Abstract
ATPases are enzymes that hydrolyze adenosine triphosphate and play a significant role in the variety of cellular functions. They produce energy through hydrolysis of ATP for mechanical functions likes protein trafficking and degradation, transport of solutes, and cellular transfers. These membrane transporters perform various essential physiological functions and are very promising drug targets. ATPase hydrolytic activity is estimated to test enzyme functionality and also provides valuable knowledge of possible inhibitory influences of molecules which obstructs hydrolytic activity. The protocol discussed here is a basic method of isolating plasma membrane and tonoplast enriched fractions along with assay of plasma membrane and tonoplast ATPase activity. Proteins hydrolyze ATP in a reaction which releases inorganic phosphate which is then measured through a colorimetric assay. This protocol is highly adjustable and can be used to estimate ATPase activity in kinetic or endpoint assays. This protocol provides a basic framework for characterization of ATPases, is rapid and can be adjusted according to requirements.
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References
Bartolommei G, Moncelli MR, Tadini-Buoninsegni F (2013) A method to measure hydrolytic activity of adenosine triphosphatases (ATPases). PLoS One 8(3):e58615. https://doi.org/10.1371/journal.pone.0058615
Sze H (1984) H+-translocating ATPases of the plasma membrane and tonoplast of plant cells. Physiol Plant 61:683–691
Sze H (1985) H+-translocating ATPases: advances using membrane vesicles. Annu Rev Plant Physiol 36:175–208
Romheld V, Moll C, Masch H (1984) Localization and capacity of proton pumps in roots of intact sunflower plants. Plant Physiol 76:603–606
Wakiuchi N, Tanimoto H, Harada H, Oji Y, Shiga H (1988) Characterization of proton pumping and ATPase activities in microsomal fractions from barley roots. Soil Sci and Plant Nut 34(4):507–518. https://doi.org/10.1080/00380768.1988.10416467
Okumura M, Kinoshita T (2016) Measurement of ATP hydrolytic activity of plasma membrane H+-ATPase from Arabidopsis thaliana leaves. BioProtocol 6(23):e2044
Janicka M, Wdowikowska A, Kłobus G (2018) Assay of plasma membrane H+-ATPase in plant tissues under abiotic stresses. Methods Mol Biol 1696:205–215. https://doi.org/10.1007/978-1-4939-7411-5_14
Shimazaki KI, Kondo N (1987) Plasma membrane H+-ATPase in guard-cell protoplasts from Vicia faba L. Plant Cell Physiol 28(5):893–900
Kinoshita T, Nishimura M, Shimazaki K (1995) Cytosolic concentration of Ca2+ regulates the plasma membrane H+-ATPase in guard cells of fava bean. Plant Cell 7(8):1333–1342
Dietz KJ, Tavakoli N, Kluge C, Mimura T, Sharma SS, Harris GC et al (2001) Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level. J Exp Bot 52(363):1969–1980
Moore TC (1981) Mineral nutrition in sunflower. In: Research experiences in plant physiology, a laboratory manual, 2nd edn. Springer-Verlag, New York
Leigh RA, Walker RR (1980a) A method for preventing sorbitol interference with the determination of inorganic phosphate. Anal Biochem 106(1):279–284
Leigh RA, Walker RR (1980b) ATPase and acid phosphatase activities associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.). Planta 150(3):222–229
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Srivastava, N. (2020). Estimation of Plasma Membrane and Tonoplast ATPase Activity in Plant Tissues. In: Gupta, N., Gupta, V. (eds) Experimental Protocols in Biotechnology. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0607-0_9
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DOI: https://doi.org/10.1007/978-1-0716-0607-0_9
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