzyme from the plasma membrane in various cell homogenates. The values are the mean±s.d. for 3 to 6 samples. Cell line Rat hepatoma AH-601 Rat liver RLC-10 RLC-18 M Monkey hepatoma NCLP-6E" Chimpanzee liver CLI Human urinary bladder carcinoma HUB-4 HUB-15 Monkey kidney V ERO Mouse adrenal tumor Y-1 Rat liver homogenate Enzyme activity (nmol/min/mg protein) 116± 4 149± ± 5 44± ±52 10± 1 13± 1 38± 3 Release 37'C 30±2 45±2 35±1 60±2 50± ± ± ±0.4 (%) o-c 2.5± ± ± ± 3 15± 1 4.4± ± ± a NCLP-6E cells were incubated with a combination of prednisolone (0.5//g/ml), butyrate (1 mm), Bt 2 camp (1 mm), and a hypertonic concentration of NaCl (final concentration, M) at 37'C for 3 days (25). CLI cells were incubated with 10% serum at 37'C for 6 days (26). Cell homogenates were incubated at 37'C for h, after which centrifugation was carried out. TABLE III. Sensitivities of alkaline phosphatase to amino acids and temperature. The concentration of the amino acids was 10 mm. The heat stability was examined at 56'C for 30min of incubation. The values are the means±s.d. for 3 to 4 analyses. : after centrifugation at 100,000 X g for 1 h, the precipitates were resuspended and homogenized in the same buffer. Cell line Rat hepatoma R-Y121B Control Serum-treated AH-601 Rat liver RLC-10 RLC-18 M Monkey hepatoma NCLP-6E Chimpanzee CLI liver Homoarginine (%of control) ±0 25±2 11±3 30±2 31±2 20±l 15±1 16±3 14±1 30±4 16±4 41±5 22±2 63±3 64±3 Phenylalanine (%of control) 60±4 59±1 62±5 59±1 69±1 68±1 70±2 71±2 62±1 63±2 65±8 62±1 70±4 69±2 11±2 7±1 Temperature (%of initial) 49±6 32±3 53±6 21±7 23±1 8±1 16±1 1±1 15±1 2±1 16±2 0±0 59±3 25±4 105 ±7 95±2 throughout the subsequent experiments, alkaline phosphatase release was investigated at ph 7.4. In order to investigate whether the release of alkaline phosphatase is specific for R-Y121B cells, 10 other cell lines were examined (Table II). Alkaline phosphatase release occurred Vol. 103, No. 1, 1988

4 K. Sorimachi and Y. Yasumura 198 A' B' C m m B B Fig. 4. Gel electrophoretic patterns of serum-treated R-Y121B cell alkaline phosphatase. The samples had been mixed with Triton X-100 (1%, v/v), and subjected to gel electrophoresis. The origins of the samples were described in the legend to Fig. 3. in the rat ascites hepatoma cell line AH-601. In addition, in the three rat liver cell lines (RLC-10, RLC-18, and M), the monkey hepatocarcinoma cell line (NCLP-6E) and the chimpanzee liver cell line (CLI), 30 to 60% of the total enzyme was released. There was no release observed in the other cell homogenates (HUB-4, VERo, and Y-l) nor in the rat liver homogenate, although a small amount of release (12% of total) was observed in the HUB-15 cell homogenate. These results indicate that the release generally takes place in cultured liver cell homogenates only. Even though R-Y121B cell homogenate was continuously incubated at 37 C, the level of release did not change after reaching the plateau level (Fig. 1), suggesting that there might be two different types of alkaline phosphatase in cultured liver cells. In order to check this point, the characteristics of the released and unreleased alkaline phosphatases were investigated by measuring the enzyme's amino acid sensitivity, and its heat-stability at 56 C. Liver-type alkaline phosphatase is more sensitive to L-homoarginine than L-phenylalanine, and it is heat-labile. Alkaline phosphatase in rat hepatoma cells and rat liver cells was heat-labile and more sensitive to L-homoarginine U). Alkaline phosphatase is stable in the presence of sodium dodecyl sulfate under certain conditions (17, 18). We also applied polyacrylamide gel electrophoresis with sodium dodecyl sulfate to the investigation of alkaline phosphatase activation (2). The addition of sodium dodecyl sulfate to the electrophoretic buffer made the alkaline phosphatase bands sharper, as shown in Fig. 5. Alkaline phosphatase extracted with 1-butanol from the whole homogenates showed two bands. In the presence of sodium dodecyl sulfate, the band with fast mobility was strongly stained but that with slow mobility was weakly stained in the control cell homogenate (Fig. 5A). The treatment of the R-Y121B cells with serum increased the enzyme activity of the slow band (Fig. 5A'). These results indicate that there are two forms of alkaline phosphatase in R-Y121B cells, although the enzyme activity of one of them was extremely low in the cells cultured at 0.5% serum. In both control and serum-treated cell homogenates only one was released from the plasma membrane by the incubation of the cell homogenates at 37 C. The mobility of the released alkaline phosphatase coincided with that of the slower band in the presence of sodium dodecyl sulfate. J. Biochem. Fig. 3. Gel electrophoretic patterns of control R-Y121B cell alkaline phosphatase. A, B, and C were obtained from the control cells, and A', B', and C were obtained from the serum-treated cells. A and A', extracted with 1-butanol from cell homogenates; B and B', extracted from the precipitate fractions after 37 C incubation (unreleased enzyme); C and C, released during 37'C incubation of the cell homogenates. Serum-treated cells, R-Y121B cells were pretreated with 10% serum for 2 days at 37'C. than L-phenylalanine (Table III). The characteristics of the released and unreleased enzymes were quite similar. Consistent data were obtained with monkey hepatocarcinoma cells (NCLP-6E) and chimpanzee liver cells (CLI), although the alkaline phosphatase in CLI cells was heatstable and more sensitive to L-phenylalanine than Lhomoarginine. These results indicate that the release of the enzyme takes place not only in liver-type alkaline phosphatase but also in placental-type alkaline phosphatase. Polyacrylamide gel electrophoresis is useful for the investigation of alkaline phosphatase. Therefore, the enzyme was extracted with 1-butanol from the cell homogenates and subjected to gel electrophoresis. Alkaline phosphatase in the control R-Y121B cells showed two bands in the gel and a small amount of enzyme activity remained at the top of the gel. The band with fast mobility was weakly stained (Fig. 3A). In the sample extracted from the precipitate fractions after incubation of cell homogenates at 37 C, three bands were also observed; however, the amount of the enzyme with fast mobility increased (Fig. 3B). On the other hand, alkaline phosphatase released from the plasma membrane showed a relatively sharp band with fast mobility (Fig. 3C). The electrophoretic pattern of alkaline phosphatase extracted from the serum-treated R-Y121B cell homogenate differed from that in the control cell homogenate. Enzyme activity in the band of enzyme with fast mobility increased and was strongly stained (Fig. 3A'). The patterns of alkaline phosphatase extracted from the precipitate fractions after the 37 C incubation, and the released were similar to those in the control cells (Fig. 3, B, C.andB', C). When the samples with Triton X-100 (1%, v/v) were subjected to gel electrophoresis, no enzyme activity was observed at the top of the gels (Fig. 4, A, B, and A', B'). However, the electrophoretic patterns of the released alkaline phosphatase were almost the same between Triton X-100-treated and untreated samples. Electrophoretic patterns (Fig. 4, A and A') show that serum treatment of R-Y121B cells increased the enzyme activity belonging to the fast band. This is consistent with our previous result

5 Autorelease of Alkaline Phosphatase in Cell Homogenates A B C A' B' C' but the fast band usually stained weakly (Fig. 6-3, -4, -5, and -6). These results indicate that the alkaline phosphatase with slow mobility during polyacrylamide gel electrophoresis with sodium dodecyl sulfate is readily released from the plasma membrane during the incubation of cultured liver cell homogenates at 37 C. DISCUSSION f- Fig. 6. Gel electrophoretic patterns of various cell lines' alkaline phosphatase in the presence of sodium dodecyl sulfate. 1, released from the CLI cell precipitate fractions during 37'C incubation; 2, NCLP-6E; 3, M; 4, AH-601; 5, RLC-10; 6, RLC-18. Judging from the electrophoretic patterns, it seems that the mobilities of the two forms of alkaline phosphatase change inversely with and without sodium dodecyl sulfate during polyacrylamide gel electrophoresis. In order to clarify this point, the enzyme activity of each band was estimated by a densitometer (FD-A4-1, Fuji Riken (Japan)). The amount of enzyme activity of the fast band without sodium dodecyl sulfate was almost equal to that of the slow band with this detergent (data not shown). Thus, the mobilities of two forms of the enzyme change inversely with and without sodium dodecyl sulfate during polyacrylamide gel electrophoresis. Alkaline phosphatase extracted from the other cell lines (CLI, NCLP-6E, M, RLC-10, RLC-18, and AH-601) was subjected to polyacrylamide gel electrophoresis. In general, alkaline phosphatase showed many bands with a smear in the absence of sodium dodecyl sulfate. However, in the presence of sodium dodecyl sulfate, the number of the bands decreased to one or two (data not shown). The released enzyme was also subjected to gel electrophoresis with sodium dodecyl sulfate (Fig. 6). The enzyme released from the CLI cell homogenate, incubated at 37 C, showed two broad bands in which the enzyme activity was almost equally distributed (Fig. 6-1). On the other hand, the released NCLP-6E alkaline phosphatase showed only one band (Fig. 6-2). The other samples showed two bands, Vol. 103, No. 1, 1988 Fig. 5. Gel electrophoretic patterns of R-Y121B cell and serum-treated R-Y121B cell alkaline phosphatase in the presence of sodium dodecyl sulfate. The origins of the samples were described in the legend to Fig. 3. Recently we showed that R-Y121B cells accumulated apoalkaline phosphatase inside the cells and the incubation of the cell homogenate at 37 C induced the binding of Mg2+ and Zn2+ to the nascent enzyme molecule accompanied by a large increase in alkaline phosphatase activity (2). In this case, the activation was observed in the partially purified enzyme as well as in the cell homogenate (or the particulate fractions). Thus, the activation of the enzyme clearly takes place in nascent alkaline phosphatase unbound to the plasma membrane. However, when using the cell homogenate or the particulate fractions, we do not know whether or not the activation of nascent alkaline phosphatase takes place in the molecule bound to the plasma membrane. We observed an increase in alkaline phosphatase activity only after incubating the homogenates or the subcellular fractions at 37 C (1-3). In the present study, the optimum ph value for enzyme release differed from the value for enzyme activation (Fig. 2). Alkaline phosphatase activity in R-Y121B cells increased about 10-fold after incubating the cell homogenate at 37 C for 10 to 20 h (1, 2). A similar result was obtained in the present study (Table I). The ratio of released enzyme activity to total enzyme activity was 0.41 in the control cell homogenate. This value was similar to the 0.67 ratio obtained with the serum-treated R-Y121B cells (Table I). In addition, the enzyme activity in the precipitate fractions obtained from the 37 Cincubated control cell homogenate was extremely higher than that before 37 C incubation: enzyme activation took place in the form bound to the plasma membrane. These results indicate that the release of alkaline phosphatase from the plasma membrane is not linked to the activation of nascent alkaline phosphatase with Mg2* and Zn2+. All alkaline phosphatase extracted from liver cells or hepatoma cells showed two bands during polyacrylamide gel electrophoresis. Kominami et al. (19) showed that rat liver alkaline phosphatase had two forms. Since two bands were observed even in the presence of sodium dodecyl sulfate, the hydrodynamic volumes of the two components differ. The difference in amino acid or carbohydrate composition might be considered to explain the existence of the two forms of alkaline phosphatase. Ito and Chou (20) showed by gel electrophoresis that there were two forms of alkaline phosphatase monomer with heterogeneous carbohydrate composition in choriocarcinoma cells. Thus, a certain difference in carbohydrate structures attached to the alkaline phosphatase molecule might contribute to the difference in the enzyme release. The present study suggests that serum not only activates the nascent alkaline phosphatase molecules but also regulates the attachment of carbohydrate chains to the enzyme molecule. In fact, the serum treatment of the cells increased the alkaline phosphatase with fast mobility on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate -(Fig. 4). Eventually the increase in this alkaline phosphatase by

6 200 K. Sorimachi and Y. Yasumura serum in R-Y121B cells induces a greater release of the enzyme from the serum-treated cell homogenate than with the control cell homogenate (Table I and Fig. 1). At present, however, we cannot determine the factor(s) that induces the difference in the mobility of alkaline phosphatase on gel electrophoresis in the presence of sodium dodecyl sulfate. In order to determine the factor, we need a large amount of purified enzyme. This subject should be clarified in a future study. Glycoproteins often migrate anomalously in molecular sieves on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (21). In the present study using polyacrylamide gels with this detergent, the mobility of the alkaline phosphatase released by 37 C incubation was almost the same as that of alkaline phosphatase extracted with 1-butanol. Moreover, the alkaline phosphatase molecule released from the precipitate fractions by the 37 C incubation bound to a concanavalin A-Sepharose column and was eluted with a-methyl-d-mannoside (unpublished data). Thus, there is little possibility of detachment of carbohydrate chains from the alkaline phosphatase molecules during incubation of the cell homogenates at 37 C. During polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the mobilities of the 1-butanol-extracted samples and the released samples of alkaline phosphatase were the same. In addition, protease inhibitors did not affect the release of alkaline phosphatase from the plasma membrane. Judging from these results, the release might not involve the cleavage of peptide bonds in the enzyme molecule. The activation of the solubilized nascent alkaline phosphatase molecule also does not involve the cleavage of the peptide bond (2). Recently it was shown that phosphatidylinositol-specific phospholipase C released alkaline phosphatase from rat liver plasma membrane (22, 23) and pig kidney microsomes (24). Therefore, phospholipase C might contribute to the release of alkaline phosphatase during the incubation of cultured liver cell homogenates at 37 C. The release of alkaline phosphatase from the plasma membrane took place during the incubation of the cell homogenates at 37 C. This phenomenon was not specific for R-Y121B cells, but was observed universally in cell lines derived from normal livers or hepatomas. However, this release was not observed in the other cell lines derived from human urinary bladder carcinoma, monkey kidney, and mouse adrenal tumor. Furthermore, rat liver alkaline phosphatase was not released during the incubation of homogenates at 37 C. Thus the release of alkaline phosphatase is specific for cultured liver cells, but not specific for either type of alkaline phosphatase or species: both heat-stable and heat-labile alkaline phosphatases were released. It has been concluded that the activation of alkaline phosphatase in R-Y121B cell homogenate is independent of the release of the enzyme, and that the physicochemical state of alkaline phosphatase bound to the plasma membrane of cultured liver cell lines is different from that of other cell lines derived from urinary bladder carcinoma, monkey kidney, mouse adrenal tumor, and rat liver homogenate. Understanding the mode of release of the alkaline phosphatase molecules would be useful for characterization of this enzyme. REFERENCES 1. Sorimachi, K. & Yasumura, Y. (1986) Biochim. Biophys. Acta 885, Sorimachi, K. (1987) J. Biol. Chem. 262, Sorimachi, K., Mizuno, H., Niwa, A., & Yasumura, Y. (1986) Dokkyo J. Med. Sci. 13, Niwa, A., Yamamoto, K., Sorimachi, K., & Yasumura, Y. 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