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Animal Sarcoplasmic/Endoplasmic Reticulum-Type Calcium Pumps on the Primary Ca*+-Atpases of Red Beet

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Animal Sarcoplasmic/Endoplasmic Reticulum-Type Calcium Pumps on the Primary Ca*+-Atpases of Red Beet Plant Physiol. (1994)104: 1295-1300 A Study of the Effect of lnhibitors of the Animal Sarcoplasmic/Endoplasmic Reticulum-Type Calcium Pumps on the Primary Ca*+-ATPases of Red Beet linda J.Thomson, J. 1. Hall, and lorraine E. Williams'* Department of Biology, Biomedical Sciences Building, University of Southampton, Downloaded from https://academic.oup.com/plphys/article/104/4/1295/6067429 by guest on 29 September 2021 Southampton, SO9 3TU, United Kingdom 10 pmol mg-', it has little effect on the,hydrolysis of ATP by The inhibitor sensitivity of the endoplasmic reticulum (ER) and the Na+,K+-ATPase, the H+,K+-ATPase, the mitochondrial plasma membrane (PM) calcium pumps of red beet (Beta vulgaris FI-ATPase, and the erythrocyte Ca2+-ATPase(Seidler et al., 1.) were studied by measuring the ATP-driven accumulation of 1989). CPA is believed to inhibit the conformational transi- "CaZ+ into isolated membrane vesicles. Both transporters were tion of the enzyme between the E1 and E2 states (Seidler et strongly inhibited by 50 pmol m-3 erythrosin 6, but only by 50% in al., 1989). It prevents the binding of Ca2+to the high-affinity the presence of 100 mmol m-3 vanadate. A number of inhibitors site of the SR calcium pump and also inhibits Ca2+-dependent considered to be specific for the sarcoplasmic reticulum (SR)/ER- formation of phosphoenzyme (Goeger and Riley, 1989). The type calcium pump in animal cells were used to further characterize the PM and ER CaZ+-ATPasesin red beet and were compared with hydrophobic reagents nonylphenol, BHQ, AHQ, thapsigar- their effect on the transport and hydrolytic activities of the PM and gin, and trilobolide are also potent inhibitors of the SR tonoplast H+-ATPases. The hydroquinones 2,5-di(tert-butyl)-l,4- calcium pump and are thought to interact with the Et con- benzohydroquinoneand 2,5-di(tert-amyl)-l,4-benzohydroquinone formation of the ATPase (Wictome, 1992). However, al- produced around 20 and 40% inhibition of adivity, respectively, though the effects of CPA and these other inhibitors have of the PM and ER calcium pumps and the PM H+-ATPase when been well characterized in animal systems and are used present at concentrationsof 30 mmol m-3. In contrast, the vacuolar routinely as specific inhibitors of the SR/ER calcium pump, proton pump displayed a much higher sensitivity to these two their effects on plant calcium transporters have not been compounds. Nonylphenol appeared to have a general inhibitory investigated thoroughly. effect on all four membrane transport proteins and gave almost The aim of this study was to evaluate the possible specific- complete inhibition when present at a concentration of 100 mmol ity of action of these compounds in a higher plant tissue, red m-3. Thapsigargin and the structurally related compound trilobol- ide produced 50% inhibition of both the ER and PM calcium pumps beet (Beta vulgarzs L.) storage roots. If there is sufficient at concentrations of 12.5 and 24 mmol m-3, respectively. The PM conservation of structure and function between the animal and tonoplast proton pumps were also sensitive to these com- and plant Ca2+-ATPases,the animal SR/ER and plant ER pounds. The ER and PM calcium pumps were almost completely calcium pumps may show selective sensitivity to the same insensitive to cyclopiazonic acid (CPA) up to a concentration of 20 compounds. Most importantly, the inhibitors have not yet mmol m-3. When present at 100 mmol m-3 CPA caused 30% been used in the study of the higher plant ER and PM calcium inhibition of the transport properties of all four ATPases. The high pumps using ER- and PM-enriched fractions, which show concentrations of all of the inhibitors of the SR/ER Ca-ATPase almost negligible cross-contamination. The emergence of a required to inhibit the red beet ER calcium pump, together with marker specific for the plant ER calcium pump, such as the similar effects on the PM calcium pump and the PM and inhibitor sensitivity, would facilitate the biochemical charac- tonoplast proton pumps, suggests that these hydrophobic com- terization, the solubilization and reconstitution, and ultimate pounds have a general nonselective action in red beet, possibly through disruption of membrane lipid-protein interactions. purification of the enzyme. Likewise, the use of such inhibi- tors in vivo may provide greater understandmg of the signal transduction pathways involved in physiological responses (Sievers and Busch, 1992), since they could be used to selec- The ER and PM E1E2-typecalcium pumps of higher plants tively interfere with the ER store of Ca2+. are responsible, in part, for the regulation of cytosolic calcium concentrations. Since they share a number of biochemical MATERIALS AND METHODS and functional characteristics, it is difficult to discriminate between them (Evans et al., 1991; Thomson et al., 1993). Plant Material Recently, it has been proposed that CPA, an indole tetramic Red beet (Betn vulgnris L.) roots were obtained either acid mycotoxin, acts as a specific inhibitor of the carrot ER commercially or harvested from the field. The tops of the calcium pump (Hsieh et al., 1991). CPA is a specific inhibitor of the SR/ER Ca2+-ATPasebut at concentrations as high as Abbreviations: AHQ,2,5-di(tert-amyl)-l,4-benzohydroquinone; BHQ,2,5-di(tert-butyl)-1,4-benzohydroquinone;BTP, 1,3-bis[tris- ' L.E.W. is a Royal Society Research Fellow. (hydroxymethy1)-methylaminolpropane;CPA, cyclopiazonic acid; * Corresponding author; fax 44-703-594269 or 44-703-594319. EB, erythrosin 8; PM, plasma membrane; SR, sarcoplasmic reticulum. 1295 1296 Thomson et al. Plant Physiol. Vol. 104, 1994 plants were removed and the roots were stored in moist m-3 KI added fresh to the homogenization medium. Proton vermiculite at 4OC until use. A11 root tissue was stored for at pumping in this microsomal fraction was not inhibited by least 10 d to ensure uniformity in membrane isolation. nitrate but was vanadate sensitive (see Table I). Membrane lsolation Enzyme and Transport Assays Microsomal fractions, Suc gradient ER- and tonoplast- ATPase Activity enriched fractions, and phase-partitioned PM fractions were prepared as previously described, whereas the final ER frac- The ATP-hydrolytic activities of the PM and Suc gradient- tion, used in calcium transport assays, was prepared by preparecl tonoplast-enriched fractions were assayed as pre- viously described (Williams et al., 1990). The PM H+-ATPase subjecting the SUCgradient ER-enriched fraction to two- Downloaded from https://academic.oup.com/plphys/article/104/4/1295/6067429 by guest on 29 September 2021 phase aqueous partitioning and collecting the lower phase hydrolylic activity was determined at pH 6.5, vvhereas the (Thomson et al., 1993). tonoplast H+-ATPase activity was measured at pH 8.0 in the presence of 1 mo1 m-3 NaN3 and 100 mo1 m-3 vanadate. Under these conditions ATPase activity within the frac- Microsomal Fractions for Proton Transport Measurements PM tion was almost completely inhibited by 100 mo1 m-3 vana- Proton transport activity due to the tonoplast H+-ATPase date (Thomson et al., 1993), and for the tonoplast fraction was measured using a microsomal fraction prepared as de- more than 90% of ATPase activity was inhibited by 100 mo1 scribed above. Proton pumping within this fraction showed me3 KN03 when included in the assay medium in the place no vanadate sensitivity (PM H+-ATPase marker), but was of KCl (see Table I). A11 assays were performed in triplicate highly sensitive to nitrate (tonoplast H+-ATPase marker) (see and the SE values within each experiment were less than 10% Table I). To measure the proton transport activity due to the of the means. The results shown are the means of at least PM H+-ATPase, the microsomal fraction was prepared ac- two experiments. The curve in Figure 2 has been fitted by cording to the method of Giannini et al. (1987) with 250 mo1 eye. Table 1. The effects of various SRIER-type Ca2+-ATPaseinhibitors on the transport properties of the ER and PM calcium pumps, and on the transport and hydrolytic activities of the PM and tonoplast H+-ATPases in red beet Assays were performed as described in “Materials and Mei.hods.”ATP-driven uptake into ER and PM fractions was measured over a 10-min incubation period at 22”C, whereas ATPase activity within the PM and tonoplast-enriched fractions was measured over a 1-h incubation period at 37°C. The inhibitors were included at the indicated concentrations. Results are expressed as the percentage of inhibition of control activity measured in the absence of any inhibitor, but in the presence of 1% (v/ v) DMSO or methanol, as appropriate. PM Ca2+ ER Ca2+ PM Tonoplast PM H+ Tonoplast H+ lnhibitor Transport Transport ATPase ATPase Transport Transport % lnhibition EB 50 kmol m-’. 73 70 6 +11 3 2 Nonylphenol 25 mmol m-’ 54 37 69 79 50 73 100 mmol m-’ 95 90 95 n.d.“ 98 83 BHQ 30 mmol m-3 9 23 15 60 17 83 AHQ 30 mmol m-3 42 44 45 92 31 97 Thapsigargin 0.2 mmol m-’ 9 +7 6 22 n.d. 8 30 mmol m-’ 48 81 75 88 70 61 Trilobolide 30 mmol m-’ 57 70 20 39 43 50 100 mmol m-’ 1 O0 97 67 91 93 97 C PA 20 mmol m-’ 7 3 n.d. n.d. n.d. n.d. 100 mmol m-’ 27 36 2 1 32 22 Vanadate 200 mmol m-’ 61 69 95 1 55 O Nitrate 100 mo1 m-’ n.d. n.d. O 94 +40 99 a n.d., Not determined. Calcium ATPase lnhibitors and Plant Membrane Pumps 1297 Proton Transport Proton transport was determined by measuring quinacrine fluorescence quenching as previously described (Williams and Hall, 1989).
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