Abstract
The inhibitor sensitivity of the endoplasmic reticulum (ER) and plasma membrane (PM) calcium pumps of red beet (Beta vulgaris L.) were studied by measuring the ATP-driven accumulation of 45Ca2+ into isolated membrane vesicles. Both transporters were strongly inhibited by 50 [mu]mol m-3 erythrosin B, but only by 50% in the presence of 100 mmol m-3 vanadate. A number of inhibitors considered to be specific for the sarcoplasmic reticulum (SR)/ER-type calcium pump in animal cells were used to further characterize the PM and ER Ca2+-ATPases in red beet and were compared with their effect on the transport and hydrolytic activities of the PM and tonoplast H+-ATPases. The hydroquinones 2,5-di(tert-butyl)-1,4-benzohydroquinone and 2,5-di(tert-amyl)-1,4-benzohydroquinone produced around 20 and 40% inhibition of activity, respectively, of the PM and ER calcium pumps and the PM H+-ATPase when present at concentrations of 30 mmol m-3. In contrast, the vacuolar proton pump displayed a much higher sensitivity to these two compounds. Nonylphenol appeared to have a general inhibitory effect on all four membrane transport proteins and gave almost complete inhibition when present at a concentration of 100 mmol m-3. Thapsigargin and the structurally related compound trilobolide produced 50% inhibition of both the ER and PM calcium pumps at concentrations of 12.5 and 24 mmol m-3, respectively. The PM and tonoplast proton pumps were also sensitive to these compounds. The ER and PM calcium pumps were almost completely insensitive to cyclopiazonic acid (CPA) up to a concentration of 20 mmol m-3. When present at 100 mmol m-3 CPA caused 30% inhibition of the transport properties of all four ATPases. The high concentrations of all of the inhibitors of the SR/ER Ca-ATPase required to inhibit the red beet ER calcium pump, together with the similar effects on the PM calcium pump and the PM and tonoplast proton pumps, suggests that these hydrophobic compounds have a general nonselective action in red beet, possibly through disruption of membrane lipid-protein interactions.
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Selected References
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- Askerlund P., Evans D. E. Reconstitution and Characterization of a Calmodulin-Stimulated Ca-Pumping ATPase Purified from Brassica oleracea L. Plant Physiol. 1992 Dec;100(4):1670–1681. doi: 10.1104/pp.100.4.1670. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Chen F. H., Ratterman D. M., Sze H. A Plasma Membrane-Type Ca2+-ATPase of 120 Kilodaltons on the Endoplasmic Reticulum from Carrot (Daucus carota) Cells (Properties of the Phosphorylated Intermediate). Plant Physiol. 1993 Jun;102(2):651–661. doi: 10.1104/pp.102.2.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Giannini J. L., Gildensoph L. H., Briskin D. P. Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue. Arch Biochem Biophys. 1987 May 1;254(2):621–630. doi: 10.1016/0003-9861(87)90145-7. [DOI] [PubMed] [Google Scholar]
- Goeger D. E., Riley R. T. Interaction of cyclopiazonic acid with rat skeletal muscle sarcoplasmic reticulum vesicles. Effect on Ca2+ binding and Ca2+ permeability. Biochem Pharmacol. 1989 Nov 15;38(22):3995–4003. doi: 10.1016/0006-2952(89)90679-5. [DOI] [PubMed] [Google Scholar]
- Hsieh W. L., Pierce W. S., Sze H. Calcium-pumping ATPases in vesicles from carrot cells : stimulation by calmodulin or phosphatidylserine, and formation of a 120 kilodalton phosphoenzyme. Plant Physiol. 1991 Dec;97(4):1535–1544. doi: 10.1104/pp.97.4.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mason M. J., Mayer B., Hymel L. J. Inhibition of Ca2+ transport pathways in thymic lymphocytes by econazole, miconazole, and SKF 96365. Am J Physiol. 1993 Mar;264(3 Pt 1):C654–C662. doi: 10.1152/ajpcell.1993.264.3.C654. [DOI] [PubMed] [Google Scholar]
- Michelangeli F., Orlowski S., Champeil P., East J. M., Lee A. G. Mechanism of inhibition of the (Ca2(+)-Mg2+)-ATPase by nonylphenol. Biochemistry. 1990 Mar 27;29(12):3091–3101. doi: 10.1021/bi00464a028. [DOI] [PubMed] [Google Scholar]
- Seidler N. W., Jona I., Vegh M., Martonosi A. Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum. J Biol Chem. 1989 Oct 25;264(30):17816–17823. [PubMed] [Google Scholar]
- Sievers A., Busch M. B. An inhibitor of the Ca(2+)-ATPases in the sarcoplasmic and endoplasmic reticula inhibits transduction of the gravity stimulus in cress roots. Planta. 1992 Nov-Dec;188(4):619–622. doi: 10.1007/BF00197057. [DOI] [PubMed] [Google Scholar]
- Wictome M., Henderson I., Lee A. G., East J. M. Mechanism of inhibition of the calcium pump of sarcoplasmic reticulum by thapsigargin. Biochem J. 1992 Apr 15;283(Pt 2):525–529. doi: 10.1042/bj2830525. [DOI] [PMC free article] [PubMed] [Google Scholar]