Isolation and characterization of plasma membranes from transplantable human astrocytoma, oat cell carcinoma, and melanomas.

Purified plasma membranes were obtained from five transplantable human tumors, a grade IV astrocytoma, an oat cell carcinoma, and three melanomas. Plasma membrane fractions were isolated from tumor homogenates by differential and discontinuous sucrose gradient centrifugation. Determination of enzyme activities indicated that the plasma membranes were enriched 10- to 20-fold with respect to 5'-nucleotidase, nicotinamide adenine dinucleotide glycohydrolase, Mg2+-activated nucleoside triphosphatase, and sialic acid. Specific activities of nearly all the enzymes varied with the individual tumors, even among tumors of the same type, i.e., the melanomas. Electron micrographs of the plasma membrane fractions showed smooth single-membrane vesicles with slight contamination by lysosomes. Therefore, these membranes are suitable for comparative biochemical studies and for the preparation of tumor-specific monoclonal antibodies. Plasma membranes from all five tumors contained very high Mg2+-adenosine triphosphatase (ATPase) activities. The Na+-K+-ATPase was a minor component of the total ATPase of these membranes (less than 30%). The major component was an ATPase exhibiting similar activity toward several nucleoside triphosphates. The activity of such a nucleoside triphosphatase has been correlated with tumorigenicity in cultured liver epithelial cells. The nucleoside triphosphatase of the plasma membranes of astrocytoma and oat cell carcinoma was stimulated from 50 to 1005 by concanavalin A, whereas ATPase of the melanoma plasma membranes was not or only slightly stimulated. The different response to concanavalin A could be due to differences in the ATPase molecules of the individual tumors or to the different environment of the ATPase.

Characteristics of adenine nucleotide fluxes and transport in human tumor mitochondria.

The efflux of adenine nucleotides from three human tumor mitochondria has been investigated with mitochondria prelabeled with radioactive ATP. Uncouplers induce a large efflux of adenine nucleotides from mitochondria from human hepatoma and oat cell carcinoma while efflux from astrocytoma mitochondria is less. This efflux does not require exchangeable anions, i.e., adenine nucleotides or pyrophosphate, in the extramitochondrial medium, and is not sensitive to atractyloside. The efflux is more extensive with dinitrophenol and CCCP than with valinomycin-K+, and may account for the differential effects of the two types of uncouplers on uncoupler-stimulated ATPase of tumor mitochondria previously reported by us. Dinitrophenol and CCCP do not elicit any efflux of adenine nucleotides from normal liver mitochondria. Efflux of orthophosphate from tumor mitochondria is also greater with dinitrophenol and CCCP; however, the more interesting finding is that the concentration of orthophosphate in these mitochondria is unusually high, i.e., 10-40-times greater than the intramitochondrial phosphate concentration of liver mitochondria. Atractyloside sensitive transport of ATP and ADP in human tumor mitochondria has also been determined. Vmax values for both ADP and ATP transport are lower than those obtained with liver mitochondria, especially with ADP transport. ATP transport in tumor mitochondria is not affected by CCCP in contrast to the 4-5-fold stimulation observed in liver mitochondria.

Differential effects of 2,4-dinitrophenol and valinomycin (+ K+) on uncoupler-stimulated ATPase of human tumor mitochondria.

The uncoupler-stimulated mitochondrial ATPase of four human tumors, mouse kidney, brain and fetal liver exhibited a characteristic behavior when preincubated with the H+-conducting uncouplers, dinitrophenol, CCCP, S-13 and gramicidin. The ATPase activity was considerably lower with preincubation than without. Preincubation with valinomycin (+ K+), on the other hand, did not result in a significant decrease of the ATPase activity. These results may be contrasted with those obtained with liver or heart mitochondria, the ATPase activity of which did not suffer any loss when preincubated with dinitrophenol. The effect of preincubation with dinitrophenol on the tumor mitochondria could not be accounted for by dinitrophenol-induced Mg2+ efflux, since the differential effects of dinitrophenol and valinomycin (+ K+) remained even when ATPase activity was determined in presence of Mg2+. Small amounts of ATP and ADP in the preincubation mixture containing dinitrophenol protected against the decay of the ATPase activity, implicating the exchangeable adenine nucleotides in the tumor mitochondria. In a model system where liver mitochondria were depleted of their adenine nucleotides, a lower ATPase activity was indeed obtained. However, direct determination of the concentrations of adenine nucleotides in dinitrophenol- and valinomycin-treated tumor mitochondria revealed only slight differences.

Reconstitution of beef heart mitochondrial F0F1 in reverse phase evaporation vesicles.

Beef heart mitochondrial F0F1 was reconstituted in proteoliposomes by a new procedure. MF0F1 was inserted in preformed reverse phase evaporation vesicles of large diameters prepared from asolectin (MF0F1-REV). Reconstitution was mediated by Triton X-100, which was subsequently removed by treatment with Bio-Beads. Parameters which resulted in optimal reconstitution were described. The MF0F1-REV proteoliposomes catalyzed an exchange between Pi and ATP and were capable of proton pumping. Both reactions were inhibited by oligomycin and uncoupler of oxidative phosphorylation. The range of Pi-ATP exchange activity of the proteoliposomes (70-110 nmol min[-1] mg[-1]) compared favorably with activities obtained in vesicles reconstituted by cholate dialysis or cholate dilution. The most important aspect of this method is that, unlike other reconstitution methods, exogenous F1 and other coupling factors are not required to obtain high Pi-ATP exchange activity by MF0F1-REV. This simple and rapid reconstitution procedure should be useful for future studies dealing with functional analysis of MF0F1.

Oxidative phosphorylation and ATPase activities of human tumor mitochondria.

Studies were carried out with intact mitochondria isolated from human astrocytoma, oat cell carcinoma and melanoma which were propagated in athymic mice. These human tumor mitochondria were capable of coupled oxidative phosphorylation. They also showed significant uncoupler-stimulated ATPase if defatted bovine serum albumin was included in the assay media. However, the uncoupler response curves were different and the magnitude of the ATPase activity was lower than could be obtained with mitochondria of a normal tissue, such as liver. Some of these characteristics were also exhibited by mitochondria from several animal hepatomas and Ehrlich ascites tumor. In the three tumors studied, mitochondria from oat cell carcinoma were more labile, whereas higher respiratory control ratios and greater stimulation of ATPase by uncouplers were obtained with melanoma mitochondria. The mitochondrial ATPase was not the major cellular ATPase in any of the three tumors. This was indicated by a low inhibition of the ATPase activity of tumor cell homogenates by oligomycin. A very large fraction of the cellular ATPase activities was recovered in the microsomal fractions.

The common occurrence of ATP diphosphohydrolase in mammalian plasma membranes.

In the plasma membranes from several mammalian tissues (including normal and tumor tissues), a Mg2+ (or Ca2+)-dependent ATP phosphohydrolase activity is present in much greater amount than the (Na+ + K+)-ATPase. The ouabain-insensitive activity can be attributed to at least two enzymes, an ATPase (EC 3.6.1.3) and an ATP diphosphohydrolase (EC 3.6.1.5). The ATPase hydrolyzes ATP and other nucleoside triphosphates and is not inhibited by azide. The ATP diphosphohydrolase hydrolyzes both ATP and ADP (and other nucleoside tri- and diphosphates) and the hydrolysis of adenine nucleotides is strongly inhibited by 10 mM azide. The ratios of these two enzymes in the various membranes (as determined by the extent of azide inhibition) vary widely. The ATP diphosphohydrolase accounts for most of the Mg2+ (or Ca2+)-dependent ATP hydrolysis activity of the plasma membranes of liver (mouse), kidney (dog), two mouse sarcomas, and a human astrocytoma (xenograft in athymic mice). The ATPase is more dominant in the plasma membranes from mouse brain and human oat cell carcinoma. The widespread presence of the ATP diphosphohydrolase in plasma membrane from various types of tissues is demonstrated for the first time and is of particular interest in view of its relatively high activity in the plasma membranes of two sarcomas. The membrane-bound ATP diphosphohydrolase is characterized with respect to its metal ion activators, substrates, and inhibitors. These results should facilitate the distinction of this enzyme from other ATP hydrolyzing enzymes of plasma membranes in future investigations.

Resolution and reconstitution of ion-transport systems.

1. Oxidative phosphorylation was reconstituted with a mitochondrial proton pump (oligomycin-sensitive ATPase) and segments of the oxidation chain (cytochrome oxidase or DPNH-Q1 reductase). A proton pump of bacteriorhodopsin substituted for the respiratory chain components, giving rise to light-induced ATP formation. 2. Since oxidative phosphorylation has thus become a special case of the problem of ion translocation in general, we have investigated and reconsituted other pumps. The reconstituted Ca++ pump of sarcoplasmic reticulum consists of two factors, the Ca++-dependent ATPase and a heat-stable coupling factor. 3. Other information obtained from reconstitution experiments include the role of asymmetry in organized membranes and the specificity of protein-phospholipid interaction. 4. Purified preparations of Ca++-ATPase catalyze the formation of ATP from Pi and ADP in a stepwise reaction stoichiometric with the enzyme and dependent on Ca++.

Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8.

Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with K (m) values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X(1-7) and P2Y(2,4,11) receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y(1,12,13) receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y(6) receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-8.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5'-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

Inhibition of growth and induction of enzyme activities in a clonal human hepatoma cell line (Li-7A): comparison of the effects of epidermal growth factor and an anti-epidermal growth factor receptor antibody.

A cloned human hepatoma cell line (Li-7A), possessing epidermal growth factor (EGF) receptors numbering in the range of 10-20 pmol/10(6) cells, was inhibited in its growth by EGF as well as an antagonist monoclonal antibody (MoAb) to the EGF receptor. The mode of action of the two ligands of EGF receptors appeared to be different as indicated by the following results: 1) EGF induced marked alteration in cell morphology, whereas the antibody did not; 2) cellular protein accumulated in the EGF-treated cells but not in the antibody treated cells; and 3) ectoATPase activities were greatly enhanced in Li-7A cells treated with EGF and cholera toxin but were unaffected in cells treated with antibody and cholera toxin. The last result also suggests that expression of ectoATPase activities is under the regulation of both EGF and cholera toxin. Li-7A cells provide an additional valuable experimental system for the study of EGF action, as well as the interactive effects of EGF and cholera toxin. The enrichment of the ATPase activities in the EGF-cholera toxin-treated cells can be exploited for the detailed study and isolation of these enzymes and elucidation of their physiological functions.

Differential expression of ectoMg2+-ATPase and ectoCa2+-ATPase activities in human hepatoma cells.

A human hepatoma cell line (Li-7A) possesses ectoATPase activity which is activated by either Mg2+ or Ca2+. Both ectoMg2+-ATPase and ectoCa2+-ATPase hydrolyze other nucleoside triphosphates, are inactive with ADP and AMP, and are inhibited by both p-chloromercuriphenyl sulfonate (pCMPS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Different Km values for ATP and pH curves are obtained for ectoMg2+-ATPase and ectoCa2+-ATPase. The specific activities of the two ATPases remain relatively constant through several days of cell growth after an initial decrease. In contrast, the specific activities of the two ATPases, especially the ectoCa2+-ATPase, increases continuously in Li-7A cells cultured in the presence of EGF, cholera toxin, and hydrocortisone. The ATPases of the factor-treated cells are also indiscriminate with respect to nucleoside triphosphate substrates; however, the kinetic constants for substrates are altered when compared to that of the untreated cells. Most strikingly, the sensitivity to inhibitors is greatly reduced. It is concluded that the long-term effect of EGF, cholera toxin, and hydrocortisone on the Li-7A cells is the induction or activation of a new or minor component of the ectoATPases, which is preferentially activated by Ca2+ and insensitive to pCMPS.

Endogenous phosphorylation of proteins and phosphatidylinositol in the plasma membranes of a human astrocytoma.

Incubation of plasma membrane preparations from several tissues with [gamma-32P]ATP resulted in the phosphorylation of phosphatidylinositol as well as of proteins. The presence of an active phosphatidylinositol kinase in these membranes was indicated by equal or greater incorporation of 32P into phosphatidylinositol phosphate than into proteins. Phosphorylation of endogenous protein and lipid substrates by protein and phosphatidylinositol kinases in the plasma membranes of a human astrocytoma was investigated in detail. Maximal protein phosphorylation required the presence of Nonidet-P40 and phosphatase inhibitors (vanadate or fluoride). The rate of protein phosphorylation was greater with Mg2+ than with Mn2+, and phosphoserine accounted for 60% of the radioactivity incorporated into proteins. In the presence of Mn2+, phosphorylation of tyrosine was increased and was equal to that of serine phosphorylation (40%). With one exception, the overall pattern of phosphorylated proteins was similar with either Mg2+ or Mn2+. Maximal phosphatidylinositol phosphorylation of the astrocytoma plasma membranes also required detergent and phosphatase inhibitors. However, the enzymatic characteristics of lipid phosphorylation differed from those of protein phosphorylation with respect to divalent cation activation, ATP dependence, and sensitivity to inhibition by p-chloromercuriphenyl sulfonate, quercetin, and nucleoside derivatives. These results suggest that phosphorylation of plasma membrane proteins and phosphatidylinositol is catalyzed by different enzymes. The fact that membrane preparations exhibited phosphatidylinositol kinase activity almost 100,000 times greater than that exhibited by the purified tyrosine kinase of ros gene would exclude this and similar oncogene proteins from making a significant contribution to the overall phosphatidylinositol phosphorylation of cell membranes.

The rat liver ecto-ATPase/C-CAM cDNA detects induction of carcinoembryonic antigen but not the mercurial-insensitive ecto-ATPase in human hepatoma Li-7A cells treated by epidermal growth factor and cholera toxin.

The rat liver ectoATPase has reportedly been cloned. The cDNA, a member of the carcinoembryonic antigen (CEA) gene family, was shown to increase aggregation of transfected cells, but ATPase activity was not evaluated. Using this cDNA as a probe to clone the mercurial-insensitive ectoATPase (MI-ectoATPase) of human hepatoma Li-7A cells, the cDNA obtained was that of CEA which has no ATPase activity. The probe also did not detect increased transcription when MI-ectoATPase activity was induced in Li-7A cells. It is concluded that the "rat liver ectoATPase cDNA" codes for a cell adhesion molecule but does not code for an ectoATPase. It was also discovered that expression of four CEA transcripts in Li-7A cells was markedly stimulated by a single growth modulator, EGF, and was further stimulated by a cAMP elevating agent, cholera toxin.

Synergistic modulation of ectoCa2(+)-ATPase activity of hepatoma (Li-7A) cells by epidermal growth factor and cyclic AMP.

A mercurial-insensitive ectoATPase, which was more active with CaATP than with MgATP, was induced when human hepatoma (Li-7A) cells were cultured in the presence of epidermal growth factor (EGF) and cholera toxin. Cholera toxin could be replaced by forskolin, 8-Br-cAMP, butyryl-cAMP, and dibutyryl-cAMP. Requirement for EGF was specific, but EGF was ineffective if added more than 24 h after the addition of forskolin or cholera toxin. It was concluded that induction of the ectoCa2(+)-ATPase was a consequence of the synergistic actions of EGF and cyclic AMP. The tyrosine kinase activity of the EGF receptor was essential for the induction of ectoCa2(+)-ATPase, since enzyme induction was abolished by a tyrosine kinase inhibitor, genistein. Cycloheximide and actinomycin D were also inhibitory to enzyme induction, indicating that enhancement of enzyme activity by EGF and cAMP was not due to post-translational modification. The results of this and previous investigations established that the two ectoATPases of Li-7A cells are under different regulation.

Butyrate induces an ectoMg2(+)-ATPase activity in Li-7A human hepatoma cells.

The human hepatoma cell line (Li-7A) possesses a high concentration of epidermal growth factor (EGF) receptors and exhibits ectoATPase activity in the presence of either MgATP or CaATP (Knowles: J. Cell. Physiol., 134:109-116, 1988). Growth for 96 hours in the presence of both EGF and cholera toxin or another cyclic AMP elevating agent induced an ectoATPase activity which was more active with CaATP and resistant to inhibition by the sulfydryl reagent, p-chloromercuriphenylsulfonate (pCMPS) (Knowles: Arch. Biochem. Biophys., 263: 264-271, 1988). In contrast, treatment of cells with butyrate, a short chain organic acid which can be derived from the analogue, dibutyryl cyclic AMP, resulted in a 4-7-fold increase of an ectoATPase which was more active with MgATP and highly sensitive to pCMPS inhibition. Maximal induction by butyrate required 48 hours and was dependent on butyrate concentration, but was independent of EGF and cyclic AMP elevating agents. Of six organic acids tested, butyrate was most effective in the induction of the ectoMg2(+)-ATPase. The increase in the ectoMg2(+)-ATPase activity could be prevented with actinomycin D and cycloheximide, indicating that both transcription and translation were necessary for induction. In addition to the induction of the ectoMg2(+)-ATPase, butyrate induced alkaline phosphatase activity, but had no effect on a third ectoenzyme 5'-nucleotidase. These data further support our proposal that two distinct ectoATPases exist in the plasma membrane of Li-7A hepatoma cells.

Enzymatic synthesis and hydrolysis of [32P]phosphatidylinositol phosphate.

Phosphatidylinositol kinase activity in plasma membrane preparations of mouse liver was found to be comparable to that in A431 cells and higher than that in three human tumor xenografts. This activity was exploited in preparing 32P-labeled phosphatidylinositol phosphate of high specific radioactivity in which approximately 4% of the radioactivity of the substrate, [gamma-32P]ATP, was incorporated into the lipid. The subcellular distribution of phosphatidylinositol phosphate phosphatase in a human astrocytoma xenograft was determined using [32P]phosphatidylinositol phosphate as a substrate. The highest phosphatase activity was found in the plasma membranes.

Purification of a low affinity Mg2+ (Ca2+)-ATPase from the plasma membranes of a human oat cell carcinoma.

Plasma membranes of many mammalian cells contain a Mg2+-dependent ATPase activity which is easily inactivated by detergents. This activity is the combined expression of at least two ATP-hydrolyzing enzymes (Knowles, A.F., Isler, R.E., and Reece, J.F. (1983) Biochim. Biophys. Acta 731, 88-96). We have purified one of these enzymes from the plasma membranes of a human oat cell carcinoma xenograft. The enzyme was extracted from the membranes by 0.5% digitonin and purified on a DE52 column. The purified enzyme contained a major protein band of Mr = 30,000 when dissociated by sodium dodecyl sulfate. It hydrolyzed all nucleoside triphosphates in the presence of Mg2+ or Ca2+, but showed little activity toward nucleoside diphosphates. The enzyme was inhibited by p-chloromercuriphenyl sulfonate, slowly inactivated by p-fluorosulfonylbenzoyl-5'-adenosine and dithiothreitol at room temperature, and lost activity readily in solutions containing low concentrations of several detergents. This knowledge of the macromolecular structure of the Mg2+(Ca2+)-ATPase and its catalytic properties is important in determining the orientation of the enzyme in the membrane and its physiological function.

Properties of a reconstituted calcium pump.

1. The translocation of 45Ca2+ in vesicles reconstituted with purified Ca2+ ATPase of sarcoplasmic reticulum and phospholipids was dependent on ATP and varied greatly with the composition of the phospholipids. 2. In contrast to sarcoplasmic reticulum fragments, the reconstituted vesicles were impermeable to 14C-labeled oxalate, 3H- or 32P-labeled ATP, or 32P-i. There was no translocation of phosphate from gamma-labeled ATP during Ca2+ uptake. These results are inconsistent with some current formulations of the mechanism of pump action. 3. Reversal of the Ca2+ pump and generation of ATP and ADP and P-i was observed when vesicles loaded with Ca2+ were exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. 4. Experiments on the formation of phosphoenzyme with 32P-labeled ATP showed that most if not all functional ATPase molecules in the reconstituted vesicles were oriented in the same direction, as in the case of sarcoplasmic reticulum fragments.

Formation of adenosine triphosphate from Pi and adenosine diphosphate by purified Ca-2+-adenosine triphosphatase.

Ca-2+-ATPase purified from sarcoplasmic reticulum of rabbit muscle forms a phsophoeznyme when exposed to inorganic phosphate in the presence of Mg-2+. On addition of ADP and Ca-2+ virtually all of the phosphate bound to the enzyme is transferred to form ATP. It has been shown previously and confirmed by us that (a) the purified ATPase contains one major polypeptide and about 30% phospholipids; (b) on removal of residual detergent by passage through Sephadex the enzyme forms vesicular membranes; and (c) these vesicles are leaky and incapable of accumulating Ca-2+. Our findings therefore indicate that we have observed ATP generation from ADP and P-i without the formation of an ion gradient across a membrane. We propose that the energy derived from ion-protein interaction drives the formation of ATP.

Prevalence of the mercurial-sensitive EctoATPase in human small cell lung carcinoma: characterization and partial purification.

Cell surface ATPase (ectoATPase) activity is detected on many mammalian cells. Previous documentation in the rat hepatocyte-hepatoma system indicated that ectoATPase activity increased during tumorigenesis with accompanying changes in enzymatic properties and localization. These results, combined with the recently established characteristics of two distinct ectoATPases, a mercurial-sensitive ectoATPase, and a mercurial-insensitive ectoATPase, suggest that the former is increased, whereas the latter is decreased, during hepatoma formation. We found that the mercurial-sensitive ecto-ATPase was also expressed at high levels in three lines of human small cell lung carcinoma (SCLC) cells. During purification of this enzyme from an SCLC xenograft, four isoforms of this enzyme, with similar biochemical properties but different ionic charges were detected. The elution of two proteins of 170 and 150 kDa from a DEAE-cellulose column appeared to correlate with elution of ATPase activity. These characterizations should be useful in the further investigation of the molecular structure and function of the SCLC mercurial-sensitive ectoATPase which may be an important cell surface marker of SCLC cells.

The epidermal growth factor/cAMP-inducible ectoCa(2+)-ATPase of human hepatoma Li-7A cells is similar to rat liver ectoATPase/hepatocyte cell adhesion molecule (cell-CAM 105).

Human hepatoma Li-7A cells exhibit two cell surface ATPase (ectoATPase) activities distinguishable by their different biochemical properties. The activity of the minor ectoATPase, ectoCa(2+)-ATPase, is enhanced severalfold when Li-7A cells are treated simultaneously by epidermal growth factor (EGF) and cAMP elevating agents (Knowles, A. F., 1990, Arch. Biochem. Biophys. 283, 114-119). Here we report that the human ectoCa(2+)-ATPase is biochemically similar to the major rat hepatocyte ectoATPase/cell adhesion molecule (cell-CAM 105) with respect to response to divalent ions and sulfhydryl reagents. Furthermore, the binding of rat liver ectoATPase antibody increased markedly in EGF/cholera toxin/hydrocortisone-treated Li-7A cells compared to untreated cells. Western blot analysis revealed cross-reactivity of the antibody with a 125-kDa protein. Partial purification of ectoCa(2+)-ATPase from EGF/cholera toxin/hydrocortisone-treated Li-7A cells confirmed that enrichment of the 125-kDa protein correlated with an increase in ATPase activity. We conclude that EGF and increased levels of cAMP lead to increased synthesis of the ectoCa(2+)-ATPase in Li-7A cells. The present demonstration of similarity between the ectoCa(2+)-ATPase and a rat liver cell adhesion molecule, cell-CAM 105, contributes significantly to an understanding of the implication of down-regulation of ectoCa(2+)-ATPase during hepatocyte-hepatoma transformation.