Adenosine triphosphatases may be involved in the transport of some ions across the plasma membrane, which is an important biological phenomenon required to maintain the constancy of the
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Adenosine Triphosphatases on Plasma Membrane and Membrane Surrounding Lipid Vacuoles in
L1210 Lymphoid Leukemic Cells
ABSTRACT. The activity of adenosine triphosphatases in L1210 lymphoid leukemic cells was demonstrated by ultracytochemical methods. The activity
of Na+,K+-ATPase was found only in the plasma membrane, but the activity of Mg2+-ATPase and Ca2+-ATPase was found in both the plasma membrane
and in the membrane surrounding lipid vacuoles in the cytoplasm. The activity of Ca2+-ATPase predominated on the periphery of the lipid vacuoles. The
localization of these enzymes is discussed in reference to its possible significance in transformed cells.
Adenosine triphosphatases may be involved in the transport of some ions across the plasma membrane, which is an important biological phenomenon required to maintain the constancy of the pH and osmotic pressure in cells, and which activates a number of enzymes and regulates the exchange and absorption of some substances.
In transformed cells, however, at early phases of transformation there is an increased influx not only of such ions as Na+, K+, PO43+, Mg2+ and Ca2+ (2), but also of nucleosides, sugars and amino acids. We recently showed that in L1210 lymphoid leukemic lymphocytes the plasma membrane has a rather low ability to bind the excess of inflowing calcium (1). This phenomenon corresponds to the reported rapid penetration of these ions into transformed cells (3, 17). This is believed to be due to adenosine triphosphatases (ATPases) activities in the membrane because of their role in the release of energy. As for the activity of adenosine triphosphatases in leukemic
cells, there are few electron-microscopis studies on this subject. Therefore we have attempted to determine the activity of Mg2+-ATPase, Na+, K+-ATPase and Ca2+-ATPase in L1210 cells. We also were interested in discovering whether there is any coincidence in the distribution of the activity of ATPases activated by various
cations with the appearance of the calcium-binding sites we found previously in L 1210 cells (1).
RESULTS
The activity of all three ATPases was found in the plasma membrane of L1210 lymphoid leukemic cells and two of the activities also were found within the cells on the periphery of lipid globules.
As regards Mg2+-ATPase, when cells were incubated in a suitable Mg-ATP medium, enzymatic activity was distinctly positive. The electron-dense reaction products were distributed regularly on the external side of the lymphocyte plasma membrane. The same products also were found, although not regularly, along the periphery of the lipid vacuoles in the cytoplasm.
Localization of Ca2+-ATPase in L1210 cells differed from that of Mg2+-ATPase. Heavy black lead deposits were seen mainly at the membrane surrounding the lipid globules. the reaction also was detected in the plasma membrane, but seemed to be rather weak.
Another situation was observed for the activity of Na+,K+-ATPase. Reaction products after incubation with Na-F,K+-ATPase medium appeared only on the external surface of the plasma membrane (Fig. 3-4); no deposits were seen in the cytoplasm. When four different concentrations, 2.5, 0.5, 0.25 and 0.1 mM of lead ions, were used
we obtained similar results for the activity and distribution of Na±,K+-ATPase.
No lead deposits were observed in the plasma membrane or around the lipid globules in any of the experimental cells when the three enzymes were incubated in the control medium without substrate. The absence of electron-dense deposits in these control L1210 lymphocytes demonstrated that there is no affinity of the plasma membrane for Pb2+. Apparently, the deposits found in our study occur only when
the plasma membrane contains free phosphate ions formed by the enzymatic hydrolysis of sodium ATP in the media. Similarly, in the control media without the cations Na+, K+, Ca2+, and Mg2+, no positive reaction for the enzymes was obtained. This indicates that no nonenzymatic hydrolysis of ATP by Pb2+ occurred in the incubation medium.
Incubation with ouabain inhibited Na+, K+-ATPase activity on the plasma membrane of the experimental cells.
Results are summarized in Table 1, but these results are for fixation in situ with 5 % glutaraldehyde. In other cases, when samples were fixed in situ with 1.5 % glutaraldehyde, the cells appeared to be severely damaged, and no precipitated products.
DISCUSSION
Na+,1(+-ATPase, Ca2+-ATPase and Mg2+-ATPase are present in the plasma membrane of L1210 cells but their distribution along the membrane profiles and the intensity of their activity differs. In the cytoplasm, in the membranes of the lipid vacuoles only Mg2+-ATPase and Ca2+-ATPase activities were found. Many discussions of ATPase cytochemistry have been devoted to the inhibitory effect of glutaraldehyde fixation as well as to lead ions and non-enzymatic ATP hydrolysis by the latter (11, 18). In our studies, the lack of any deposits in cells incubated
in medium containing ATP and Pb(NO3)2 indicates that non-enzymatic hydrolysis is not responsible for the splitting of ATP in L 1210 cells. As for Na+,K+-ATPase in L1210 cells, its activity does not seem to be inhibited by lead ions at concentrations up to 2.5 mM.
Our results agree with those of Russo et al. (20) who detected Na+,K+-ATPase and Mg2+-ATPase in mammary carcinoma when using a concentration of 3.6 mM Pb(NO3)2. Marchesi and Palade have reported that such a high concentration of Pb ion in the incubation medium inhibits Na±,K+-ATPase activity (11). The discrepancy
between their findings and ours and Russo et al. have yet to be explained. As to the influence of fixation, an application of 5 % glutaraldehyde was more favorable for the protection of all three ATPase activities than was an application of 1.5 % glutaraldehyde when the fixative was injected into the animals body. Probably under these conditions an injection of a low concentration of glutaraldehyde, further diluted by body fluids, does not preserve the cell constituents well ; this is reflected in serious damage to the structure of the cell and in the loss of enzyme activity. Recently, studies with other tumor cells have shown fixation in 5 % glutaraldehyde does not disturb the activity of ATPase (6).
The localization of Ca2+-ATPase and Mg2+-ATPase around the lipid globules is of special interest. The presence of numerous calcium-binding sites has been recently revealed in the membrane surrounding these globules (1). In both cases the applied cytochemical reactions were completely negative when proper control conditions were used. No Ca2+-ATPase activity was found when either Ca2+ or ATP was lacking in the incubation medium ; this confirms the truly enzymatic character of the observed reaction. When we visualized Ca-binding sites according to the method of Oschman and Wall (12), no Ca-dependent deposits appeared when the experimental medium contained a Ca-chelating agent (1). Thus, localization at the lipid vacuole membrane of both Ca2+-binding sites and Ca2+ and Mg2+-activated ATPase is not accidental. The two phenomena appear to be intimately related to the regulation of the intracellular level of calcium ions in L1210 cells.
Probably the lipid globules are involved in the sequestration of calcium (1), and the reverse process, extrusion of calcium requires energy provided, not by the sodium electron chemical gradient, but from ATPase activity sensitive to changes in the free calcium concentration. Such a mechanism for calcium extrusion has been proposed by several researchers who studied the plasma membranes of hepatocytes (19), human erythrocytes (10) and Ehrlich ascites tumor cells (8). However, the energy required for the extrusion of calcium also may be supplied by the activation of Mg2+-ATPase. At present, a Ca pump linked to the high activity of Mg2+-ATPase in mouse and rat livers (4, 7), in embryonic chick fibroblasts (14), in rat kidney cortex (13), and in guinea pig placenta (21) has been demonstrated. Thus, in L1210 cells the high activities of Ca2+-ATPase and Mg2+-ATPase, but not of Na+,K+-ATPase in the membrane surrounding the lipid globules may be connected with the release of calcium ions from these globules.
Moreover, the possibility that phospholipids, abundant in lipid globules, play a role in the activation of Ca2+-ATPase and Mg2+-ATPase, as demonstrated in erythrocytes (5) has not been excluded.
Recently it has been shown that Ca2+-ATPase is activated by calmodulin, which binds reversibly to the internal surface of the plasma membrane (9). A high concentration of calcium in the cytosol induces the detachment of calmodulin from the membrane (15). A relationship between the low activity of Ca2+-ATPase found by us in the membrane of L1210 cells and the rapid calcium influx characterizing these cells (19) which results in an increase in intracellular calcium and the detachment of calmodulin from the membrane cannot be excluded. In contrast, perhaps in the extrusion of calcium across the membrane of these cells Na±,K+-ATPase and Mg2+ -ATPase are engaged the former by stimulating the exchange of ions across the membrane and the latter by supplying the energy for this process. Both enzymes display rather high activity in the plasma membrane of L1210 cells, as shown in this study.
Acknowledgements. I am very grateful to Professor A. Przelcka for her continuous guidance during this study and for her critical reading of the manuscript; to Associate Professor B. Grzelakowska- Sztabert for her critical reading of the manuscript; to Mrs. K. Mrozinska for her technical assistance; and to Professor J. Kawiak for kindly providing the experimental animals and for many interesting discussions.
This study was supported by a research grant to MR.II.1. from the M. Nencki Institute of Experimental Biology of the Polish Academy of Sciences.
ATPase in L1210 Cells
Dai Duy Ban was in Department of Cell Biology, Nencki Institute of Experimental Biology, 02-093, Warsaw, Poland* and Department of Biophysics and Biomathematics, Medical Center of Postgraduate Education, 01-813, Warsaw, Poland