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. 1994 Oct;106(2):731–737. doi: 10.1104/pp.106.2.731

Direct Measurement of ATP-Dependent Proton Concentration Changes and Characterization of a K+-Stimulated ATPase in Pea Chloroplast Inner Envelope Vesicles.

R Shingles 1, R E McCarty 1
PMCID: PMC159581  PMID: 12232365

Abstract

Inner envelope membrane vesicles prepared from pea (Pisum sativum L. var Laxton's Progress No. 9) chloroplasts have K+-stimulated ATPase activity with a pH optimum of 8.4. ATP addition to inner envelope vesicles loaded with pyranine caused a decrease in pyranine fluorescence that was consistent with internal acidification. The transmembrane pH change induced by the addition of 5 mM ATP was about 0.4 unit. Measurement of phosphate released by ATP hydrolysis paralleled the pH change, indicating that intravesicular acidification was linked to ATPase activity. Vanadate, molybdate, N-ethylmaleimide, and dithiothreitol inhibited ATP-dependent vesicle acidification completely, whereas ATPase activity was only partially inhibited. These data indicate that pea chloroplast inner envelope vesicles contain a proton translocating ATPase and that the pyranine-loading method can be utilized to study directly ATP-dependent H+ transport across these membranes.

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Selected References

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  1. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  2. Berkowitz G. A., Peters J. S. Chloroplast Inner-Envelope ATPase Acts as a Primary H+ Pump. Plant Physiol. 1993 May;102(1):261–267. doi: 10.1104/pp.102.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Biegel C. M., Gould J. M. Kinetics of hydrogen ion diffusion across phospholipid vesicle membranes. Biochemistry. 1981 Jun 9;20(12):3474–3479. doi: 10.1021/bi00515a026. [DOI] [PubMed] [Google Scholar]
  4. Briskin D. P., Poole R. J. Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet. Plant Physiol. 1983 Feb;71(2):350–355. doi: 10.1104/pp.71.2.350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chow D. C., Browning C. M., Forte J. G. Gastric H(+)-K(+)-ATPase activity is inhibited by reduction of disulfide bonds in beta-subunit. Am J Physiol. 1992 Jul;263(1 Pt 1):C39–C46. doi: 10.1152/ajpcell.1992.263.1.C39. [DOI] [PubMed] [Google Scholar]
  6. Douce R., Holtz R. B., Benson A. A. Isolation and properties of the envelope of spinach chloroplasts. J Biol Chem. 1973 Oct 25;248(20):7215–7222. [PubMed] [Google Scholar]
  7. Howitz K. T., McCarty R. E. Measurement of proton-linked transport activities in pyranine loaded chloroplast inner envelope vesicles. Plant Physiol. 1988 Apr;86(4):999–1001. doi: 10.1104/pp.86.4.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Maury W. J., Huber S. C., Moreland D. E. Effects of Magnesium on Intact Chloroplasts : II. CATION SPECIFICITY AND INVOLVEMENT OF THE ENVELOPE ATPase IN (SODIUM) POTASSIUM/PROTON EXCHANGE ACROSS THE ENVELOPE. Plant Physiol. 1981 Dec;68(6):1257–1263. doi: 10.1104/pp.68.6.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. McCarty D. R., Keegstra K., Selman B. R. Characterization and localization of the ATPase associated with pea chloroplast envelope membranes. Plant Physiol. 1984 Nov;76(3):584–588. doi: 10.1104/pp.76.3.584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Robinson S. P., Wiskich J. T. Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in exchange for endogenous adenine nucleotides. Plant Physiol. 1977 Mar;59(3):422–427. doi: 10.1104/pp.59.3.422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Sabnis D. D., Gordon M., Galston A. W. Localization of adenosine triphosphatase activity on the chloroplast envelope in tendrils of Pisum sativum. Plant Physiol. 1970 Jan;45(1):25–32. doi: 10.1104/pp.45.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Smolarsky M., Teitelbaum D., Sela M., Gitler C. A simple fluorescent method to determine complement-mediated liposome immune lysis. J Immunol Methods. 1977;15(3):255–265. doi: 10.1016/0022-1759(77)90063-1. [DOI] [PubMed] [Google Scholar]
  13. Villalba J. M., Palmgren M. G., Berberián G. E., Ferguson C., Serrano R. Functional expression of plant plasma membrane H(+)-ATPase in yeast endoplasmic reticulum. J Biol Chem. 1992 Jun 15;267(17):12341–12349. [PubMed] [Google Scholar]
  14. Young X. K., McCarty R. E. Assay of Proton-Coupled Glycolate and D-Glycerate Transport into Chloroplast Inner Envelope Membrane Vesicles by Stopped-Flow Fluorescence. Plant Physiol. 1993 Mar;101(3):793–799. doi: 10.1104/pp.101.3.793. [DOI] [PMC free article] [PubMed] [Google Scholar]

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