Calcif Tissue Int (1992) 51:312-316

Calcified Tissue International 9 1992 Springer-Verlag New York Inc.

Demonstration of Calmodulin-Sensitive Calcium Translocation by Isolated Osteoclast Plasma Membrane Vesicles Petrus J. Bekker 1 and Carol V. Gay 1'2 ~Departments of Molecular and Cell Biology and 2poultry Science, 468A North Frear, The Pennsylvania State University, University Park, Pennsylvania 16802, USA Received November 14, 1991, and in revised form January 23, 1992

Summary. Plasma membrane vesicles were prepared from chicken osteoclasts, and active calcium transport was demonstrated in a spectrofluorimetric assay using the fluorescent calcium concentration indicator, fura-2. Transport activity was inhibited by quercetin (10 ~LM), sodium vanadate (10 pLM), and the anticalmodulin agents, compound 48/80 (20 and 200 txg/ml) and calmidazolium (10 and 20 ~LM). The transport rate (Vm~x, 1.3 nmol/mg protein/min) was not altered in the presence of the protonophore, nigericin (1 ~LM), indicating that proton transport was not driving calcium transport. Release of accumulated calcium in the vesicles occurred with the addition of bromo-A23187 (5 ~LM) or ionomycin (5 IxM). Increasing calcium transport occurred with increasing calcium concentration. Finally, the calmodulin content of the vesicles was demonstrated to be 54-134 U/mg protein. These results d e m o n s t r a t e that a calmodulin-sensitive, ATPdependent calcium transporter is present in the osteoclast plasma membrane.

Key words: Osteoclasts - Plasma membrane vesicles - Calcium transport.

The osteoclast plasma membrane has a polarized distribution of calcium-dependent ATPase activity; specifically, the pump is situated in the plasma membrane opposite the ruffled border membrane [I]. Other cells reported to contain asymmetrically distributed calcium-dependent ATPase include renal [2, 3] and intestinal [4, 5] epithelial cells, placental trophoblastic cells [6], and osteoblasts [1]. We have previously described an isolation and characterization procedure for chicken osteoclast plasma membrane vesicles [7]. In that study, a calcium-stimulated, magnesium-dependent ATPase activity in those vesicles was characterized. The present study was performed to demonstrate ATP-dependent calcium transport and to define the characteristics of a plasma membrane calcium pump in osteoclast plasma membrane vesicles. A method for demonstrating calcium transport using the calcium-sensitive fluorescent indicator, fura-2, is described. The results indicate that the osteoclast plasma membrane contains an outwardly directed energy-dependent calcium transporter, with characteristics resembling a plasma membrane calcium transporter found in many other cells.

Offprint requests to: C. V. Gay

Materials and Methods Fura-2 (base) was purchased from Molecular Probes, Inc. (Eugene, OR). Other chemicals, obtained from Sigma Chemical Co. (St. Louis, MO), included 5'-nucleotidase from Crotalus adamanteus venom (N-4005), phosphodiesterase 3':5'-cyclic nucleotide (activator-deficient) from bovine heart (P0520), and bovine brain calmodulin (P2277) were used in the calmodulin assay. Osteoclasts were routinely isolated from 3- to 3.5-week-old chicks (Peterson-Arbor Acres strain), on a low calcium (0.3%) diet for 2-2.5 weeks prior to sacrifice. The complete cell isolation and vesicle preparation procedure was described earlier [7]. Briefly, osteoclasts were harvested from tibial endosteal surfaces by scraping with a rubber policeman. After sequential filtration of the cell suspension to remove debris, osteoclasts were purified using a Percoll step gradient. This procedure yielded 77% intact osteoclasts, 2-4% alkaline phosphatase positive cells, 15% acid phosphatase positive debris, and the remainder was unidentifiable debris. Homogenization was performed by passage through a thin bore needle (30G), and plasma membrane vesicles were isolated by centrifugation in a continuous Percoll gradient (20%). The plasma membrane suspension was characterized by methods described in reference [7]: the plasma membrane markers, 5'-nucleotidase and ouabain-sensitive Na +K+-ATPase, were enriched five- and 10-fold, respectively; the membranes were vesicular, as determined by electron microscopy; SDS latency of 5'-nucleotidase activity revealed that 10-15% of vesicles were sealed in the inside-out orientation. Only freshly isolated vesicles were used in the calcium transport assays. Only sealed inside-out vesicles would accumulate calcium in an active calcium transport assay, as sealed right side-out vesicles would not be able to bind ATP and leaky vesicles would not accumulate calcium. The plasma membrane preparations employed corresponded to fractions A1 and B1 in reference [7]. Fraction A1 contained 1.7 times more 5'-nucleotidase and 1.4 times more Na§ activity than fraction B1. Fraction A1 was used throughout this study. Calcium transport rates were measured in fraction B1 for comparison. Calcium transport was demonstrated spectrofluorimetrically using the fluorescent calcium concentration indicator, fura-2, developed by Grynkiewicz et al. [8]. The reaction medium contained 150 mM KCI, 0.5 mM MgC12, 5 mM Hepes (pH 7.3), 1 mM ouabain, to block Na+-K+-ATPase activity, and 20 ~g/ml oligomycin, to block mitochondrial ATPase activity. A stock solution of fura-2 (base) in 0.1 M Tris/HC1 (pH 7.2) was added to this medium just prior to transport assay to make a final fura-2 concentration of 2 IxM. This solution was protected from light to prevent fluorescence decay. To 60 ~Llof fura-2 solution, 60 pJ of plasma membrane vesicles in 0.25 M sucrose, 5 mM Hepes, pH 7.3 was added (10-40 p~gprotein) and mixed in a quartz microcuvette (105.250-QS, Hellma, Forest Hills, NY). A 6-ram Teflon spacer was inserted in the cuvette holder before insertion of the cuvette into the holder to insure that the cuvette window was at the proper height. The vesicles were equilibrated at room temperature for at least 15 minutes. Samples were excited alternately at I = 340 and I = 380 nm using a chopper in a dual excitation spectrofluorimeter (Spex Fluorolog-2). Emission was

P. J. Bekker and C. V. Gay: Calcium Translocation by Osteoclast Plasma Membrane continuously monitored at I = 505 nm. The excitation slit widths were adjusted to 1 mm or less, and the emission slit width was set at 1.5 mm. Data were collected in the signal/reference mode as photon counts per second, using a rhodamine-B reference quantum counter. The excitation slits and high voltage were adjusted to obtain a 340nm signal in the region of 106 counts per second and a current of 0.1-1 mA. The change in ratio of the signals at Iex~ = 340 nm and Iexc = 380 nm is an accurate estimate of the change in calcium concentration [8]. The relatively membrane-impermeable form of fura-2 was employed and therefore sealed plasma membrane vesicles would not accumulate a significant amount of dye over a short period of time (30 minutes or less). Changes in the intravesicular calcium concentration were measured after addition of 1.2 I~1 MgATP, 2 mM final concentration. Before use, the pH of the MgATP solution was adjusted to 7.3 with 2 M Tris. Bromo-A23187, a nonfluorescent analog of the calcium ionophore, A23187, was added at a final concentration of 5 ixM to release accumulated calcium from vesicles. Rates of transport were estimated as tangents to the decrease in the I34o/I38o ratio over the first 150-250 seconds after MgATP addition to the vesicle suspension. Transport assays were also performed in the presence and absence of NaVO3, Na3VO4, quercetin, nigericin, and the anticalmodulin drugs, compound 48/80 and calmidazolium (R24571). The calcium transport rate at various calcium concentrations was determined by adding increasing amounts of CaC12 or EGTA to the vesicle suspension just prior to assay and equilibrating at room temperature for 15 minutes. Transport was initiated by MgATP addition, release was mediated by Br-A23187, after which CaC12 was added to a final concentration of 10 mM to saturate fura-2 and to obtain Rmax, the 340/380 ratio at dye saturation. When stable signals were observed, EGTA (5 mM final concentration) was added to obtain Rmin, the 340/380 ratio at zero-free calcium. The Rmin obtained experimentally was 1.17 --- 0.22 and the Rmax 19.66 -+ 3.04. Autofluorescence was not significant. The calmodulin content of the isolated plasma membranes was determined based on the method described by Teo et al. [9]. The procedure involved measurement of the degree of phosphodiesterase (PDE) activation by calmodutin. Cyclic-AMP is converted to 5'-AMP by PDE, and 5'-AMP is hydrolyzed by 5'-nucleotidase. The amount of inorganic phosphate released is measured by the Fiske and Subbarow method [10]. In the presence of an increasing amount of calmodulin, PDE is increasingly activated until a plateau is reached. As the plasma membrane preparation contained 5'nucleotidase and possibly PDE activity, the plasma membranes were boiled for 3 minutes prior to calmodulin assay to destroy these enzyme activities. Endogenous calmodulin, which is notably heat resistant, would not be damaged by this treatment [11]. The reaction medium contained 72 mM Tris, 72 mM imidazole, 36 mM MgCI2, and 40 IxM CaC12 (pH 7.5). To aliquots of this medium, 3.2 mU PDE, 0.4 U 5'-nucleotidase and increasing concentrations of calmodulin (0-12 U/ml) were added in a final volume of 0.5 ml. These samples were preincubated at 37~ for 10 minutes. Then, 0.5 ml cyclic-AMP in the Tris-imidazole buffer solution (at 37~ was added to the samples at 1.8 mM final concentration. After incubation at 37~ for 30 minutes, reactions were terminated by addition of ice-cold trichloroacetic acid, and the amount of inorganic phosphate released was measured spectrophotometrically [ 10]. A standard curve of calmodulin versus inorganic phosphate released was constructed. The endogenous calmodulin content was determined by using the plasma membrane suspension (pretreated at 100~ for 3 minutes), instead of calmodulin, in the procedure described above.

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Time Isec] Fig. 1. Change in calcium concentration in medium which contained 2 ~M fura-2 and 60 ml vesicles (10-40 mg protein) as a function of time. The points of additions of 2 mM MgATP, (41-) and 5 IxM Br-A23187 (y), are shown.

ratio signal, indicating a d e c r e a s e in the e x t r a v e s i c u l a r calcium concentration. The increase in the ratio after addition of the nonfluorescent b r o m o - a n a l o g o f the calcium ionophore, A23187, indicated a release of a c c u m u l a t e d calcium. I o n o m y c i n (5 IxM), another calcium ionophore, caused similar calcium release (not shown). In Figure 2, the inhibition of calcium transport in the p r e s e n c e of q u e r c e t i n is shown. The inhibition o b s e r v e d w h e n the anticalmodulin drug, compound 48/80, was added is depicted in Figure 3. As proton transport also o c c u r r e d with the addition of M g A T P [12], the possibility that proton m o v e m e n t might be responsible for calcium uptake was tested. To obliterate the developing proton gradient after M g A T P addition, the calcium transport assay was run in the p r e s e n c e of the protonophore, nigericin. As can be seen f r o m Figure 4, there was no difference b e t w e e n calcium transport in the absence or presence of nigericin. Sodium vanadate (both the m e t a and the ortho forms) and another anticatmodulin drug, calmidazolium, caused partial inhibition of calcium transport. A s u m m a r y of results obtained with different inhibitors is shown in Table 1. The effect of increasing calcium c o n c e n t r a t i o n in the reaction medium is shown in Figure 5. T h e r e was an increase in transport rate with increasing free calcium. F r o m a standard curve of calmodulin versus inorganic phosphate released, the calmodulin c o n c e n t r a t i o n was estimated to be 54-134 U / m g protein. The ratio of calcium transport rate in fraction A1 versus B1 was 1.7; for 5'-nucleotidase, the A1/B1 ratio was also 1.7, and for N a + - K + - A T P a s e it was 1.4.

Discussion Results Figure 1 shows the change in calcium c o n c e n t r a t i o n as function of time as vesicles r e m o v e calcium f r o m the surrounding medium. Before A T P addition, the 340/380 ratio baseline was stable and c o r r e s p o n d e d to a free calcium concentration of 0.1-0.5 txM, as d e t e r m i n e d f r o m the S p e x program (Beta 2.45 version). A f t e r A T P addition, there was a decline in the

We h a v e s h o w n p r e v i o u s l y that a M g - d e p e n d e n t Ca 2+A T P a s e activity is p r e s e n t in isolated o s t e o c l a s t p l a s m a m e m b r a n e s at a level of 19.8 m M / m g protein/hour [7]; this activity is 2- to 25-fold greater than values r e p o r t e d for other tissues [11]. Also, in an e n z y m e histochemical study from this laboratory, v a n a d a t e - s e n s i t i v e C a Z + - A T P a s e activity was demonstrated in the osteoclast plasma m e m b r a n e opposite the ruffled b o r d e r m e m b r a n e [1].

314

P.J. Bekker and C. V. Gay: Calcium Translocation by Osteoclast Plasma Membrane 2 -

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Fig. 4. Calcium transport in the absence or presence of the protonophore, nigericin (1 txM). The points of addition of 2 mM MgATP (-II-) and 5 ixM Br-A23187 (y) are shown.

Table 1. Summary of inhibitor effects on active calcium transport in

osteoclast plasma membrane vesicles

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Concentration

% Inhibition

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35.6 29.6 17.1 64.4 45.5 54.6 35.2 72.0

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free calcium concentration, M Fig. 5. Calcium transport at increasing free calcium concentration. Data were obtained from three separate experiments.

sium concentration [8]. Changes in fluorescence due to changes in magnesium concentration is therefore insignificant. The membrane preparation used to demonstrate calcium transport was enriched in the plasma membrane markers ouabain-sensitive Na+-K+-ATPase and 5'-nucleotidase [7], but to ensure that we were looking at plasma membrane calcium pumping activity, we compared the rate of calcium transport observed with fraction A1 to fraction B1. The former was higher in plasma membrane markers than the latter [7]. The A1/B1 ratio of calcium transport rate and 5'nucleotidase activity were both found to be 1.7, and that of Na +-K +-ATPase, 1.4. We concluded that the calcium transport activity was of plasma membrane origin. Vanadate (both the meta and the ortho forms) caused partial inhibition of calcium transport activity; the reason for this incomplete inhibition is not clear. Higher concentrations of vanadate did not lead to increased inhibition. It was also observed that NaVO3 also did not completely inhibit the Ca 2+-ATPase activity in osteoclast plasma membranes prepared similarly [7]. Vanadate inhibition of the calcium pump is indicative of a phosphorylated intermediate in the enzymatic reaction cycle. It binds to the E 2 conformation and prevents the E 2 to E1 conformational change, similar to the mechanism for inhibition of Na+-K+-ATPase [13]. The erythrocyte plasma membrane calcium is highly sensitive to vanadate (Ki = 5 p~m). However, calcium pumps described in other cells (e.g., hepatocytes) are not as sensitive to vanadate as the erythrocyte pump. Epping and Bygrave [ 14] saw no inhibition by vanadate of the liver plasma membrane calcium pump, and Chan and Junger [15] found a Ki of - 2 0 ~M. Dog heart sarcolemma calcium transport was inhibited with a Ki of 0.5 ~M [16] and erythrocyte calcium transport with a K i of 0.4--0.9 ~M, although the maximum inhibition was -80% [17]. Quercetin, or 3,3',4',5,7-pentahydroxyflavone, is a potent inhibitor (Ki = 4-6 FM) of the erythrocyte calcium pump [18]. It is a noncompetitive inhibitor with respect to Ca 2§ or ATP activation of the Ca 2§ Because it is highly membrane-permeable, it is difficult to study the sidedness of inhibition. Quercetin is not a specific inhibitor of the plasma membrane calcium pump, however, because it also inhibits the Na § +-ATPase [19] and the sarcoplasmic

315

reticulum Ca2+-ATPase [20]. Nevertheless, our finding of strong inhibition of calcium transport in osteoclast plasma membrane vesicles support the evidence indicating the presence of an active calcium pump in the membranes. Calmodulin influences many cellular enzymes; however, it regulates only one transport ATPase, namely, the plasma membrane calcium pump. It is an acidic protein which binds to a positively charged - 2 7 amino acid sequence near the carboxyl terminus of the enzyme [21], and it seems to expose a calcium binding site on the pump. This mechanism of activation is similar to activation by partial proteolytic degradation of the enzyme [22-25], which causes removal of the calmodulin-binding domain of the pump and also results in exposure of the active site. Anticalmodulin drugs compete with the CaZ+-ATPase for calmodulin and therefore cause inhibition of Ca z +-ATPase and calcium transport activity. Calmidazolium (compound R24571) is a highly lipophylic derivative of the antimicotic drug, miconazole, with potent anticalmodulin properties [26]. It inhibited active calcium transport activity in erythrocyte plasma membrane vesicles with a K i of 2 IxM [26]. Our finding of partial inhibition of calcium transport activity in osteoclast plasma membrane vesicles emphasized that this pump is calmodulin-sensitive. Partial inhibition was not surprising, as the calcium pump is still functional, although at a lower rate, in the absence of calmodulin. Compound 48/80, a condensation product of N-methyl-pmethoxy-phenethylamine with formaldehyde, is a histaminereleasing agent. It consists of a mixture of homologous hydrophobic polycations [26]. This compound inhibited the calmodulin-dependem calcium-translocating component of the enzyme far more specifically than basal enzyme activity [26]. The Ki of calcium transport in erythrocytes was determined to be 7 p~g/ml. Although compound 48/80 inhibited calcium transport in osteoclast plasma membrane vesicles with slightly lower affinity (35% inhibition at 20 txg/ml), our results strongly suggest calmodulin involvement with the calcium pump. The data presented in Figure 5 was not sufficient to obtain Kca and Vmax values, mainly because near saturation of fura-2, the sensitivity of the assay is decreased. Therefore, calcium transport at high free calcium concentrations could not be determined effectively. However, the concentration range and the shape of the curve resembled that of the highaffinity component of the Ca2+-ATPase activity in the osteoclast plasma membrane [7]. The maximum rate of calcium transport observed in osteoclast plasma membrane vesicles was 1.3 nmol/mg protein/minute. This is similar to the Vm~x of other cells with a polarized distribution of the calcium pump: 2 nmol/mg/minute for placental trophoblast pump [6], 1 nmol/mg/minute for rat kidney cortex pump [3], and 0.34.7 nmol/mg/minute for small intestine epithelial cell membranes [4]. The rate for rat liver plasma membranes varied from 35 pmol/mg/minute [15] to 30 nmol/mg/minute [27], corpus luteal membranes had a Vmax of 85 pmol/mg/minute [28], and rat parotid gland basolateral membranes [29] and neutrophil membranes [30] showed a rate of 17 nmol/mg/minute. Erythrocyte inside-out vesicles had a Vm~ of 5-10 nmol/mg/ minute [31, 32]. This large variability is understandable considering that different tissues, isolation techniques, and methods for demonstrating calcium transport are used. The trifluoperazine inhibition and lack of stimulation of Ca2+-ATPase activity with calmodulin addition [7], as well as results with anticalmodulin agents in the present study, suggested that endogenous calmodulin was still present in the vesicles after preparation. Therefore, calmodulin assays were performed on the isolated vesicles. The strong associ-

316

P. J. Bekker and C. V. Gay: Calcium Translocation by Osteoclast Plasma Membrane

ation of calmodulin with the osteoclast plasma membrane, even after centrifugation in 1.2 mM E G T A , was confirmed. From the evidence presented, it is concluded that a calmodulin-sensitive energy-dependent plasma membrane calcium pump is present in osteoclasts. The role of the pump is likely to be maintenance of low intracellular calcium levels; in addition, the assymetric distribution of the pump [1] suggests a role in the transcellular movement of calcium from bone to blood, similar to its transceUular role in the placental trophoblast and intestinal epithelium.

15. 16. 17.

18.

Acknowledgments. The authors are indebted to Dr. David J. Hurley, Susan E. Lingenfelter, Nancy L. Kief, and Virginia R. Gilman for technical support and expertise. This work was supported by NIH Grant DE04345 to C.V.G. and a NASA Grant, NAGW-1196 to the Center for Cell Research at Penn State University.

19. 20. 21.

References 1. Akisaka T, Yamamoto T, Gay CV (1988) Ultracytochemical investigation of calcium-activated adenosine triphosphatase (Ca + +-ATPase) in chick tibia. J Bone Miner Res 3:19-25 2. Kinne-Saffran E, Kinne R (1974) Localization of a calciumstimulated ATPase in the basal-lateral plasma membranes of the proximal tubule of rat kidney cortex. J Membr Biol 17:263-274 3. Gmaj P, Murer H, Kinne R (1979) Calcium ion transport across plasma membranes isolated from rat kidney cortex. Biochem J 178:54%557 4. Ghijsen WEJM, Van Os CH (1979) Ca-stimulated ATPase in brush border and basolateral membranes of rat duodenum with high affinity sites for Ca ions. Nature 279:802-803 5. Nellans HN, Popovitch JF (1981) Calmodulin-regulated, ATPdriven calcium transport by basolateral membranes of rat small intestine. J Biol Chem 256:9932-9936 6. Fisher GJ, Kelley LK, Smith CH (1987) ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast. Am J Physiol 252:C38-C46 7. Bekker PJ, Gay CV (1990) Characterization of a Ca + +-ATPase in osteoclast plasma membrane. J Bone Miner Res 5:557-567 8. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450 9. Teo TS, Wang TH, Wang JH (1973) Purification and properties of the protein activator of bovine heart cyclic adenosine 3',5'monophosphate phosphodiesterase. J Biol Chem 248:588-595 10. Fiske CH, Subbarow Y (1925) The colorimetric determination of phosphorus. J Biol Chem 66:375-400 11. Rega AF, Garrahan PJ (1986) The Ca2+ pump of plasma membranes. CRC Press, Boca Raton, FL, pp 27 and 56 12. Bekker PJ, Gay CV (1990) Biochemical characterization of an electrogenic vacuolar proton pump in purified chicken osteoclast plasma membrane vesicles. J Bone Miner Res 5:569-579 13. Karlish SJD, Beauge LA, Glynn IM (1979) Vanadate inhibits (Na + +K+)-ATPase by blocking a conformational change of the unphosphorylated form. Nature 282:333-335 14. Epping RJ, Bygrave FL (1984) A procedure for the rapid isolation from rat liver of plasma membrane vesicles exhibiting

22.

23.

24.

25.

26.

27. 28. 29. 30. 31. 32.

Ca2+-transport and Ca2+-ATPase activities. Biochem J 223: 733-745 Chan K-M, Junger KD (1983) Calcium transport and phosphorylated intermediate of (Caz+ + Mg2+)-ATPase in plasma membranes of rat liver. J Biol Chem 258:4404--4410 Caroni P, Carafoli E (1981) The Caz+ pumping ATPase from heart sarcolemma. Characterization, calmodulin dependence and partial purification. J Biol Chem 256:3263-3270 Niggli V, Adunyah ES, Penniston JT, Carafoli E (1981) Purified (Ca2+ +MgZ+)-ATPase of the erythrocyte membrane. Reconstitution and effect of calmodulin and phospholipids. J Biol Chem 256:395-401 W~ithrich A, Schatzmann HJ (1980) Inhibition of the red cell calcium pump by quercetin. Cell Calcium 1:21-35 Kuriki Y, Racher E (1976) Inhibition of (Na+,K +) adenosine triphosphatase and its partial reactions by quercetin. Biochemistry 15:4951--4956 Fewtrell CMS, Gomperts BD (1977) Effect of flavone inhibitors of transport ATPase on histamine secretion from rat mast cells. Nature 265:635-636 Verma AK, Filoteo AG, Stanford DR, Wieben ED, Penniston JT, Strehler EE, Fischer R, Hiem R, Vogel G, Mathews S, Strehler-Page M-A, James P, Vorherr T, Krebs J, Carafoli E (1988) Complete primary structure of a human plasma membrane Ca2+ pump. J Biol Chem 263:14152-14159 Papp B, Sarkadi B, Enyedi A, Caride AJ, Penniston JT, Gardos G (1989) Functional domains of the in situ red cell membrane calcium pump revealed by proteolysis and monoclonal antibodies. J Biol Chem 264:4577-4582 James P, Vorherr T, Krebs J, MoreUi A, Castello G, McCormick DJ, Penniston JT, Deflora A, Carafoli E (1989) Modulation of erythrocyte CaZ+-ATPase by selective calpain cleavage of the calmodulin-binding domain. J Biol Chem 264:8289-8296 Wang KKW, Villalobo A, Roufoglas BD (1988) Activation of the CaZ+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease. Arch Biochem Biophys 260:696--704 Taverna RD, Hanahan DJ (1980) Modification of human CaZ+/ MgZ+-ATPase activity by phospholipase A2 and proteases. A comparison with calmodulin. Biochim Biophys Res Comm 94:652-659 Gietzen K, Wtithrich A, Bader H (1981) R24571: a new powerful inhibitor of red blood cell Ca + +-transport ATPase and of calmodulin-regulated function. Biochim Biophys Res Comm 101:418--425 Kraus-Friedmann N, Biber J, Murer H, Carafoli E (1982) Calcium uptake in isolated hepatic plasma-membrane vesicles. Eur J Biochem 129:7-12 Minami J, Penniston JT (1987) Caz+ uptake by corpus-luteum plasma membranes. Biochem J 242:889-894 Takuma T, Kuyatt BL, Baum BJ (1985) Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland. Biochem J 227:239-245 Ochs DL, Reed PW (1983) ATP-dependent calcium transport in plasma membrane vesicles from neutrophil leukocytes. J Biol Chem 258:10116-10122 Caride AJ, Rega AF, Garrahan PJ (1983) Effects of p-nitrophenyl phosphatase on Ca2+ transport in inside out vesicles from human red cell membranes. Biochim Biophys Acta 734:363-367 Mollman JE, Pleasure DE (1980) Calcium transporting human inside-out erythrocyte vesicles. J Biol Chem 255:56%574