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Title: Biochemical and proton NMR characterization of the isolated functional beta-subunit of coupling factor one from spinach chloroplasts

Abstract

Beta subunits have been dissociated from CF1 of spinach chloroplasts, purified by HPLC and characterized by two-dimensional electrophoresis and fluorescence emission. The solutions of isolated beta subunits are able to hydrolyze MgATP; this ATPase activity is an intrinsic property of the beta molecule. From proton NMR at 300 and 500 MHz, it is shown that the preparations are fully reproducible and that beta subunits remain monomeric with 75% aliphatic protons associated with rigid parts of the molecule. The other 25% give rise to separate resonances and belong to mobile side-chains and/or to flexible regions. The measurement of the transverse relaxation times T2 has permitted a detailed characterization of the molecular dynamics of the isolated beta subunits.

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
C.E.N. Saclay, Gif Sur Yvette, France
OSTI Identifier:
6355130
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochem. Biophys. Res. Commun.; (United States); Journal Volume: 2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CHLOROPLASTS; MOLECULAR STRUCTURE; NUCLEAR MAGNETIC RESONANCE; ATP; ELECTROPHORESIS; FLUORESCENCE SPECTROSCOPY; PLANTS; PROTONS; RELAXATION TIME; SPINACH; BARYONS; CELL CONSTITUENTS; ELEMENTARY PARTICLES; EMISSION SPECTROSCOPY; FERMIONS; FOOD; HADRONS; MAGNETIC RESONANCE; NUCLEONS; NUCLEOTIDES; ORGANIC COMPOUNDS; RESONANCE; SPECTROSCOPY; VEGETABLES; 550300* - Cytology

Citation Formats

Roux-Fromy, M., Neumann, J.M., Andre, F., Berger, G., Girault, G., Galmiche, J.M., and Remy, R.. Biochemical and proton NMR characterization of the isolated functional beta-subunit of coupling factor one from spinach chloroplasts. United States: N. p., 1987. Web. doi:10.1016/S0006-291X(87)80024-4.
Roux-Fromy, M., Neumann, J.M., Andre, F., Berger, G., Girault, G., Galmiche, J.M., & Remy, R.. Biochemical and proton NMR characterization of the isolated functional beta-subunit of coupling factor one from spinach chloroplasts. United States. doi:10.1016/S0006-291X(87)80024-4.
Roux-Fromy, M., Neumann, J.M., Andre, F., Berger, G., Girault, G., Galmiche, J.M., and Remy, R.. 1987. "Biochemical and proton NMR characterization of the isolated functional beta-subunit of coupling factor one from spinach chloroplasts". United States. doi:10.1016/S0006-291X(87)80024-4.
@article{osti_6355130,
title = {Biochemical and proton NMR characterization of the isolated functional beta-subunit of coupling factor one from spinach chloroplasts},
author = {Roux-Fromy, M. and Neumann, J.M. and Andre, F. and Berger, G. and Girault, G. and Galmiche, J.M. and Remy, R.},
abstractNote = {Beta subunits have been dissociated from CF1 of spinach chloroplasts, purified by HPLC and characterized by two-dimensional electrophoresis and fluorescence emission. The solutions of isolated beta subunits are able to hydrolyze MgATP; this ATPase activity is an intrinsic property of the beta molecule. From proton NMR at 300 and 500 MHz, it is shown that the preparations are fully reproducible and that beta subunits remain monomeric with 75% aliphatic protons associated with rigid parts of the molecule. The other 25% give rise to separate resonances and belong to mobile side-chains and/or to flexible regions. The measurement of the transverse relaxation times T2 has permitted a detailed characterization of the molecular dynamics of the isolated beta subunits.},
doi = {10.1016/S0006-291X(87)80024-4},
journal = {Biochem. Biophys. Res. Commun.; (United States)},
number = ,
volume = 2,
place = {United States},
year = 1987,
month = 4
}
  • Incubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of {sup 35}SO{sub 4}{sup 2{minus}} resulted in the light-dependent formation of a chloroform-soluble sulfur-containing compound distinct from sulfolipid. The authors have identified this compound as the most stable form (S{sub 8}) of elemental sulfur (S{sup 0}, valence state for S = O) by mass spectrometry. It is possible that elemental sulfur (S{sup 0}) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S{sub 8} under the experimental conditions used for analysis.
  • The time course of light-induced O/sub 2/ exchange by isolated intact chloroplasts and cells from spinach was determined under various conditions using isotopically labeled O/sub 2/ and a mass spectrometer. In dark-adapted chloroplasts and cells supplemented with saturating amounts of bicarbonate, O/sub 2/ evolution began immediately upon illumination. However, this initial rate of O/sub 2/ evolution began immediately upon illumination. However, this initial rate of O/sub 2/ evolution was counterbalanced by a simultaneous increase in the rate of O/sub 2/ uptake, so that little net O/sub 2/ was evolved or consumed during the first approx. 1 minute of illumination. Aftermore » this induction (lag) phase, the rate of O/sub 2/ evolution increased 3- to 4-fold while the rate of O/sub 2/ uptake diminshed to a very low level. Inhibition of the Calvin cycle had negligible effects on the initial rate of O/sub 2/ evolution or O/sub 2/ uptake; both rates were sustained for several minutes, and about balanced so that no net O/sub 2/ was produced. Uncouplers had an effect similar to that observed with Calvin cycle inhibitors, except that rates of O/sub 2/ evolution and photoreduction were stimulated 40 to 50%. These results suggest that higher plant photosynthetic preparations which retain the ability to reduce CO/sub 2/ also have a significant capacity to photoreduce O/sub 2/. With near-saturating light and sufficient CO/sub 2/, O/sub 2/ reduction appears to take place primarily via a direct interaction between O/sub 2/ and reduced electron transport carriers, and occurs principally when CO/sub 2/ fixation reactions are suboptimal. The inherent maximum endogenous rate of O/sub 2/ reduction is approximately 25 to 50% of the maximum rate of noncyclic electron transport coupled to CO/sub 2/ fixation. Although the photoreduction of O/sub 2/ is coupled to ion transport and/or phosophorylation, this process does not appear to supply significant amounts of ATP directly during steady-state CO/sub 2/ fixation in strong light.« less
  • Labeled sulfur dioxide was found to be extensively absorbed by spinach (Spinacea oleracea L.) leaves. Labeled sulfides detected in leaf blades following fumigations with sulfur dioxide in light indicated that photoreduction of sulfur dioxide had occurred. Measurable proportions of this labeled sulfur was localized within the chloroplast fraction. Suspensions of isolated chloroplasts supplied with labeled sulfur dioxide contained labeled sulfides following a 30-minute illumination period in water-cooled reaction vessels. With reference to recent studies of the chloroplast sulfur reduction pathway, probable points of entry for sulfur dioxide and the subsequent release of hydrogen sulfide are discussed. 21 references, 2 tables.
  • Penetration of ozone into spinach chloroplasts and some of the physiological effects of the oxidant have been studied. Ozone fumigation of class II coupled and uncoupled plastids showed that 9 x 10/sup -7/ moles O/sub 3/ reduced the Hill reaction by 36% and 43% respectively though 4.4 x 10/sup -7/ moles produced no reduction in coupled plastids and 41% reduction in uncoupled ones. The oxidant did not uncouple the coupled chloroplasts indicating that gross membrane permeability changes had not taken place. Both light-induced absorbance and volume changes were reduced by the oxidant suggesting that more specific damage had been causedmore » as compared to general membrane disruption. It was found that ozone reduced CO/sub 2/ stimulated evolution of O/sub 2/ though ozonation and subsequent rupturing of class I plastids indicated that the oxidant did not penetrate the stroma to inhibit the photosystems. These data indicate that ozone at the concentrations used does not indiscriminately disrupt the membrane system and produce general increased permeability.« less