Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0

Siegfried Engelbrecht, H Lill, Wolfgang Junge

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].

Original languageEnglish
Pages (from-to)635-43
Number of pages9
JournalEuropean Journal of Biochemistry
Volume160
Issue number3
DOIs
Publication statusPublished - 3 Nov 1986

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Chloroplast Proton-Translocating ATPases
Thylakoids
Edetic Acid
Dicyclohexylcarbodiimide
Protons
Spinacia oleracea
Peas
Membranes
Zea mays
Alcohols
Photophosphorylation
Delta functions
Chloroplasts
Chromatography
Washing
Surface-Active Agents
Anions
Ion exchange
Buffers
High Pressure Liquid Chromatography

Keywords

  • Chloroplasts
  • Immunoelectrophoresis
  • Kinetics
  • Macromolecular Substances
  • Peptide Fragments
  • Plants
  • Proton-Translocating ATPases
  • Protons
  • Species Specificity
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

@article{e4e31bb1cc0b436b8e4f0236d9e0974c,
title = "Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0",
abstract = "Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].",
keywords = "Chloroplasts, Immunoelectrophoresis, Kinetics, Macromolecular Substances, Peptide Fragments, Plants, Proton-Translocating ATPases, Protons, Species Specificity, Journal Article, Research Support, Non-U.S. Gov't",
author = "Siegfried Engelbrecht and H Lill and Wolfgang Junge",
year = "1986",
month = "11",
day = "3",
doi = "10.1111/j.1432-1033.1986.tb10085.x",
language = "English",
volume = "160",
pages = "635--43",
journal = "European Journal of Biochemistry",
issn = "0014-2956",
publisher = "Wiley-Blackwell",
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}

Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0. / Engelbrecht, Siegfried; Lill, H; Junge, Wolfgang.

In: European Journal of Biochemistry, Vol. 160, No. 3, 03.11.1986, p. 635-43.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0

AU - Engelbrecht, Siegfried

AU - Lill, H

AU - Junge, Wolfgang

PY - 1986/11/3

Y1 - 1986/11/3

N2 - Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].

AB - Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].

KW - Chloroplasts

KW - Immunoelectrophoresis

KW - Kinetics

KW - Macromolecular Substances

KW - Peptide Fragments

KW - Plants

KW - Proton-Translocating ATPases

KW - Protons

KW - Species Specificity

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1111/j.1432-1033.1986.tb10085.x

DO - 10.1111/j.1432-1033.1986.tb10085.x

M3 - Article

VL - 160

SP - 635

EP - 643

JO - European Journal of Biochemistry

JF - European Journal of Biochemistry

SN - 0014-2956

IS - 3

ER -